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WO2025232118A1 - Fused ring compound as well as preparation method therefor and use thereof - Google Patents

Fused ring compound as well as preparation method therefor and use thereof

Info

Publication number
WO2025232118A1
WO2025232118A1 PCT/CN2024/129466 CN2024129466W WO2025232118A1 WO 2025232118 A1 WO2025232118 A1 WO 2025232118A1 CN 2024129466 W CN2024129466 W CN 2024129466W WO 2025232118 A1 WO2025232118 A1 WO 2025232118A1
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WO
WIPO (PCT)
Prior art keywords
alkyl
seq
ala
cdr
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/129466
Other languages
French (fr)
Chinese (zh)
Inventor
田强
张毅涛
叶健
刘万里
李怡乐
宋攀
周琴
龙虎
杨禹
袁晓曦
宋宏梅
葛均友
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Original Assignee
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
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Filing date
Publication date
Application filed by Sichuan Kelun Biotech Biopharmaceutical Co Ltd filed Critical Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Publication of WO2025232118A1 publication Critical patent/WO2025232118A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application relates to the pharmaceutical field, specifically to a fused-ring compound and its preparation method and uses.
  • Antibody-drug conjugates (ADCs) for cancer treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a cytotoxic agent that kills tumor cells), and a linker.
  • the bioactive molecule is covalently coupled to the antibody via the linker.
  • the antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells.
  • the ADC then enters the cancer cells via endocytosis.
  • the bioactive molecule is then released within the cancer cells, killing them by inhibiting microtubules or damaging their DNA, thereby minimizing damage to normal tissue cells.
  • Pyrrolobenzodiazepine PBD protonated bromine
  • PBD dimer is a sequence-selective DNA minor groove binder and a member of the anthraxmycin family of antibiotics.
  • This PBD dimer exhibits pmol/L activity in various tumor cell lines.
  • This application relates to a fused-ring compound having the structure shown in the general formula D-E-L-M'.
  • the fused-ring compound can be used to prepare antibody-drug conjugates, and the conjugates exhibit excellent targeted killing effects against tumors.
  • this application provides a compound or a pharmaceutically acceptable salt thereof having the structure shown in formula D-E-L-M’, wherein:
  • M' is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site;
  • L is the structural segment connecting M and E;
  • E is a structural segment connecting L and D;
  • D is a cytotoxic drug fragment.
  • M is selected from the following substituted or unsubstituted structural segments:
  • M is selected from the following substituted or unsubstituted structural segments:
  • M is selected from the following substituted or unsubstituted structural segments:
  • Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxy (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments.
  • halogens e.g., F, Cl, Br, I
  • the halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, and halogenated C1-6 alkyl sulfonyl groups are further specified.
  • the ester group, C1-6 alkyl sulfinate group, C1-6 alkyl sulfoxide group, halophenoxy group, alkenyl group, alkynyl group and alkynyl-containing structural segments are optionally replaced by one or more suitable substituents.
  • Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkyl sulfonyl, halogenated C1-6 alkyl sulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl , and alkynyl-containing structural fragments.
  • halogens e.g., F, Cl, Br, I
  • Lg is selected from halogens, substituted or unsubstituted C1-6 alkylsulfonyl groups, halophenoxy groups, hydroxyl groups (-OH), mercapto groups (-SH), or amino groups ( -NH2 ).
  • Lg is selected from halogen, substituted or unsubstituted methanesulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH) or amino ( -NH2 ).
  • Lg is selected from methanesulfonyl.
  • L is selected from one or more of the following substituted or unsubstituted structural fragments or stereoisomers thereof: C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic, -N(R')-, -NH(R'), -N(R') 2 , carbonyl, -O-, natural or non-natural amino acids and their analogs (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( COCH2CH2 ( OCH2CH2 ) rOCH3 ) (), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro
  • R' is composed of one or more of the following groups, including but not limited to Hydrogen, C1-6 alkyl, C
  • DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues
  • DOTAGA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, ⁇ -propionyl
  • NOA 1,4,7-triazacyclononane-N,N',N”-triacetic acid residues
  • L is selected from one or more substituted or unsubstituted structural fragments composed of: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( COCH2CH2 ( OCH2CH2 ) rOCH3 )) , Lys(R'), and short peptides composed of amino acids (e.g.,
  • R' is composed of one or more of the following groups, including but not limited to hydrogen, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, ⁇ -propionyl), or NOA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), wherein s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s
  • L is selected from one or more substituted or unsubstituted structural fragments composed of: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), and short peptides composed of amino acids (e.g., Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala).
  • natural or non-natural amino acids and their analogues e.g., Ala, Arg, Asn
  • r is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;
  • s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, 9, or 10.
  • L is selected from one or more of the following substituted or unsubstituted structural fragments: Val, Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala, Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.
  • L is selected from one or more of the following substituted or unsubstituted structural segments: Val, Such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, 9, or 10.
  • L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • L is selected from the following substituted or unsubstituted structures:
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • E is a single bond, substituted or unsubstituted -NH- CH2- or
  • E is a single bond, substituted or unsubstituted -NH- CH2- .
  • E is a single bond or -NH- CH2- .
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • the cytotoxic agent is selected from anti-microtubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors.
  • the anti-microtubule agent is an oliguriatin class, a maytansine class, or an eribulin class.
  • the DNA intercalating agent is a pyrrolobenzodiazepine (PBD) class, trabectedin, or rubotecan.
  • the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecone, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide).
  • the RNA polymerase inhibitor is ⁇ -amanitin.
  • the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogs.
  • the cytotoxic drug is selected from pyrrolobenzodiazepine (PBD) compounds.
  • the cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino ( -NH2 ), secondary amino ( -NRaH ), and tertiary amino ( -NRbRc ), where Ra , Rb , and Rc represent only non-hydrogen substituents on N, or thiol (-SH). These functional groups can react with suitable functional groups in the remainder of the conjugate to achieve linkage.
  • functional groups such as hydroxyl (-OH), carboxyl (-COOH), primary amino ( -NH2 ), secondary amino ( -NRaH ), and tertiary amino ( -NRbRc ), where Ra , Rb , and Rc represent only non-hydrogen substituents on N, or thiol (-SH).
  • the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary amino, or tertiary amino group, or a -SH group.
  • D is a monovalent structure obtained by losing an H from the -OH, -NH2 , or secondary amino group on the cytotoxic drug.
  • D is a monovalent structure obtained by losing an H from the -OH or -NH2 group on the cytotoxic drug.
  • the cytotoxic drug is selected from compounds with the structure shown in formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs thereof:
  • R1 and R2 are each independently selected from hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl; wherein the alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl groups;
  • R4 and R4 ' are each independently selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halo- C1-6 alkyl and C1-6 alkoxy, and R5 and R5 ' are both hydrogen;
  • R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms;
  • R4 and R5 may be bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5 , and R5 ' and R4 ' may be independently selected from hydrogen, halogen, -NH2, C1-6 alkyl, halo -C1-6 alkyl and C1-6 alkoxy;
  • R6 is hydrogen, C1-6 alkyl, or...
  • R6 ' , R7 , and R7 ' are all hydrogen; or R6 ' is bonded to R7 , and R7 ' is hydrogen.
  • Ra is selected from H, C1-6 alkyl, or C3-6 cycloalkyl
  • Ring A is selected from 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups; the 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups may optionally be substituted by one or more of the following substituents: hydrogen, - CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen,
  • R3 is selected from hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;
  • X and Y are independently -CH 2- and -CD 2- , respectively. and -C(O)-;
  • t is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • R3 is not methoxy
  • R1 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is selected from C1-6 alkyl, deuterated C1-6 alkyl, and C3-6 cycloalkyl.
  • R1 is selected from -ORa , wherein Ra is selected from C1-6 alkyl, deuterated C1-6 alkyl and C3-6 cycloalkyl.
  • R1 is selected from methoxy, trideuteroxy, and -O-cyclopropyl.
  • R1 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is a C1-6 alkyl or C3-6 cycloalkyl.
  • R1 is -ORa , where Ra is a C1-6 alkyl or C3-6 cycloalkyl.
  • R1 is methoxy or -O-cyclopropyl.
  • R2 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is a C1-6 alkyl or C3-6 cycloalkyl.
  • R2 is -ORa , where Ra is a C1-6 alkyl group.
  • R2 is a methoxy group.
  • R4 is hydrogen or a halogen
  • R4 ' is selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halogenated C1-6 alkyl and C1-6 alkoxy, R5 and R5 ' are both hydrogen; or, R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ', R5 and R5 ' are both hydrogen; or, R4 and R5 are bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5 , R5 ' and R4 ' are both hydrogen.
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , methyl, trifluoromethyl, and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5' are both hydrogen
  • R4 and R5 are bonded together or together form a 3 membered carbon ring with the carbon atom connecting R4 and R5 , and R5' and R4 ' are both hydrogen.
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , methyl, trifluoromethyl, and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' together form a carbon-carbon double bond with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5 , and R5' and R4 ' are both hydrogen.
  • R4 and R4 ' form a ternary carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms.
  • R6 is
  • Ring A is selected from 3-6 membered cycloalkyl, C6-10 aryl, or 5-10 membered heteroaryl;
  • R3 is selected from hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;
  • Ra is selected from H and C1-6 alkyl groups.
  • R6 is
  • Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;
  • R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.
  • R6 is
  • Ring A is phenyl
  • R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.
  • ring A is C6-10 aryl or 5-10 heteroaryl.
  • ring A is selected from phenyl, pyrimidinyl, pyridinyl, furanyl, and thiophenyl.
  • R3 is selected from -OH, methoxy, -NH2 and hydroxymethyl.
  • R6 is selected from phenyl, pyridyl,
  • R6 is selected from phenyl, pyridyl,
  • R6 ' and R7 are bonded together, and R7 ' is hydrogen.
  • R6 is
  • R6 ' , R7 and R7 ' are all hydrogen.
  • t is 3-8, such as 3, 4, 5, 6, 7 or 8.
  • X is -CH2- or -CD2- ; optionally, Y is selected from -CH2- , -CD2- , And -C(O)-.
  • X is -CH2- and Y is -CH2- ;
  • X is -CH2- and Y is -CD2- or -C(O)-; or
  • X is -CD2- and Y is -CH2- .
  • X is -CH 2 - and Y is -CH 2 -.
  • R1 is selected from methoxy, trideuteroxy, and -O-cyclopropyl
  • R2 is a methoxy group
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , trifluoromethyl and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' form a carbon-carbon double bond or a 3-membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5
  • R5 ' and R4 ' are both hydrogen.
  • Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;
  • R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl;
  • R1 and R2 are both methoxy groups
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , trifluoromethyl and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' form a carbon-carbon double bond or a 3-membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5
  • R5 ' and R4 ' are both hydrogen.
  • the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
  • the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
  • the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
  • the cytotoxic drug fragment (D) has the structure shown in formula (I'):
  • rings A, R1 , R2 , R4 , R4' , R5 , R5' , R6' , R7 , R7 ' , X, Y, and t are as described in any of the preceding items;
  • R 3A is selected from chemical bonds, -NH-, -N(C 1-6 alkyl)-, -O-, -C 1-6 alkylene-O-, and -C 1-6 alkylene-O-.
  • the cytotoxic drug fragment (D) has the structure shown below:
  • the compound has the following structure:
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • a second aspect of the invention provides a coupling as shown in formula (II), wherein:
  • M, L, E, and D are as described in any of the preceding items; x is 1-10;
  • A represents the target group, whose targets are selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, and O.
  • A is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.
  • a small molecule ligand such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.
  • A represents an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies (e.g., scFv), non-human antibodies, humanized antibodies, chimeric antibodies, fully human antibodies, and proboscis. Bispecific antibodies or multispecific antibodies.
  • A is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase family.
  • the antibody-drug conjugates described in this application can be prepared in a modular manner.
  • the compound M'-LED i.e., the free form of the "drug-linker" described in any of the above-mentioned claims can be obtained first, wherein M' is the structural form of M before covalently linking it with an antibody or its antigen-binding fragment, and then it can be covalently linked with the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.
  • M' is linked to one or more thiol (-SH) or amino ( -NH2 ) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me or pentafluorophenol structure) or an addition reaction.
  • a substitution reaction e.g., removal of the -SO2Me or pentafluorophenol structure
  • -MLED can be formed from the following structure, for example, through a substitution reaction (e.g., removal of the methanesulfonyl group structure).
  • the antibody-drug conjugates of this application are formed by conjugating the compounds A-1 to A-9, B-1 to B-19 or G-1 to G-16 described above with an antibody or antigen-binding fragment.
  • the antibody-drug conjugate of this application is formed by desaturating the methylsulfonyl group of the compounds A-1 to A-9, B-1 to B-19 or G-1 to G-16 described above and then conjugating them with an antibody or antigen-binding fragment.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in either (1a) or (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or the variant having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originated; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH Heavy chain variable region
  • VL Light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with sequence SEQ ID NO:34 or a variant thereof
  • CDR-H3 with sequence SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • CDR-H2 with sequence SEQ ID NO:14 or a variant thereof
  • CDR-H2 with sequence SEQ ID NO:15 or a variant thereof.
  • CDR-H3 and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:30, CDR-H3 of SEQ ID NO:30, CDR-H1 ... CDR-L2 of SEQ ID NO:25 has the sequence CDR-L3.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment comprises:
  • the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the antibody or its antigen-binding fragment comprises:
  • the antibody or its antigen-binding fragment comprises:
  • the antibody or its antigen-binding fragment comprises:
  • the antibody or its antigen-binding fragment further comprises:
  • the light chain constant region (CL) of human immunoglobulin or a variant thereof which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).
  • the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.
  • the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.
  • CH heavy chain constant region
  • the antibody or its antigen-binding fragment includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.
  • the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.
  • CL light chain constant region
  • the antibody or its antigen-binding fragment includes a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibody or its antigen-binding fragment comprises:
  • a heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or
  • a heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.
  • the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT/mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT/mAb-DB ID) of 80.
  • the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide.
  • the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.
  • pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.
  • compositions comprising the antibody or antigen-binding fragment disclosed herein, wherein Various antibody or antigen-binding fragments may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine, and/or contain N-terminal glutamine or glutamate, N-terminal amino acid cyclized to pyroglutamic acid, or N-terminal amino acid cyclized to pyroglutamate salt.
  • the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification/storage.
  • post-translational modifications thereof e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain
  • the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39 undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.
  • the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine residue.
  • the antibody-drug conjugate is selected from:
  • HA-(S- or) in each antibody-drug conjugate It is an antibody or its antigen-binding fragment
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH Heavy chain variable region
  • VL Light chain variable region
  • CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof
  • CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof
  • CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in either (2a) or (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has at least 70%, at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has at least 70%, at least 80%, at least 85%, at least 90 ...
  • it has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • CDR-H2 with sequence SEQ ID NO:29 or a variant thereof
  • CDR-H2 with sequence SEQ ID NO:30 or a variant thereof
  • CDR-H3 and/or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:25 or a variant thereof;
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:5, CDR-H2 with sequence SEQ ID NO:6, and CDR-H3 with sequence SEQ ID NO:7; and/or, comprising the following three The light chain variable region (VL) of each CDR: CDR-L1 with sequence SEQ ID NO:8, CDR-L2 with sequence SEQ ID NO:9, and CDR-L3 with sequence SEQ ID NO:10; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the HA in each antibody-drug conjugate includes:
  • the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the HA in each antibody-drug conjugate includes:
  • the HA in each antibody-drug conjugate includes:
  • the HA in each antibody-drug conjugate includes:
  • the HA in each antibody-drug conjugate further comprises:
  • the light chain constant region (CL) of human immunoglobulin or a variant thereof which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).
  • the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.
  • the HA in each antibody-drug conjugate contains a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions of amino acids compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conserved substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conserved substitutions of amino acids).
  • CH heavy chain constant region
  • the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.
  • the HA in each antibody-drug conjugate contains a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions of amino acids compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conserved substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conserved substitutions of amino acids).
  • CL light chain constant region
  • the HA in each antibody-drug conjugate includes a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • CH heavy chain constant region
  • CL light chain constant region
  • the HA in each antibody-drug conjugate includes:
  • a heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or
  • a heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.
  • the HA in each antibody-drug conjugate is selected from Trastuzumab or Pertuzumab, wherein the amino acid sequence of Trastuzumab has a lookup accession number (IMGT/mAb-DB ID) of 97 in the IMGT database, and the amino acid sequence of Pertuzumab has a lookup accession number (IMGT/mAb-DB ID) of 80 in the IMGT database.
  • the HA in each antibody-drug conjugate may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine and/or contain N-terminal glutamine or glutamate, cyclize the N-terminal amino acid to pyroglutamic acid or cyclize the N-terminal amino acid to pyroglutamate salt.
  • the HA in each antibody-drug conjugate may include its post-translational modifications (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate in the heavy or light chain being converted to pyroglutamate or pyroglutamate salt), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification/storage.
  • post-translational modifications e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate in the heavy or light chain being converted to pyroglutamate or pyroglutamate salt
  • the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39 undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.
  • the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine residue.
  • the HA in each antibody-drug conjugate represents trastuzumab, pertuzumab, or their antigen-binding fragments.
  • the HA in each antibody-drug conjugate represents the following antibody or antigen-binding fragment:
  • a heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or
  • x is 1-10, for example 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.
  • x is 5-8, such as 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.
  • x is 1-6, such as 2-6, 3-6, 4-6.
  • x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the antibody-drug conjugates of the present invention may optionally be substituted with one or more suitable substituents.
  • compositions of antibody-drug conjugates as described herein.
  • Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10.
  • DAR drug-antibody ratio
  • the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.
  • the DAR of the ADC compositions described herein is about 3 to 9, such as about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5 to 6, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.
  • 6.0 approximately 4.0 to 6.5, approximately 4.0 to 7.0, approximately 4.0 to 8.0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 7.5, approximately 5.0 to 8.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.5, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 7.5, approximately 6.5 to 8.5, approximately 7.0 to 7.5.
  • the DAR of the ADC composition described herein is about 3 to 8, for example, about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0, or about 7.5 to 8.0.
  • the DAR of the ADC composition described herein is 7.5 to 8.5, for example, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5.
  • the DAR of the ADC composition described herein is about 6.0 to 9.0, preferably about 6.0-8.0, for example about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.09, about 6.10, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19 ...
  • this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates or compositions described in any of the preceding claims, and one or more pharmaceutical excipients.
  • the antibody-drug conjugates or compositions described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A.Ed.).
  • the antibody-drug conjugates described herein or pharmaceutical compositions containing said antibody-drug conjugates can be administered via any route appropriate for the individual to be treated.
  • the antibody-drug conjugates, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.
  • Her2-expressing cancers including solid tumors or hematologic malignancies such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.
  • this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of a medicament for treating Her2-expressing cancers.
  • this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the conjugates described above in a medicament for treating Her2-expressing cancer.
  • This application also provides a method for treating Her2-expressing cancers, comprising administering [treatment] to a subject in need of [treatment].
  • the steps involve using an effective amount of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the antibody-drug conjugate described in any of the preceding statements.
  • the antibody-drug conjugate, compound, drug-linker, or pharmaceutical composition containing it is sufficient (e.g., in a subject):
  • the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.
  • gastric cancer breast cancer
  • lung cancer e.g., non-small cell lung cancer, specifically lung adenocarcinoma
  • urothelial carcinoma e.g., non-small cell lung cancer, specifically lung adenocarcinoma
  • antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as ⁇ (kappa) and ⁇ (lambda) light chains. Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain does not directly participate in antibody-antigen binding but exhibits [a specific function/characteristic].
  • Multiple effector functions such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus.
  • the variable regions (VH and VL) of each heavy/light chain pair form antigen-binding sites.
  • the allocation of amino acids in each region or domain can follow various numbering systems known in the art.
  • antibody also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide.
  • the term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and/or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.
  • CDR complementarity-determining region
  • CDR1 complementarity-determining region
  • CDR2 complementarity-determining region
  • CDR3 complementarity-determining region
  • the precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; C).
  • the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.
  • the complete amino acid sequence of V H is usually numbered according to Kabat, while the three CDRs in the variable region can be numbered according to the above. Any numbering system may be used for definition.
  • the amino acid sites in VH may be sequentially numbered starting from amino acid site 1 and continuing to the end of the sequence, or they may be numbered according to Kabat. Unless otherwise stated, the amino acid sites in VH and VL described herein are defined according to sequential numbering.
  • the amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu.
  • the amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330.
  • the corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.
  • framework region or "FR” residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.
  • antigen-binding fragment in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety".
  • Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab)' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • Fd refers to an antibody fragment composed of VH and CH1 domains
  • dAb fragment refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989))
  • Fab fragment refers to an antibody fragment composed of VL, VH, CL and CH1 domains
  • F(ab') 2 fragment refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region
  • Fab'fragment refers to the fragment obtained by reducing the disulfide bonds of the two heavy chain fragments in the F(ab') 2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).
  • Fv refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.
  • Fc refers to the region formed by the second and third constant regions of the first heavy chain of the antibody and the second and third constant regions of the second heavy chain.
  • Antibody fragments are formed by binding via disulfide bonds.
  • the Fc fragment of an antibody has various functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have a general structure: NH2 - VL-linker-VH-COOH or NH2 - VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:46) amino acid sequence or a variant thereof.
  • a linker having the amino acid sequence (GGGGS) 4 (SEQ ID NO:47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al.
  • a disulfide bond may also exist between VH and VL of scFv.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFv containing NH2 -VH-VH-COOH and NH2 - VL-VL-COOH.
  • single-domain antibody has the meaning commonly understood by those skilled in the art, referring to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen.
  • antibody includes not only the complete antibody but also the antigen-binding fragment of the antibody.
  • Antigen-binding fragments e.g., the antibody fragments described above
  • a given antibody e.g., the antibody provided in this invention
  • Antigen-binding fragments of a given antibody can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.
  • identity refers to the sequence matching between two polypeptides or two nucleic acids. It occurs when a position in two compared sequences is occupied by the same base or amino acid monomeric subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). Then the molecules are identical at that position.
  • the "percentage identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match).
  • two sequences are compared to produce the maximum identity.
  • Such comparisons can be made conveniently using, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453.
  • the percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.
  • the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.
  • conservative substitution refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein/peptide containing an amino acid sequence.
  • conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid and glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine
  • ⁇ -branched side chains e.g., threonine, valine, and isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, and histidine
  • amino acids are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • linker refers to a structural segment that links a cytotoxic drug to an antibody or antigen-binding fragment. For example, it refers to the -MLE- structural segment in the formula Ab-[MLED] x .
  • drug-linker refers to the structure of the cytotoxic drug and linker described in this invention before they are covalently linked to an antibody or its antigen-binding fragment.
  • drug-linker refers to M'-L-E-D, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment.
  • drug-linker is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.
  • drug-linker also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the “drug-linker” compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature.
  • isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H , 3H , deuterium D, tritium T); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 37Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); and isotopes of sulfur (e.g., 35S ).
  • isotopes of hydrogen e.g., 2H , 3H , deuterium D, tritium T
  • isotopes of carbon e.g., 11C
  • isotopically labeled compound means that the compound has the same structure as the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but different atomic mass or mass number from the dominant atomic mass or mass number in nature.
  • isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H , 3H , deuterium D, tritium T); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 37Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); and isotopes of sulfur (e.g., 35S ).
  • isotopes of hydrogen e.g., 2H , 3H , deuterium D, tritium T
  • isotopes of carbon e.g., 11C
  • substitution refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only if such combinations form a stable compound.
  • a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted.
  • FIG. 1 Efficacy of antibody-drug conjugate (ADC) in a mouse model of NCI-N87 subcutaneous tumor-bearing cells.
  • NMR nuclear magnetic resonance
  • MS Mass spectrometry
  • Step 1 Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5-2)
  • reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.82 g, 4.99 mmol), which was used directly in the next step without purification.
  • Step 2 Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5-3)
  • Step 3 Preparation of (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-5-5)
  • Tetra(triphenylphosphine)palladium (114 mg, 0.099 mmol) was added, and the mixture was heated at 80 °C for 3 hours under nitrogen purging twice and protection.
  • the reaction solution was then added to ethyl acetate (20 mL) and water (10 mL), stirred, filtered, and the filtrate was allowed to stand and separated.
  • Step 4 Preparation of tert-butyl carbamate (7-5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (7-5-6)
  • Step 5 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-5-7)
  • Step Six Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-1)
  • the reaction was carried out at 80 °C for 3 hours. After the reaction was completed, the mixture was extracted with water and ethyl acetate. The organic phases were combined, dried, and concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give the title compound (303 mg, 461.04 ⁇ mol).
  • Step 2 Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-10-(2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-2)
  • Step 3 Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-3-3)
  • reaction was carried out at room temperature for 16 hours. After the reaction was complete, formic acid (0.5 mL) was added to quench the reaction. Stirring was continued for 10 minutes. The intermediate state conversion was confirmed. The reaction solution was prepared and purified to obtain the title compound (14 mg, 23.03 ⁇ mol).
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 4 (S)-8-((5-(((S)-2-(4-(benzyloxy)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo] Preparation of [e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-3-4)
  • the reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography to obtain the title compound (10 mg, 12.32 ⁇ mol).
  • Step 5 Preparation of (S)-8-((5-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-3)
  • Mobile phase A Acetonitrile
  • Mobile phase B Water (0.05% ammonium bicarbonate)
  • Step 1 Preparation of 8,8”-(pentane-1,5-diylbis(oxy))(11aS,11a”S)-bis(7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-10(5H)-carboxylic acid diallyl ester)(7-9-2)
  • Step 2 Preparation of (11aS,11a”S)-8,8”-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo][e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one)(7-9)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-10-4)
  • Methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.1 g, 3.23 mmol) was dissolved in tetrahydrofuran (30 mL), and glacial acetic acid (3.75 mL) was added at 0 °C. Hydrogen peroxide (7 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution, and the mixture was stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found.
  • Methyl 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzene (688 mg, 2.15 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL), and lithium hydroxide (103.1 mg, 4.3 mmol) was added. The mixture was reacted at room temperature for 3 hours, and the solution was adjusted with dilute hydrochloric acid. The system was at pH 5, and a solid precipitated. It was extracted with 30 mL of ethyl acetate, and after separation, it was washed with saturated brine. The organic phase was dried with anhydrous sodium sulfate and filtered. The concentration yielded the title compound (400 mg, 1.31 mmol).
  • Step 7 Preparation of (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (7-10-8)
  • Step 8 Preparation of (S)-8-(benzyloxy)-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-10-9)
  • Methyl (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate (570 mg, 1.29 mmol) was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (4 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine.
  • the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.
  • Step 11 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-(methoxy-d3)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-10-12)
  • Step 12 (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]) Preparation of pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-(methoxy-d3)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-10)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-11-2)
  • Methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.6 g, 4.41 mmol) was dissolved in tetrahydrofuran (40 mL), and glacial acetic acid (5 mL) was added at 0 °C. Hydrogen peroxide (10 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (50 mL) was added to the reaction solution and stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found. 30 mL of ethyl acetate was added for extraction.
  • Step 7 Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-11-7)
  • Methyl (S)-5-(4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate 910 mg, 1.95 mmol was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (6 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine.
  • the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.
  • Step 8 Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-11-8)
  • Step 11 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-11)
  • tert-butyl carbamate (4-((S)-8-((5-((((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.6 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21-1)
  • Step 2 Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21-2)
  • Step 4 Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-22-3)
  • Step 3 Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22-4)
  • Step 5 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-23-2)
  • Step 2 Preparation of (2R,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-3)
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-4)
  • Step 4 Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-23-5)
  • Step 5 Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23-6)
  • Step Six (4-((S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5),10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate Preparation of (7-23-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23)
  • tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5),10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (3 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-24-2)
  • Step 2 Preparation of (2S,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-3)
  • the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-4)
  • Step 4 Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5--(7-24-5)
  • Step 5 Preparation of (2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24-6)
  • Step 7 (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexadecyl) Preparation of hydrogen-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24)
  • tert-butyl carbamate (4-((S)-8-((5-((((2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl carbamate was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (2 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-5,11(10H)-dione (7-25-4)
  • Step 4 Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-5)
  • Step 5 Preparation of (2R,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-6)
  • Step Six Preparation of (4-((S)-8-((5-(((2R,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H)-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(methoxy)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-26-4)
  • Step 4 Preparation of (2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-5)
  • Step 5 Preparation of (2S,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-6)
  • Step Six Preparation of (4-((S)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26)
  • Trifluoroacetic acid (0.5 mL) was added dropwise with stirring, and stirring was continued for 1 hour after the addition was complete.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylic acid methyl ester (7-27-2)
  • Step 2 Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-3)
  • Step 3 Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-4)
  • tert-butyl carbamate ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-2-yl) in tetrahydrofuran (100 mL), add sodium borohydride (843.15 mg, 11.55 mmol), and then add trifluoroacetic acid (877.8 mg, 7.7 mmol) dropwise at room temperature until no large amount of gas is released. Then, reflux the mixture at 75 °C for 5 hours.
  • reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.4 g, 3.09 mmol), which was used directly in the next step without purification.
  • Step 4 Preparation of ((2R,11aS)-8-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-5)
  • Step 5 (4-((S)-8-((5-(((2R,11aS)-2-((tert-butoxycarbonyl)amino)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo) Preparation of -5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-27-6)
  • Step Six Preparation of (S)-8-((5-(((2R,11aS)-2-amino-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-aminophenyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-27)
  • tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butoxycarbonyl)amino)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester (7-29-3)
  • Step 3 Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-29-4)
  • Step 4 Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29-5)
  • Step 5 Preparation of (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-5-one (7-29-6)
  • Step Six Preparation of (4-((S)-8-((5-((((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro)-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-29-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylic acid methyl ester (7-30-2)
  • Step 2 Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-5,11(10H)-dione (7-30-3)
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-4)
  • Step 4 Preparation of (2S,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-5)
  • Step 5 (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H- Preparation of benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-31-3)
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-4)
  • Step 4 Preparation of (2R,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-5)
  • Step 5 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-32-3)
  • Methyl (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (820.79 mg, 3.51 mmol, CL) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol, FR) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation.
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-32-4)
  • Step 4 Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-5)
  • Step 5 Preparation of (2S,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-33-3)
  • Methyl (2S,4R)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (824.79 mg, 3.53 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation.
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-33-4)
  • Step 4 Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-5)
  • reaction solution was concentrated, and water (50 mL) was added.
  • the mixture was extracted twice with ethyl acetate (50 mL x 2).
  • the combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.1 g, 2.71 mmol), which was used directly in the next step without purification.
  • Step 5 Preparation of (2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • Step 1 Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-1)
  • Furan-3-ylboronic acid (40.15 mg, 358.85 ⁇ mol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (100 mg, 179.43 ⁇ mol), tetra(triphenylphosphine)palladium (20.73 mg, 17.94 ⁇ mol), and sodium carbonate (76.07 mg, 717.70 ⁇ mol) were mixed together, and water (0.5 mL), toluene (2 mL), and ethanol (0.5 mL) were added.
  • Step 2 Preparation of (S)-2-(furan-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-2)
  • Step 3 Preparation of (S)-8-((5-(((S)-2-(furan-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-36)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-1)
  • Step 2 (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrole)[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-(thiophene-3-yl)-1,11a-dihydro-5H-benzo[e] Preparation of pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-2)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-37)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-((5-bromopentyl)oxy)-2-(5-(hydroxymethyl)thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-1)
  • Step 2 Preparation of (S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-2)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (S)-8-((5-(((S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spirocyclic[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-38)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of allyl (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid ester (7-39-1)
  • Step 2 Preparation of (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-39)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-40-1)
  • Step 2 Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40-2)
  • Step 3 Preparation of (S)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40)
  • the mixture was stirred for 1 hour.
  • the solution was diluted with ethyl acetate, washed twice with saturated ammonium chloride aqueous solution, and twice with saturated brine.
  • the solution was then concentrated under reduced pressure.
  • the title compound (17.11 mg, 24.24 ⁇ mol) was obtained by preparative high performance liquid chromatography purification followed by freeze drying.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)

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Abstract

Provided are a fused ring compound and a preparation method therefor and the use thereof. The fused ring compound has a structure shown as formula M'-L-E-D, the compound can be used for preparing an antibody-drug conjugate, the conjugate has a better drug antibody coupling ratio, and has an excellent targeted killing effect on solid tumors such as gastric cancer, breast cancer, lung cancer and urothelial carcinoma.

Description

稠环类化合物及其制备方法和用途Fused ring compounds, their preparation methods and uses

本申请是以CN申请号为202410585326.9，申请日为2024年5月10日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。This application is based on and claims priority to CN application number 202410585326.9, filed on May 10, 2024, the disclosure of which is incorporated herein by reference in its entirety.

技术领域Technical Field

本申请涉及药物领域，具体涉及一种稠环类化合物及其制备方法和用途。This application relates to the pharmaceutical field, specifically to a fused-ring compound and its preparation method and uses.

背景技术Background Technology

治疗肿瘤的抗体药物偶联物(antibody drug conjugate,ADC)通常由单克隆抗体、生物活性分子(以杀伤肿瘤的细胞毒素为主)及连接体(Linker)组成。生物活性分子通过连接体共价偶联到抗体上；抗体能够识别肿瘤细胞表面的特异性靶点，进而引导ADC到达肿瘤微环境及癌细胞表面，并使ADC通过内吞效应进入癌细胞；然后生物活性分子在癌细胞内释放，并通过抑制癌细胞的微管蛋白或损伤癌细胞的DNA等作用，达到杀灭癌细胞而尽量不损伤正常组织细胞的目的。Antibody-drug conjugates (ADCs) for cancer treatment typically consist of a monoclonal antibody, a bioactive molecule (primarily a cytotoxic agent that kills tumor cells), and a linker. The bioactive molecule is covalently coupled to the antibody via the linker. The antibody recognizes specific targets on the surface of tumor cells, guiding the ADC to the tumor microenvironment and the surface of cancer cells. The ADC then enters the cancer cells via endocytosis. The bioactive molecule is then released within the cancer cells, killing them by inhibiting microtubules or damaging their DNA, thereby minimizing damage to normal tissue cells.

吡咯并苯二氮杂(PBD)是一种序列选择性的DNA小沟结合剂，是抗生素炭疽霉素家族中的一员。科学家们发现，通过亚烷基基链将两个PBD单元连接在一起，可以使PBD二聚体与DNA之间产生高度致命的链间交联。而且这种PBD二聚体在多种肿瘤细胞系中的活性可达到pmol/L的水平。Pyrrolobenzodiazepine PBD (protonated bromine) is a sequence-selective DNA minor groove binder and a member of the anthraxmycin family of antibiotics. Scientists have discovered that linking two PBD units together via an alkylene chain can create a highly lethal interstrand cross-link between the PBD dimer and DNA. Furthermore, this PBD dimer exhibits pmol/L activity in various tumor cell lines.

作为药物-连接体(linker)是抗体偶联药物的重要组成部分，其中药物部分和连接体部分的结构均对ADC药物整体的有效性和安全性有重要影响。因此，开发新型的PBD药物连接体化合物对于提高这类抗体偶联药物的药效及安全性具有重要意义。As a crucial component of antibody-drug conjugates (ADCs), both the drug and linker components significantly impact the overall efficacy and safety of the ADC. Therefore, developing novel PBD drug linker compounds is of great importance for improving the efficacy and safety of these ADCs.

发明内容Summary of the Invention

本申请涉及一种稠环化合物，具有通式D-E-L-M’所示结构。所述稠环化合物能够用于制备抗体药物偶联物，并且所述偶联物对肿瘤具有很好的靶向杀伤效果。This application relates to a fused-ring compound having the structure shown in the general formula D-E-L-M'. The fused-ring compound can be used to prepare antibody-drug conjugates, and the conjugates exhibit excellent targeted killing effects against tumors.

化合物compound

在一个方面，本申请提供一种化合物或其药学上可接受的盐，其具有式D-E-L-M’所示结构，其中：In one aspect, this application provides a compound or a pharmaceutically acceptable salt thereof having the structure shown in formula D-E-L-M’, wherein:

M’为-M-Lg，其中Lg为亲核取代反应的离去基团，M是和靶向部位结合的结构片段； M' is -M-Lg, where Lg is the leaving group of the nucleophilic substitution reaction, and M is the structural fragment that binds to the target site;

L是连接M和E之间的结构片段；L is the structural segment connecting M and E;

E是连接L和D的结构片段；E is a structural segment connecting L and D;

D是细胞毒性药物片段。D is a cytotoxic drug fragment.

在一些实施方案中，M选自以下取代或未取代的结构片段：
In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

在一些实施方案中，M选自以下取代或未取代的结构片段：
In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

在一些实施方案中，M选自以下取代或未取代的结构片段：
In some embodiments, M is selected from the following substituted or unsubstituted structural segments:

在一些实施方案中，Lg选自卤素(例如F、Cl、Br、I)、卤代C1-6烷基、C1-6烷基磺酰基、卤代C1-6烷基磺酰基、卤代磺酰基、C1-6烷基磺酸酯基、卤代C1-6烷基磺酸酯基、C1-6烷基亚磺酸酯基、C1-6烷基亚砜基、卤代苯氧基、羟基(-OH)、巯基(-SH)、氨基(-NH2)、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段，所述的卤代C1-6烷基、C1-6烷基磺酰基、卤代C1-6烷基磺酰基、卤代磺酰基、C1-6烷基磺酸酯基、卤代C1-6烷基磺 酸酯基、C1-6烷基亚磺酸酯基、C1-6烷基亚砜基、卤代苯氧基、烯基、炔基及含炔基的结构片段任选地被一个或多个合适的取代基所取代。In some embodiments, Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxy (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments. The halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, and halogenated C1-6 alkyl sulfonyl groups are further specified. The ester group, C1-6 alkyl sulfinate group, C1-6 alkyl sulfoxide group, halophenoxy group, alkenyl group, alkynyl group and alkynyl-containing structural segments are optionally replaced by one or more suitable substituents.

在一些实施方案中，Lg选自卤素(例如F、Cl、Br、I)、卤代C1-6烷基、C1-6烷基磺酰基、卤代C1-6烷基磺酰基、卤代磺酰基、C1-6烷基磺酸酯基、卤代C1-6烷基磺酸酯基、C1-6烷基亚磺酸酯基、C1-6烷基亚砜基、卤代苯氧基、羟基(-OH)、巯基(-SH)、氨基(-NH2)、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段。In some embodiments, Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkyl sulfonyl, halogenated C1-6 alkyl sulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl , and alkynyl-containing structural fragments.

在一些实施方案中，Lg选自卤素、取代或未取代的C1-6烷基磺酰基、卤代苯氧基、羟基(-OH)、巯基(-SH)或氨基(-NH2)。In some embodiments, Lg is selected from halogens, substituted or unsubstituted C1-6 alkylsulfonyl groups, halophenoxy groups, hydroxyl groups (-OH), mercapto groups (-SH), or amino groups ( -NH2 ).

在一些实施方案中，Lg选自卤素、取代或未取代的甲基磺酰基、卤代苯氧基、羟基(-OH)、巯基(-SH)或氨基(-NH2)。In some embodiments, Lg is selected from halogen, substituted or unsubstituted methanesulfonyl, halophenoxy, hydroxy (-OH), mercapto (-SH) or amino ( -NH2 ).

在一些实施方案中，Lg选自甲基磺酰基。In some implementations, Lg is selected from methanesulfonyl.

在一些实施方案中，L选自由下述的一个或多个组成的取代或未取代的结构片段或其立体异构体：C1-6亚烷基、6-10元芳基、5-6元杂芳基、9-12元含氮杂环基、-N(R’)-、-NH(R’)、-N(R’)2、羰基、-O-、天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH2CH2(OCH2CH2)rOCH3))、Lys(R’)、以及氨基酸组成的短肽(比如Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Gly-Lys、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Cit、Val-Lys、Val-Lys(Ac)、Ala-Ala-Ala、Ala-D-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly(GGFG,SEQ ID NO:41)、Gly-Gly-Val-Ala(GGVA,SEQ ID NO:42)、Gly-Phe-Leu-Gly(GFLG,SEQ ID NO:43)、Glu-Ala-Ala-Ala(EAAA,SEQ ID NO:44)、Gly-Gly-Gly-Gly-Gly(GGGGG,SEQ ID NO:45))、 其中R’由下述一个或多个基团组成，包括但不限于 氢、C1-6烷基、C1-6亚烷基、胺基、羟基、羧基、酰基、-O-、-C1-6亚烷基CO2H、-C1-6亚烷基SO3H、-SO3H、-PO3H2、-C1-6亚烷基-NHC1-6烷基、-C1-6亚烷基-N(C1-6烷基)2、-CH2N(C1- 6烷基)-C(＝O)C1-6亚烷基-杂环、-CH2NH-SO3H、-CH2N(C1-6烷基)-SO3H、-CH2NHC1-6亚烷基-SO3H、-CH2N(C1-6烷基)C1-6亚烷基-SO3H、-CH2N(C1-6亚烷基-SO3H)2、-CH2N+(C1-6亚烷基-SO3H)3、-CH2N+(C1-6烷基)2-C1-6亚烷基-SO3H、-CH2N(C1-6烷基)-C(＝O)C1-6亚烷基-N+(C1-6亚烷基-SO3H)3、-CH2NH-C(＝O)C1-6亚烷基-N+(C1-6亚烷基-SO3H)3、-CH2N(C1-6烷基)-C(＝O)C1-6亚烷基-N+(C1-6烷基)3、-CH2NH-C(＝O)C1-6亚烷基-N+(C1-6烷基)3、-CH2N(C1- 6烷基)-C(＝O)OC2-6亚烷基-N+(C1-6烷基)3、-CH2N(C1-6烷基)-C(＝O)OC2-6亚烷基-N+(C1-6烷基)2-CH2CO2H、-CH2N(C1-6烷基)-C1-6亚烷基-CO2H、-CH2N+(C1-6烷基)2-C1-6亚烷基-CO2H、葡萄糖基、半乳糖基、葡萄糖醛酸基、半乳糖醛酸基、-CH2N(C1-6烷基)-C(＝O)-(CH2CH2O)r-C1-6烷基、-CH2N(C1-6烷基)-C(＝O)-(OCH2CH2)r-OC1-6烷基、-(CH2N(Me)-C(＝O))r-C1-6烷基、或含1-10个EO单元的聚乙二醇片段(即-(CH2CH2O)r-C1-6烷基)、 DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸残基)、DOTAGA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸,α-丙酰基)、或NOTA(1,4,7-三氮杂环壬烷-N,N’,N”-三乙酸残基)，其中r选自1-20的整数；s选自1-20的整数。In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments or stereoisomers thereof: C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic, -N(R')-, -NH(R'), -N(R') 2 , carbonyl, -O-, natural or non-natural amino acids and their analogs (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( COCH2CH2 ( OCH2CH2 ) rOCH3 ) (), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Gly-Lys, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-A). sn, Ala-Ala-Gly, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Al a, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:41), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:42), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:43), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:44), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:45)), R' is composed of one or more of the following groups, including but not limited to Hydrogen, C1-6 alkyl, C1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O- , -C1-6 alkylene CO2H, -C1-6 alkylene SO3H , -SO3H , -PO3H2, -C1-6 alkylene -NHC1-6 alkyl , -C1-6 alkylene -N(C1-6 alkyl) 2 , -CH2N ( C1-6 alkyl)-C(= O)C1-6 alkylene-heterocyclic, -CH2NH-SO3H, -CH2N(C1-6 alkyl ) -SO3H , -CH2NHC1-6 alkylene - SO3H , -CH2N ( C1-6 alkyl) C1-6 alkylene- SO3H , -CH2N ( C1-6 alkyl - SO3H ) 2 , -CH2 N + (C 1-6 alkylene-SO 3 H) 3 , -CH 2 N + (C 1-6 alkyl) 2 -C 1-6 alkylene-SO 3 H, -CH 2 N(C 1-6 alkyl)-C(＝O)C 1-6 alkylene-N + (C 1-6 alkylene-SO 3 H) 3 , -CH 2 NH-C(＝O)C 1-6 alkylene-N + (C 1-6 alkylene-SO 3 H) 3 , -CH 2 N(C 1-6 alkyl)-C(＝O)C 1-6 alkylene-N + (C 1-6 alkyl) 3 , -CH 2 NH-C(＝O)C 1-6 alkylene-N + (C 1-6 alkyl) 3 , -CH 2 N(C 1-6 alkyl )-C(＝O)OC 2-6 alkylene-N + (C 1-6 alkyl) 3 , -CH 2 N(C 1-6 alkyl)-C(＝O)OC 2-6 alkylene-N + (C 1-6 alkyl) 2 -CH 2 CO 2 H, -CH 2 N(C 1-6 alkyl)-C 1-6 alkylene-CO 2 H, -CH 2 N + (C 1-6 alkyl) 2 -C 1-6 alkylene-CO 2 H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH 2 N(C 1-6 alkyl)-C(＝O)-(CH 2 CH 2 O) r -C 1-6 alkyl, -CH 2 N(C 1-6 alkyl)-C(＝O)-(OCH 2 CH 2 ) r -OC 1-6 alkyl, -(CH 2 N(Me)-C(＝O)) r -C 1-6 alkyl, or polyethylene glycol fragments containing 1-10 EO units (i.e. , -( CH₂CH₂O ) r - C₁-6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), where r is selected from an integer from 1 to 20; s is selected from an integer from 1 to 20.

在一些实施方案中，“-NOTA”是指 In some implementations, "-NOTA" refers to

在一些实施方案中，“-DOTA”是指 In some implementations, "-DOTA" refers to

在一些实施方案中，“-DOTAGA”是指 In some implementations, "-DOTAGA" refers to

在一些实施方案中，L选自由下述的一个或多个组成的取代或未取代的结构片段：天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH2CH2(OCH2CH2)rOCH3))、Lys(R’)、以及氨基酸组成的短肽(比如Gly- Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala)、其中R’由下述一个或多个基团组成，包括但不限于氢、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸残基)、DOTAGA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸,α-丙酰基)、或NOTA(1,4,7-三氮杂环壬烷-N,N’,N”-三乙酸残基)，其中s选自1-20的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；优选地，s为3-10，例如，s为5、8、或10。In some embodiments, L is selected from one or more substituted or unsubstituted structural fragments composed of: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( COCH2CH2 ( OCH2CH2 ) rOCH3 )) , Lys(R'), and short peptides composed of amino acids (e.g. , Gly- Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala), R' is composed of one or more of the following groups, including but not limited to hydrogen, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), or NOA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), wherein s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, or 10.

在一些实施方案中，L选自由下述的一个或多个组成的取代或未取代的结构片段：天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH2CH2(OCH2CH2)rOCH3))、以及氨基酸组成的短肽(比如Gly-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala)、 其中r选自1-20的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；s选自1-20的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；优选地，s为3-10，例如，s为5、8、9或10。In some embodiments, L is selected from one or more substituted or unsubstituted structural fragments composed of: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), and short peptides composed of amino acids (e.g., Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). Where r is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, 9, or 10.

在一些实施方案中，L选自由下述的一个或多个组成的取代或未取代的结构片段：Val、Gly-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala、 其中s选自1-20的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；优选地，s为3-10，例如，s为5、8、9或10。 In some embodiments, L is selected from one or more of the following substituted or unsubstituted structural fragments: Val, Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala, Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.

在一些实施方案中，L选自由下述的一个或多个组成的取代或未取代的结构片段：Val、如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；优选地，s为3-10，例如，s为5、8、9或10。In some implementations, L is selected from one or more of the following substituted or unsubstituted structural segments: Val, Such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, 9, or 10.

在一些实施方案中，L选自以下取代或未取代的结构：Gly-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala、


In some embodiments, L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,


其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

在一些实施方案中，L选自以下取代或未取代的结构：Gly-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala、 In some embodiments, L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,

其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

在一些实施方案中，L选自以下取代或未取代的结构：


In some implementations, L is selected from the following substituted or unsubstituted structures:


其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

在一些实施方案中，E为单键，取代或未取代的-NH-CH2-或 In some implementations, E is a single bond, substituted or unsubstituted -NH- CH2- or

在一些实施方案中，E为单键，取代或未取代的-NH-CH2-。In some implementations, E is a single bond, substituted or unsubstituted -NH- CH2- .

在一些实施方案中，E为单键或-NH-CH2-。In some implementations, E is a single bond or -NH- CH2- .

在一些实施方案中，选自以下取代或未取代的结构：



In some implementation schemes, Selected from the following substituted or unsubstituted structures:



其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

在一些实施方案中，选自以下取代或未取代的结构：

In some implementation schemes, Selected from the following substituted or unsubstituted structures:

其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

在一些实施方案中，选自以下取代或未取代的结构：

In some implementation schemes, Selected from the following substituted or unsubstituted structures:

在一些实施方案中，所述细胞毒性药物选自抗微管蛋白剂，DNA嵌入剂，DNA拓扑异构酶抑制剂，RNA聚合酶抑制剂和基因转录抑制剂。在一些实施方案中，所述抗微管蛋白剂为奥瑞他汀类化合物、美登素类化合物或艾日布林类化合物。在一些实施方案中，所述DNA嵌入剂为吡咯并苯二氮卓(PBD)类化合物、曲贝替定或卢比替定。在一些实施方案中，所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、多柔比星、PNU-159682及其类似物、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)。在一些实施方案中，所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)。在一些实施方案中，所述基因转录抑制剂为雷公藤甲素及其药学上可接受的盐、酯和类似物。In some embodiments, the cytotoxic agent is selected from anti-microtubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors. In some embodiments, the anti-microtubule agent is an oliguriatin class, a maytansine class, or an eribulin class. In some embodiments, the DNA intercalating agent is a pyrrolobenzodiazepine (PBD) class, trabectedin, or rubotecan. In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecone, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin. In some embodiments, the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogs.

在一些实施方案中，所述细胞毒性药物选自吡咯并苯二氮卓(PBD)类化合物。In some embodiments, the cytotoxic drug is selected from pyrrolobenzodiazepine (PBD) compounds.

本申请中所公开的细胞毒性药物通常含有多种官能团，例如羟基(-OH)、羧基(-COOH)，一级氨基(-NH2)，二级胺基(-NRaH)，三级胺基(-NRbRc)，其中Ra、Rb、Rc在此仅代表N上的非氢取代基，或巯基(-SH)，这些官能团可与偶联物其余部分中合适的官能团反应以实现连接。 The cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino ( -NH2 ), secondary amino ( -NRaH ), and tertiary amino ( -NRbRc ), where Ra , Rb , and Rc represent only non-hydrogen substituents on N, or thiol (-SH). These functional groups can react with suitable functional groups in the remainder of the conjugate to achieve linkage.

在一些实施方案中，细胞毒性药物通过其上的-OH，一级氨基，二级胺基或三级胺基，或-SH与所述抗体药物偶联物中的E连接。在一些实施方案中，D为所述细胞毒性药物上的-OH、-NH2或二级胺基失掉一个H得到的一价结构。在一些实施方案中，D为所述细胞毒性药物上的-OH或-NH2失掉一个H得到的一价结构。In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary amino, or tertiary amino group, or a -SH group. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2 , or secondary amino group on the cytotoxic drug. In some embodiments, D is a monovalent structure obtained by losing an H from the -OH or -NH2 group on the cytotoxic drug.

在一些实施方案中，所述细胞毒性药物选自下式(I)所示结构的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：
In some embodiments, the cytotoxic drug is selected from compounds with the structure shown in formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs thereof:

其中，R1和R2各自独立地选自氢、-CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1- 6烷基)2、C1-6烷基、C2-6烯基、C2-6炔基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C2-6烯基、C2-6炔基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代； R1 and R2 are each independently selected from hydrogen, -CN, halogen, -ORα, -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl; wherein the alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl groups;

R4和R4’各自独立地选自氢、卤素、-OH、-NH2、C1-6烷基、卤代C1-6烷基和C1-6烷氧基，R5和R5’均为氢； R4 and R4 ' are each independently selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halo- C1-6 alkyl and C1-6 alkoxy, and R5 and R5 ' are both hydrogen;

或者，R4和R4’一起与连接R4和R4’的碳原子成碳碳双键或者3-6元碳环，R5和R5’均为氢；Alternatively, R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms;

或者，R4和R5连接成键或一起与连接R4和R5的碳原子成3-6元碳环，R5’和R4’各自独立地选自氢、卤素、-NH2、C1-6烷基、卤代C1-6烷基和C1-6烷氧基；Alternatively, R4 and R5 may be bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5 , and R5 ' and R4 ' may be independently selected from hydrogen, halogen, -NH2, C1-6 alkyl, halo -C1-6 alkyl and C1-6 alkoxy;

R6为氢、C1-6烷基或 R6 is hydrogen, C1-6 alkyl, or...

R6’、R7和R7’均为氢；或者R6’与R7连接成键，R7’为氢；R6 ' , R7 , and R7 ' are all hydrogen; or R6 ' is bonded to R7 , and R7 ' is hydrogen.

Ra选自H、C1-6烷基或C3-6环烷基； Ra is selected from H, C1-6 alkyl, or C3-6 cycloalkyl;

环A选自3-6元环烷基、C6-10芳基、5-10元杂芳基、3-6元杂环基；所述3-6元环烷基、C6-10芳基、5-10元杂芳基、3-6元杂环基任选被以下一个或多个取代基所取代：氢、- CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、C1-6烷基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代；Ring A is selected from 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups; the 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups may optionally be substituted by one or more of the following substituents: hydrogen, - CN, halogen, -ORα, -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;

R3选自氢、-CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、C1-6烷基、C3- 6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代； R3 is selected from hydrogen, -CN, halogen, -ORα, -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;

X和Y各自独立地为-CH2-、-CD2-、和-C(O)-；X and Y are independently -CH 2- and -CD 2- , respectively. and -C(O)-;

t选自1-10，例如1、2、3、4、5、6、7、8、9或10。t is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

在一些实施方案中，当式(I)中的n为5，环A为苯基时，R3不为甲氧基。In some implementations, when n in formula (I) is 5 and ring A is phenyl, R3 is not methoxy.

在一些实施方案中，R1选自氢、-CN、卤素、-ORa和C1-6烷基，其中Ra选自C1-6烷基、氘代C1-6烷基和C3-6环烷基。In some embodiments, R1 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is selected from C1-6 alkyl, deuterated C1-6 alkyl, and C3-6 cycloalkyl.

在一些实施方案中，R1选自-ORa，其中Ra选自C1-6烷基、氘代C1-6烷基和C3-6环烷基。In some embodiments, R1 is selected from -ORa , wherein Ra is selected from C1-6 alkyl, deuterated C1-6 alkyl and C3-6 cycloalkyl.

在一些实施方案中，R1选自甲氧基、三氘代甲氧基和-O-环丙基。In some embodiments, R1 is selected from methoxy, trideuteroxy, and -O-cyclopropyl.

在一些实施方案中，R1选自氢、-CN、卤素、-ORa和C1-6烷基，其中Ra为C1-6烷基或C3-6环烷基。In some embodiments, R1 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is a C1-6 alkyl or C3-6 cycloalkyl.

在一些实施方案中，R1为-ORa，其中Ra为C1-6烷基或C3-6环烷基。In some embodiments, R1 is -ORa , where Ra is a C1-6 alkyl or C3-6 cycloalkyl.

在一些实施方案中，R1为甲氧基或-O-环丙基。In some embodiments, R1 is methoxy or -O-cyclopropyl.

在一些实施方案中，R2选自氢、-CN、卤素、-ORa和C1-6烷基，其中Ra为C1-6烷基或C3-6环烷基。In some embodiments, R2 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is a C1-6 alkyl or C3-6 cycloalkyl.

在一些实施方案中，R2为-ORa，其中Ra为C1-6烷基。In some implementations, R2 is -ORa , where Ra is a C1-6 alkyl group.

在一些实施方案中，R2为甲氧基。In some implementations, R2 is a methoxy group.

在一些实施方案中，R4为氢或卤素，R4’选自氢、卤素、-OH、-NH2、C1-6烷基、卤代 C1-6烷基和C1-6烷氧基，R5和R5’均为氢；或者，R4和R4’一起与连接R4和R4’的碳原子成碳碳双键或者3-6元碳环，R5和R5’均为氢；或者，R4与R5连接成键或一起与连接R4和R5的碳原子成3-6元碳环，R5’和R4’均为氢。In some embodiments, R4 is hydrogen or a halogen, and R4 ' is selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halogenated C1-6 alkyl and C1-6 alkoxy, R5 and R5 ' are both hydrogen; or, R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ', R5 and R5 ' are both hydrogen; or, R4 and R5 are bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5 , R5 ' and R4 ' are both hydrogen.

在一些实施方案中，R4为氢或氟，R4’选自氢、氟、-OH、-NH2、甲基、三氟甲基和甲氧基，R5和R5’均为氢；或者，R4和R4’一起与连接R4和R4’的碳原子成碳碳双键或者3-6元碳环，R5和R5’均为氢；或者，R4与R5连接成键或一起与连接R4和R5的碳原子成3元碳环，R5’和R4’均为氢。In some embodiments, R4 is hydrogen or fluorine, R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , methyl, trifluoromethyl, and methoxy, and R5 and R5 ' are both hydrogen; or, R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ', and R5 and R5' are both hydrogen; or, R4 and R5 are bonded together or together form a 3 membered carbon ring with the carbon atom connecting R4 and R5 , and R5' and R4 ' are both hydrogen.

在一些实施方案中，R4为氢或氟，R4’选自氢、氟、-OH、-NH2、甲基、三氟甲基和甲氧基，R5和R5’均为氢；或者，R4和R4’一起与连接R4和R4’的碳原子成碳碳双键，R5和R5’均为氢；或者，R4与R5连接成键或一起与连接R4和R5的碳原子成3元碳环，R5’和R4’均为氢。In some embodiments, R4 is hydrogen or fluorine, R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , methyl, trifluoromethyl, and methoxy, and R5 and R5 ' are both hydrogen; or, R4 and R4 ' together form a carbon-carbon double bond with the carbon atom connecting R4 and R4 ', and R5 and R5 ' are both hydrogen; or, R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5 , and R5' and R4 ' are both hydrogen.

在一些实施方案中，R4和R4’与连接R4和R4’的碳原子成3元碳环，R5和R5’均为氢。In some implementations, R4 and R4 ' form a ternary carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms.

在一些实施方案中，R6为 In some implementations, R6 is

环A选自3-6元环烷基、C6-10芳基或5-10元杂芳基；Ring A is selected from 3-6 membered cycloalkyl, C6-10 aryl, or 5-10 membered heteroaryl;

R3选自氢、-CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、C1-6烷基、C3- 6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代； R3 is selected from hydrogen, -CN, halogen, -ORα, -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;

Ra选自H和C1-6烷基。 Ra is selected from H and C1-6 alkyl groups.

在一些实施方案中，R6为 In some implementations, R6 is

环A选自环丙基、苯基、呋喃基、噻吩基、嘧啶基和吡啶基；Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;

R3选自氢、氟、-OH、甲氧基、-NH2和羟甲基。 R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.

在一些实施方案中，R6为 In some implementations, R6 is

环A为苯基；Ring A is phenyl;

R3选自氢、氟、-OH、甲氧基、-NH2和羟甲基。 R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.

在一些实施方案中，环A为C6-10芳基或5-10元杂芳基。In some implementations, ring A is C6-10 aryl or 5-10 heteroaryl.

在一些实施方案中，环A选自苯基、嘧啶基、吡啶基、呋喃基和噻吩基。In some embodiments, ring A is selected from phenyl, pyrimidinyl, pyridinyl, furanyl, and thiophenyl.

在一些实施方案中，R3选自-OH、甲氧基、-NH2和羟甲基。In some embodiments, R3 is selected from -OH, methoxy, -NH2 and hydroxymethyl.

在一些实施方案中，R6选自苯基、吡啶基、 In some embodiments, R6 is selected from phenyl, pyridyl,

在一些实施方案中，R6选自苯基、吡啶基、 In some embodiments, R6 is selected from phenyl, pyridyl,

R6’与R7连接成键，R7’为氢。R6 ' and R7 are bonded together, and R7 ' is hydrogen.

在一些实施方案中，R6为 In some implementations, R6 is

R6’、R7和R7’均为氢。R6 ' , R7 and R7 ' are all hydrogen.

在一些实施方案中，t为3-8，例如3、4、5、6、7或8。In some implementations, t is 3-8, such as 3, 4, 5, 6, 7 or 8.

在一些实施方案中，X为-CH2-或-CD2-；任选的，Y选自-CH2-、-CD2-、和-C(O)-。In some implementations, X is -CH2- or -CD2- ; optionally, Y is selected from -CH2- , -CD2- , And -C(O)-.

在一些实施方案中，X为-CH2-，Y为-CH2-；X为-CH2-，Y为-CD2-或-C(O)-；或者X为-CD2-，Y为-CH2-。In some implementations, X is -CH2- and Y is -CH2- ; X is -CH2- and Y is -CD2- or -C(O)-; or X is -CD2- and Y is -CH2- .

在一些实施方案中，X为-CH2-，Y为-CH2-。In some implementations, X is -CH 2 - and Y is -CH 2 -.

在一些实施方案中，In some implementation schemes,

R1选自甲氧基、三氘代甲氧基和-O-环丙基； R1 is selected from methoxy, trideuteroxy, and -O-cyclopropyl;

R2为甲氧基； R2 is a methoxy group;

R4为氢或氟，R4’选自氢、氟、-OH、-NH2、三氟甲基和甲氧基，R5和R5’均为氢；或者，R4和R4’与连接R4和R4’的碳原子成碳碳双键或者3元碳环，R5和R5’均为氢；或者，R4与R5连接成键或一起与连接R4和R5的碳原子成3元碳环，R5’和R4’均为氢； R4 is hydrogen or fluorine, R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , trifluoromethyl and methoxy, and R5 and R5 ' are both hydrogen; or, R4 and R4 ' form a carbon-carbon double bond or a 3-membered carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen; or, R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5 , and R5 ' and R4 ' are both hydrogen.

环A选自环丙基、苯基、呋喃基、噻吩基、嘧啶基和吡啶基；Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;

R3选自氢、氟、-OH、甲氧基、-NH2和羟甲基； R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl;

t为5。t is 5.

在一些实施方案中，In some implementation schemes,

R1和R2均为甲氧基； R1 and R2 are both methoxy groups;

R4为氢或氟，R4’选自氢、氟、-OH、-NH2、三氟甲基和甲氧基，R5和R5’均为氢；或者，R4和R4’与连接R4和R4’的碳原子成碳碳双键或者3元碳环，R5和R5’均为氢；或者，R4与R5连接成键或一起与连接R4和R5的碳原子成3元碳环，R5’和R4’均为氢； R4 is hydrogen or fluorine, R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , trifluoromethyl and methoxy, and R5 and R5 ' are both hydrogen; or, R4 and R4 ' form a carbon-carbon double bond or a 3-membered carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen; or, R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5 , and R5 ' and R4 ' are both hydrogen.

选自 Selected from

t为5。t is 5.

在一些实施方案中，所述细胞毒性药物选自以下化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：



In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:



在一些实施方案中，所述细胞毒性药物选自以下化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：
In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:

在一些实施方案中，所述细胞毒性药物选自以下化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：
In some embodiments, the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:

在一些实施方案中，所述细胞毒性药物片段(D)为以下式(I’)所示结构：
In some embodiments, the cytotoxic drug fragment (D) has the structure shown in formula (I'):

其中：环A、R1、R2、R4、R4’、R5、R5’、R6’、R7、R7’、X、Y、t如前文任意一项所述；Wherein: rings A, R1 , R2 , R4 , R4' , R5 , R5' , R6' , R7 , R7 ' , X, Y, and t are as described in any of the preceding items;

R3A选自化学键、-NH-、-N(C1-6烷基)-、-O-、-C1-6亚烷基-O-、-C1-6亚烷基-O-。R 3A is selected from chemical bonds, -NH-, -N(C 1-6 alkyl)-, -O-, -C 1-6 alkylene-O-, and -C 1-6 alkylene-O-.

在一些实施方案中，所述细胞毒性药物片段(D)为以下所示结构：
In some embodiments, the cytotoxic drug fragment (D) has the structure shown below:

在一些实施方案中，所述化合物结构如下：









In some embodiments, the compound has the following structure:









其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

抗体药物偶联物Antibody-drug conjugates

本发明的第二方面提供一种如式(II)所示的偶联物，其中：A second aspect of the invention provides a coupling as shown in formula (II), wherein:

A-[M-L-E-D]xA-[M-L-E-D]x

式(II) Equation (II)

M、L、E和D如前文任意一项所述；x为1-10；M, L, E, and D are as described in any of the preceding items; x is 1-10;

A是靶向部分，A的靶标选自表皮生长因子、Trop-2、CD37、HER2、CD70、EGFRvIII、Mesothelin、Folate eceoptor1、Mucin 1、CD138、CD20、CD19、CD30、SLTRK6、Nectin 4、Tissue factor、Mucin16、Endothelinreceoptor、STEAP1、SLC39A6、Guanylylcyclase C、PSMA、CCD79b、CD22、Sodium phosphate cotransporter 2B、GPNMB、Trophoblast glycoprotein、AGS-16、EGFR、CD33、CD66e、CD74、CD56、PD-L1、TACSTD2、DR5、E16、STEAP1、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、BAFF-R、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2、TENB2、整合素α5β6，α4β7、FGF2、FGFR2、Her3、CD70、CA6、DLL3、DLL4、P-cadherin、EpCAM、pCAD、CD223、LYPD3、LY6E、EFNA4、ROR1、SLITRK6、5T4、ENPP3、SLC39A6、Claudin18.2、BMPR1B、E16、STEAP1、Tyro7、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2，c-Met,ApoE、CD1 lc、CD40、CD45(PTPRC)、CD49D(ITGA4)、CD80、CSF1R、CTSD、GZMB、Ly86、MS4A7、PIK3AP1、PIK3CD、CCR5、IFNG、IL10RA1、IL-6、ACTA2、COL7A1、LOX、LRRC15、MCPT8、MMP10、NOG、SERPINEl、STAT1、TGFBR1、CTSS、PGF、VEGFA、C1QA、C1QB、ANGPTL4、EGLN、ANGPTL4、EGLN3、BNIP3、AIF1、CCL5、CXCL10、CXCL11、IFI6、PLOD2、KISS1R、STC2、DDIT4、PFKFB3、PGK1、PDK1、AKR1C1、AKR1C2、CADM1、CDH11、COL6A3、CTGF、HMOX1、KRT33A、LUM、WNT5A、IGFBP3、MMP14、CDCP1、PDGFRA、TCF4、TGF、TGFB1、TGFB2、CDl lb、ADGRE1、EMR2、TNFRSF21、UPK1B、TNFSF9、MMP16、MFI2、IGF-1R、RNF43、NaPi2b和TENB2。A represents the target group, whose targets are selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, and O. 772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72 LY64, FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR1B, E16, ST EAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79 b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5 , CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD , GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, M MP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNI P3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2 , CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGF B1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and TENB2.

在一些优选实施方案中，A为小分子配体，例如叶酸衍生物、谷氨酸脲衍生物、生长抑素衍生物、芳基磺酰胺类衍生物(例如碳酸酐酶IX抑制剂)、连接两个脂肪族吲哚的多烯，花青染料或IR-783或其衍生物。In some preferred embodiments, A is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.

在一些优选实施方案中，A为抗体，例如单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段包括Fab、Fab'、F(ab’)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、非人抗体、人源化抗体、嵌合抗体、全人抗体、前抗(Probody)、 双特异性抗体或多特异性抗体。In some preferred embodiments, A represents an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies (e.g., scFv), non-human antibodies, humanized antibodies, chimeric antibodies, fully human antibodies, and proboscis. Bispecific antibodies or multispecific antibodies.

在一些优选实施方案中，A是与ErbB家族受体酪氨酸激酶成员表皮生长因子受体2(Her2)特异性结合的抗体或其抗原结合片段。In some preferred embodiments, A is an antibody or antigen-binding fragment thereof that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase family.

本领域技术人员应当理解，可模块化制备本申请所述抗体药物偶联物。例如，先获得上文任意一项所述的化合物M’-L-E-D(即游离形式的“药物-连接体”)，其中M’为M与抗体或其抗原结合片段共价连接前的结构形式，而后将其与抗体或其抗原结合片段共价连接得到本申请所述抗体药物偶联物。相应地，所述游离形式的“药物-连接体”中M’通过取代反应(例如脱除其上的-SO2Me或五氟苯酚等结构)或者通过加成反应等方式与抗体或其抗原结合片段上的一个或多个巯基(-SH)或氨基(-NH2)连接。在一些实施方案中，-M-L-E-D能够由以下结构形成，例如通过取代反应(例如脱除其上的甲基磺酰基结构)形成。Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, the compound M'-LED (i.e., the free form of the "drug-linker") described in any of the above-mentioned claims can be obtained first, wherein M' is the structural form of M before covalently linking it with an antibody or its antigen-binding fragment, and then it can be covalently linked with the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application. Accordingly, in the free form of the "drug-linker", M' is linked to one or more thiol (-SH) or amino ( -NH2 ) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me or pentafluorophenol structure) or an addition reaction. In some embodiments, -MLED can be formed from the following structure, for example, through a substitution reaction (e.g., removal of the methanesulfonyl group structure).

在一些实施方案中，本申请的抗体药物偶联物由上文所述的化合物A-1～A-9、B-1～B-19或G-1～G-16与抗体或抗原结合片段相偶联而形成。In some embodiments, the antibody-drug conjugates of this application are formed by conjugating the compounds A-1 to A-9, B-1 to B-19 or G-1 to G-16 described above with an antibody or antigen-binding fragment.

在一些实施方案中，本申请的抗体药物偶联物由上文所述的化合物A-1～A-9、B-1～B-19或G-1～G-16脱除其上的甲基磺酰基后与抗体或抗原结合片段相偶联而形成。In some embodiments, the antibody-drug conjugate of this application is formed by desaturating the methylsulfonyl group of the compounds A-1 to A-9, B-1 to B-19 or G-1 to G-16 described above and then conjugating them with an antibody or antigen-binding fragment.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or its antigen-binding fragment comprises:

(1)下述重链可变区(VH)和/或轻链可变区(VL)：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5或其变体的CDR-H1，序列为SEQ ID NO:6或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20或其变体的CDR-H1，序列为SEQ ID NO:21或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(1a)、(1b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少 96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in either (1a) or (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or the variant having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originated; preferably, the substitutions are conservative substitutions;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18或其变体的CDR-H1，序列为SEQ ID NO:19或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33或其变体的CDR-H1，序列为SEQ ID NO:34或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:33 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(2a)、(2b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11或其变体的CDR-H1，序列为SEQ ID NO:12或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26或其变体的 CDR-H1，序列为SEQ ID NO:27或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(3b) Heavy chain variable region (VH) containing the following 3 CDRs: sequence of SEQ ID NO:26 or a variant thereof CDR-H1, CDR-H2 with the sequence of SEQ ID NO:27 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(3a)、(3b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13或其变体的CDR-H1，序列为SEQ ID NO:14或其变体的CDR-H2，序列为SEQ ID NO:15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16或其变体的CDR-L1，序列为SEQ ID NO:17或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28或其变体的CDR-H1，序列为SEQ ID NO:29或其变体的CDR-H2，序列为SEQ ID NO:30或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31或其变体的CDR-L1，序列为SEQ ID NO:32或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:28 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:29 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:30 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:31 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:32 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(4a)、(4b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or its antigen-binding fragment comprises:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统 定义：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDRs are numbered according to the Chothia numbering system. definition:

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5或其变体的CDR-H1，序列为SEQ ID NO:6或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20或其变体的CDR-H1，序列为SEQ ID NO:21或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(1a)、(1b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18或其变体的CDR-H1，序列为SEQ ID NO:19或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33或其变体的CDR-H1，序列为SEQ ID NO:34或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3； (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

其中，(2a)、(2b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11或其变体的CDR-H1，序列为SEQ ID NO:12或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26或其变体的CDR-H1，序列为SEQ ID NO:27或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:26 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:27 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(3a)、(3b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13或其变体的CDR-H1，序列为SEQ ID NO:14或其变体的CDR-H2，序列为SEQ ID NO:15或其变体 的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16或其变体的CDR-L1，序列为SEQ ID NO:17或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(4a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H2 with sequence SEQ ID NO:15 or a variant thereof. CDR-H3; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28或其变体的CDR-H1，序列为SEQ ID NO:29或其变体的CDR-H2，序列为SEQ ID NO:30或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31或其变体的CDR-L1，序列为SEQ ID NO:32或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:28 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:29 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:30 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:31 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:32 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(4a)、(4b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or its antigen-binding fragment comprises:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5的CDR-H1，序列为SEQ ID NO:6的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20的CDR-H1，序列为SEQ ID NO:21的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18的CDR-H1， 序列为SEQ ID NO:19的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(2a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:18, The CDR-H2 sequence of SEQ ID NO:19, the CDR-H3 sequence of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 sequence of SEQ ID NO:8, CDR-L2 sequence of SEQ ID NO:9, and CDR-L3 sequence of SEQ ID NO:10; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33的CDR-H1，序列为SEQ ID NO:34的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33, CDR-H2 with sequence SEQ ID NO:34, and CDR-H3 with sequence SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23, CDR-L2 with sequence SEQ ID NO:24, and CDR-L3 with sequence SEQ ID NO:25;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11的CDR-H1，序列为SEQ ID NO:12的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11, CDR-H2 of SEQ ID NO: 12, and CDR-H3 of SEQ ID NO: 7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and CDR-L3 of SEQ ID NO: 10; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26的CDR-H1，序列为SEQ ID NO:27的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of sequence SEQ ID NO:26, CDR-H2 of sequence SEQ ID NO:27, and CDR-H3 of sequence SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of sequence SEQ ID NO:23, CDR-L2 of sequence SEQ ID NO:24, and CDR-L3 of sequence SEQ ID NO:25;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13的CDR-H1，序列为SEQ ID NO:14的CDR-H2，序列为SEQ ID NO:15的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16的CDR-L1，序列为SEQ ID NO:17的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28的CDR-H1，序列为SEQ ID NO:29的CDR-H2，序列为SEQ ID NO:30的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31的CDR-L1，序列为SEQ ID NO: 32的CDR-L2，序列为SEQ ID NO:25的CDR-L3。(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:30, CDR-H3 of SEQ ID NO:30, CDR-H1 ... CDR-L2 of SEQ ID NO:25 has the sequence CDR-L3.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or its antigen-binding fragment comprises:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5的CDR-H1，序列为SEQ ID NO:6的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20的CDR-H1，序列为SEQ ID NO:21的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18的CDR-H1，序列为SEQ ID NO:19的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33的CDR-H1，序列为SEQ ID NO:34的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33, CDR-H2 with sequence SEQ ID NO:34, and CDR-H3 with sequence SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23, CDR-L2 with sequence SEQ ID NO:24, and CDR-L3 with sequence SEQ ID NO:25;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11的CDR-H1， 序列为SEQ ID NO:12的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(3a) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:11, The sequences are CDR-H2 of SEQ ID NO:12 and CDR-H3 of SEQ ID NO:7; and, a light chain variable region (VL) containing the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26的CDR-H1，序列为SEQ ID NO:27的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of sequence SEQ ID NO:26, CDR-H2 of sequence SEQ ID NO:27, and CDR-H3 of sequence SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of sequence SEQ ID NO:23, CDR-L2 of sequence SEQ ID NO:24, and CDR-L3 of sequence SEQ ID NO:25;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13的CDR-H1，序列为SEQ ID NO:14的CDR-H2，序列为SEQ ID NO:15的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16的CDR-L1，序列为SEQ ID NO:17的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28的CDR-H1，序列为SEQ ID NO:29的CDR-H2，序列为SEQ ID NO:30的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31的CDR-L1，序列为SEQ ID NO:32的CDR-L2，序列为SEQ ID NO:25的CDR-L3。(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or its antigen-binding fragment comprises:

(a)SEQ ID NO：1所示的VH或其变体，和/或，SEQ ID NO：2所示的VL或其变体；或(a) VH or a variant thereof shown in SEQ ID NO: 1, and/or VL or a variant thereof shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH或其变体，和/或，SEQ ID NO：4所示的VL或其变体；(b) VH or a variant thereof shown in SEQ ID NO: 3, and/or VL or a variant thereof shown in SEQ ID NO: 4;

其中，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。 The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or its antigen-binding fragment comprises:

(a)SEQ ID NO：1所示的VH，和/或，SEQ ID NO：2所示的VL；或(a) VH as shown in SEQ ID NO: 1, and/or VL as shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH，和/或，SEQ ID NO：4所示的VL。(b) VH shown in SEQ ID NO: 3, and/or VL shown in SEQ ID NO: 4.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or its antigen-binding fragment comprises:

(a)SEQ ID NO：1所示的VH或其变体，和，SEQ ID NO：2所示的VL或其变体；或(a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH或其变体，和，SEQ ID NO：4所示的VL或其变体。(b) VH or a variant thereof shown in SEQ ID NO: 3, and VL or a variant thereof shown in SEQ ID NO: 4.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or its antigen-binding fragment comprises:

(a)SEQ ID NO：1所示的VH，和，SEQ ID NO：2所示的VL；或(a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH，和，SEQ ID NO：4所示的VL。(b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

在一些实施方案中，所述抗体或其抗原结合片段进一步包含：In some embodiments, the antibody or its antigen-binding fragment further comprises:

(a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和(a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

(b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。(b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

在一些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区，例如人IgG1重链恒定区或人IgG4重链恒定区。In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：35所示的重链恒定区(CH)或其变体，所述变体与SEQ ID NO：35相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。 In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：35所示的重链恒定区(CH)。In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.

在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：36所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO：36相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：36所示的轻链恒定区(CL)。In some embodiments, the antibody or its antigen-binding fragment includes a light chain constant region (CL) as shown in SEQ ID NO: 36.

在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：35所示的重链恒定区(CH)和如SEQ ID NO：36所示的轻链恒定区(CL)。In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or its antigen-binding fragment comprises:

(1)包括SEQ ID NO：1所示序列的VH和SEQ ID NO：35所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：2所示序列的VL和SEQ ID NO：36所示的轻链恒定区(CL)的轻链；或(1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

(2)包括SEQ ID NO：3所示序列的VH和SEQ ID NO：35所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：4所示序列的VL和SEQ ID NO：36所示的轻链恒定区(CL)的轻链。(2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

在一些实施方案中，所示抗体或其抗原结合片段选自Trastuzumab或Pertuzumab，所述Trastuzumab的氨基酸序列在IMGT数据库中的查询登录号(IMGT/mAb-DB ID)：97，所述Pertuzumab的氨基酸序列在IMGT数据库中的查询登录号(IMGT/mAb-DB ID)：80。In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT/mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT/mAb-DB ID) of 80.

在本文公开的抗体或抗原结合片段的某些实施方案中，重链恒定结构域可包含C-末端赖氨酸或缺乏C-末端赖氨酸或C-末端甘氨酸-赖氨酸二肽。在抗体或其抗原结合片段的一些实施方案中，抗体或其抗原结合片段的N端氨基酸可以环化成焦谷氨酸。In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

如本领域技术人员已知的，焦谷氨酸是焦谷氨酸盐的共轭酸，并且与溶液中的焦谷氨酸盐相互平衡。As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

在某些实施方案中，本文提供了包含本文公开的抗体或抗原结合片段的组合物，其中 的各种抗体或抗原结合片段可以独立地包含C端赖氨酸、缺乏C端赖氨酸、缺乏C端甘氨酸-赖氨酸和/或包含N端谷氨酰胺或谷氨酸、N端氨基酸环化为焦谷氨酸或N端氨基酸环化为焦谷氨酸盐。In some embodiments, this document provides compositions comprising the antibody or antigen-binding fragment disclosed herein, wherein Various antibody or antigen-binding fragments may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine, and/or contain N-terminal glutamine or glutamate, N-terminal amino acid cyclized to pyroglutamic acid, or N-terminal amino acid cyclized to pyroglutamate salt.

在某些实施方案中，本文公开的抗体或抗原结合片段包括特异性结合抗原的抗体或抗原结合片段，并且可以包括其翻译后修饰(例如，重链中的C端赖氨酸剪切，重链或轻链中的N-末端谷氨酰胺或谷氨酸转化为焦谷氨酸或焦谷氨酸盐)，这可以在宿主细胞(例如，CHO细胞)中重组表达时或在纯化/储存期间发生。In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification/storage.

在某些实施方案中，所述如SEQ ID NO:1或3所示序列的VH或其变体或者所述如SEQ ID NO:37或39所示序列的重链或其变体的N端谷氨酰胺经历环化形成焦谷氨酸或焦谷氨酸盐。In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

在某些实施方案中，所述如SEQ ID NO：35所示序列的重链恒定区(CH)或其变体或者所述如SEQ ID NO:37或39所示序列的重链或其变体缺乏C-末端赖氨酸。In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

在一些实施方案中，所述抗体药物偶联物选自：










In some embodiments, the antibody-drug conjugate is selected from:










其中，各抗体药物偶联物中的HA-(S-或为抗体或其抗原结合片段；Among them, HA-(S- or) in each antibody-drug conjugate It is an antibody or its antigen-binding fragment;

其中，-(S-或表示抗体或其抗原结合片段中的巯基与M片段的具体连接方式；Wherein, -(S- or represents the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the M fragment;

n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.

在一些实施方案中，各抗体药物偶联物中的HA包含:In some implementations, the HA in each antibody-drug conjugate includes:

(1)下述重链可变区(VH)和/或轻链可变区(VL)：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5或其变体的 CDR-H1，序列为SEQ ID NO:6或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(1a) Heavy chain variable region (VH) containing the following 3 CDRs: sequence of SEQ ID NO:5 or a variant thereof CDR-H1, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20或其变体的CDR-H1，序列为SEQ ID NO:21或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(1a)、(1b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18或其变体的CDR-H1，序列为SEQ ID NO:19或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33或其变体的CDR-H1，序列为SEQ ID NO:34或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:33 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(2a)、(2b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源 自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in either (2a) or (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has at least 70%, at least 80%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has at least 70%, at least 80%, at least 85%, at least 90 ... Compared to the original sequence, it has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); preferably, the substitutions are conservative substitutions;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11或其变体的CDR-H1，序列为SEQ ID NO:12或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26或其变体的CDR-H1，序列为SEQ ID NO:27或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:26 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:27 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(3a)、(3b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL):

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13或其变体的CDR-H1，序列为SEQ ID NO:14或其变体的CDR-H2，序列为SEQ ID NO:15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16或其变体的CDR-L1，序列为SEQ ID NO:17或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28或其变体的CDR-H1，序列为SEQ ID NO:29或其变体的CDR-H2，序列为SEQ ID NO:30或其变体 的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31或其变体的CDR-L1，序列为SEQ ID NO:32或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(4b) A heavy chain variable region (VH) containing the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H2 with sequence SEQ ID NO:30 or a variant thereof. CDR-H3; and/or, a light chain variable region (VL) containing the following three CDRs: CDR-L1 with the sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:25 or a variant thereof;

其中，(4a)、(4b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

在一些实施方案中，各抗体药物偶联物中的HA包含:In some implementations, the HA in each antibody-drug conjugate includes:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5或其变体的CDR-H1，序列为SEQ ID NO:6或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20或其变体的CDR-H1，序列为SEQ ID NO:21或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(1a)、(1b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定 义：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is determined according to the AbM numbering system. righteous:

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18或其变体的CDR-H1，序列为SEQ ID NO:19或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33或其变体的CDR-H1，序列为SEQ ID NO:34或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:33 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(2a)、(2b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11或其变体的CDR-H1，序列为SEQ ID NO:12或其变体的CDR-H2，序列为SEQ ID NO:7或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或其变体的CDR-L1，序列为SEQ ID NO:9或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26或其变体的CDR-H1，序列为SEQ ID NO:27或其变体的CDR-H2，序列为SEQ ID NO:22或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23或其变体的CDR-L1，序列为SEQ ID NO:24或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3； (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

其中，(3a)、(3b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13或其变体的CDR-H1，序列为SEQ ID NO:14或其变体的CDR-H2，序列为SEQ ID NO:15或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16或其变体的CDR-L1，序列为SEQ ID NO:17或其变体的CDR-L2，序列为SEQ ID NO:10或其变体的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28或其变体的CDR-H1，序列为SEQ ID NO:29或其变体的CDR-H2，序列为SEQ ID NO:30或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31或其变体的CDR-L1，序列为SEQ ID NO:32或其变体的CDR-L2，序列为SEQ ID NO:25或其变体的CDR-L3；(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:28 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:29 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:30 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:31 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:32 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

其中，(4a)、(4b)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

在一些实施方案中，各抗体药物偶联物中的HA包含:In some implementations, the HA in each antibody-drug conjugate includes:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5的CDR-H1，序列为SEQ ID NO:6的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和/或，包含如下3 个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:5, CDR-H2 with sequence SEQ ID NO:6, and CDR-H3 with sequence SEQ ID NO:7; and/or, comprising the following three The light chain variable region (VL) of each CDR: CDR-L1 with sequence SEQ ID NO:8, CDR-L2 with sequence SEQ ID NO:9, and CDR-L3 with sequence SEQ ID NO:10; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20的CDR-H1，序列为SEQ ID NO:21的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18的CDR-H1，序列为SEQ ID NO:19的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33的CDR-H1，序列为SEQ ID NO:34的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33, CDR-H2 with sequence SEQ ID NO:34, and CDR-H3 with sequence SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23, CDR-L2 with sequence SEQ ID NO:24, and CDR-L3 with sequence SEQ ID NO:25;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11的CDR-H1，序列为SEQ ID NO:12的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26的CDR-H1，序列为SEQ ID NO:27的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3； (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13的CDR-H1，序列为SEQ ID NO:14的CDR-H2，序列为SEQ ID NO:15的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16的CDR-L1，序列为SEQ ID NO:17的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28的CDR-H1，序列为SEQ ID NO:29的CDR-H2，序列为SEQ ID NO:30的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31的CDR-L1，序列为SEQ ID NO:32的CDR-L2，序列为SEQ ID NO:25的CDR-L3。(4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

在一些实施方案中，各抗体药物偶联物中的HA包含:In some implementations, the HA in each antibody-drug conjugate includes:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(1) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

(1a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:5的CDR-H1，序列为SEQ ID NO:6的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(1b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:20的CDR-H1，序列为SEQ ID NO:21的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：(2) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

(2a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:18的CDR-H1，序列为SEQ ID NO:19的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和，包含如下3个 CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and, comprising the following three The light chain variable region (VL) of the CDR: CDR-L1 with sequence SEQ ID NO:8, CDR-L2 with sequence SEQ ID NO:9, and CDR-L3 with sequence SEQ ID NO:10; or,

(2b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:33的CDR-H1，序列为SEQ ID NO:34的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33, CDR-H2 with sequence SEQ ID NO:34, and CDR-H3 with sequence SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23, CDR-L2 with sequence SEQ ID NO:24, and CDR-L3 with sequence SEQ ID NO:25;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(3) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

(3a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11的CDR-H1，序列为SEQ ID NO:12的CDR-H2，序列为SEQ ID NO:7的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8的CDR-L1，序列为SEQ ID NO:9的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

(3b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:26的CDR-H1，序列为SEQ ID NO:27的CDR-H2，序列为SEQ ID NO:22的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:23的CDR-L1，序列为SEQ ID NO:24的CDR-L2，序列为SEQ ID NO:25的CDR-L3；(3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of sequence SEQ ID NO:26, CDR-H2 of sequence SEQ ID NO:27, and CDR-H3 of sequence SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of sequence SEQ ID NO:23, CDR-L2 of sequence SEQ ID NO:24, and CDR-L3 of sequence SEQ ID NO:25;

或者，or,

(4)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(4) The following heavy chain variable regions (VH) and/or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

(4a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:13的CDR-H1，序列为SEQ ID NO:14的CDR-H2，序列为SEQ ID NO:15的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:16的CDR-L1，序列为SEQ ID NO:17的CDR-L2，序列为SEQ ID NO:10的CDR-L3；或，(4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

(4b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28的CDR-H1，序列为SEQ ID NO:29的CDR-H2，序列为SEQ ID NO:30的CDR-H3；和，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:31的CDR-L1，序列为SEQ ID NO:32的CDR-L2，序列为SEQ ID NO:25的CDR-L3。 (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

在一些实施方案中，各抗体药物偶联物中的HA包含：In some implementations, the HA in each antibody-drug conjugate includes:

(a)SEQ ID NO：1所示的VH或其变体，和/或，SEQ ID NO：2所示的VL或其变体；或(a) VH or a variant thereof shown in SEQ ID NO: 1, and/or VL or a variant thereof shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH或其变体，和/或，SEQ ID NO：4所示的VL或其变体；(b) VH or a variant thereof shown in SEQ ID NO: 3, and/or VL or a variant thereof shown in SEQ ID NO: 4;

其中，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

在一些实施方案中，各抗体药物偶联物中的HA包含：In some implementations, the HA in each antibody-drug conjugate includes:

(a)SEQ ID NO：1所示的VH，和/或，SEQ ID NO：2所示的VL；或(a) VH as shown in SEQ ID NO: 1, and/or VL as shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH，和/或，SEQ ID NO：4所示的VL。(b) VH shown in SEQ ID NO: 3, and/or VL shown in SEQ ID NO: 4.

在一些实施方案中，各抗体药物偶联物中的HA包含：In some implementations, the HA in each antibody-drug conjugate includes:

(a)SEQ ID NO：1所示的VH或其变体，和，SEQ ID NO：2所示的VL或其变体；或(a) VH or a variant thereof shown in SEQ ID NO: 1, and VL or a variant thereof shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH或其变体，和，SEQ ID NO：4所示的VL或其变体。(b) VH or a variant thereof shown in SEQ ID NO: 3, and VL or a variant thereof shown in SEQ ID NO: 4.

在一些实施方案中，各抗体药物偶联物中的HA包含：In some implementations, the HA in each antibody-drug conjugate includes:

(a)SEQ ID NO：1所示的VH，和，SEQ ID NO：2所示的VL；或(a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

(b)SEQ ID NO：3所示的VH，和，SEQ ID NO：4所示的VL。(b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4.

在一些实施方案中，各抗体药物偶联物中的HA进一步包含：In some implementations, the HA in each antibody-drug conjugate further comprises:

(a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和 (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

(b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。(b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

在一些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区，例如人IgG1重链恒定区或人IgG4重链恒定区。In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

在一些实施方案中，各抗体药物偶联物中的HA包含如SEQ ID NO：35所示的重链恒定区(CH)或其变体，所述变体与SEQ ID NO：35相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。In some embodiments, the HA in each antibody-drug conjugate contains a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions of amino acids compared to SEQ ID NO: 35 (e.g., up to 15, up to 10, or up to 5 conserved substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conserved substitutions of amino acids).

在一些实施方案中，各抗体药物偶联物中的HA包含如SEQ ID NO：35所示的重链恒定区(CH)。In some implementations, the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.

在一些实施方案中，各抗体药物偶联物中的HA包含如SEQ ID NO：36所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO：36相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。In some embodiments, the HA in each antibody-drug conjugate contains a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions of amino acids compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conserved substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conserved substitutions of amino acids).

在一些实施方案中，各抗体药物偶联物中的HA包含如SEQ ID NO：36所示的轻链恒定区(CL)。In some implementations, the HA in each antibody-drug conjugate includes a light chain constant region (CL) as shown in SEQ ID NO: 36.

在一些实施方案中，各抗体药物偶联物中的HA包含如SEQ ID NO：35所示的重链恒定区(CH)和如SEQ ID NO：36所示的轻链恒定区(CL)。In some embodiments, the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

在一些实施方案中，各抗体药物偶联物中的HA包含：In some implementations, the HA in each antibody-drug conjugate includes:

(1)包括SEQ ID NO：1所示序列的VH和SEQ ID NO：35所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：2所示序列的VL和SEQ ID NO：36所示的轻链恒定区(CL)的轻链；或(1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

(2)包括SEQ ID NO：3所示序列的VH和SEQ ID NO：35所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：4所示序列的VL和SEQ ID NO：36所示的轻链恒定区(CL)的轻链。 (2) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

在一些实施方案中，各抗体药物偶联物中的HA选自Trastuzumab或Pertuzumab，所述Trastuzumab的氨基酸序列在IMGT数据库中的查询登录号(IMGT/mAb-DB ID)：97，所述Pertuzumab的氨基酸序列在IMGT数据库中的查询登录号(IMGT/mAb-DB ID)：80。In some embodiments, the HA in each antibody-drug conjugate is selected from Trastuzumab or Pertuzumab, wherein the amino acid sequence of Trastuzumab has a lookup accession number (IMGT/mAb-DB ID) of 97 in the IMGT database, and the amino acid sequence of Pertuzumab has a lookup accession number (IMGT/mAb-DB ID) of 80 in the IMGT database.

在某些实施方案中，各抗体药物偶联物中的HA可以独立地包含C端赖氨酸、缺乏C端赖氨酸、缺乏C端甘氨酸-赖氨酸和/或包含N端谷氨酰胺或谷氨酸、N端氨基酸环化为焦谷氨酸或N端氨基酸环化为焦谷氨酸盐。In some embodiments, the HA in each antibody-drug conjugate may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine and/or contain N-terminal glutamine or glutamate, cyclize the N-terminal amino acid to pyroglutamic acid or cyclize the N-terminal amino acid to pyroglutamate salt.

在某些实施方案中，各抗体药物偶联物中的HA可以包括其翻译后修饰(例如，重链中的C端赖氨酸剪切，重链或轻链中的N-末端谷氨酰胺或谷氨酸转化为焦谷氨酸或焦谷氨酸盐)，这可以在宿主细胞(例如，CHO细胞)中重组表达时或在纯化/储存期间发生。In some embodiments, the HA in each antibody-drug conjugate may include its post-translational modifications (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate in the heavy or light chain being converted to pyroglutamate or pyroglutamate salt), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification/storage.

在某些实施方案中，所述如SEQ ID NO:1或3所示序列的VH或其变体或者所述如SEQ ID NO:37或39所示序列的重链或其变体的N端谷氨酰胺经历环化形成焦谷氨酸或焦谷氨酸盐。In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

在某些实施方案中，所述如SEQ ID NO：35所示序列的重链恒定区(CH)或其变体或者所述如SEQ ID NO:37或39所示序列的重链或其变体缺乏C-末端赖氨酸。In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof, lacks a C-terminal lysine residue.

在一些实施方案中，各抗体药物偶联物中的HA代表曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、或它们的抗原结合片段。In some implementations, the HA in each antibody-drug conjugate represents trastuzumab, pertuzumab, or their antigen-binding fragments.

在一些实施方案中，各抗体药物偶联物中的HA代表以下抗体或抗原结合片段：In some implementations, the HA in each antibody-drug conjugate represents the following antibody or antigen-binding fragment:

(1)包括SEQ ID NO：1所示序列的VH和SEQ ID NO：35所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：2所示序列的VL和SEQ ID NO：36所示的轻链恒定区(CL)的轻链；或(1) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

(2)包括SEQ ID NO：3所示序列的VH和SEQ ID NO：35所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：4所示序列的VL和SEQ ID NO：36所示的轻链恒定区(CL)的轻链；(2) A heavy chain including the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain including the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36;

其中，表示抗体或其抗原结合片段中的巯基与连接体的具体连接方式。 in, This indicates the specific way in which the thiol group in the antibody or its antigen-binding fragment is linked to the linker.

在一些实施方案中，x为1-10，例如1-2，1-3，1-4，1-5，1-6，1-7，1-8，1-9，1-10，2-3，2-4，2-5，2-6，2-7，2-8，2-9，2-10，3-4，3-5，3-6，3-7，3-8，3-9，3-10，4-5，4-6，4-7，4-8，4-9，4-10，5-6，5-7，5-8，5-9，5-10，6-7，6-8，6-9，6-10，7-8，7-9，7-10，8-9，8-10或9-10。In some implementations, x is 1-10, for example 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

在一些实施方案中，x为5-8，例如5-6，5-7，5-8，6-7，6-8或7-8。In some implementations, x is 5-8, such as 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.

在一些实施方案中，x为1-6，例如2-6、3-6、4-6。In some implementations, x is 1-6, such as 2-6, 3-6, 4-6.

在一些实施方案中，x为1、2、3、4、5、6、7、8、9或10。In some implementations, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

本发明所述的抗体药物偶联物任选地被一个或多个合适的取代基所取代。The antibody-drug conjugates of the present invention may optionally be substituted with one or more suitable substituents.

组合物Composition

在另一方面，本申请提供了如本文所述的抗体药物偶联物(ADC)的组合物。这种组合物可包含多个本文所述的ADC，其中每个ADC包含本文所述的药物-连接体，其中x独立地为1、2、3、4、5、6、7、8、9或10。换言之，所述组合物中的每个抗体分子可以与1、2、3、4、5、6、7、8、9或10个药物-连接体缀合。因此，所述组合物的特征在于“药物-抗体”比(DAR)在约1至约10的范围内。测定DAR的方法是技术人员熟知的，包括使用反相色谱或HPLC-MS的方法。On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

例如，在任意实施方案中，本文所述的ADC组合物具有约1至约10或其间任何子范围的DAR，例如:约1至2、约1至3、约1至4、约1至5、约1至6、约1至7、约1至8、约1至9、约1至10、约2至3、约2至4、约2至5、约2至6，约2至7，约2至8，约2至9，约2至10，约3至4，约3至5，约3至6，约3至7，约3至8，约3至9，约3至10，约4至5，约4至6，约4至7，约4至8，约4至9，约4至10，约5至6，约5至7，约5至8，约5至9、约5至10、约6至7、约6至8、约6至9、约6至10、约7至8、约7至9、约7至10、约8至9、约8至10或约9至10。For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

在某些实施方案中，本文所述ADC组合物的DAR为约3至9，例如约3.0至3.5、约3.0至4.0、约3.0至4.5、约3.0至5.0、约3.0至6.0、约3.5至4.0、约3.5至4.5、约3.5至5.0、约3.5至5.5、约3.5至6.0、约3.5至6.5至6约4.0至4.5，约4.0至5.0，约4.0至5.5，约4.0至6.0，约4.0至6.5，约4.0至7.0，约4.0至8.0，约4.5至5.0，约4.5至5.5，约4.5至6.0，约4.5至6.5，约4.5至7.0，约4.5至7.5约5.0至8.0，约5.5至6.0，约5.5至6.5，约5.5至7.0，约5.5至7.5，约5.5至8.0，约6.0至6.5，约6.0至7.0，约 6.0至7.5，约6.0至8.5，约6.5至7.0，约6.5至7.5，约6.5至7.5，约6.5至8.5，约7.0至7.5。In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, such as about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5 to 6, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4. 0 to 6.0, approximately 4.0 to 6.5, approximately 4.0 to 7.0, approximately 4.0 to 8.0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 7.5, approximately 5.0 to 8.0, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.5, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 7.5, approximately 6.5 to 8.5, approximately 7.0 to 7.5.

在一些实施方案中，本文所述ADC组合物的DAR为约3至8，例如，约3.0至3.5，约3.0至4.0，约3.0至4.5，约3.0至5.0，约6.0至6.5，约6.0至7.0，约6.0至7.5，约6.0至8.0，约6.0至8.5，约6.5至7.0，约6.5至7.5，约6.5至8.0，约6.5至8.5，约7.0至7.5，约7.0至8.0或约7.5至8.0。In some embodiments, the DAR of the ADC composition described herein is about 3 to 8, for example, about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0, or about 7.5 to 8.0.

在一些实施方案中，本文所述ADC组合物的DAR为7.5至8.5，例如，约7.5，约7.6，约7.7，约7.8，约7.9，约8.0，约8.1，约8.2，约8.3，约8.4，约8.5。In some embodiments, the DAR of the ADC composition described herein is 7.5 to 8.5, for example, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5.

在一些实施方案中，本文所述ADC组合物的DAR为约6.0至9.0，优选为约6.0-8.0，例如约6.0、约6.01、约6.02、约6.03、约6.04、约6.05、约6.06、约6.07、约6.08、约6.09、约6.1、约6.11、约6.12、约6.13、约6.14、约6.15、约6.16、约6.17、约6.18、约6.19、约6.2、约6.21、约6.22、约6.23、约6.24、约6.25、约6.26、约6.27、约6.28、约6.29、约6.3、约6.31、约6.32、约6.33、约6.34、约6.35、约6.36、约6.37、约6.38、约6.39、约6.4、约6.41、约6.42、约6.43、约6.44、约6.45、约6.46、约6.47、约6.48、约6.49、约6.5、约6.51、约6.52、约6.53、约6.54、约6.55、约6.56、约6.57、约6.58、约6.59、约6.6、约6.61、约6.62、约6.63、约6.64、约6.65、约6.66、约6.67、约6.68、约6.69、约6.7、约6.71、约6.72、约6.73、约6.74、约6.75、约6.76、约6.77、约6.78、约6.79、约6.8、约6.81、约6.82、约6.83、约6.84、约6.85、约6.86、约6.87、约6.88、约6.89、约6.9、约6.91、约6.92、约6.93、约6.94、约6.95、约6.96、约6.97、约6.98、约6.99、约7.0、约7.01、约7.02、约7.03、约7.04、约7.05、约7.06、约7.07、约7.08、约7.09、约7.1、约7.11、约7.12、约7.13、约7.14、约7.15、约7.16、约7.17、约7.18、约7.19、约7.2、约7.21、约7.22、约7.23、约7.24、约7.25、约7.26、约7.27、约7.28、约7.29、约7.3、约7.31、约7.32、约7.33、约7.34、约7.35、约7.36、约7.37、约7.38、约7.39、约7.4、约7.41、约7.42、约7.43、约7.44、约7.45、约7.46、约7.47、约7.48、约7.49、约7.5、约7.51、约7.52、约7.53、约7.54、约7.55、约7.56、约7.57、约7.58、约7.59、约7.6、约7.61、约7.62、约7.63、约7.64、约7.65、约7.66、约7.67、约7.68、约7.69、约7.7、约7.71、约7.72、约7.73、约7.74、约7.75、约7.76、约7.77、约7.78、约7.79、约7.8、约7.81、约7.82、约7.83、约7.84、约7.85、约7.86、约7.87、约7.88、约7.89、约7.9、约7.91、约7.92、约7.93、约7.94、约7.95、约7.96、约7.97、约7.98、约7.99、约8.0、约8.01、约8.02、约8.03、约8.04、约8.05、约8.06、约8.07、约8.08、约 8.09、约8.1、约8.11、约8.12、约8.13、约8.14、约8.15、约8.16、约8.17、约8.18、约8.19、约8.2、约8.21、约8.22、约8.23、约8.24、约8.25、约8.26、约8.27、约8.28、约8.29、约8.3、约8.31、约8.32、约8.33、约8.34、约8.35、约8.36、约8.37、约8.38、约8.39、约8.4、约8.41、约8.42、约8.43、约8.44、约8.45、约8.46、约8.47、约8.48、约8.49、约8.5、约8.51、约8.52、约8.53、约8.54、约8.55、约8.56、约8.57、约8.58、约8.59、约8.6、约8.61、约8.62、约8.63、约8.64、约8.65、约8.66、约8.67、约8.68、约8.69、约8.7、约8.71、约8.72、约8.73、约8.74、约8.75、约8.76、约8.77、约8.78、约8.79、约8.8、约8.81、约8.82、约8.83、约8.84、约8.85、约8.86、约8.87、约8.88、约8.89、约8.9、约8.91、约8.92、约8.93、约8.94、约8.95、约8.96、约8.97、约8.98、约8.99、约9.0。In some embodiments, the DAR of the ADC composition described herein is about 6.0 to 9.0, preferably about 6.0-8.0, for example about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.09, about 6.10, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19 ... 6.19, approximately 6.2, approximately 6.21, approximately 6.22, approximately 6.23, approximately 6.24, approximately 6.25, approximately 6.26, approximately 6.27, approximately 6.28, approximately 6.29, approximately 6.3, approximately 6.31, approximately 6.32, approximately 6.33, approximately 6.34, approximately 6.35, approximately 6.36, approximately 6.37, approximately 6.38, approximately 6.39, approximately 6.4, approximately 6.41, approximately 6.42, approximately 6.43, approximately 6.44, approximately 6.45, approximately 6 .46, about 6.47, about 6.48, about 6.49, about 6.5, about 6.51, about 6.52, about 6.53, about 6.54, about 6.55, about 6.56, about 6.57, about 6.58, about 6.59, about 6.6, about 6.61, about 6.62, about 6.63, about 6.64, about 6.65, about 6.66, about 6.67, about 6.68, about 6.69, about 6.7, about 6.71, about 6.72, about 6. 73, approximately 6.74, approximately 6.75, approximately 6.76, approximately 6.77, approximately 6.78, approximately 6.79, approximately 6.8, approximately 6.81, approximately 6.82, approximately 6.83, approximately 6.84, approximately 6.85, approximately 6.86, approximately 6.87, approximately 6.88, approximately 6.89, approximately 6.9, approximately 6.91, approximately 6.92, approximately 6.93, approximately 6.94, approximately 6.95, approximately 6.96, approximately 6.97, approximately 6.98, approximately 6.99, approximately 7. 0, approximately 7.01, approximately 7.02, approximately 7.03, approximately 7.04, approximately 7.05, approximately 7.06, approximately 7.07, approximately 7.08, approximately 7.09, approximately 7.1, approximately 7.11, approximately 7.12, approximately 7.13, approximately 7.14, approximately 7.15, approximately 7.16, approximately 7.17, approximately 7.18, approximately 7.19, approximately 7.2, approximately 7.21, approximately 7.22, approximately 7.23, approximately 7.24, approximately 7.25, approximately 7.26, approximately 7.2 7, approximately 7.28, approximately 7.29, approximately 7.3, approximately 7.31, approximately 7.32, approximately 7.33, approximately 7.34, approximately 7.35, approximately 7.36, approximately 7.37, approximately 7.38, approximately 7.39, approximately 7.4, approximately 7.41, approximately 7.42, approximately 7.43, approximately 7.44, approximately 7.45, approximately 7.46, approximately 7.47, approximately 7.48, approximately 7.49, approximately 7.5, approximately 7.51, approximately 7.52, approximately 7.53, approximately 7.54 Approximately 7.55, approximately 7.56, approximately 7.57, approximately 7.58, approximately 7.59, approximately 7.6, approximately 7.61, approximately 7.62, approximately 7.63, approximately 7.64, approximately 7.65, approximately 7.66, approximately 7.67, approximately 7.68, approximately 7.69, approximately 7.7, approximately 7.71, approximately 7.72, approximately 7.73, approximately 7.74, approximately 7.75, approximately 7.76, approximately 7.77, approximately 7.78, approximately 7.79, approximately 7.8, approximately 7.81. Approximately 7.82, 7.83, 7.84, 7.85, 7.86, 7.87, 7.88, 7.89, 7.9, 7.91, 7.92, 7.93, 7.94, 7.95, 7.96, 7.97, 7.98, 7.99, 8.0, 8.01, 8.02, 8.03, 8.04, 8.05, 8.06, 8.07, 8.08, approximately 8.09, approximately 8.1, approximately 8.11, approximately 8.12, approximately 8.13, approximately 8.14, approximately 8.15, approximately 8.16, approximately 8.17, approximately 8.18, approximately 8.19, approximately 8.2, approximately 8.21, approximately 8.22, approximately 8.23, approximately 8.24, approximately 8.25, approximately 8.26, approximately 8.27, approximately 8.28, approximately 8.29, approximately 8.3, approximately 8.31, approximately 8.32, approximately 8.33, approximately 8.34, approximately 8.35, approximately 8.36, approximately 8.37, approximately 8.38, approximately 8.39, approximately 8.4, approximately 8.41, approximately 8.42, approximately 8.43, approximately 8.44, approximately 8.45, approximately 8.46, approximately 8.47, approximately 8.48, approximately 8.49, approximately 8.5, approximately 8.51, approximately 8.52, approximately 8.53, approximately 8.54, Approximately 8.55, 8.56, 8.57, 8.58, 8.59, 8.6, 8.61, 8.62, 8.63, 8.64, 8.65, 8.66, 8.67, 8.68, 8.69, 8.7, 8.71, 8.72, 8.73, 8.74, 8.75, 8.76, 8.77 Approximately 8.78, 8.79, 8.8, 8.81, 8.82, 8.83, 8.84, 8.85, 8.86, 8.87, 8.88, 8.89, 8.9, 8.91, 8.92, 8.93, 8.94, 8.95, 8.96, 8.97, 8.98, 8.99, 9.0.

药物组合物Pharmaceutical Composition

在另一个方面，本申请提供一种药物组合物，其含有前文任一项所述的抗体药物偶联物或任一项所述的组合物，以及一种或多种药用辅料。In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates or compositions described in any of the preceding claims, and one or more pharmaceutical excipients.

本文所述抗体药物偶联物或组合物通常与药学上可接受的胃肠外媒介一起以单位可注射形式配制，供胃肠外使用，例如推注、静脉注射、肿瘤内注射等。任选地，以冻干剂或溶液剂的形式将具有期望纯度的抗体药物偶联物与药学上可接受的稀释剂、载体、赋型剂或稳定剂混合(Remington’s Pharmaceutical Sciences(1980)16th edition,Osol,A.Ed.)。可以通过对于要治疗的个体适宜的任何路径施用本文所述抗体药物偶联物或含有所述抗体药物偶联物的药物组合物。The antibody-drug conjugates or compositions described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing said antibody-drug conjugates can be administered via any route appropriate for the individual to be treated.

应用application

本文所述抗体药物偶联物、组合物、或其药物组合物可以用于治疗多种疾病或病症，例如Her2表达癌症，包括实体瘤或血液系统恶性肿瘤，例如尿路上皮癌，胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)，或淋巴癌。The antibody-drug conjugates, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as urothelial carcinoma, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

因此，本申请提供前文任一项所述的抗体药物偶联物、组合物、或含有其的药物组合物在制备治疗Her2表达癌症的药物中的用途。Therefore, this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of a medicament for treating Her2-expressing cancers.

同时，本申请提供前文任一项所述的抗体药物偶联物、组合物、或含有其的药物组合物，其用于治疗Her2表达癌症的药物中的用途。Additionally, this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the conjugates described above in a medicament for treating Her2-expressing cancer.

同时，本申请还提供一种治疗Her2表达癌症的方法，其包括向由此需要的受试者施 用有效量的前文任一项所述的抗体药物偶联物、组合物、或含有其的药物组合物的步骤。This application also provides a method for treating Her2-expressing cancers, comprising administering [treatment] to a subject in need of [treatment]. The steps involve using an effective amount of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the antibody-drug conjugate described in any of the preceding statements.

在一些实施方案中，所述抗体药物偶联物、化合物、药物-连接体或含有其的药物组合物足以(例如，在受试者中):In some embodiments, the antibody-drug conjugate, compound, drug-linker, or pharmaceutical composition containing it is sufficient (e.g., in a subject):

(1)抑制细胞(如肿瘤细胞)的增殖；(1) Inhibits the proliferation of cells (such as tumor cells);

(2)抑制肿瘤生长；(2) Inhibits tumor growth;

(3)诱导和/或增加抗体依赖性细胞毒性活性；(3) Inducing and/or increasing antibody-dependent cytotoxic activity;

(4)抑制HER2介导的信号转导；(4) Inhibit HER2-mediated signal transduction;

(5)预防和/或治疗HER2介导的疾病/障碍；或者(5) Prevention and/or treatment of HER2-mediated diseases/disorders; or

(6)上述(1)-(5)的任何组合。(6) Any combination of (1)-(5) above.

在一些实施方案中，所述癌症选自实体瘤或血液系统恶性肿瘤；例如选自胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)和尿路上皮癌。In some implementations, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.

定义definition

除非在下文中另有定义，本文中所使用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。并且，本文中所用的基因组学、核酸化学、分子生物学等实验室操作步骤均为相应领域内广泛使用的常规步骤。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出 多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循本领域已知的各种编号系统。The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits [a specific function/characteristic]. Multiple effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen-binding sites. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

术语“抗体”还包括重链恒定区包含C-末端赖氨酸、或缺少C-末端赖氨酸或C-末端甘氨酸-赖氨酸二肽的实施方案。该术语还包括抗体可变区的N-末端氨基酸已经环化成焦谷氨酸盐的实施方案。因此，在包含本文公开的抗体的组合物中，其中的各种抗体可以独立地包含C-末端赖氨酸、缺少C-末端赖氨酸、缺少C-末端甘氨酸-赖氨酸和/或包含N-末端谷氨酷胺或谷氨酸或N-末端氨基酸环化为焦谷氨酸。The term "antibody" also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and/or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)、IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)或AbM编号系统(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; C...). The definitions in the CDRs defined by hothia et al. (1989) Nature 342:878-883, the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

在本发明中，抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定，例如通过Kabat、Chothia、IMGT或AbM编号系统确定。在某些实施方案中，抗体或其抗原结合片段含有的CDR通过Chothia编号系统定义。In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

下述通用规则(公开于www.bioinf.org.uk:Andrew C.R.Martin教授研究小组)可用于定义抗体序列中的CDR，其中包括与抗体结合的抗原表位组成的氨基酸发生特异性相互作用的氨基酸。在极少数情况下，该等一般恒定特征不会出现；但Cys残基是最保守的特征。
The following general rules (published at www.bioinf.org.uk: Professor Andrew C. Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids that form the antigenic epitope that the antibody binds to. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

VH的全部氨基酸序列通常根据Kabat进行编号，而可变区内的三个CDR可根据上述 任何一种编号系统进行定义。在某些实施方案中，VH中的氨基酸位点可从氨基酸位点1开始依次顺序编号，直至序列末端，也可根据Kabat进行编号。除非另有说明，本文所述VH和VL中的氨基酸位点根据顺序编号定义。The complete amino acid sequence of V H is usually numbered according to Kabat, while the three CDRs in the variable region can be numbered according to the above. Any numbering system may be used for definition. In some embodiments, the amino acid sites in VH may be sequentially numbered starting from amino acid site 1 and continuing to the end of the sequence, or they may be numbered according to Kabat. Unless otherwise stated, the amino acid sites in VH and VL described herein are defined according to sequential numbering.

重链恒定区中氨基酸位点的编号可从氨基酸位点1开始依次顺序编号，直至序列末端，也可根据Eu进行编号。IgG1重链恒定区的氨基酸序列有330个氨基酸，编号依次为1至330。根据Eu编号的相应序列从位点118开始，至位点447结束。除非另有说明，本文所述重链和轻链的氨基酸位点根据顺序编号定义。The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

术语抗体的“抗原结合片段”是指抗体的片段的多肽，例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab片段、Fab’片段、F(ab)’2片段、F(ab)’3片段、Fd、Fv、scFv、di-scFv、(scFv)2、二硫键稳定的Fv蛋白(“dsFv”)、单结构域抗体(sdAb，纳米抗体)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab)' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab') 2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab'fragment" refers to the fragment obtained by reducing the disulfide bonds of the two heavy chain fragments in the F(ab') 2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDRs赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDRs)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定 区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。The term "Fc" refers to the region formed by the second and third constant regions of the first heavy chain of the antibody and the second and third constant regions of the second heavy chain. Antibody fragments are formed by binding via disulfide bonds. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS(SEQ ID NO:46)氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4(SEQ ID NO:47)的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在某些实施方案中，VH和VL结构域可以以任何合适的排列彼此相对定位。例如，包含NH2-VH-VH-COOH、NH2-VL-VL-COOH的scFv。The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH₂ - VL-linker-VH-COOH or NH₂ - VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:46) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS) 4 (SEQ ID NO:47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFv containing NH₂ -VH-VH-COOH and NH₂ - VL-VL-COOH.

术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体也称为纳米抗体(nanobody)。The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, referring to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen.

在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)， 那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. It occurs when a position in two compared sequences is occupied by the same base or amino acid monomeric subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). Then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made conveniently using, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein/peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。 The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

术语“连接体”指将细胞毒性药物与抗体或抗原结合片段连接起来的结构片段。例如，指式Ab-[M-L-E-D]x中的-M-L-E-结构片段。The term "linker" refers to a structural segment that links a cytotoxic drug to an antibody or antigen-binding fragment. For example, it refers to the -MLE- structural segment in the formula Ab-[MLED] x .

术语“药物-连接体”指本发明所述的细胞毒性药物及连接体与抗体或其抗原结合片段连接前的结构。例如，“药物-连接体”指M’-L-E-D，其中M’为M与抗体或其抗原结合片段共价连接前的结构形式。“药物-连接体”与抗体或其抗原结合片段共价连接得到本申请所述抗体药物偶联物。The term "drug-linker" refers to the structure of the cytotoxic drug and linker described in this invention before they are covalently linked to an antibody or its antigen-binding fragment. For example, "drug-linker" refers to M'-L-E-D, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment. The "drug-linker" is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.

所述“药物-连接体”还包括其所有药学上可接受的同位素标记的化合物，其与本发明的“药物-连接体”化合物相同，除了一个或多个原子被具有相同原子序数但原子质量或质量数不同于在自然界中占优势的原子质量或质量数的原子替代。适合包含入本发明的同位素的实例包括但不限于氢的同位素(例如2H、3H、氘D、氚T)；碳的同位素(例如11C、13C及14C)；氯的同位素(例如37Cl)；氟的同位素(例如18F)；碘的同位素(例如123I及125I)；氮的同位素(例如13N及15N)；氧的同位素(例如15O、17O及18O)；及硫的同位素(例如35S)。The “drug-linker” also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the “drug-linker” compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H , 3H , deuterium D, tritium T); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 37Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); and isotopes of sulfur (e.g., 35S ).

术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

术语“同位素标记的化合物”表示该化合物除了一个或多个原子被具有相同原子序数但原子质量或质量数不同于在自然界中占优势的原子质量或质量数的原子替代之外，其余结构与本发明的化合物相同。适合包含入本发明的同位素的实例包括但不限于氢的同位素(例如2H、3H、氘D、氚T)；碳的同位素(例如11C、13C及14C)；氯的同位素(例如37Cl)；氟的同位素(例如18F)；碘的同位素(例如123I及125I)；氮的同位素(例如13N及15N)；氧的同位素(例如15O、17O及18O)；及硫的同位素(例如35S)。The term "isotopically labeled compound" means that the compound has the same structure as the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but different atomic mass or mass number from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H , 3H , deuterium D, tritium T); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 37Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); and isotopes of sulfur (e.g., 35S ).

术语“取代”指所指定的化合物或结构片段上的一个或多个(例如1个、2个、3个、4个或5个)氢被取代基所代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。取代基和/或变量的组合仅仅当这种组合形成稳定的化合物时才是允许的。例如，所述取代基各自独立地由一个或多个以下结构构成：-O-、-S-、-NR’-、卤素、-CN、-OH、-NH2、-NO2、-CN、＝O、C1-C6(亚)烷基、C1-C6卤代(亚)烷基、C1-C6烷氧基、C2-C6(亚)烯基、C2-C6(亚)炔基、C3-C8(亚)环烷基、3-8元(亚)杂环基、C6-C10(亚)芳基和5-10元(亚)杂芳基等。The term "substitution" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only if such combinations form a stable compound. For example, each of the substituents is independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH , -NH2 , -NO2, -CN, =O, C1 - C6 (alkylene), C1 - C6 haloalkylene, C1 - C6 alkoxy, C2 - C6 (alkenylene), C2 - C6 (alkynylene), C3 - C8 (cycloalkylene), 3-8 membered (heterocyclic), C6 - C10 (aryl), and 5-10 membered (heteroaryl), etc.

如果官能团或结构片段被描述为“取代或未取代”，则该官能团或结构片段可(1)未被取代或(2)被取代。 If a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted.

附图说明Attached Figure Description

图1抗体偶联药物ADC对NCI-N87细胞皮下荷瘤鼠模型的药效结果Figure 1. Efficacy of antibody-drug conjugate (ADC) in a mouse model of NCI-N87 subcutaneous tumor-bearing cells.

图2人胃癌细胞NCI-N87 CDX模型中各组小鼠体重的变化情况Figure 2. Changes in body weight of mice in different groups in the NCI-N87 CDX model of human gastric cancer cells.

图3抗体偶联药物ADC对JIMT-1模型的药效结果Figure 3. Efficacy results of antibody-drug conjugates (ADCs) on the JIMT-1 model.

图4JIMT-1模型中各组小鼠体重的变化情况Figure 4. Changes in body weight of mice in each group in the JIMT-1 model.

图5抗体偶联药物ADC对乳腺癌移植瘤模型的药效结果Figure 5. Efficacy results of antibody-drug conjugates (ADCs) in a breast cancer xenograft model.

图6乳腺癌移植瘤模型中各组小鼠体重的变化情况Figure 6. Changes in body weight of mice in different groups in the breast cancer xenograft model.

具体实施方式Detailed Implementation

下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，决不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制。本领域技术人员根据本发明的教导，可以做出各种修改或改进，而不脱离本发明的基本思想和范围。The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

本发明涉及的序列的信息描述于下面的表中：






The sequence information involved in this invention is described in the table below:






本文中所使用的缩写具有以下含义：
The abbreviations used in this article have the following meanings:

以下的实施例中记载的化合物的结构通过核磁共振(1H NMR)或质谱(MS)来确定。The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( ¹H NMR) or mass spectrometry (MS).

核磁共振(1H NMR)的测定使用Bruker 400MHz核磁共振仪；氘代试剂为六氘代二甲基亚砜(DMSO-d6)；内标物质为四甲基硅烷(TMS)。Nuclear magnetic resonance (¹H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

实施例中使用的核磁共振(NMR)图谱中的缩写示于以下。The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

s：单峰(singlet)、d：二重峰(doublet)、t：三重峰(triplet)、q：四重峰(quartet)、m：多重峰(multiplet)、br：宽峰(broad)、J：偶合常数、Hz：赫兹、DMSO-d6：氘化二甲基亚砜。δ值用ppm值表示。 s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values are expressed in ppm.

质谱(MS)的测定使用Agilent(ESI)质谱仪，型号为Agilent 6120B。Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

一、化合物的制备I. Preparation of Compounds

制备例一:(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11-四氢-3H，5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-5)
Preparation Example 1: (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-5)

步骤一:(S)-8-(苄氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-5-2)的制备Step 1: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5-2)

将(S)-8-(苄氧基)-7-甲氧基-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(2g,5.29mmol,FR)溶于四氢呋喃(100mL)中，加入硼氢化钠(599.85mg,15.86mmol)，然后室温滴加三氟乙酸(1.21g,10.57mmol)，直至无大量气体溢出，升温至75℃回流反应5小时(S)-8-(benzyloxy)-7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (2 g, 5.29 mmol, FR) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (599.85 mg, 15.86 mmol) was added, and then trifluoroacetic acid (1.21 g, 10.57 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then heated to 75 °C and refluxed for 5 hours.

将反应液浓缩，加水(50mL)后，用乙酸乙酯(50mL X 2)萃取两次，合并有机相用饱和氯化钠水溶液洗涤后，用无水硫酸钠干燥，过滤浓缩得标题化合物粗品(1.82g,4.99mmol)，未经纯化直接用于下一步。The reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.82 g, 4.99 mmol), which was used directly in the next step without purification.

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):365.2[M+H]+ ESI-MS (m/z): 365.2 [M+H] +

步骤二:(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-5-3)的制备Step 2: Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5-3)

将(S)-8-(苄氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(100mg,0.274mmol,FR)溶于二氯甲烷(0.8mL)中，降温至0℃。然后滴加甲磺酸(0.4mL)。加毕保温反应1小时。向反应液加入水(2ml)，然后用饱和碳酸氢钠水溶液调节pH＝8。用二氯甲烷(3mL X 5)萃取5次，合并有机相用饱和氯化钠水溶液(3mL)洗涤，无水硫酸钠干燥后过滤浓缩得标题化合物粗品(75mg,0.273mmol)，未经纯化直接用于下一步。(S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (100 mg, 0.274 mmol, FR) was dissolved in dichloromethane (0.8 mL) and cooled to 0 °C. Then, methanesulfonic acid (0.4 mL) was added dropwise. The reaction mixture was kept at this temperature for 1 hour. Water (2 mL) was added to the reaction mixture, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. The mixture was extracted five times with dichloromethane (3 mL x 5), and the combined organic phases were washed with saturated sodium chloride solution (3 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (75 mg, 0.273 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):275.2[M+H]+ ESI-MS (m/z): 275.2 [M+H] +

步骤三:(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧基-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-5-5)的制备Step 3: Preparation of (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-5-5)

将(S)-8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基-三氟甲磺酸酯(550mg,0.987mmol)和4-(叔丁氧基羰基)氨基苯硼酸(468mg,1.97mmol)，碳酸钠(418mg,3.95mmol)溶于甲苯(10mL)，乙醇(1mL)和水(1mL)混合溶剂中，加入四(三苯基膦)钯(114mg,0.099mmol)，氮气置换两次并保护下80℃加热反应3小时。将反应液加入乙酸乙酯(20mL)和水(10mL)中搅拌，过滤，滤液静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)再次浓缩得标题化合物(397mg，0.661mmol)。(S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl-trifluoromethanesulfonate (550 mg, 0.987 mmol) and 4-(tert-butoxycarbonyl)aminophenylboronic acid (468 mg, 1.97 mmol), sodium carbonate (418 mg, 3.95 mmol) were dissolved in a mixture of toluene (10 mL), ethanol (1 mL), and water (1 mL). Tetra(triphenylphosphine)palladium (114 mg, 0.099 mmol) was added, and the mixture was heated at 80 °C for 3 hours under nitrogen purging twice and protection. The reaction solution was then added to ethyl acetate (20 mL) and water (10 mL), stirred, filtered, and the filtrate was allowed to stand and separated. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was then purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated again to obtain the title compound (397 mg, 0.661 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):600.7[M+H]+ ESI-MS (m/z): 600.7 [M+H] +

步骤四:(4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-5,11-二氧代-5,10,11,11a-四氢-1-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-5-6)的制备Step 4: Preparation of tert-butyl carbamate (7-5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (7-5-6)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧基-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(100mg,0.167mmol)和(S)-8-羟基-7-甲氧基- 1,10,11,11a-四氢-3H，5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2，1’-环丙烷]-5-酮(46mg,0.167mmol)溶于DMF(1mL)中，加入碳酸钾(69mg,0.500mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(130mg，0.165mmol)(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (100 mg, 0.167 mmol) and (S)-8-hydroxy-7-methoxy- 1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (46 mg, 0.167 mmol) was dissolved in DMF (1 mL), and potassium carbonate (69 mg, 0.500 mmol) was added. The mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH/DCM = 0%–5%) yielded the title compound (130 mg, 0.165 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):794.4[M+H]+ ESI-MS (m/z): 794.4 [M+H] +

步骤五:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-5-7)的制备Step 5: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-5-7)

将(4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-5,11-二氧代-5,10,11,11a-四氢-1-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(200mg,0.122mmol)溶于二氯甲烷(2mL)中，搅拌下滴入三氟乙酸(0.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(31mg,0.045mmol)。200 mg (0.122 mmol) of tert-butyl carbamate (4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (31 mg, 0.045 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):694.4[M+H]+ESI-MS (m/z): 694.4 [M+H]+

步骤六:(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-5)的制备Step Six: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5)

将(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(31mg,0.045mmol)溶于干燥四氢呋喃(5mL)，加入硼氢化钠(8.5mg,0.224mmol),缓慢滴入三氟乙酸(20.4mg,0.179mmol)，产生大量气体。气体散尽后缓慢升温至70℃回流反应16小时。反应液冷却至室温，滴入甲醇淬灭反应，减压浓缩。经反相柱色谱(乙腈/1％甲酸水溶液＝0％～50％)纯化后冷冻干燥得标题化合物(4.65mg,0.007mmol)。(S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (31 mg, 0.045 mmol) was dissolved in dry tetrahydrofuran (5 mL), sodium borohydride (8.5 mg, 0.224 mmol) was added, and trifluoroacetic acid (20.4 mg, 0.179 mmol) was slowly added dropwise, producing a large amount of gas. After the gas dissipated, the temperature was slowly raised to 70 °C and refluxed for 16 hours. The reaction solution was cooled to room temperature, and the reaction was quenched dropwise with methanol. The solution was then concentrated under reduced pressure. After purification by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–50%), the solution was freeze-dried to give the title compound (4.65 mg, 0.007 mmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):680.4[M+H]+ ESI-MS (m/z): 680.4 [M+H] +

1H NMR(400MHz,DMSO):δ7.31(d,J＝13.4Hz,2H),7.26(s,1H),7.11(d,J＝8.4Hz,2H),6.53(d,J＝8.5Hz,3H),6.29(d,J＝8.1Hz,3H),5.19(s,2H),4.13(d,J＝4.4Hz,1H),3.93(dd,J＝12.0,6.1Hz,4H),3.83(d,J＝7.6Hz,1H),3.65(d,J＝7.5Hz,6H),3.56-3.46(m,3H),3.31-3.11(m,4H),2.69(dd,J＝16.7,3.9Hz,1H),1.95(dd,J＝12.5,7.3Hz,1H),1.79(dd,J＝12.5,7.4Hz,5H),1.56(d,J＝6.8Hz,2H),0.67-0.62(m,1H),0.60-0.50(m,3H). 1 H NMR (400MHz, DMSO): δ7.31(d,J＝13.4Hz,2H),7.26(s,1H),7.11(d,J＝8.4Hz,2H),6.53(d,J＝8.5Hz,3H),6. 29(d,J＝8.1Hz,3H),5.19(s,2H),4.13(d,J＝4.4Hz,1H),3.93(dd,J＝12.0,6.1Hz,4H),3.83(d,J＝7.6Hz,1H ),3.65(d,J＝7.5Hz,6H),3.56-3.46(m,3H),3.31-3.11(m,4H),2.69(dd,J＝16.7,3.9Hz,1H),1.95(dd,J＝1 2.5,7.3Hz,1H),1.79(dd,J＝12.5,7.4Hz,5H),1.56(d,J＝6.8Hz,2H),0.67-0.62(m,1H),0.60-0.50(m,3H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二:(S)-8-((5-(((S)-2-(4-羟基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-3)的制备
Preparation Example 2: Preparation of (S)-8-((5-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-3)

步骤一:(S)-2-(4-(苄氧基)苯基)-8-((5-溴戊基)氧基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-3-1)的制备Step 1: Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-1)

将(S)-8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基三氟甲磺酸酯(500mg,897.13μmol),(4-(苄氧基)苯基)硼酸(409.18mg,1.79mmol),四三苯基膦钯(103.67mg,89.71μmol)和碳酸钠(380.34mg,3.59mmol)溶于甲苯(8mL),水(2mL)和乙醇(2mL)中。反应在80℃温度下反应3小时。反应完毕后加水和乙酸乙酯萃取。有机相合并干燥浓缩得到粗品。粗品经硅胶柱色谱纯化得到标题化合物(303mg,461.04μmol)。(S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (500 mg, 897.13 μmol), (4-(benzyloxy)phenyl)boronic acid (409.18 mg, 1.79 mmol), tetraphenylphosphine palladium (103.67 mg, 89.71 μmol), and sodium carbonate (380.34 mg, 3.59 mmol) were dissolved in toluene (8 mL), water (2 mL), and ethanol (2 mL). The reaction was carried out at 80 °C for 3 hours. After the reaction was completed, the mixture was extracted with water and ethyl acetate. The organic phases were combined, dried, and concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give the title compound (303 mg, 461.04 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):591.4[M+H]+ ESI-MS (m/z): 591.4 [M+H] +

步骤二:(S)-2-(4-(苄氧基)苯基)-8-((5-溴戊基)氧基)-7-甲氧基-10-(2-(三甲基甲硅烷基)乙氧基)甲基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-3-2)的制备Step 2: Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-10-(2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-2)

将(S)-2-(4-(苄氧基)苯基)-8-((5-溴戊基)氧基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(186mg,314.46μmol)溶于四氢呋喃(5mL)中，降温至- 78℃。然后滴正丁基锂(294.81μL,471.69μmol)。加毕保温反应0.5小时。向反应液里滴加2-(三甲基硅烷基)乙氧甲基氯(78.64mg,471.69μmol)。加完后继续在该温度下反应1小时。反应结束后，加入饱和氯化铵淬灭后乙酸乙酯萃取。有机相干燥后浓缩得到粗品。粗品经硅胶柱色谱纯化得到标题化合物(200mg,263.25μmol)。Dissolve (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (186 mg, 314.46 μmol) in tetrahydrofuran (5 mL) and cool to - The temperature was 78℃. Then, n-butyllithium (294.81 μL, 471.69 μmol) was added dropwise. The reaction was maintained at this temperature for 0.5 hours. 2-(trimethylsilyl)ethoxymethyl chloride (78.64 mg, 471.69 μmol) was added dropwise to the reaction solution. After the addition was complete, the reaction was continued at this temperature for 1 hour. After the reaction was completed, saturated ammonium chloride was added to quench the reaction, followed by extraction with ethyl acetate. The organic phase was dried and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the title compound (200 mg, 263.25 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):721.3[M+H]+ ESI-MS (m/z): 721.3 [M+H] +

步骤三:(S)-2-(4-(苄氧基)苯基)-8-((5-溴戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮(7-3-3)的制备Step 3: Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-3-3)

将硼氢化钠(52.42mg,1.39mmol)和氯化钙(153.77mg,1.39mmol)溶解在四氢呋喃(2mL)和乙醇(2mL)的混合体系中。再将(S)-2-(4-(苄氧基)苯基)-8-((5-溴戊基)氧基)-7-甲氧基-10-(2-(三甲基甲硅烷基)乙氧基)甲基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(100mg,138.55μmol)的四氢呋喃(2mL)溶液缓慢滴加到上述体系中。反应在室温反应16小时。反应完毕后加入甲酸(0.5mL)淬灭。继续搅拌10分钟。经检测中间态转化完毕。反应液经制备纯化得到标题化合物(14mg,23.03μmol)。Sodium borohydride (52.42 mg, 1.39 mmol) and calcium chloride (153.77 mg, 1.39 mmol) were dissolved in a mixture of tetrahydrofuran (2 mL) and ethanol (2 mL). A solution of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-10-(2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (100 mg, 138.55 μmol) in tetrahydrofuran (2 mL) was slowly added dropwise to the above system. The reaction was carried out at room temperature for 16 hours. After the reaction was complete, formic acid (0.5 mL) was added to quench the reaction. Stirring was continued for 10 minutes. The intermediate state conversion was confirmed. The reaction solution was prepared and purified to obtain the title compound (14 mg, 23.03 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):577.3[M+H]+ ESI-MS (m/z): 577.3 [M+H] +

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤四:(S)-8-((5-(((S)-2-(4-(苄氧基)苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并 [e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-3-4)的制备Step 4: (S)-8-((5-(((S)-2-(4-(benzyloxy)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo] Preparation of [e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-3-4)

将(S)-2-(4-(苄氧基)苯基)-8-((5-溴戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮(12mg,20.78μmol)和(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(5.70mg,20.78μmol)溶于DMF(2mL)中，加入碳酸钾(8.62mg,62.34μmol)后室温反应4小时。反应完毕后加水淬灭反应，用乙酸乙酯萃取后合并有机相，饱和食盐水洗涤后干燥浓缩。粗品经硅胶柱色谱纯化得标题化合物(10mg,12.32μmol)。(S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (12 mg, 20.78 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (5.70 mg, 20.78 μmol) were dissolved in DMF (2 mL), and potassium carbonate (8.62 mg, 62.34 μmol) was added. The reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography to obtain the title compound (10 mg, 12.32 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):771.4[M+H]+ ESI-MS (m/z): 771.4 [M+H] +

步骤五:(S)-8-((5-(((S)-2-(4-羟基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-3)的制备Step 5: Preparation of (S)-8-((5-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-3)

将(S)-8-((5-(((S)-2-(4-(苄氧基)苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(10.00mg,12.97μmol)溶于二氯甲烷(2mL)中，搅拌下滴入甲磺酸(0.5mL)，滴加完毕后继续搅拌16小时。反应完毕后，反应液经制备纯化后冻干得到标题化合物(0.47mg,0.69μmol)。(S)-8-((5-(((S)-2-(4-(benzyloxy)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (10.00 mg, 12.97 μmol) was dissolved in dichloromethane (2 mL), and methanesulfonic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 16 hours. After the reaction was completed, the reaction solution was prepared, purified, and lyophilized to obtain the title compound (0.47 mg, 0.69 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):681.3[M+H]+ ESI-MS (m/z): 681.3 [M+H] +

1H NMR(400MHz,DMSO):δ1H NMR(400MHz,DMSO)δ9.49(s,1H),7.37(s,1H),7.32(s,1H),7.29(s,1H),7.26(d,J＝8.8Hz,2H),6.73(d,J＝8.4Hz,2H),6.54(d,J＝6.4Hz,1H),6.31-6.28(m,3H),4.20-4.12(m,1H),3.95-3.89(m,3H),3.86-3.81(m,1H),3.66(s,3H),3.64(s,3H),3.57-3.46(m,3H),3.30-3.28(m,1H),3.26-3.20(m,1H),3.17 -3.11(m,1H),2.76-2.67(m,2H),1.98-1.93(m,1H),1.83-1.77(m,4H),1.59-1.52(m,2H),0.67-0.62(m,1H),0.60-0.56(m,1H),0.56-0.50(m,2H). 1H NMR (400MHz, DMSO): δ1H NMR(400MHz,DMSO)δ9.49(s,1H),7.37(s,1H),7.32(s,1H),7.29(s,1H),7.2 6(d,J＝8.8Hz,2H),6.73(d,J＝8.4Hz,2H),6.54(d,J＝6.4Hz,1H),6.31-6.28( m,3H),4.20-4.12(m,1H),3.95-3.89(m,3H),3.86-3.81(m,1H),3.66(s,3H) ,3.64(s,3H),3.57-3.46(m,3H),3.30-3.28(m,1H),3.26-3.20(m,1H),3.17 -3.11(m,1H),2.76-2.67(m,2H),1.98-1.93(m,1H),1.83-1.77(m,4H),1. 59-1.52(m,2H),0.67-0.62(m,1H),0.60-0.56(m,1H),0.56-0.50(m,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μm Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％碳酸氢铵)
Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

制备例三:(11aS,11a”S)-8,8”-(戊烷-1,5-二基双(氧基))双(7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并][e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮)(7-9)的制备
Preparation Example 3: Preparation of (11aS,11a”S)-8,8”-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo][e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one)(7-9)

步骤一:8,8”-(戊烷-1,5-二基双(氧基))(11aS,11a”S)-双(7-甲氧基-5-氧代-11,11a-二氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-10(5H)-羧酸二烯丙酯)(7-9-2)的制备Step 1: Preparation of 8,8”-(pentane-1,5-diylbis(oxy))(11aS,11a”S)-bis(7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-10(5H)-carboxylic acid diallyl ester)(7-9-2)

将(S)-8-羟基-7-甲氧基-5-氧代-11,11a-二氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-10(5H)-羧酸烯丙酯(55mg,153.47μmol)和1,5-二溴戊烷(17.64mg,76.73μmol)加入到DMF(2mL)中，然后加入碳酸钾(63.63mg,460.40μmol),室温搅拌反应20小时。反应完毕后，反应液加水10mL，乙酸乙酯萃取3次(5mL x 3)，有机相合并后用饱和食盐水5mL洗涤，无水硫酸钠干燥后减压浓缩。粗品制备薄层色谱法纯化(石油醚/乙酸乙酯＝1:2)后再次减压浓缩得标题化合物(100mg,127.41μmol)。(S)-8-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-10(5H)-carboxylic acid allyl ester (55 mg, 153.47 μmol) and 1,5-dibromopentane (17.64 mg, 76.73 μmol) were added to DMF (2 mL), followed by potassium carbonate (63.63 mg, 460.40 μmol). The mixture was stirred at room temperature for 20 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (5 mL x 3). The combined organic phases were washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by thin-layer chromatography (petroleum ether/ethyl acetate = 1:2) and then concentrated under reduced pressure to obtain the title compound (100 mg, 127.41 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):785.4[M+H]+ ESI-MS (m/z): 785.4 [M+H] +

步骤二:(11aS,11a”S)-8,8”-(戊烷-1,5-二基双(氧基))双(7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并][e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮)(7-9)的制备 Step 2: Preparation of (11aS,11a”S)-8,8”-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo][e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one)(7-9)

将8,8”-(戊烷-1,5-二基双(氧基))(11aS,11a”S)-双(7-甲氧基-5-氧代-11,11a-二氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-10(5H)-羧酸二烯丙酯)(80mg,101.92μmol)和四氢吡咯(14.50mg,203.85μmol)溶于DMF(1.5mL)中，氮气保护下加入Pd(PPh3)4(11.78mg,10.19μmol)，氮气置换三次，然后在氮气保护下室温搅拌反应2小时。反应完成后，向反应液中加水5mL，乙酸乙酯5mL X 3萃取，有机相饱和食盐水5mL洗涤，无水硫酸钠干燥后减压浓缩。粗品先经制备薄层色谱法纯化(乙酸乙酯:甲醇＝20:1)除去三苯基氧磷后再次减压浓缩。再经制备高效液相色谱纯化后冷冻干燥得标题化合物(12.83mg,20.18μmol)。8,8”-(pentane-1,5-diylbis(oxy))(11aS,11a”S)-bis(7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-carboxylic acid diallyl ester (80 mg, 101.92 μmol) and tetrahydropyrrole (14.50 mg, 203.85 μmol) were dissolved in DMF (1.5 mL). Pd( PPh3 ) 4 (11.78 mg, 10.19 μmol) was added under nitrogen protection, followed by three nitrogen purgings. The reaction mixture was then stirred at room temperature for 2 hours under nitrogen protection. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was first purified by preparative thin-layer chromatography (ethyl acetate:methanol = 20:1) to remove triphenylphosphine oxide, and then concentrated under reduced pressure. After further purification by preparative high-performance liquid chromatography, it was freeze-dried to obtain the title compound (12.83 mg, 20.18 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):617.4[M+H]+；309.3[M/2+1]+ ESI-MS(m/z):617.4[M+H] + ;309.3[M/2+1] +

1H NMR(400MHz,CDCl3)δ7.54(s,2H),6.10(s,2H),3.99(dd,J＝16.0,10.4Hz,3H),3.88(d,J＝30.0Hz,3H),3.74-3.38(m,4H),2.04(dd,J＝17.2,7.6Hz,1H),1.96-1.83(m,2H),1.71(ddd,J＝21.6,12.0,5.6Hz,2H),0.77-0.66(m,1H),0.66-0.50(m,3H). 1 H NMR (400MHz, CDCl 3 )δ7.54(s,2H),6.10(s,2H),3.99(dd,J＝16.0,10.4Hz,3H),3.88(d,J＝30.0Hz,3H),3.74-3.38(m,4H),2.04(dd,J ＝17.2,7.6Hz,1H),1.96-1.83(m,2H),1.71(ddd,J＝21.6,12.0,5.6Hz,2H),0.77-0.66(m,1H),0.66-0.50(m,3H).

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例四:(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-(甲氧基-d3)-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-10)的制备
Preparation Example 4: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-(methoxy-d3)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-10)

步骤一:4-溴-5-氟-2-硝基苯甲酸甲酯(7-10-2)的制备Step 1: Preparation of methyl 4-bromo-5-fluoro-2-nitrobenzene (7-10-2)

将4-溴-5-氟-2-硝基苯甲酸(5.9g,22.35mmol,FR)溶于甲醇(30mL)中，加入氯化亚砜(30mL)，于室温反应16小时，将反应液浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)再次浓缩得标题化合物(3.05g,10.97mmol)。4-Bromo-5-fluoro-2-nitrobenzoic acid (5.9 g, 22.35 mmol, FR) was dissolved in methanol (30 mL), thionyl chloride (30 mL) was added, and the mixture was reacted at room temperature for 16 hours. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated again to obtain the title compound (3.05 g, 10.97 mmol).

步骤二:4-溴-5-(甲氧基-d3)-2-硝基苯甲酸甲酯(7-10-3)的制备Step 2: Preparation of methyl 4-bromo-5-(methoxy-d3)-2-nitrobenzene (7-10-3)

将4-溴-5-氟-2-硝基苯甲酸甲酯(1.5g,5.4mmol,FR)溶于DMF(6mL)中，加入氘代甲醇(388.5mg,10.8mmol)，碳酸铯(2.64mg,8.09mmol)，氮气置换3次后于室温反应16小时，将反应液加入乙酸乙酯(20mL)和水(30mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(1.08 g，3.68mmol)。Methyl 4-bromo-5-fluoro-2-nitrobenzene (1.5 g, 5.4 mmol, FR) was dissolved in DMF (6 mL), and deuterated methanol (388.5 mg, 10.8 mmol) and cesium carbonate (2.64 mg, 8.09 mmol) were added. After purging with nitrogen three times, the reaction was carried out at room temperature for 16 hours. The reaction solution was then added to ethyl acetate (20 mL) and water (30 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.08 g). g, 3.68 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

1H NMR(400MHz,DMSO):δ8.35(s,1H),7.51(s,1H). 1 H NMR (400MHz, DMSO): δ8.35(s,1H),7.51(s,1H).

步骤三:5-(甲氧基-d3)-2-硝基-4-(4,4,5,5-四甲基-1,3,2-二氧硼杂硼烷-2-基)苯甲酸甲酯(7-10-4)的制备Step 3: Preparation of methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-10-4)

将4-溴-5-(甲氧基-d3)-2-硝基苯甲酸甲酯(1.08g，3.68mmol)，4,4,4’,4’,5,5,5’,5’-八甲基-2,2’-双(1,3,2-二氧硼杂硼烷)(2.34g,9.21mmol)，双三苯基磷二氯化钯(775.94mg,1.11mmol)，醋酸钾(1.08g,11.05mmol)加入单口瓶中，加入1，4-二氧六环(15mL)中，氮气置换3次，升温至101℃反应16小时，冷却至室温，经硅藻土过滤后浓缩得粗品(1.1g，3.23mmol)，未经纯化直接用于下一步。Methyl 4-bromo-5-(methoxy-d3)-2-nitrobenzene (1.08 g, 3.68 mmol), 4,4,4’,4’,5,5,5’,5’-octamethyl-2,2’-bis(1,3,2-dioxoboronane) (2.34 g, 9.21 mmol), bis(triphenylphosphine)-palladium dichloride (775.94 mg, 1.11 mmol), and potassium acetate (1.08 g, 11.05 mmol) were added to a single-necked flask, followed by 1,4-dioxane (15 mL). The mixture was purged with nitrogen three times, heated to 101 °C, and reacted for 16 hours. After cooling to room temperature, the mixture was filtered through diatomaceous earth and concentrated to obtain the crude product (1.1 g, 3.23 mmol), which was used directly in the next step without purification.

步骤四:4-羟基-5-(甲氧基-d3)-2-硝基苯甲酸甲酯(7-10-5)的制备Step 4: Preparation of methyl 4-hydroxy-5-(methoxy-d3)-2-nitrobenzene (7-10-5)

将5-(甲氧基-d3)-2-硝基-4-(4,4,5,5-四甲基-1,3,2-二氧硼杂硼烷-2-基)苯甲酸甲酯(1.1g，3.23mmol)溶于四氢呋喃(30mL)中，于0℃下加入冰醋酸(3.75mL)，缓慢滴加双氧水(7mL)，于室温反应3小时，向反应液中加入饱和亚硫酸钠溶液(30mL)搅拌1h，减压浓缩至无四氢呋喃残余，加入30mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)浓缩得标题化合物(680mg，2.95mmol)。Methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.1 g, 3.23 mmol) was dissolved in tetrahydrofuran (30 mL), and glacial acetic acid (3.75 mL) was added at 0 °C. Hydrogen peroxide (7 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution, and the mixture was stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found. 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and concentrated to obtain the title compound (680 mg, 2.95 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):230.2[M-H]- ESI-MS (m/z): 230.2 [MH] -

步骤五:4-(苄氧基)-5-(甲氧基-d3)-2-硝基苯甲酸甲酯(7-10-6)的制备Step 5: Preparation of methyl 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzene (7-10-6)

将4-羟基-5-(甲氧基-d3)-2-硝基苯甲酸甲酯(680mg，2.95mmol)溶于DMF(5mL)中，加入碳酸钾(815.3mg，5.91mmol)，苄溴(1.01g，5.91mmol)，于室温反应15小时，加入30mL水后，加入30mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(688mg，2.15mmol)。Methyl 4-hydroxy-5-(methoxy-d3)-2-nitrobenzoate (680 mg, 2.95 mmol) was dissolved in DMF (5 mL), potassium carbonate (815.3 mg, 5.91 mmol) and benzyl bromide (1.01 g, 5.91 mmol) were added, and the mixture was reacted at room temperature for 15 hours. After adding 30 mL of water, 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (688 mg, 2.15 mmol).

步骤六:4-(苄氧基)-5-(甲氧基-d3)-2-硝基苯甲酸(7-10-7)的制备Step Six: Preparation of 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoic acid (7-10-7)

将4-(苄氧基)-5-(甲氧基-d3)-2-硝基苯甲酸甲酯(688mg，2.15mmol)溶于四氢呋喃(5mL)，水(1mL)中，加入氢氧化锂(103.1mg，4.3mmol)，于室温反应3小时，用稀盐酸调 体系PH＝5,固体析出，加入30mL乙酸乙酯萃取，分层后，饱和食盐水洗涤，有机相用无水硫酸钠干燥后过滤，浓缩得标题化合物(400mg，1.31mmol)。Methyl 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzene (688 mg, 2.15 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL), and lithium hydroxide (103.1 mg, 4.3 mmol) was added. The mixture was reacted at room temperature for 3 hours, and the solution was adjusted with dilute hydrochloric acid. The system was at pH 5, and a solid precipitated. It was extracted with 30 mL of ethyl acetate, and after separation, it was washed with saturated brine. The organic phase was dried with anhydrous sodium sulfate and filtered. The concentration yielded the title compound (400 mg, 1.31 mmol).

步骤七:(S)-5-(4-(苄氧基)-5-(甲氧基-d3)-2-硝基苯甲酰基)-5-氮杂螺[2.4]庚烷-6-甲酸甲酯(7-10-8)的制备Step 7: Preparation of (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (7-10-8)

将4-(苄氧基)-5-(甲氧基-d3)-2-硝基苯甲酸(400mg，1.31mmol)，(S)-5-氮杂螺[2.4]庚烷-6-甲酸甲酯(203mg，1.31mmol)，溶于DMF(5mL)中，加入HATU(597.3mg，1.57mmol)，DIPEA(507mg，3.93mmol)，于室温反应15小时，加入30mL水后，加入30mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(570mg，1.29mmol)。4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoic acid (400 mg, 1.31 mmol) and methyl (S)-5-azaspiro[2.4]heptane-6-carboxylate (203 mg, 1.31 mmol) were dissolved in DMF (5 mL), and HATU (597.3 mg, 1.57 mmol) and DIPEA (507 mg, 3.93 mmol) were added. The mixture was reacted at room temperature for 15 hours. After adding 30 mL of water, 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (570 mg, 1.29 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):444.1[M+H]+ ESI-MS (m/z): 444.1 [M+H] +

步骤八:(S)-8-(苄氧基)-7-(甲氧基-d3)-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(7-10-9)的制备Step 8: Preparation of (S)-8-(benzyloxy)-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-10-9)

将(S)-5-(4-(苄氧基)-5-(甲氧基-d3)-2-硝基苯甲酰基)-5-氮杂螺[2.4]庚烷-6-甲酸甲酯(570mg，1.29mmol)，溶于甲醇(30mL)中，加入10％的氯化铵水溶液(4mL)，锌粉(1.28g，19.51mmol)，升温至40℃反应1小时后，再次升温至80℃反应过夜，浓缩得粗品，加入35mL乙酸乙酯溶清，饱和食盐水洗涤3次，有机相用无水硫酸钠干燥后过滤浓缩得粗品，反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(306mg,0.8mmol)。Methyl (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate (570 mg, 1.29 mmol) was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (4 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (306 mg, 0.8 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):382.2[M+H]+ ESI-MS (m/z): 382.2 [M+H] +

步骤九:(S)-8-羟基-7-(甲氧基-d3)-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(7-10-10)的制备Step Nine: Preparation of (S)-8-hydroxy-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-10-10)

将(S)-8-(苄氧基)-7-(甲氧基-d3)-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(132mg,0.29mmol)溶于二氯甲烷(1mL)中，降温至0℃。然后滴加甲磺酸(0.5mL)。加毕保温反应1小时。向反应液加入水(3ml)，然后用饱和碳酸氢钠水溶液调节pH＝8。用二氯甲烷(5mL X 3)萃取3次，合并有机相用饱和氯化钠水溶液(10mL)洗涤，无水硫酸钠干燥后过滤浓缩得标题化合物粗品(100mg,0.27mmol)，未经纯 化直接用于下一步。(S)-8-(benzyloxy)-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (132 mg, 0.29 mmol) was dissolved in dichloromethane (1 mL) and cooled to 0 °C. Then, methanesulfonic acid (0.5 mL) was added dropwise. The reaction mixture was kept at this temperature for 1 hour. Water (3 mL) was added to the reaction mixture, and the pH was adjusted to 8 with a saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane (5 mL x 3), and the combined organic phases were washed with a saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (100 mg, 0.27 mmol), which was not purified. The chemical can be directly applied to the next step.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):292.1[M+H]+ ESI-MS (m/z): 292.1 [M+H] +

步骤十:(4-((S)-7-甲氧基-8-((5-(((S)-7-(甲氧基-d3))-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-10-11)的制备Step 10: Preparation of tert-butyl carbamate (7-10-11)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧基-50,11,11-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(167mg,0.27mmol)和(S)-8-羟基-7-(甲氧基-d3)-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(100mg,0.27mmol)溶于DMF(2mL)中，加入碳酸钾(60mg,0.54mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(120mg，0.136mmol)(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (167 mg, 0.27 mmol) and (S)-8-hydroxy-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (100 mg, 0.27 mmol) were dissolved in DMF (2 mL), potassium carbonate (60 mg, 0.54 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH/DCM = 0%–5%) yielded the title compound (120 mg, 0.136 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):797.3[M+H]+ ESI-MS (m/z): 797.3 [M+H] +

步骤十一:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((S)-7-(甲氧基-d3)-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-10-12)的制备Step 11: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-(methoxy-d3)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-10-12)

将(4-((S)-7-甲氧基-8-((5-(((S)-7-(甲氧基-d3))-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(120mg，0.136mmol)溶于二氯甲烷(3mL)中，搅拌下滴入三氟乙酸(0.6mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(35mg,0.05mmol)。120 mg (0.136 mmol) of tert-butyl carbamate (4-((S)-7-methoxy-8-((5-((((S)-7-(methoxy-d3))-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (3 mL) was dissolved in dichloromethane. Trifluoroacetic acid (0.6 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (35 mg, 0.05 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):697.3[M+H]+ ESI-MS (m/z): 697.3 [M+H] +

步骤十二:(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e] 吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-(甲氧基-d3)-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-10)的制备Step 12: (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]) Preparation of pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-(methoxy-d3)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-10)

将(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((S)-7-(甲氧基-d3)-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(35mg,0.05mmol)溶于干燥四氢呋喃(5mL)，加入硼氢化钠(8.8mg,0.225mmol),缓慢滴入三氟乙酸(20.9mg,0.18mmol)，产生大量气体。气体散尽后缓慢升温至70℃回流反应16小时。反应液冷却至室温，滴入甲醇淬灭反应，减压浓缩。经反相柱色谱(乙腈/1％甲酸水溶液＝0％～50％)纯化后冷冻干燥得标题化合物(2.9mg,0.004mmol)。(S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-(methoxy-d3)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (35 mg, 0.05 mmol) was dissolved in dry tetrahydrofuran (5 mL), sodium borohydride (8.8 mg, 0.225 mmol) was added, and trifluoroacetic acid (20.9 mg, 0.18 mmol) was slowly added dropwise, producing a large amount of gas. After the gas dissipated, the temperature was slowly raised to 70 °C and refluxed for 16 hours. The reaction solution was cooled to room temperature, and the reaction was quenched dropwise with methanol. The solution was then concentrated under reduced pressure. After purification by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–50%), the solution was freeze-dried to give the title compound (2.9 mg, 0.004 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):683.3[M+H]+ ESI-MS (m/z): 683.3 [M+H] +

1H NMR(400MHz,DMSO):δ7.33(s,1H),7.30(s,1H),7.27(s,1H),7.13(d,J＝8.4Hz,1H),6.58-6.53(m,2H),6.34-6.26(m,2H),4.20-4.10(m,1H),4.0-3.92(m,4H),3.86-3.80(m,2H),3.67(s,3H),3.56(s,3H),3.3-3.22(m,3H),3.20-3.12(m,2H),2.76-2.66(m,2H),2.02-1.94(m,1H),1.85-1.77(m,4H),1.63-1.52(m,2H),0.62-0.52(m,3H). 1 H NMR (400MHz, DMSO): δ7.33(s,1H),7.30(s,1H),7.27(s,1H),7.13(d,J＝8.4Hz,1H), 6.58-6.53(m,2H),6.34-6.26(m,2H),4.20-4.10(m,1H),4.0-3.92(m,4H),3.86-3.8 0(m,2H),3.67(s,3H),3.56(s,3H),3.3-3.22(m,3H),3.20-3.12(m,2H),2.76-2.66 (m,2H),2.02-1.94(m,1H),1.85-1.77(m,4H),1.63-1.52(m,2H),0.62-0.52(m,3H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例五:(S)-2-(4-氨基苯基)-8-((5-(((S)-7-环丙氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-11)的制备
Preparation Example 5: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-11)

步骤一:4-溴-5-环丙氧基-2-硝基苯甲酸甲酯(7-11-1)的制备Step 1: Preparation of methyl 4-bromo-5-cyclopropoxy-2-nitrobenzene (7-11-1)

将4-溴-5-氟-2-硝基苯甲酸甲酯(1.5g,5.4mmol,FR)溶于DMF(6mL)中，加入环丙醇(635mg,10.97mmol)，碳酸铯(2.68mg,8.3mmol)，氮气置换3次后于室温反应16小时，将反应液加入乙酸乙酯(30mL)和水(30mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(750mg，2.37mmol)。Methyl 4-bromo-5-fluoro-2-nitrobenzoate (1.5 g, 5.4 mmol, FR) was dissolved in DMF (6 mL), and cyclopropanol (635 mg, 10.97 mmol) and cesium carbonate (2.68 mg, 8.3 mmol) were added. After purging with nitrogen three times, the reaction was carried out at room temperature for 16 hours. The reaction solution was then added to ethyl acetate (30 mL) and water (30 mL) and stirred. The mixture was allowed to stand and separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (750 mg, 2.37 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

1H NMR(400MHz,DMSO):δ8.41(s,1H),7.70(s,1H),4.21(tt,J＝5.8,2.8Hz,1H),3.88(s,3H),0.92(m,2H),0.80(m,2H). 1 H NMR (400MHz, DMSO): δ8.41 (s, 1H), 7.70 (s, 1H), 4.21 (tt, J = 5.8, 2.8Hz, 1H), 3.88 (s, 3H), 0.92 (m, 2H), 0.80 (m, 2H).

步骤二:5-环丙氧基-2-硝基-4-(4,4,5,5-四甲基-1,3,2-二氧硼杂硼烷-2-基)苯甲酸甲酯(7-11-2)的制备Step 2: Preparation of methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-11-2)

将4-溴-5-环丙氧基-2-硝基苯甲酸甲酯(750mg，2.37mmol)，4,4,4’,4’,5,5,5’,5’-八甲基-2,2’-双(1,3,2-二氧硼杂硼烷)(1.5g,5.93mmol)，双三苯基磷二氯化钯(499.6mg,0.71mmol)，醋酸钾(700mg,7.12mmol)加入单口瓶中，加入1，4-二氧六环(15mL)中，氮气置换3次， 升温至101℃反应16小时，冷却至室温，经硅藻土过滤后浓缩得粗品(1.6g，4.41mmol)，未经纯化直接用于下一步。Methyl 4-bromo-5-cyclopropoxy-2-nitrobenzene (750 mg, 2.37 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxoboronane) (1.5 g, 5.93 mmol), bis(triphenylphosphine) palladium dichloride (499.6 mg, 0.71 mmol), and potassium acetate (700 mg, 7.12 mmol) were added to a single-necked flask, followed by 1,4-dioxane (15 mL). The mixture was then purged with nitrogen three times. The mixture was heated to 101°C and reacted for 16 hours. After cooling to room temperature, the product was filtered through diatomaceous earth and concentrated to obtain crude product (1.6 g, 4.41 mmol). It was used directly in the next step without purification.

步骤三:5-环丙氧基-4-羟基-2-硝基苯甲酸甲酯(7-11-3)的制备Step 3: Preparation of methyl 5-cyclopropoxy-4-hydroxy-2-nitrobenzene (7-11-3)

将5-环丙氧基-2-硝基-4-(4,4,5,5-四甲基-1,3,2-二氧硼杂硼烷-2-基)苯甲酸甲酯(1.6g，4.41mmol)溶于四氢呋喃(40mL)中，于0℃下加入冰醋酸(5mL)，缓慢滴加双氧水(10mL)，于室温反应3小时，向反应液中加入饱和亚硫酸钠溶液(50mL)搅拌1h，减压浓缩至无四氢呋喃残余，加入30mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)浓缩得标题化合物(1.05g，4.15mmol)。Methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.6 g, 4.41 mmol) was dissolved in tetrahydrofuran (40 mL), and glacial acetic acid (5 mL) was added at 0 °C. Hydrogen peroxide (10 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (50 mL) was added to the reaction solution and stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found. 30 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and concentrated to obtain the title compound (1.05 g, 4.15 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):253.2[M-H]- ESI-MS (m/z): 253.2 [MH] -

步骤四:4-(苄氧基)-5-环丙氧基-2-硝基苯甲酸甲酯(7-11-4)的制备Step 4: Preparation of methyl 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzene (7-11-4)

将5-环丙氧基-4-羟基-2-硝基苯甲酸甲酯(1.05g，4.15mmol)溶于DMF(5mL)中，加入碳酸钾(1.2g，8.6mmol)，苄溴(1.49g，8.6mmol)，于室温反应15小时，加入30mL水后，加入50mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(1.09g，3.17mmol)。Methyl 5-cyclopropoxy-4-hydroxy-2-nitrobenzoate (1.05 g, 4.15 mmol) was dissolved in DMF (5 mL), potassium carbonate (1.2 g, 8.6 mmol) and benzyl bromide (1.49 g, 8.6 mmol) were added, and the mixture was reacted at room temperature for 15 hours. After adding 30 mL of water, 50 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.09 g, 3.17 mmol).

步骤五:4-(苄氧基)-5-环丙氧基-2-硝基苯甲酸(7-11-5)的制备Step 5: Preparation of 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoic acid (7-11-5)

将4-(苄氧基)-5-环丙氧基-2-硝基苯甲酸甲酯(1.09g，3.17mmol)溶于四氢呋喃(8mL)，水(2mL)中，加入氢氧化锂(152.1mg，6.35mmol)，于室温反应3小时，用稀盐酸调体系PH＝5,固体析出，加入60mL乙酸乙酯萃取，分层后，饱和食盐水洗涤，有机相用无水硫酸钠干燥后过滤，浓缩得标题化合物(743mg，2.26mmol)，未经纯化直接用于下一步。Methyl 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoate (1.09 g, 3.17 mmol) was dissolved in tetrahydrofuran (8 mL) and water (2 mL). Lithium hydroxide (152.1 mg, 6.35 mmol) was added, and the mixture was reacted at room temperature for 3 hours. The pH of the system was adjusted to 5 with dilute hydrochloric acid, and a solid precipitated out. The solid was extracted with 60 mL of ethyl acetate, and after separation, the layers were washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The concentrate yielded the title compound (743 mg, 2.26 mmol), which was used directly in the next step without purification.

步骤六:(S)-5-(4-(苄氧基)-5-环丙氧基-2-硝基苯甲酰基)-5-氮杂螺[2.4]庚烷-6-甲酸甲酯(7-11-6)的制备Step Six: Preparation of (S)-5-(4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (7-11-6)

将4-(苄氧基)-5-环丙氧基-2-硝基苯甲酸(743mg，2.26mmol)，(S)-5-氮杂螺[2.4]庚烷-6-甲酸甲酯(700mg，4.51mmol)，溶于DMF(5mL)中，加入HATU(1.71g，4.51mmol)，DIPEA(875mg，6.77mmol)，于室温反应2小时，加入30mL水后，加入50mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(910mg，1.95mmol)。 4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoic acid (743 mg, 2.26 mmol) and methyl (S)-5-azaspiro[2.4]heptane-6-carboxylate (700 mg, 4.51 mmol) were dissolved in DMF (5 mL), and HATU (1.71 g, 4.51 mmol) and DIPEA (875 mg, 6.77 mmol) were added. The mixture was reacted at room temperature for 2 hours. After adding 30 mL of water, 50 mL of ethyl acetate was added for extraction. After separation, the organic phase was dried over anhydrous sodium sulfate and then filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (910 mg, 1.95 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):467.1[M+H]+ ESI-MS (m/z): 467.1 [M+H] +

步骤七:(S)-8-(苄氧基)-7-环丙氧基-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(7-11-7)的制备Step 7: Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-11-7)

将(S)-5-(4-(苄氧基)-5-环丙氧基-2-硝基苯甲酰基)-5-氮杂螺[2.4]庚烷-6-甲酸甲酯(910mg，1.95mmol)，溶于甲醇(30mL)中，加入10％的氯化铵水溶液(6mL)，锌粉(1.28g，19.51mmol)，升温至40℃反应1小时后，再次升温至80℃反应过夜，浓缩得粗品，加入35mL乙酸乙酯溶清，饱和食盐水洗涤3次，有机相用无水硫酸钠干燥后过滤浓缩得粗品，反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(467mg,1.15mmol)。Methyl (S)-5-(4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate (910 mg, 1.95 mmol) was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (6 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (467 mg, 1.15 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):405.2[M+H]+ ESI-MS (m/z): 405.2 [M+H] +

步骤八:(S)-8-(苄氧基)-7-环丙氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-11-8)的制备Step 8: Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-11-8)

将(S)-8-(苄氧基)-7-环丙氧基-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(467mg,1.15mmol)溶于干燥四氢呋喃(20mL)，加入硼氢化钠(128mg,3.46mmol),缓慢滴入三氟乙酸(264mg,2.31mmol)，产生大量气体。气体散尽后缓慢升温至70℃回流反应16小时。反应液冷却至室温，滴入甲醇淬灭反应，减压浓缩。经反相柱色谱(乙腈/1％甲酸水溶液＝0％～80％)纯化后冷冻干燥得标题化合物(380mg,0.97mmol)。(S)-8-(benzyloxy)-7-cyclopropoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (467 mg, 1.15 mmol) was dissolved in dry tetrahydrofuran (20 mL), sodium borohydride (128 mg, 3.46 mmol) was added, and trifluoroacetic acid (264 mg, 2.31 mmol) was slowly added dropwise, producing a large amount of gas. After the gas dissipated, the mixture was slowly heated to 70 °C and refluxed for 16 hours. The reaction solution was cooled to room temperature, and the reaction was quenched dropwise with methanol and concentrated under reduced pressure. After purification by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–80%), the solution was freeze-dried to give the title compound (380 mg, 0.97 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):363.3[M+H]+ ESI-MS (m/z): 363.3 [M+H] +

步骤九:(S)-7-环丙氧基-8-羟基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-11-9)的制备Step Nine: Preparation of (S)-7-cyclopropoxy-8-hydroxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-11-9)

将(S)-8-(苄氧基)-7-环丙氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(380mg,0.97mmol)溶于二氯甲烷(1.5mL)中，降温至0℃。然后滴加甲磺酸(0.6mL)。加毕保温反应1小时。向反应液加入水(10ml)，然后用饱和碳酸氢钠水溶液调节pH＝8。用二氯甲烷(8mL X 3)萃取3次，合并有机相用饱和氯化钠水溶液(20mL) 洗涤，无水硫酸钠干燥后过滤浓缩得标题化合物粗品(118mg,0.31mmol)，未经纯化直接用于下一步。(S)-8-(benzyloxy)-7-cyclopropoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (380 mg, 0.97 mmol) was dissolved in dichloromethane (1.5 mL) and cooled to 0 °C. Then, methanesulfonic acid (0.6 mL) was added dropwise. The reaction mixture was kept at this temperature for 1 hour. Water (10 mL) was added to the reaction mixture, and the pH was adjusted to 8 with a saturated sodium bicarbonate solution. The mixture was extracted three times with dichloromethane (8 mL x 3), and the combined organic phases were extracted with a saturated sodium chloride solution (20 mL). After washing, drying with anhydrous sodium sulfate, filtration and concentration yielded the crude title compound (118 mg, 0.31 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):301.1[M+H]+ ESI-MS (m/z): 301.1 [M+H] +

步骤十:(4-((S)-8-((5-(((S)-7-环丙氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-11-10)的制备Step 10: Preparation of tert-butyl carbamate (7-11-10)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧基-50,11,11-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(141mg,0.23mmol)和(S)-7-环丙氧基-8-羟基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(118mg,0.31mmol)溶于DMF(2mL)中，加入碳酸钾(49mg,0.47mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(20mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(81mg，0.09mmol)。(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (141 mg, 0.23 mmol) and (S)-7-cyclopropoxy-8-hydroxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (118 mg, 0.31 mmol) were dissolved in DMF (2 mL), potassium carbonate (49 mg, 0.47 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (MeOH/DCM = 0%–5%) to give the title compound (81 mg, 0.09 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):806.3[M+H]+ ESI-MS (m/z): 806.3 [M+H] +

步骤十一:(S)-2-(4-氨基苯基)-8-((5-(((S)-7-环丙氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-11)的制备Step 11: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-11)

将(4-((S)-8-((5-(((S)-7-环丙氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(81mg，0.09mmol)溶于二氯甲烷(1mL)中，搅拌下滴入三氟乙酸(0.6mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(3.5mg,0.005mmol)。81 mg (0.09 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.6 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (3.5 mg, 0.005 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):706.3[M+H]+ESI-MS (m/z): 706.3 [M+H]+

1H NMR(400MHz,DMSO):δ7.58(s,1H),7.27(d,J＝11.8Hz,2H),7.11(d,J＝8.4Hz,2H),6.57-6.51(m,2H),6.29(d,J＝9.0Hz,2H),5.19(s,2H),4.18-4.07(m,1H),4.01-3.88(m,4H), 3.86-3.80(m,1H),3.67(s,3H),3.57-3.51(m,4H),3.27-3.10(m,4H),2.73-2.65(m,1H),2.02-1.93(m,1H),1.88-1.71(m,5H),1.61-1.48(m,2H),1.24(s,2H),0.88-0.75(m,1H),0.69-0.48(m,6H). 1 H NMR (400MHz, DMSO): δ7.58(s,1H),7.27(d,J＝11.8Hz,2H),7.11(d,J＝8.4Hz,2H),6.57- 6.51(m,2H),6.29(d,J＝9.0Hz,2H),5.19(s,2H),4.18-4.07(m,1H),4.01-3.88(m,4H), 3.86-3.80(m,1H),3.67(s,3H),3.57-3.51(m,4H),3.27-3.10(m,4H),2.73-2.65(m,1H),2.02-1.9 3(m,1H),1.88-1.71(m,5H),1.61-1.48(m,2H),1.24(s,2H),0.88-0.75(m,1H),0.69-0.48(m,6H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例六:(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮-11,11-d2(7-21)的制备
Preparation Example 6: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one-11,11-d2(7-21)

步骤一:(S)-8-(苄氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮-11,11-d2(7-21-1)的制备Step 1: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21-1)

将(S)-8-(苄氧基)-7-甲氧基-1,11a-二氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5,11(10H)-二酮(1.00g,2.64mmol)溶于四氢呋喃(3.00mL)中，冷却到0℃后，在 氮气保护下向反应体系中滴加氘代硼烷的四氢呋喃溶液(0.75M,7.05mL)。恢复至25℃继续搅拌2小时。向反应体系中加入甲醇(100mL)后继搅拌半小时后，直接浓缩得标题化合物粗品(1.00g,crude)，未经纯化直接用于下一步。(S)-8-(benzyloxy)-7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5,11(10H)-dione (1.00 g, 2.64 mmol) was dissolved in tetrahydrofuran (3.00 mL), cooled to 0 °C, and then... Under nitrogen protection, a tetrahydrofuran solution of deuterated borane (0.75 M, 7.05 mL) was added dropwise to the reaction system. The temperature was restored to 25 °C and stirring was continued for 2 hours. After adding methanol (100 mL) to the reaction system and stirring for another half hour, the mixture was directly concentrated to obtain the crude title compound (1.00 g), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):367.2[M+H]+ ESI-MS (m/z): 367.2 [M+H] +

步骤二:(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮-11,11-d2(7-21-2)的制备Step 2: Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21-2)

将(S)-8-(苄氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮-11,11-d2(1.00g,2.73mmol)溶于甲醇(10.0mL)中，氮气置换3次后，加入Pd/C(0.20g,187μmol,10％purity)通入氢气后继续反应2小时。反应液经硅胶垫过滤后，直接浓缩得粗品。经硅胶柱纯化(二氯甲烷/甲醇＝100/1～97/3)后，再次浓缩得标题化合物(500mg,1.77mmol)。(S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2 (1.00 g, 2.73 mmol) was dissolved in methanol (10.0 mL), purged three times with nitrogen, and then Pd/C (0.20 g, 187 μmol, 10% purity) was added. Hydrogen gas was then introduced, and the reaction was continued for 2 hours. The reaction solution was filtered through a silica gel pad and directly concentrated to obtain the crude product. After purification by silica gel column chromatography (dichloromethane/methanol = 100/1 to 97/3), it was concentrated again to obtain the title compound (500 mg, 1.77 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):277.1[M+H]+ ESI-MS (m/z): 277.1 [M+H] +

1H NMR:(400MHz,CD3OD)δ7.36(s,1H),6.52(s,1H),3.95-4.04(m,1H),3.86(s,3H),3.63(d,J＝11.6Hz,1H),3.49(d,J＝12.0Hz,1H),2.26(dd,J＝12.8,8.0Hz,1H),1.68(dd,J＝12.8,4.4Hz,1H),0.69-0.79(m,2H),0.62-0.69(m,2H) 1 H NMR: (400MHz, CD3OD) δ7.36(s,1H),6.52(s,1H),3.95-4.04(m,1H),3.86(s,3H),3.63(d,J＝11.6Hz,1H),3.49(d ,J＝12.0Hz,1H),2.26(dd,J＝12.8,8.0Hz,1H),1.68(dd,J＝12.8,4.4Hz,1H),0.69-0.79(m,2H),0.62-0.69(m,2H)

步骤三:(4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基-11,11-d2)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-21-3)的制备Step 3: Preparation of tert-butyl carbamate (7-21-3)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(21.23mg,36.19μmol)和(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮-11,11-d2(10mg,36.19μmol)溶于DMF(1mL)中，加入碳酸钾(14.98mg,108.57μmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(20mg，25.58μmol)。(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (21.23 mg, 36.19 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2 (10 mg, 36.19 μmol) were dissolved in DMF (1 mL), potassium carbonate (14.98 mg, 108.57 μmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The solution was purified by silica gel column chromatography (MeOH/DCM = 0%–5%) to give the title compound (20 mg, 25.58 μmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):794.4[M+H]+ ESI-MS (m/z): 794.4 [M+H] +

步骤四:(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮-11,11-d2(7-21)的制备Step 4: Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21)

将(4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基-11,11-d2)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(20mg，25.58μmol)溶于二氯甲烷(1mL)中，搅拌下滴入三氟乙酸(0.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(4.7mg,6.82μmol)。20 mg (25.58 μmol) of tert-butyl carbamate was dissolved in 1 mL of dichloromethane. Trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (4.7 mg, 6.82 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):682.4[M+H]+ ESI-MS (m/z): 682.4 [M+H] +

1H NMR(400MHz,DMSO)δ7.28(d,J＝13.4Hz,2H),7.27(s,1H),7.15(d,J＝8.5Hz,2H),6.59(d,J＝8.6Hz,2H),6.30(d,J＝10.2Hz,2H),3.95(dd,J＝12.0,6.1Hz,4H),3.66(d,J＝7.5Hz,6H),3.59-3.51(m,2H),3.37-3.15(m,4H),2.76-2.67(m,2H),2.03-1.92(m,2H),1.85-1.75(m,4H),1.62-1.52(m,2H),0.68-0.58(m,2H),0.56-0.48(m,2H). 1 H NMR (400MHz, DMSO) δ7.28(d,J＝13.4Hz,2H),7.27(s,1H),7.15(d,J＝8.5Hz,2H),6. 59(d,J＝8.6Hz,2H),6.30(d,J＝10.2Hz,2H),3.95(dd,J＝12.0,6.1Hz,4H),3.66(d,J ＝7.5Hz,6H),3.59-3.51(m,2H),3.37-3.15(m,4H),2.76-2.67(m,2H),2.03-1.92( m,2H),1.85-1.75(m,4H),1.62-1.52(m,2H),0.68-0.58(m,2H),0.56-0.48(m,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例七:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-22)的制备
Preparation Example 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22)

步骤一：(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-L-脯氨酸甲酯(7-22-2)的制备Step 1: Preparation of (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-L-proline methyl ester (7-22-2)

将4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(3.00g,9.89mmol)、L-脯氨酸甲酯盐酸盐(1.97g,11.87mmol)溶于DMF(30mL)中，依次加入HATU(5.64g,14.84mmol)和DIPEA(3.84g,29.68mmol)，25℃反应1小时，将反应液倒入水(100ml)中，EA/PE＝1/1(200ml)萃取产品，盐水(30ml)洗，浓缩得到标题化合物(4.10g,9.89mmol)，未经纯化直接用于下一步。4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (3.00 g, 9.89 mmol) and L-proline methyl ester hydrochloride (1.97 g, 11.87 mmol) were dissolved in DMF (30 mL), and HATU (5.64 g, 14.84 mmol) and DIPEA (3.84 g, 29.68 mmol) were added sequentially. The mixture was reacted at 25 °C for 1 hour. The reaction solution was poured into water (100 mL), and the product was extracted with EA/PE = 1/1 (200 mL). The product was washed with brine (30 mL) and concentrated to obtain the title compound (4.10 g, 9.89 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI):415.1[M+H]+ MS m/z(ESI): 415.1 [M+H] +

步骤二：(S)-8-羟基-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-22-3)的制备Step 2: Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-22-3)

将化合物(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-L-脯氨酸甲酯(3.80g,8.98mmol)溶于甲醇(40mL)、四氢呋喃(40mL)中，加入10％钯碳(380mg)、HOAc(1.08g,17.95mmol)，真空抽除空气，通入氢气，25℃反应16小时，滤除钯碳，滤液浓缩得到标题化合物(2.35g,8.98mmol)，未经纯化直接用于下一步。The compound (4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-L-proline methyl ester (3.80 g, 8.98 mmol) was dissolved in methanol (40 mL) and tetrahydrofuran (40 mL), and 10% palladium on carbon (380 mg) and HOAc (1.08 g, 17.95 mmol) were added. The air was removed under vacuum, hydrogen was introduced, and the reaction was carried out at 25 °C for 16 hours. The palladium on carbon was filtered off, and the filtrate was concentrated to obtain the title compound (2.35 g, 8.98 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI)：263.1[M+H]+ MS m/z (ESI): 263.1 [M+H] +

步骤三：(S)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-22-4)的制备 Step 3: Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22-4)

将化合物(S)-8-羟基-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(100.0mg,381.30μmol)溶于THF(3mL)中，加入硼烷四氢呋喃溶液(4M,476.62μL)，25℃搅拌反应1小时，将反应液倒入甲醇(3ml)中破坏，20％甲酸水溶液调PH＝5～6，浓缩得到粗品，通过C18反向柱纯化(H2O:ACN＝0～30％,0.05％FA)后冻干得到标题化合物(95.0mg,306.11μmol)。The compound (S)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (100.0 mg, 381.30 μmol) was dissolved in THF (3 mL), and a boranetetrahydrofuran solution (4 M, 476.62 μL) was added. The mixture was stirred at 25 °C for 1 hour. The reaction solution was then poured into methanol (3 mL) to destroy the compound. The pH was adjusted to 5-6 with 20% formic acid aqueous solution, and the crude product was concentrated. The crude product was purified by C18 reverse column chromatography (H2O:ACN = 0-30%, 0.05% FA) and then lyophilized to obtain the title compound (95.0 mg, 306.11 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI):249.1[M+H]+ MS m/z(ESI): 249.1 [M+H] +

步骤四：(4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-22-5)的制备Step 4: Preparation of tert-butyl carbamate (7-22-5)

将(S)-8-((5-溴戊基)氧基)-2-(4-((叔丁氧基羰基)氨基)苯基)-7-甲氧基-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(20mg,34.10μmol)、化合物(S)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(12.70mg,51.15μmol,FR)溶于DMF(1mL)中，加入Cs2CO3(33.33mg,102.30μmol)，升温至40℃反应16小时，向体系中加入EA(20ml)、H2O(6ml)搅拌5分钟，分出EA相，盐水洗，浓缩得到粗品通过薄层层析法纯化(MeOH/DCM＝10％)得到标题化合物(15.0mg,19.90μmol)。The following compounds were prepared: (S)-8-((5-bromopentyl)oxy)-2-(4-((tert-butoxycarbonyl)amino)phenyl)-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (20 mg, 34.10 μmol), and (S)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4] Diaza-5-one (12.70 mg, 51.15 μmol, FR) was dissolved in DMF (1 mL), and Cs2CO3 (33.33 mg, 102.30 μmol) was added. The mixture was heated to 40 °C and reacted for 16 hours. EA (20 mL) and H2O (6 mL) were added to the system and stirred for 5 minutes. The EA phase was separated, washed with brine, and concentrated to obtain the crude product. The crude product was purified by thin-layer chromatography (MeOH/DCM = 10%) to give the title compound (15.0 mg, 19.90 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI):754.4[M+H]+ MS m/z(ESI): 754.4 [M+H] +

步骤五：(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-22)的制备Step 5: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22)

将化合物(4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(15.0mg,19.90μmol)溶于DCM(0.5mL)中，加入三氟乙酸(226.87mg,1.99mmol)，25℃反应1小时，浓缩得到粗品，经制备高效液相色谱纯化后冷冻干燥得标题化合物(7.10mg,10.53μmol)。The compound (4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl) tert-butyl carbamate (15.0 mg, 19.90 μmol) was dissolved in DCM (0.5 mL), and trifluoroacetic acid (226.87 mg, 1.99 mmol) was added. The mixture was reacted at 25 °C for 1 hour, concentrated to obtain the crude product, purified by preparative high performance liquid chromatography, and freeze-dried to obtain the title compound (7.10 mg, 10.53 μmol).

其制备方法如下： Its preparation method is as follows:

色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI):654.4[M+H]+ MS m/z(ESI): 654.4 [M+H] +

1H NMR(400MHz,MeOD):δ7.38(dd,J＝8.2,2.3Hz,3H),6.91(d,J＝8.9Hz,2H),6.27(d,J＝9.3Hz,2H),4.24(s,1H),4.00(dd,J＝9.9,6.3Hz,4H),3.80(s,3H),3.77(s,3H),3.76(s,3H),3.64(s,1H),3.61(s,1H),3.56(d,J＝11.1Hz,2H),3.50-3.46(m,2H),3.41(s,1H),3.40-3.33(m,3H),2.80(d,J＝13.5Hz,1H),2.08(d,J＝5.0Hz,1H),1.95-1.86(m,5H),1.69(d,J＝6.7Hz,2H),0.71-0.68(m,1H),0.61(dd,J＝12.4,6.0Hz,3H). 1 H NMR (400MHz, MeOD): δ7.38 (dd, J＝8.2, 2.3Hz, 3H), 6.91 (d, J＝8.9Hz, 2H), 6.27 (d, J＝9.3Hz, 2H), 4.24 (s, 1H),4.00(dd,J＝9.9,6.3Hz,4H),3.80(s,3H),3.77(s,3H),3.76(s,3H),3.64(s,1H),3.61(s,1H),3.56 (d,J＝11.1Hz,2H),3.50-3.46(m,2H),3.41(s,1H),3.40-3.33(m,3H),2.80(d,J＝13.5Hz,1H),2.08(d,J ＝5.0Hz,1H),1.95-1.86(m,5H),1.69(d,J＝6.7Hz,2H),0.71-0.68(m,1H),0.61(dd,J＝12.4,6.0Hz,3H).

制备例八:(S)-2-(4-氨基苯基)-8-((5-(((2R,11aS)-2-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-23)的制备
Preparation Example 8: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23)

步骤一:(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-羟基吡咯烷-2-甲酸甲酯(7-23-2)的制备Step 1: Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-23-2)

将4-苄氧基-5-甲氧基-2-硝基-苯甲酸(10g,32.97mmol)，(2S,4R)-4-羟基吡咯烷-2-甲酸甲酯盐酸盐(5.27g,36.27mmol)，HATU(18.81g,49.46mmol)溶于DMF(50mL)，滴入DIPEA(12.78g,98.92mmol)，搅拌反应2小时。加水和EA搅拌，分液，水相用EA萃取2次，合并有机相，饱和食盐水洗涤2次，干燥，浓缩。硅胶柱纯化(洗脱剂30-65％乙酸乙酯/石油醚)后再次浓缩得标题化合物(14g,32.53mmol)。4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (10 g, 32.97 mmol), (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester hydrochloride (5.27 g, 36.27 mmol), and HATU (18.81 g, 49.46 mmol) were dissolved in DMF (50 mL), and DIPEA (12.78 g, 98.92 mmol) was added dropwise. The mixture was stirred for 2 hours. Water and EA were added and stirred. The mixture was separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed twice with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (eluent 30-65% ethyl acetate/petroleum ether), the mixture was concentrated again to give the title compound (14 g, 32.53 mmol).

步骤二:(2R,11aS)-8-(苄氧基)-2-羟基-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-23-3)的制备Step 2: Preparation of (2R,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-3)

将(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-羟基吡咯烷-2-甲酸甲酯(14g,32.53mmol)溶于甲醇(700mL)，加入饱和氯化铵水溶液(70mL)，再分批加入锌粉(21.27g,325.27mmol)，升温至80℃回流反应16小时。冷却至室温，过滤，滤液减压浓缩后硅胶柱纯化(洗脱剂：乙酸乙酯)得标题化合物(5.3g,14.39mmol)。Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylate (14 g, 32.53 mmol) was dissolved in methanol (700 mL), and saturated ammonium chloride aqueous solution (70 mL) was added. Zinc powder (21.27 g, 325.27 mmol) was then added in portions, and the mixture was refluxed at 80 °C for 16 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate) to give the title compound (5.3 g, 14.39 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):368.5[M+H]+ MS m/z(ESI): 368.5 [M+H] +

步骤三:(2R,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-23-4)的制备Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-4)

将(2R,11aS)-8-(苄氧基)-2-羟基-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(1g,2.71mmol)溶于DMF(25mL)，加入咪唑(1.85g,27.15mmol)和TBSCl(1.12g,13.57mmol)，反应16小时。加入水和乙酸乙酯搅拌，静置分液，有机相用饱和食盐水洗涤3次后减压浓缩。硅胶柱纯化(洗脱剂：50％乙酸乙酯/石油醚)后再次浓缩得标题化合物(1.02g,2.11mmol)。(2R,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1 g, 2.71 mmol) was dissolved in DMF (25 mL), and imidazole (1.85 g, 27.15 mmol) and TBSCl (1.12 g, 13.57 mmol) were added. The reaction was allowed to proceed for 16 hours. Water and ethyl acetate were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed three times with saturated brine and concentrated under reduced pressure. The solution was purified by silica gel column chromatography (eluent: 50% ethyl acetate/petroleum ether) and then concentrated again to give the title compound (1.02 g, 2.11 mmol).

步骤四:(2R,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮(7-23-5)的制备Step 4: Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-23-5)

将(2R,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(500mg,1.04mmol)溶于四氢呋喃(5mL)中，降温至0℃。然后滴加四氢呋喃硼烷络合物(10.3mL)。加毕升温至室温反应3小时。向反应液滴入甲醇(10ml)，然后浓缩得标题化合物粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)后浓缩得标题化合物(120mg，0.26mmol)(2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (500 mg, 1.04 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and then concentrated to obtain the title compound (120 mg, 0.26 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):453.3[M+H]+ ESI-MS (m/z): 453.3 [M+H] +

步骤五:(2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-23-6)的制备Step 5: Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23-6)

将(2R,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮(120mg，0.26mmol)溶于四氢呋喃(3mL)中，然后加入20％钯碳(12mg)，氢气置换三次后升温至45℃搅拌反应3小时。经硅藻土过滤后浓缩得标题化合物粗品(90mg,0.23mmol)，未经纯化直接用于下一步。(2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (120 mg, 0.26 mmol) was dissolved in tetrahydrofuran (3 mL), then 20% palladium on carbon (12 mg) was added. After three hydrogen purgings, the mixture was heated to 45 °C and stirred for 3 hours. The solution was filtered through diatomaceous earth and concentrated to obtain the crude title compound (90 mg, 0.23 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):379.2[M+H]+ ESI-MS (m/z): 379.2 [M+H] +

步骤六:(4-((S)-8-((5-(((2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5),10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯 (7-23-7)的制备Step Six: (4-((S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5),10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate Preparation of (7-23-7)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧基-50,11,11-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(135mg,0.23mmol)和(2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(90mg,0.23mmol)溶于DMF(2mL)中，加入碳酸钾(64mg,0.47mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(20mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)后再次浓缩得标题化合物(82mg，0.092mmol)。(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (135 mg, 0.23 mmol) and (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (90 mg, 0.23 mmol) were dissolved in DMF (2 mL), potassium carbonate (64 mg, 0.47 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH/DCM = 0%–5%), the mixture was concentrated again to give the title compound (82 mg, 0.092 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):884.3[M+H]+ ESI-MS (m/z): 884.3 [M+H] +

步骤七:(S)-2-(4-氨基苯基)-8-((5-(((2R,11aS)-2-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-23)的制备Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23)

将(4-((S)-8-((5-(((2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5),10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(82mg，0.092mmol)溶于二氯甲烷(5mL)中，搅拌下滴入三氟乙酸(3mL)，滴加完毕后继续搅拌5小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(8.4mg,0.012mmol)。82 mg (0.092 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5),10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (3 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 5 hours. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (8.4 mg, 0.012 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):670.3[M+H]+ ESI-MS (m/z): 670.3 [M+H] +

1H NMR(400MHz,DMSO):δ7.37(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J＝8.4Hz,2H),6.53(d,J＝8.4Hz,2H),6.31(s,1H),6.26(s,1H),5.28-5.10(m,2H),5.08-4.79(m,1H),4.21-4.11(m,2H),3.97-3.88(m,4H),3.89-3.76(m,1H),3.66(s,3H),3.64(s,3H),3.55-3.47(m,4H),3.25-3.20(m,2H),3.01-2.93(m,1H),2.68-2.65(m,1H),2.12-2.04(m,1H),1.85-1.76(m,4H),1.74-1.66(m,1H),1.61-1.52(m,2H). 1 H NMR (400MHz, DMSO): δ7.37(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J＝8.4Hz,2H),6.53(d,J＝8.4Hz, 2H),6.31(s,1H),6.26(s,1H),5.28-5.10(m,2H),5.08-4.79(m,1H),4.21-4.11(m,2H),3.97-3.88(m ,4H),3.89-3.76(m,1H),3.66(s,3H),3.64(s,3H),3.55-3.47(m,4H),3.25-3.20(m,2H),3.01-2.93( m,1H),2.68-2.65(m,1H),2.12-2.04(m,1H),1.85-1.76(m,4H),1.74-1.66(m,1H),1.61-1.52(m,2H).

其制备方法如下： Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例九:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-24)的制备
Preparation Example 9: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24)

步骤一:(2S,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-羟基吡咯烷-2-甲酸甲酯(7-24-2)的制备Step 1: Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-24-2)

将4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.0g，3.3mmol)，(2S,4S)-4-羟基吡咯烷-2-甲酸甲酯(503mg，3.46mmol)，溶于DMF(10mL)中，加入HATU(2.51g，6.51mmol)，DIPEA(1.28 g，9.9mmol)，于室温反应2小时，加入50mL水后，加入70mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(1.36g，3.16mmol)。Dissolve 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.0 g, 3.3 mmol) and (2S,4S)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (503 mg, 3.46 mmol) in DMF (10 mL), then add HATU (2.51 g, 6.51 mmol) and DIPEA (1.28 g). The compound was reacted at room temperature for 2 hours (g, 9.9 mmol), 50 mL of water was added, followed by extraction with 70 mL of ethyl acetate. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.36 g, 3.16 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):431.1[M+H]+ ESI-MS (m/z): 431.1 [M+H] +

步骤二:(2S,11aS)-8-(苄氧基)-2-羟基-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-24-3)的制备Step 2: Preparation of (2S,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-3)

将(2S,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-羟基吡咯烷-2-甲酸甲酯(1.36g，3.16mmol)，溶于甲醇(52mL)中，加入10％的氯化铵水溶液(10mL)，锌粉(2.43g，37.2mmol)，升温至40℃反应1小时后，再次升温至80℃反应过夜，浓缩得粗品，加入50mL乙酸乙酯溶清，饱和食盐水洗涤3次，有机相用无水硫酸钠干燥后过滤浓缩得粗品，反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(1.3g,3.53mmol)。Methyl (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylate (1.36 g, 3.16 mmol) was dissolved in methanol (52 mL), and 10% ammonium chloride aqueous solution (10 mL) and zinc powder (2.43 g, 37.2 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 50 mL of ethyl acetate. The mixture was washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (1.3 g, 3.53 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):369.2[M+H]+ ESI-MS (m/z): 369.2 [M+H] +

步骤三:(2S,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-24-4)的制备Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-4)

将(2S,11aS)-8-(苄氧基)-2-羟基-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(1.3g,3.53mmol)，溶于DMF(15mL)中，加入叔丁基二甲基氯硅烷(2.66g，17.64mmol)，咪唑(2.4g，35.3mmol)，于室温下反应16小时后，，加入60mL水后，加入80mL乙酸乙酯萃取，分层后，有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～50％)浓缩得标题化合物(1.38g，2.86mmol)(2S,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.3 g, 3.53 mmol) was dissolved in DMF (15 mL), and tert-butyldimethylchlorosilane (2.66 g, 17.64 mmol) and imidazole (2.4 g, 35.3 mmol) were added. After reacting at room temperature for 16 hours, 60 mL of water was added, followed by extraction with 80 mL of ethyl acetate. After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–50%) and concentrated to obtain the title compound (1.38 g, 2.86 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):483.2[M+H]+ ESI-MS (m/z): 483.2 [M+H] +

步骤四:(2S,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-一(7-24-5)的制备Step 4: Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5--(7-24-5)

将(2S,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,11a-四氢-5H- 苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(500mg,1.04mmol)溶于四氢呋喃(5mL)中，降温至0℃。然后滴加四氢呋喃硼烷络合物(10.3mL)。加毕升温至室温反应3小时。向反应液滴入甲醇(10ml)，然后浓缩得标题化合物粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)浓缩得标题化合物(60mg，0.13mmol)(2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H- Benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (500 mg, 1.04 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and concentrated to obtain the title compound (60 mg, 0.13 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):469.2[M+H]+ ESI-MS (m/z): 469.2 [M+H] +

步骤五:(2S,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-24-6)的制备Step 5: Preparation of (2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24-6)

将(2S,11aS)-8-(苄氧基)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮(60mg，0.13mmol)溶于四氢呋喃(3mL)中，然后加入20％钯碳(6mg)，氢气置换三次后保温至25℃搅拌反应2小时。经硅藻土过滤后浓缩得标题化合物粗品(46mg,0.12mmol)，未经纯化直接用于下一步。(2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (60 mg, 0.13 mmol) was dissolved in tetrahydrofuran (3 mL), and then 20% palladium on carbon (6 mg) was added. After three hydrogen purgings, the mixture was kept at 25 °C and stirred for 2 hours. After filtration through diatomaceous earth, the crude product of the title compound (46 mg, 0.12 mmol) was obtained and used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):379.2[M+H]+ ESI-MS (m/z): 379.2 [M+H] +

步骤六:(4-((S)-8-((5-(((2S,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-24-6)的制备Step Six: Preparation of tert-butyl carbamate (7-24-6)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧基-50,11,11-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(71mg,0.12mmol)和(2S,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(46mg,0.12mmol)溶于DMF(2mL)中，加入碳酸钾(33mg,0.24mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(20mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(39mg，0.04mmol)(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-50,11,11-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (71 mg, 0.12 mmol) and (2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (46 mg, 0.12 mmol) were dissolved in DMF (2 mL), potassium carbonate (33 mg, 0.24 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (20 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH/DCM = 0%–5%) yielded the title compound (39 mg, 0.04 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):884.3[M+H]+ ESI-MS (m/z): 884.3 [M+H] +

步骤七:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六 氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-24)的制备Step 7: (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexadecyl) Preparation of hydrogen-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24)

将(4-((S)-8-((5-(((2S,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(39mg，0.04mmol)溶于二氯甲烷(3mL)中，搅拌下滴入三氟乙酸(2mL)，滴加完毕后继续搅拌5小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(7mg,0.01mmol)。39 mg (0.04 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl carbamate was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (2 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 5 hours. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (7 mg, 0.01 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):670.3[M+H]+ESI-MS (m/z): 670.3 [M+H]+

1H NMR(400MHz,DMSO):δ7.28(d,J＝4.0Hz,2H),7.26(s,1H),7.12(d,J＝8.4Hz,2H),6.54(d,J＝8.4Hz,2H),6.30(s,1H),6.26(s,1H),5.37-5.14(m,1H),5.10-5.03(m,1H),4.24-4.06(m,2H),3.96-3.84(m,2H),3.66(s,3H),3.63(s,3H),3.61-3.56(m,1H),3.55-3.48(m,1H),3.47-3.40(m,3H),3.26-3.21(m,3H),2.68-2.64(m,1H),2.34-2.30(m,1H),1.85-1.76(m,4H),1.71-1.63(m,1H),1.62-1.51(m,2H). 1 H NMR (400MHz, DMSO): δ7.28(d,J＝4.0Hz,2H),7.26(s,1H),7.12(d,J＝8.4Hz,2H),6.54(d,J＝8.4Hz,2H ),6.30(s,1H),6.26(s,1H),5.37-5.14(m,1H),5.10-5.03(m,1H),4.24-4.06(m,2H),3.96-3.84(m,2 H),3.66(s,3H),3.63(s,3H),3.61-3.56(m,1H),3.55-3.48(m,1H),3.47-3.40(m,3H),3.26-3.21(m ,3H),2.68-2.64(m,1H),2.34-2.30(m,1H),1.85-1.76(m,4H),1.71-1.63(m,1H),1.62-1.51(m,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2S,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-25)的制备
Preparation Example 10: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25)

步骤一:(2S,4R)-4-甲氧基吡咯烷-2-甲酸甲酯(7-25-2)的制备Step 1: Preparation of (2S,4R)-4-methoxypyrrolidine-2-carboxylic acid methyl ester (7-25-2)

将1-(叔丁基)2-甲基(2S,4R)-4-甲氧基吡咯烷-1,2-二甲酸(1.0g,3.53mmol,FR)溶于甲醇(10mL)中，加入HCl(4M in EA)(5mL)，室温反应1小时。浓缩得粗品，未经纯化直接用于下一步(800.6mg，5.01mmol)1-(tert-butyl)-2-methyl(2S,4R)-4-methoxypyrrolidine-1,2-dicarboxylic acid (1.0 g, 3.53 mmol, FR) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (800.6 mg, 5.01 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):160.1[M+H]+ ESI-MS (m/z): 160.1 [M+H] +

步骤二:(2R,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-甲氧基吡咯烷-2-甲酸甲酯(7-25-3)的制备Step 2: Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methoxypyrrolidine-2-carboxylic acid methyl ester (7-25-3)

将(2S,4R)-4-甲氧基吡咯烷-2-甲酸甲酯(800.6mg，5.01mmol))和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.18g,3.88mmol,FR)溶于DMF(10mL)中，加入HATU(1.48g,3.88mmol)和DIPEA(1.36g,10.59mmol)，室温反应1小时。将反应液加入乙酸乙酯(100mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(1.56g，3.51mmol)Methyl (2S,4R)-4-methoxypyrrolidine-2-carboxylate (800.6 mg, 5.01 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol, FR) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.56 g, 3.51 mmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):445.2[M+H]+ ESI-MS (m/z): 445.2 [M+H] +

步骤三:(2R,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5,11(10H)-二酮(7-25-4)的制备Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-5,11(10H)-dione (7-25-4)

将(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(1.56g，3.1mmol)溶于甲醇(30mL)中，加入锌粉(2.4g,31.1mmol)，然后加入氯化铵饱和水溶液(40mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.13g,2.95mmol)，未经纯化直接用于下一步。Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (1.56 g, 3.1 mmol) was dissolved in methanol (30 mL), zinc powder (2.4 g, 31.1 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.13 g, 2.95 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):383.2[M+H]+ ESI-MS (m/z): 383.2 [M+H] +

步骤四:(2R,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-25-5)的制备Step 4: Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-5)

将(2R,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(0.5g,1.3mmol,FR)溶于四氢呋喃(5mL)中，降温至0℃。然后滴加四氢呋喃硼烷络合物(10.3mL)。加毕升温至室温反应3小时。向反应液滴入甲醇(10ml)，然后浓缩得标题化合物粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)浓缩得标题化合物(80mg，0.22mmol)。(2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (0.5 g, 1.3 mmol, FR) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and concentrated to obtain the title compound (80 mg, 0.22 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):369.1[M+H]+ ESI-MS (m/z): 369.1 [M+H] +

步骤五:(2R,11aS)-8-羟基-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-25-6)的制备Step 5: Preparation of (2R,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-6)

将(2R,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(80mg，0.22mmol)溶于四氢呋喃(3mL)中，加入钯碳(8mg)然后在氢气氛围下反应1小时。反应完成后过滤，滤液无水硫酸钠干燥后过滤浓缩得标题化合物粗品(49mg,0.17mmol)，未经纯化直接用于下一步。(2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (80 mg, 0.22 mmol) was dissolved in tetrahydrofuran (3 mL), palladium on carbon (8 mg) was added, and the reaction was carried out under a hydrogen atmosphere for 1 hour. After the reaction was completed, the mixture was filtered, dried over anhydrous sodium sulfate, and then concentrated to give the crude title compound (49 mg, 0.17 mmol), which was used directly in the next step without purification.

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):279.2[M+H]+ ESI-MS (m/z): 279.2 [M+H] +

步骤六:(4-((S)-8-((5-(((2R,11aS)-2,7-二甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H)-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-26-7)的制备Step Six: Preparation of (4-((S)-8-((5-(((2R,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H)-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(30.8mg,0.053mmol)和(2R,11aS)-8-羟基-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(15mg,0.05mmol)溶于DMF(1.5mL)中，加入碳酸钾(13.8mg,0.1mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)后再次浓缩得标题化合物(19.6mg，0.25mmol)(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (30.8 mg, 0.053 mmol) and (2R,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (15 mg, 0.05 mmol) were dissolved in DMF (1.5 mL), potassium carbonate (13.8 mg, 0.1 mmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. The resulting product was purified by silica gel column chromatography (MeOH/DCM = 0%–5%) and then concentrated again to give the title compound (19.6 mg, 0.25 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):784.4[M+H]+ ESI-MS (m/z): 784.4 [M+H] +

步骤七:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2S,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-25)的制备Step 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25)

将(4-((S)-8-((5-(((2R,11aS)-2,7-二甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H)-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(19.6mg，0.25mmol)溶于二氯甲烷(2mL)中，搅拌下滴入三氟乙酸(1mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(9.2mg,13.16μmol)。19.6 mg (0.25 mmol) of tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H)-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (9.2 mg, 13.16 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):684.4[M+H]+ ESI-MS (m/z): 684.4 [M+H] +

1H NMR(400MHz,DMSO):δ7.37(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J＝8.4Hz,2H),6.53(d,J＝8.4Hz,2H),6.30(s,1H),6.26(s,1H),5.19(s,2H),4.19-4.08(m,1H),3.96-3.84(m,5H),3.74-3.68(m,2H),3.66(s,3H),3.64(s,3H),3.56-3.46(m,3H),3.29-3.22(m,2H),3.22(s,3H),3.02-2.93(m,1H),2.73-2.66(m,1H),1.85-1.76(m,4H),1.75-1.66(m,1H),1.61-1.50(m,2H). 1 H NMR (400MHz, DMSO): δ7.37(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J＝8.4Hz,2H),6.53(d,J ＝8.4Hz,2H),6.30(s,1H),6.26(s,1H),5.19(s,2H),4.19-4.08(m,1H),3.96-3.84(m,5H),3.7 4-3.68(m,2H),3.66(s,3H),3.64(s,3H),3.56-3.46(m,3H),3.29-3.22(m,2H),3.22(s,3H),3 .02-2.93(m,1H),2.73-2.66(m,1H),1.85-1.76(m,4H),1.75-1.66(m,1H),1.61-1.50(m,2H).

其制备方法如下： Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十一:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2,7-二甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-26)的制备
Preparation Example 11: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26)

步骤一:(2S,4S)-4-(甲氧基)吡咯烷-2-甲酸甲酯(7-26-2)的制备Step 1: Preparation of (2S,4S)-4-(methoxy)pyrrolidine-2-carboxylic acid methyl ester (7-26-2)

将1-(叔丁基)2-甲基(2S,4S)-4-(甲氧基)吡咯烷-1,2-二甲酸(1.0g,4.08mmol)溶于甲醇(10mL)中，加入HCl(4M in EA)(5mL)，室温反应1小时。浓缩得粗品，未经纯化直接用于下一步(797.66mg，4.08mmol) 1-(tert-butyl)-2-methyl(2S,4S)-4-(methoxy)pyrrolidine-1,2-dicarboxylic acid (1.0 g, 4.08 mmol) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (797.66 mg, 4.08 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):196.2[M+H]+ ESI-MS (m/z): 196.2 [M+H] +

步骤二:(2S,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(甲氧基)吡咯烷-2-甲酸甲酯(7-26-3)的制备Step 2: Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(methoxy)pyrrolidine-2-carboxylic acid methyl ester (7-26-3)

将(2S,4S)-4-(甲氧基)吡咯烷-2-甲酸甲酯(797.66mg，4.08mmol)和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.24g,4.08mmol)溶于DMF(10mL)中，加入HATU(1.86g,4.89mmol)和DIPEA(1.58g,12.23mmol)，室温反应1小时。将反应液加入乙酸乙酯(100mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(1.8g，4.05mmol)Methyl (2S,4S)-4-(methoxy)pyrrolidine-2-carboxylate (797.66 mg, 4.08 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.24 g, 4.08 mmol) were dissolved in DMF (10 mL), and HATU (1.86 g, 4.89 mmol) and DIPEA (1.58 g, 12.23 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.8 g, 4.05 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):445.2[M+H]+ ESI-MS (m/z): 445.2 [M+H] +

步骤三:(2S,11aS)-8-(苄氧基)-7-甲氧基-2-(甲氧基)-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-26-4)的制备Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(methoxy)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-26-4)

将(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(甲氧基)吡咯烷-2-甲酸甲酯(1.8g，4.05mmol)溶于甲醇(30mL)中，加入锌粉(2.63g,40.50mmol)，然后加入氯化铵饱和水溶液(40mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.5g,3.92mmol)，未经纯化直接用于下一步。Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(methoxy)pyrrolidine-2-carboxylate (1.8 g, 4.05 mmol) was dissolved in methanol (30 mL), zinc powder (2.63 g, 40.50 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.5 g, 3.92 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):383.2[M+H]+ ESI-MS (m/z): 383.2 [M+H] +

步骤四:(2S,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-26-5)的制备Step 4: Preparation of (2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-5)

将(2S,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(0.8g,2.09mmol)溶于四氢呋喃(10mL)中，加入硼氢化钠(158.13mg,41.8mmol)，然后室温滴加三氟乙酸(596mg,5.23mmol)，直至无大量气体溢出，升温至75℃回流反应5小时,将反应液浓缩，加水(50mL)后，用乙酸乙酯(50mL X 2)萃取两次，合并有机相用饱和氯化钠水溶液洗涤后，用无水硫酸钠干燥，减压浓缩后反相柱色谱纯化(乙腈/1％ 甲酸水溶液＝0％～70％)后冻干得标题化合物(640mg,1.73mmol)。其结构表征数据如下：(2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (0.8 g, 2.09 mmol) was dissolved in tetrahydrofuran (10 mL), sodium borohydride (158.13 mg, 41.8 mmol) was added, and then trifluoroacetic acid (596 mg, 5.23 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then refluxed at 75 °C for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The mixture was then extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by reversed-phase column chromatography (acetonitrile/1%). The title compound (640 mg, 1.73 mmol) was obtained by lyophilization after being dissolved in an aqueous formic acid solution (0%–70%). Its structural characterization data are as follows:

ESI-MS(m/z):369.1[M+H]+ ESI-MS (m/z): 369.1 [M+H] +

步骤五:(2S,11aS)-8-羟基-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-26-6)的制备Step 5: Preparation of (2S,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-6)

将(2S,11aS)-8-(苄氧基)-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(640mg,1.73mmol)溶于甲醇(10mL)中，加入钯碳(30mg)然后在氢气氛围下反应1小时。反应完成后过滤，滤液无水硫酸钠干燥后过滤浓缩得标题化合物粗品(434mg,1.56mmol)，未经纯化直接用于下一步。(2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (640 mg, 1.73 mmol) was dissolved in methanol (10 mL), palladium on carbon (30 mg) was added, and the reaction was carried out under a hydrogen atmosphere for 1 hour. After the reaction was completed, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (434 mg, 1.56 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):279.2[M+H]+ ESI-MS (m/z): 279.2 [M+H] +

步骤六:(4-((S)-8-((5-(((2S,11aS)-2,7-二甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-26-7)的制备Step Six: Preparation of (4-((S)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(21.07mg,35.93μmol)和(2S,11aS)-8-羟基-2,7-二甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(10mg,35.93μmol)溶于DMF(1mL)中，加入碳酸钾(14.88mg,107.80μmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)后再次浓缩得标题化合物(20mg，25.51μmol)(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (21.07 mg, 35.93 μmol) and (2S,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10 mg, 35.93 μmol) were dissolved in DMF (1 mL), potassium carbonate (14.88 mg, 107.80 μmol) was added, and the mixture was stirred for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH/DCM = 0%–5%), the mixture was concentrated again to give the title compound (20 mg, 25.51 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):784.4[M+H]+ ESI-MS (m/z): 784.4 [M+H] +

步骤七:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2,7-二甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-26)的制备Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26)

将(4-((S)-8-((5-(((2S,11aS)-2,7-二甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(20mg，25.51μmol)溶于二氯甲烷(2mL)中， 搅拌下滴入三氟乙酸(0.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(13.51mg,19.56μmol)。20 mg (25.51 μmol) of tert-butyl carbamate (4-((S)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL). Trifluoroacetic acid (0.5 mL) was added dropwise with stirring, and stirring was continued for 1 hour after the addition was complete. The mixture was concentrated under reduced pressure and purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%), followed by lyophilization to obtain the title compound (13.51 mg, 19.56 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):684.4[M+H]+ ESI-MS (m/z): 684.4 [M+H] +

1H NMR(400MHz,DMSO):δ7.39(s,1H),7.28(d,J＝9.3Hz,2H),6.85-6.75(m,2H),6.48-6.63(m,2H),6.31(s,1H),6.29(s,1H)4.22-4.15(m,1H),3.97-3.86(m,4H),3.75-3.65(m,2H),3.66(s,3H),3.64(s,3H),3.62-3.50(m,6H),3.24(s,3H),3.32-3.16(m,2H),2.76-2.67(m,1H),2.43-2.36(m,1H),1.87-1.73(m,4H),1.60-1.49(m,4H). 1 H NMR (400MHz, DMSO): δ7.39 (s, 1H), 7.28 (d, J = 9.3Hz, 2H), 6.85-6.75 (m, 2H), 6.48- 6.63(m,2H),6.31(s,1H),6.29(s,1H)4.22-4.15(m,1H),3.97-3.86(m,4H),3.75-3 .65(m,2H),3.66(s,3H),3.64(s,3H),3.62-3.50(m,6H),3.24(s,3H),3.32-3.16( m,2H),2.76-2.67(m,1H),2.43-2.36(m,1H),1.87-1.73(m,4H),1.60-1.49(m,4H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十二:(S)-8-((5-(((2R,11aS)-2-氨基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-2-(4-氨基苯基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-27)的制备
Preparation Example 12: Preparation of (S)-8-((5-(((2R,11aS)-2-amino-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-aminophenyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-27)

步骤一:(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-((叔丁氧基羰基)氨基)吡咯烷-2-甲酸甲酯(7-27-2)的制备Step 1: Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylic acid methyl ester (7-27-2)

将(2S,4R)-4-((叔丁氧基羰基)氨基)吡咯烷-2-甲酸甲酯(1g,4.09mmol,FR)和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.37g,4.50mmol,FR)溶于DMF(10mL)中，加入HATU(1.71g,4.50mmol)和DIPEA(1.59g,12.28mmol)，室温反应1小时。将反应液加入乙酸乙酯(50mL)和饱和食盐水(50mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(2.1g，3.97mmol)Methyl (2S,4R)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylate (1 g, 4.09 mmol, FR) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.37 g, 4.50 mmol, FR) were dissolved in DMF (10 mL), and HATU (1.71 g, 4.50 mmol) and DIPEA (1.59 g, 12.28 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (50 mL) and saturated brine (50 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (2.1 g, 3.97 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):530.2[M+H]+ ESI-MS (m/z): 530.2 [M+H] +

步骤二:((2R,11aS)-8-(苄氧基)-7-甲氧基-5,11-二氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)氨基甲酸叔丁酯(7-27-3)的制备 Step 2: Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-3)

将(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-((叔丁氧基羰基)氨基)吡咯烷-2-甲酸甲酯(2.1g，3.97mmol,FR)溶于甲醇(50mL)中，加入锌粉(2.58g,15.86mmol)，然后加入氯化铵饱和水溶液(20mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.8g,3.85mmol)，未经纯化直接用于下一步。Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylate (2.1 g, 3.97 mmol, FR) was dissolved in methanol (50 mL), zinc powder (2.58 g, 15.86 mmol) was added, followed by 20 mL of saturated ammonium chloride aqueous solution. The mixture was refluxed at 85 °C for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.8 g, 3.85 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):468.3[M+H]+ESI-MS (m/z): 468.3 [M+H]+

步骤三:((2R,11aS)-8-(苄氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)氨基甲酸叔丁酯(7-27-4)的制备Step 3: Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-4)

将((2R,11aS)-8-(苄氧基)-7-甲氧基-5,11-二氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)氨基甲酸叔丁酯(1.8g,3.85mmol,FR)溶于四氢呋喃(100mL)中，加入硼氢化钠(843.15mg,11.55mmol)，然后室温滴加三氟乙酸(877.8mg,7.7mmol)，直至无大量气体溢出，升温至75℃回流反应5小时。将反应液浓缩，加水(50mL)后，用乙酸乙酯(50mL X 2)萃取两次，合并有机相用饱和氯化钠水溶液洗涤后，用无水硫酸钠干燥，过滤浓缩得标题化合物粗品(1.4g,3.09mmol)，未经纯化直接用于下一步。Dissolve tert-butyl carbamate ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-2-yl) in tetrahydrofuran (100 mL), add sodium borohydride (843.15 mg, 11.55 mmol), and then add trifluoroacetic acid (877.8 mg, 7.7 mmol) dropwise at room temperature until no large amount of gas is released. Then, reflux the mixture at 75 °C for 5 hours. The reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.4 g, 3.09 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):454.2[M+H]+ ESI-MS (m/z): 454.2 [M+H] +

步骤四:((2R,11aS)-8-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)氨基甲酸叔丁酯(7-27-5)的制备Step 4: Preparation of ((2R,11aS)-8-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-5)

将((2R,11aS)-8-(苄氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)氨基甲酸叔丁酯(100mg,220.50μmol,FR)溶于甲醇(5mL)中，加入钯碳(5mg)，置于氢气氛围下反应1小时。反应完成后过滤，滤液经无水硫酸钠干燥后过滤浓缩得标题化合物粗品(80.13mg,220.50μmol)，未经纯化直接用于下一步。((2R,11aS)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (100 mg, 220.50 μmol, FR) was dissolved in methanol (5 mL), and palladium on carbon (5 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (80.13 mg, 220.50 μmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):364.2[M+H]+ ESI-MS (m/z): 364.2 [M+H] +

步骤五:(4-((S)-8-((5-(((2R,11aS)-2-((叔丁氧基羰基)氨基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代 -5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-27-6)的制备Step 5: (4-((S)-8-((5-(((2R,11aS)-2-((tert-butoxycarbonyl)amino)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo) Preparation of -5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-27-6)

将((2R,11aS)-8-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)氨基甲酸叔丁酯(10mg,27.52μmol)和(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(16.14mg,27.52μmol)溶于DMF(1mL)中，加入碳酸钾(11.39mg,82.55μmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(20mg,23.01μmol)((2R,11aS)-8-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (10 mg, 27.52 μmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (16.14 mg, 27.52 μmol) were dissolved in DMF (1 mL), and potassium carbonate (11.39 mg, 82.55 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification by silica gel column chromatography (MeOH/DCM = 0%–5%) yielded the title compound (20 mg, 23.01 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):869.8[M+H]+ ESI-MS (m/z): 869.8 [M+H] +

步骤六:(S)-8-((5-(((2R,11aS)-2-氨基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-2-(4-氨基苯基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-27)的制备Step Six: Preparation of (S)-8-((5-(((2R,11aS)-2-amino-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-aminophenyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-27)

将(4-((S)-8-((5-(((2R,11aS)-2-((叔丁氧基羰基)氨基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(20mg,23.01μmol)溶于二氯甲烷(2mL)中，搅拌下滴入三氟乙酸(0.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(5.41mg,0.045mmol)。20 mg (23.01 μmol) of tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butoxycarbonyl)amino)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (5.41 mg, 0.045 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):670.4[M+H]+ ESI-MS (m/z): 670.4 [M+H] +

1H NMR(400MHz,DMSO):δ7.36(s,1H),7.29(s,1H),7.25(s,1H),7.11(d,J＝8.5Hz,2H),6.53(d,J＝8.6Hz,2H),6.29(d,J＝9.3Hz,2H),4.17-4.09(m,1H),3.96-3.90(m,4H),3.65(d,J＝7.1Hz,6H),3.51(d,J＝4.9Hz,2H),3.30-3.20(m,4H),2.99(dd,J＝12.9,8.8Hz,2H),2.23-2.14(m,2H),1.96-1.87(m,2H),1.84-1.76(m,4H),1.56(d,J＝6.4Hz,2H). 1 H NMR (400MHz, DMSO): δ7.36 (s, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 7.11 (d, J = 8.5Hz, 2H), 6 .53(d,J＝8.6Hz,2H),6.29(d,J＝9.3Hz,2H),4.17-4.09(m,1H),3.96-3.90(m,4H),3.6 5(d,J＝7.1Hz,6H),3.51(d,J＝4.9Hz,2H),3.30-3.20(m,4H),2.99(dd,J＝12.9,8.8Hz ,2H),2.23-2.14(m,2H),1.96-1.87(m,2H),1.84-1.76(m,4H),1.56(d,J＝6.4Hz,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μm Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十三:(S)-2-(4-氨基苯基)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-29)的制备
Preparation Example 13: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29)

步骤一：(S)-4,4-二氟吡咯烷-2-甲酸甲酯(7-29-2)的制备Step 1: Preparation of (S)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester (7-29-2)

将(S)-1-(叔丁氧基羰基)-4,4-二氟吡咯烷-2-甲酸(0.6g,2.39mmol,)溶于MeOH(3mL)中，滴入4M HCl/dioxane溶液(4mL)后室温搅拌反应20小时。反应完成后，反应液直接减压 浓缩即得标题化合物的盐酸盐粗品(460mg,2.28mmol)，未经纯化直接用于下一步反应。(S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid (0.6 g, 2.39 mmol) was dissolved in MeOH (3 mL), and 4M HCl/dioxane solution (4 mL) was added dropwise. The mixture was stirred at room temperature for 20 hours. After the reaction was complete, the reaction solution was directly subjected to reduced pressure. The crude hydrochloride of the title compound was obtained by concentration (460 mg, 2.28 mmol) and used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):166.1[M+H]+ ESI-MS (m/z): 166.1 [M+H] +

步骤二：(S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4,4-二氟吡咯烷-2-甲酸甲酯(7-29-3)的制备Step 2: Preparation of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester (7-29-3)

将4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(743.76mg,2.45mmol)溶于DMF(5mL)中加入HATU(1.03g,2.70mmol)和DIPEA(316.96mg,2.45mmol)，室温搅拌10分钟。另取一反应瓶，将(S)-4,4-二氟吡咯烷-2-甲酸甲酯盐酸盐(450mg,2.45mmol)溶于DMF(5mL)，加入DIPEA(316.96mg,2.45mmol)使其呈碱性。然后将两反应液合并，室温搅拌反应2小时。待反应完成后，向反应液中加水30mL，乙酸乙酯15mL X 3萃取，有机相用饱和食盐水15mL洗涤，无水硫酸钠干燥后减压浓缩得标题化合物的粗品(1.4g,2.18mmol),未经纯化直接用于下一步反应。4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (743.76 mg, 2.45 mmol) was dissolved in DMF (5 mL), and HATU (1.03 g, 2.70 mmol) and DIPEA (316.96 mg, 2.45 mmol) were added. The mixture was stirred at room temperature for 10 minutes. In a separate reaction flask, (S)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester hydrochloride (450 mg, 2.45 mmol) was dissolved in DMF (5 mL), and DIPEA (316.96 mg, 2.45 mmol) was added to make the solution alkaline. The two reaction solutions were then combined and stirred at room temperature for 2 hours. After the reaction was complete, 30 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL x 3). The organic phase was washed with 15 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (1.4 g, 2.18 mmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):468.2[M+H]+ ESI-MS (m/z): 468.2 [M+H] +

步骤三：(S)-8-(苄氧基)-2,2-二氟-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-29-4)的制备Step 3: Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-29-4)

向(S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4,4-二氟吡咯烷-2-甲酸甲酯(700mg,1.09mmol)中加入甲醇(12mL)和饱和氯化铵水溶液(3mL)，体系未溶清。加入锌粉(711.41mg,10.88mmol)，直接放入预热至75℃的油浴锅中加热反应16小时。反应完成后，将反应液降温至室温，过滤除去不溶物，滤饼用适量甲醇洗涤，滤液减压浓缩。残余物加水10mL,乙酸乙酯萃取10mL X 3，有机相合并用盐水10mL洗涤，无水硫酸钠干燥后减压浓缩得标题化合物的粗品(580mg,1.05mmol),未经纯化直接用于下一步反应。Methanol (12 mL) and saturated ammonium chloride aqueous solution (3 mL) were added to methyl (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxylate (700 mg, 1.09 mmol), and the mixture remained insoluble. Zinc powder (711.41 mg, 10.88 mmol) was added, and the mixture was directly placed in an oil bath preheated to 75 °C and heated for 16 hours. After the reaction was complete, the reaction solution was cooled to room temperature, filtered to remove insoluble matter, and the filter cake was washed with an appropriate amount of methanol. The filtrate was concentrated under reduced pressure. The residue was extracted with 10 mL of water and ethyl acetate (10 mL x 3). The combined organic phases were washed with 10 mL of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (580 mg, 1.05 mmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):389.2[M+H]+ ESI-MS (m/z): 389.2 [M+H] +

步骤四：(S)-8-(苄氧基)-2,2-二氟-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-29-5)的制备 Step 4: Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29-5)

将(S)-8-(苄氧基)-2,2-二氟-7-甲氧基-1,2,3,11-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(100mg,257.49μmol)溶于THF(2mL)中，氮气保护下降温至0℃滴加硼烷(1M in THF,2.57mL)。加完后，自然升至室温反应1.5小时。反应完成后，反应液降温至0℃，滴加适量甲醇淬灭反应，室温搅拌1小时。向反应液中加水10mL，乙酸乙酯10mL X 3萃取，有机相盐水10mL洗涤，无水硫酸钠干燥后减压浓缩。粗品经硅胶柱色谱纯化(乙酸乙酯-石油醚＝10～70％)后再次减压浓缩得标题化合物(23mg,61.43μmol)。(S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,11-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (100 mg, 257.49 μmol) was dissolved in 2 mL of THF. Under nitrogen protection, the mixture was cooled to 0 °C and borane (1 M in THF, 2.57 mL) was added dropwise. After the addition was complete, the mixture was allowed to rise naturally to room temperature for 1.5 hours. Once the reaction was complete, the reaction solution was cooled to 0 °C, and the reaction was quenched dropwise with an appropriate amount of methanol. The mixture was stirred at room temperature for 1 hour. 10 mL of water was added to the reaction solution, and the mixture was extracted with 10 mL of ethyl acetate (3 times). The extract was washed with 10 mL of organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate-petroleum ether = 10-70%) and then concentrated under reduced pressure to obtain the title compound (23 mg, 61.43 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):385.1[M+H]+ ESI-MS (m/z): 385.1 [M+H] +

步骤五:(S)-2,2-二氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5-酮(7-29-6)的制备Step 5: Preparation of (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-5-one (7-29-6)

将(S)-8-(苄氧基)-2,2-二氟-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(23mg,61.43μmol)混悬于甲醇(2mL)中，加乙酸乙酯(1mL)，使原料完全溶解。加入Pd/C(7.46mg,6.14μmol,10％purity)，然后置换氢气三次。在氢气球氛围下室温搅拌反应3小时。反应完成后，反应液过滤，滤饼用适量乙酸乙酯洗涤，滤液直接减压浓缩得标题化合物粗品(23mg,59.88μmol)，未经纯化直接用于下一步。(S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (23 mg, 61.43 μmol) was suspended in methanol (2 mL), and ethyl acetate (1 mL) was added to completely dissolve the starting material. Pd/C (7.46 mg, 6.14 μmol, 10% purity) was added, followed by purging with hydrogen three times. The reaction was stirred at room temperature for 3 hours under a hydrogen balloon atmosphere. After the reaction was complete, the reaction solution was filtered, the filter cake was washed with an appropriate amount of ethyl acetate, and the filtrate was directly concentrated under reduced pressure to obtain the crude title compound (23 mg, 59.88 μmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):285.2[M+H]+ ESI-MS (m/z): 285.2 [M+H] +

步骤六:(4-((S)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢)-1H-苯并[e]吡咯并[1,2-a][1,4]二氮卓-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(7-29-7)的制备Step Six: Preparation of (4-((S)-8-((5-((((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro)-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-29-7)

将(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(20.63mg,35.18μmol)和(S)-2,2-二氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(10mg,35.18μmol)溶于DMF(0.5mL)中，加入K2CO3(14.59mg,105.54μmol)，室温搅拌18小时。反应完成后，反应液中加水4mL，乙酸乙酯5mL X 3萃取，有机相饱和食盐水5mL洗涤，无水硫酸钠干燥后减压浓缩得标题化合物粗品(28mg,26.59μmol)，未经纯化直接用于下一步。(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (20.63 mg, 35.18 μmol) and (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10 mg, 35.18 μmol) were dissolved in DMF (0.5 mL), and K2CO3 (14.59 mg, 105.54 μmol) was added. The mixture was stirred at room temperature for 18 hours. After the reaction was complete, 4 mL of water was added to the reaction solution, and 5 mL of ethyl acetate was extracted three times. The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (28 mg, 26.59 μmol), which was used directly in the next step without purification.

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):790.4[M+H]+ ESI-MS (m/z): 790.4 [M+H] +

步骤七：(S)-2-(4-氨基苯基)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-29)的制备Step 7: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29)

室温下向叔丁基(4-((S)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢)-1H-苯并[e]吡咯并[1,2-a][1,4]二氮卓-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸酯(28mg,35.45μmol)中加入二氯甲烷(1mL)，然后加入TFA(0.2mL)，加毕，室温搅拌反应2小时。反应完成后，反应液直接减压浓缩除去溶剂，残余物直接经制备高效液相色谱纯化后冷冻干燥得标题化合物(4.5mg,6.29μmol)。Dichloromethane (1 mL) was added to tert-butyl(4-((S)-8-((5-((((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro)-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (28 mg, 35.45 μmol) at room temperature, followed by the addition of TFA (0.2 mL). After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the solvent, and the residue was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (4.5 mg, 6.29 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):690.4[M+H]+；345.9[M/2+1]+ ESI-MS(m/z):690.4[M+H] + ;345.9[M/2+1] +

1H NMR(400MHz,DMSO-d6)δ7.33(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J＝8.4Hz,1H),6.53(d,J＝8.4Hz,2H),6.49(brs,1H),6.30(s,2H),5.19(brs,1H),4.14-4.10(m,1H),3.98-3.84(m,7H),3.67(s,3H),3.64(s,3H),3.57-3.50(m,2H),3.25-3.14(m,3H),2.75-2.72(m,2H),1.82-1.78(m,3H),1.82-1.78(m,4H),1.59-1.53(m,2H). 1H NMR (400MHz, DMSO-d6 ) )δ7.33(s,1H),7.29(s,1H),7.26(s,1H),7.11(d,J＝8.4Hz,1H),6.53(d,J＝8. 4Hz,2H),6.49(brs,1H),6.30(s,2H),5.19(brs,1H),4.14-4.10(m,1H),3.98 -3.84(m,7H),3.67(s,3H),3.64(s,3H),3.57-3.50(m,2H),3.25-3.14(m,3H) ,2.75-2.72(m,2H),1.82-1.78(m,3H),1.82-1.78(m,4H),1.59-1.53(m,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十四:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-30)的制备
Preparation Example 14: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30)

步骤一:(2S,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-氟吡咯烷-2-甲酸甲酯(7-30-2)的制备Step 1: Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylic acid methyl ester (7-30-2)

将(2S,4S)-4-氟吡咯烷-2-甲酸甲酯(1.07g,7.25mmol)和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(2g,6.59mmol)溶于DMF(10mL)中，加入HATU(2.76g,7.25mmol)和DIPEA(2.56g,19.78mmol)，室温反应1小时。将反应液加入乙酸乙酯(100mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(2.3g，5.32mmol)。Methyl (2S,4S)-4-fluoropyrrolidine-2-carboxylate (1.07 g, 7.25 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2 g, 6.59 mmol) were dissolved in DMF (10 mL), and HATU (2.76 g, 7.25 mmol) and DIPEA (2.56 g, 19.78 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (2.3 g, 5.32 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):433.2[M+H]+ ESI-MS (m/z): 433.2 [M+H] +

步骤二:(2S,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5,11(10H)-二酮(7-30-3)的制备 Step 2: Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-5,11(10H)-dione (7-30-3)

将(2S,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-氟吡咯烷-2-甲酸甲酯(2.3g，5.32mmol)溶于甲醇(50mL)中，加入锌粉(3.46g,53.19mmol)，然后加入氯化铵饱和水溶液(10mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.8g,4.86mmol)，未经纯化直接用于下一步。Methyl (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylate (2.3 g, 5.32 mmol) was dissolved in methanol (50 mL), zinc powder (3.46 g, 53.19 mmol) was added, followed by saturated aqueous solution of ammonium chloride (10 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.8 g, 4.86 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):371.2[M+H]+ ESI-MS (m/z): 371.2 [M+H] +

步骤三:(2S,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-30-4)的制备Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-4)

将(2S,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(1.8g,4.86mmol)溶于四氢呋喃(100mL)中，加入硼氢化钠(1064.34mg,14.58mmol)，然后室温滴加三氟乙酸(1108.1mg,9.72mmol)，直至无大量气体溢出，升温至75℃回流反应5小时。将反应液浓缩，加水(50mL)后，用乙酸乙酯(50mL X 2)萃取两次，合并有机相用饱和氯化钠水溶液洗涤后，用无水硫酸钠干燥，过滤浓缩得标题化合物粗品(1.4g,3.93mmol)，未经纯化直接用于下一步。(2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.8 g, 4.86 mmol) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (1064.34 mg, 14.58 mmol) was added, and then trifluoroacetic acid (1108.1 mg, 9.72 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then refluxed at 75 °C for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.4 g, 3.93 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):357.3[M+H]+ ESI-MS (m/z): 357.3 [M+H] +

步骤四:(2S,11aS)-2-氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-30-5)的制备Step 4: Preparation of (2S,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-5)

将(2S,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(200mg,561.18μmol)溶于甲醇(10mL)中，加入钯碳(5mg)，置于氢气氛围下反应1小时。反应完成后过滤，滤液经无水硫酸钠干燥后过滤浓缩得标题化合物粗品(0.14g,525.79μmol)，未经纯化直接用于下一步。(2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (200 mg, 561.18 μmol) was dissolved in methanol (10 mL), and palladium on carbon (5 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (0.14 g, 525.79 μmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):267.1[M+H]+ ESI-MS (m/z): 267.1 [M+H] +

步骤五:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H- 苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-30-6)的制备Step 5: (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H- Preparation of benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-6)

将(2S,11aS)-2-氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(10mg,37.56μmol)和(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(22.03mg,37.56μmol)溶于DMF(1mL)中，加入碳酸钾(15.55mg,112.67μmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)后再次浓缩得标题化合物(20mg,25.91μmol)。(2S,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10 mg, 37.56 μmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (22.03 mg, 37.56 μmol) were dissolved in DMF (1 mL), and potassium carbonate (15.55 mg, 112.67 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH/DCM = 0%–5%), the mixture was concentrated again to give the title compound (20 mg, 25.91 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):772.3[M+H]+ ESI-MS (m/z): 772.3 [M+H] +

步骤六:(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-30)的制备Step Six: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30)

将(S)-2-(4-氨基苯基)-8-((5-(((2S,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(20mg,25.91μmol)溶于二氯甲烷(2mL)中，搅拌下滴入三氟乙酸(0.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(8.14mg,12.00μmol)。(S)-2-(4-aminophenyl)-8-((5-((((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (20 mg, 25.91 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (8.14 mg, 12.00 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):673.3[M+H]+ ESI-MS (m/z): 673.3 [M+H] +

1H NMR(400MHz,DMSO)δ7.40(s,1H),7.29(s,1H),7.24(s,1H),6.87-6.70(m,2H),6.60-6.45(m,2H),6.31(s,2H),4.17(s,1H),4.03-3.87(m,4H),3.87-3.76(m,2H),3.67(s,3H),3.65(s,3H),3.58-3.45(m,4H),3.29-3.20(m,2H),2.82-2.61(m,2H),2.20-2.06(m,2H),1.86-1.73(m,4H),1.60-1.53(m,2H).其制备方法如下： ¹H NMR (400MHz, DMSO) δ 7.40 (s, ¹H), 7.29 (s, ¹H), 7.24 (s, ¹H), 6.87–6.70 (m, 2H), 6.60–6.45 (m, 2H), 6.31 (s, 2H), 4.17 (s, ¹H), 4.03–3.87 (m, 4H), 3.87–3.76 (m, 2H), 3.67 (s, 3H), 3.65 (s, 3H), 3.58–3.45 (m, 4H), 3.29–3.20 (m, 2H), 2.82–2.61 (m, 2H), 2.20–2.06 (m, 2H), 1.86–1.73 (m, 4H), 1.60–1.53 (m, 2H). The preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十五:(S)-2-(4-氨基苯基)-8-((5-(((2R,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-31)的制备
Preparation Example 15: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31)

步骤一:(2R,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-氟吡咯烷-2-甲酸甲酯(7-31-2)的制备Step 1: Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylic acid methyl ester (7-31-2)

将(2R,4S)-4-氟吡咯烷-2-甲酸甲酯(1.1g,7.25mmol,FR)和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(2.05g,6.59mmol,FR)溶于DMF(10mL)中，加入HATU(2.76g,7.25mmol)和DIPEA(2.56g,19.78mmol)，室温反应1小时。将反应液加入乙酸乙酯(100mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(2.1g，4.85mmol)。Methyl (2R,4S)-4-fluoropyrrolidine-2-carboxylate (1.1 g, 7.25 mmol, FR) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (2.05 g, 6.59 mmol, FR) were dissolved in DMF (10 mL), and HATU (2.76 g, 7.25 mmol) and DIPEA (2.56 g, 19.78 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (2.1 g, 4.85 mmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):433.2[M+H]+ ESI-MS (m/z): 433.2 [M+H] +

步骤二:(2R,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-31-3)的制备Step 2: Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-31-3)

将(2R,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-氟吡咯烷-2-甲酸甲酯(2.1g，4.85mmol)溶于甲醇(50mL)中，加入锌粉(3.4g,53.1mmol)，然后加入氯化铵饱和水溶液(10mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.7g,4.7mmol)，未经纯化直接用于下一步。Methyl (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylate (2.1 g, 4.85 mmol) was dissolved in methanol (50 mL), zinc powder (3.4 g, 53.1 mmol) was added, followed by saturated aqueous solution of ammonium chloride (10 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.7 g, 4.7 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):371.2[M+H]+ ESI-MS (m/z): 371.2 [M+H] +

步骤三:(2R,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-31-4)的制备Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-4)

将(2R,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(400mg,1.04mmol)溶于四氢呋喃(5mL)中，降温至0℃。然后滴加四氢呋喃硼烷络合物(10.3mL)。加毕升温至室温反应3小时。向反应液滴入甲醇(10ml)，然后浓缩得标题化合物粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)浓缩得标题化合物(110mg，0.31mmol)。(2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (400 mg, 1.04 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and concentrated to obtain the title compound (110 mg, 0.31 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):357.3[M+H]+ ESI-MS (m/z): 357.3 [M+H] +

步骤四:(2R,11aS)-2-氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-31-5)的制备Step 4: Preparation of (2R,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-5)

将(2S,11aS)-8-(苄氧基)-2-氟-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(110mg，0.31mmol)溶于四氢呋喃(5mL)中，加入钯碳(10mg)，置于氢气氛围下反应1小时。反应完成后经硅藻土过滤，滤液经无水硫酸钠干燥后过滤浓缩得标题化合物粗品(50mg,190.1μmol)，未经纯化直接用于下一步。(2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (110 mg, 0.31 mmol) was dissolved in tetrahydrofuran (5 mL), and palladium on carbon (10 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (50 mg, 190.1 μmol), which was used directly in the next step without purification.

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):267.1[M+H]+ ESI-MS (m/z): 267.1 [M+H] +

步骤五:(S)-2-(4-氨基苯基)-8-((5-(((2R,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-31-6)的制备Step 5: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-6)

将(2R,11aS)-2-氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(50mg,190.1μmol)和叔丁基(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸酯(117.3mg,199.5μmol)溶于DMF(3mL)中，加入碳酸钾(52.4mg,380μmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(20mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)后再次浓缩得标题化合物(88mg,114.1μmol)(2R,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (50 mg, 190.1 μmol) and tert-butyl(S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (117.3 mg, 199.5 μmol) were dissolved in DMF (3 mL), and potassium carbonate (52.4 mg, 380 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH/DCM = 0%–5%), the mixture was concentrated again to give the title compound (88 mg, 114.1 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):772.3[M+H]+ ESI-MS (m/z): 772.3 [M+H] +

步骤六:(S)-2-(4-氨基苯基)-8-((5-(((2R,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-31)的制备Step Six: Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31)

将(S)-2-(4-氨基苯基)-8-((5-(((2R,11aS)-2-氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(88mg,114.1μmol))溶于二氯甲烷(3mL)中，搅拌下滴入三氟乙酸(1.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(8.0mg,11.7μmol)。(S)-2-(4-aminophenyl)-8-((5-((((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (88 mg, 114.1 μmol)) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (8.0 mg, 11.7 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):673.3[M+H]+ ESI-MS (m/z): 673.3 [M+H] +

1H NMR(400MHz,DMSO):δ7.39(s,1H),7.29(s,2H),7.14(d,J＝7.8Hz,2H),6.58(d,J＝7.4Hz,2H),6.29(d,J＝10.0Hz,2H),5.26(d,J＝53.6Hz,1H),4.21-4.08(m,1H),4.01-3.89(m,4H),3.85-3.76(m,2H),3.65(d,J＝5.8Hz,6H),3.60-3.49(m,4H),3.29-3.11(m,4H),3.04-2.95(m,1H),2.74-2.63(m,1H),1.88-1.73(m,4H),1.62-1.50(m,2H). 1 H NMR (400MHz, DMSO): δ7.39 (s, 1H), 7.29 (s, 2H), 7.14 (d, J = 7.8Hz, 2H), 6.58 (d, J = 7. 4Hz,2H),6.29(d,J＝10.0Hz,2H),5.26(d,J＝53.6Hz,1H),4.21-4.08(m,1H),4.01-3 .89(m,4H),3.85-3.76(m,2H),3.65(d,J＝5.8Hz,6H),3.60-3.49(m,4H),3.29-3.11 (m,4H),3.04-2.95(m,1H),2.74-2.63(m,1H),1.88-1.73(m,4H),1.62-1.50(m,2H).

其制备方法如下： Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十六:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2S,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-32)的制备
Preparation Example 16: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32)

步骤一:(2S,4S)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(7-32-2)的制备Step 1: Preparation of (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylate methyl ester (7-32-2)

将1-(叔丁基)2-甲基(2S,4S)-4-(三氟甲基)吡咯烷-1,2-二甲酸(1.0g,3.53mmol,FR)溶于甲醇(10mL)中，加入HCl(4M in EA)(5mL)，室温反应1小时。浓缩得粗品，未经纯化直接用于下一步(820.79mg，3.51mmol)。 1-(tert-butyl)-2-methyl(2S,4S)-4-(trifluoromethyl)pyrrolidine-1,2-dicarboxylic acid (1.0 g, 3.53 mmol, FR) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (820.79 mg, 3.51 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):234.2[M+H]+ ESI-MS (m/z): 234.2 [M+H] +

步骤二:(2R,4S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(7-32-3)的制备Step 2: Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-32-3)

将(2S,4S)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(820.79mg，3.51mmol,CL)和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.18g,3.88mmol,FR)溶于DMF(10mL)中，加入HATU(1.48g,3.88mmol)和DIPEA(1.36g,10.59mmol)，室温反应1小时。将反应液加入乙酸乙酯(100mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(1.5g，3.1mmol)。Methyl (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (820.79 mg, 3.51 mmol, CL) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol, FR) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.5 g, 3.1 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):483.2[M+H]+ ESI-MS (m/z): 483.2 [M+H] +

步骤三:(2S,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-32-4)的制备Step 3: Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-32-4)

将(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(1.5g，3.1mmol)溶于甲醇(30mL)中，加入锌粉(2.2g,35.1mmol)，然后加入氯化铵饱和水溶液(40mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.4g,3.3mmol)，未经纯化直接用于下一步。Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (1.5 g, 3.1 mmol) was dissolved in methanol (30 mL), zinc powder (2.2 g, 35.1 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.4 g, 3.3 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):421.2[M+H]+ ESI-MS (m/z): 421.2 [M+H] +

步骤四:(2S,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-32-5)的制备Step 4: Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-5)

将(2S,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(400mg,0.95mmol)溶于四氢呋喃(5mL)中，降温至0℃。然后滴加四氢呋喃硼烷络合物(10.3mL)。加毕升温至室温反应3小时。向反应液滴入甲醇(10ml)，然后浓缩得标题化合物粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝0％～70％)浓缩得标题化合物(100mg，0.24mmol)。 (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (400 mg, 0.95 mmol) was dissolved in tetrahydrofuran (5 mL) and cooled to 0 °C. Then, a tetrahydrofuran borane complex (10.3 mL) was added dropwise. After the addition was complete, the mixture was heated to room temperature and reacted for 3 hours. Methanol (10 mL) was added dropwise to the reaction mixture, and the solution was concentrated to obtain the crude title compound. The crude compound was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and concentrated to obtain the title compound (100 mg, 0.24 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):408.1[M+H]+ ESI-MS (m/z): 408.1 [M+H] +

步骤五:(2S,11aS)-8-羟基-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-32-6)的制备Step 5: Preparation of (2S,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-6)

将(2S,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(100mg，0.24mmol)溶于四氢呋喃(3mL)中，加入钯碳(10mg)，置于氢气氛围下反应1小时。反应完成后过滤，滤液经无水硫酸钠干燥后过滤浓缩得标题化合物粗品(70mg,0.22mmol)，未经纯化直接用于下一步。(2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (100 mg, 0.24 mmol) was dissolved in tetrahydrofuran (3 mL), and palladium on carbon (10 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (70 mg, 0.22 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):317.1[M+H]+ ESI-MS (m/z): 317.1 [M+H] +

步骤六:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2S,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-32-7)的制备Step Six: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-7)

将(2R,11aS)-8-羟基-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(70mg,0.22mmol)和(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(136mg,0.23mmol)溶于DMF(2mL)中，加入碳酸钾(61mg,0.44mmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)纯化后再次浓缩得标题化合物(72mg,88.3μmol)。(2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (70 mg, 0.22 mmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (136 mg, 0.23 mmol) were dissolved in DMF (2 mL), and potassium carbonate (61 mg, 0.44 mmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. The mixture was purified by silica gel column chromatography (MeOH/DCM = 0%–5%) and then concentrated again to give the title compound (72 mg, 88.3 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):822.5[M+H]+ ESI-MS (m/z): 822.5 [M+H] +

步骤七:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2S,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-32)的制备Step 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32)

将(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2S,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(72mg,88.3μmol)溶于二氯甲烷 (3mL)中，搅拌下滴入三氟乙酸(1.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(9.8mg,13.6μmol)。(S)-2-(4-aminophenyl)-7-methoxy-8-((5-((((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (72 mg, 88.3 μmol) was dissolved in dichloromethane. In a 3 mL solution, trifluoroacetic acid (1.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. The solution was concentrated under reduced pressure and purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%), followed by lyophilization to obtain the title compound (9.8 mg, 13.6 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):723.3[M+H]+ ESI-MS (m/z): 723.3 [M+H] +

1H NMR(400MHz,DMSO):δ7.41(s,1H),7.27(d,J＝12.2Hz,2H),7.11(d,J＝8.2Hz,2H),6.53(d,J＝8.4Hz,2H),6.29(d,J＝12.4Hz,2H),5.20(s,2H),4.19-4.08(m,1H),3.97-3.88(m,4H),3.87-3.76(m,2H),3.66(s,3H),3.64(s,3H),3.59-3.46(m,4H),3.28-3.16(m,4H),3.04-2.94(m,1H),2.73-2.64(m,1H),1.84-1.76(m,4H),1.61-1.50(m,2H). 1 H NMR (400MHz, DMSO): δ7.41(s,1H),7.27(d,J＝12.2Hz,2H),7.11(d,J＝8.2Hz,2H),6.5 3(d,J＝8.4Hz,2H),6.29(d,J＝12.4Hz,2H),5.20(s,2H),4.19-4.08(m,1H),3.97-3.8 8(m,4H),3.87-3.76(m,2H),3.66(s,3H),3.64(s,3H),3.59-3.46(m,4H),3.28-3.16 (m,4H),3.04-2.94(m,1H),2.73-2.64(m,1H),1.84-1.76(m,4H),1.61-1.50(m,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十七:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2R,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-33)的制备
Preparation Example 17: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33)

步骤一:(2S,4R)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(7-33-2)的制备Step 1: Preparation of (2S,4R)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (7-33-2)

将1-(叔丁基)2-甲基(2S,4R)-4-(三氟甲基)吡咯烷-1,2-二甲酸(1.0g,3.53mmol)溶于甲醇(10mL)中，加入HCl(4M in EA)(5mL)，室温反应1小时。浓缩得粗品，未经纯化直接用于下一步(824.79mg，3.53mmol)1-(tert-butyl)-2-methyl(2S,4R)-4-(trifluoromethyl)pyrrolidine-1,2-dicarboxylic acid (1.0 g, 3.53 mmol) was dissolved in methanol (10 mL), and HCl (4 M in EA) (5 mL) was added. The reaction was carried out at room temperature for 1 hour. The crude product was concentrated and used directly in the next step without purification (824.79 mg, 3.53 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):234.2[M+H]+ ESI-MS (m/z): 234.2 [M+H] +

步骤二:(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(7-33-3)的制备Step 2: Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-33-3)

将(2S,4R)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(824.79mg，3.53mmol)和4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.18g,3.88mmol)溶于DMF(10mL)中，加入HATU(1.48g,3.88mmol)和DIPEA(1.36g,10.59mmol)，室温反应1小时。将反应液加入乙酸乙酯(100mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(乙酸乙酯/石油醚＝20％～60％)再次浓缩得标题化合物(1.7g，3.52mmol)Methyl (2S,4R)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (824.79 mg, 3.53 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. This crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 20%–60%) and concentrated again to obtain the title compound (1.7 g, 3.52 mmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):483.2[M+H]+ ESI-MS (m/z): 483.2 [M+H] +

步骤三:(2R,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-33-4)的制备Step 3: Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-33-4)

将(2S,4R)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-(三氟甲基)吡咯烷-2-甲酸甲酯(1.7g，3.52mmol)溶于甲醇(30mL)中，加入锌粉(2.29g,35.24mmol)，然后加入氯化铵饱和水溶液(40mL)，升温至85℃回流反应16小时。将反应液加入乙酸乙酯(150mL)和饱和食盐水(100mL)中搅拌，静置分液。有机相用无水硫酸钠干燥后浓缩得标题化合物粗品(1.48g,3.52mmol)，未经纯化直接用于下一步。Methyl (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (1.7 g, 3.52 mmol) was dissolved in methanol (30 mL), zinc powder (2.29 g, 35.24 mmol) was added, followed by saturated aqueous solution of ammonium chloride (40 mL). The mixture was heated to 85 °C and refluxed for 16 hours. The reaction solution was then added to ethyl acetate (150 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude title compound (1.48 g, 3.52 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):421.2[M+H]+ ESI-MS (m/z): 421.2 [M+H] +

步骤四:(2R,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-33-5)的制备Step 4: Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-5)

将(2R,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(1.48g,3.52mmol)溶于四氢呋喃(30mL)中，加入硼氢化钠(770.88mg,10.56mmol)，然后室温滴加三氟乙酸(802.56mg,7.04mmol)，直至无大量气体溢出，升温至75℃回流反应5小时。将反应液浓缩，加水(50mL)后，用乙酸乙酯(50mL X 2)萃取两次，合并有机相用饱和氯化钠水溶液洗涤后，用无水硫酸钠干燥，过滤浓缩得标题化合物粗品(1.1g,2.71mmol)，未经纯化直接用于下一步。(2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.48 g, 3.52 mmol) was dissolved in tetrahydrofuran (30 mL), sodium borohydride (770.88 mg, 10.56 mmol) was added, and then trifluoroacetic acid (802.56 mg, 7.04 mmol) was added dropwise at room temperature until no large amount of gas was released. The mixture was then refluxed at 75 °C for 5 hours. The reaction solution was concentrated, and water (50 mL) was added. The mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.1 g, 2.71 mmol), which was used directly in the next step without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):408.1[M+H]+ ESI-MS (m/z): 408.1 [M+H] +

步骤五:(2R,11aS)-8-羟基-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-33-6)的制备Step 5: Preparation of (2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-6)

将(2R,11aS)-8-(苄氧基)-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(100mg,246.06μmol)溶于甲醇(10mL)中，加入钯碳(5mg)，置于氢气氛围下反应1小时。反应完成后过滤，滤液经无水硫酸钠干燥后过滤浓缩得标题化合物粗品(77.82mg,246.05μmol)，未经纯化直接用于下一步。(2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (100 mg, 246.06 μmol) was dissolved in methanol (10 mL), and palladium on carbon (5 mg) was added. The mixture was reacted under a hydrogen atmosphere for 1 hour. After the reaction was complete, the mixture was filtered, and the filtrate was dried over anhydrous sodium sulfate and then concentrated to obtain the crude title compound (77.82 mg, 246.05 μmol), which was used directly in the next step without purification.

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):317.1[M+H]+ ESI-MS (m/z): 317.1 [M+H] +

步骤六:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2R,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-33-7)的制备Step Six: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-7)

将(2R,11aS)-8-羟基-7-甲氧基-2-(三氟甲基)-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(10.78mg,34.10μmol)和(S)-(4-(8-((5-溴戊基)氧基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-2-基)苯基)氨基甲酸叔丁酯(20.00mg,34.10μmol)溶于DMF(1mL)中，加入碳酸钾(14.12mg,102.30μmol)后搅拌反应16小时。加水淬灭反应，用乙酸乙酯萃取3次(10mL x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)后再次浓缩得标题化合物(20mg,24.33μmol)(2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (10.78 mg, 34.10 μmol) and (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl ester (20.00 mg, 34.10 μmol) were dissolved in DMF (1 mL), and potassium carbonate (14.12 mg, 102.30 μmol) was added. The mixture was stirred and reacted for 16 hours. The reaction was quenched with water, and the mixture was extracted three times (10 mL x 3) with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (MeOH/DCM = 0%–5%), the mixture was concentrated again to give the title compound (20 mg, 24.33 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):822.5[M+H]+ ESI-MS (m/z): 822.5 [M+H] +

步骤七:(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2R,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-33)的制备Step 7: Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33)

将(S)-2-(4-氨基苯基)-7-甲氧基-8-((5-(((2R,11aS)-7-甲氧基-5-氧代-2-(三氟甲基)-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(20mg,24.33μmol)溶于二氯甲烷(2mL)中，搅拌下滴入三氟乙酸(0.5mL)，滴加完毕后继续搅拌1小时。减压浓缩后反相柱色谱纯化(乙腈/1％甲酸水溶液＝0％～70％)后冻干得标题化合物(9.33mg,12.80μmol)。(S)-2-(4-aminophenyl)-7-methoxy-8-((5-((((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (20 mg, 24.33 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. After concentration under reduced pressure, the compound was purified by reversed-phase column chromatography (acetonitrile/1% formic acid aqueous solution = 0%–70%) and then lyophilized to obtain the title compound (9.33 mg, 12.80 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):723.3[M+H]+ ESI-MS (m/z): 723.3 [M+H] +

1H NMR(400MHz,DMSO)δ7.39(s,1H),7.28(s,1H),7.25(s,1H),6.82(s,2H),6.59-6.43(m,2H),6.31(s,2H),4.18-4.08(m,1H),3.97-3.85(m,4H),3.82-3.74(m,2H),3.68(s,3H),3.66(s,3H),3.56-3.44(m,4H),3.28-3.19(m,2H),2.81-2.65(m,2H),2.21-2.04(m,2H),1.87-1.70(m,4H),1.59-1.52(m,2H). 1 H NMR(400MHz,DMSO)δ7.39(s,1H),7.28(s,1H),7.25(s,1H),6.82(s,2H),6. 59-6.43(m,2H),6.31(s,2H),4.18-4.08(m,1H),3.97-3.85(m,4H),3.82-3. 74(m,2H),3.68(s,3H),3.66(s,3H),3.56-3.44(m,4H),3.28-3.19(m,2H), 2.81-2.65(m,2H),2.21-2.04(m,2H),1.87-1.70(m,4H),1.59-1.52(m,2H).

其制备方法如下： Its preparation method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例十八和十九：(S)-8-((5-(((2S,11aS)-2-(4-氨基苯基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-34)和(S)-8-((5-(((2R,11aS)-2-(4-氨基苯基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-35)的制备
Preparation Examples 18 and 19: (S)-8-((5-(((2S,11aS)-2-(4-aminophenyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7- 34) and the preparation of (S)-8-((5-(((2R,11aS)-2-(4-aminophenyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-35)

将(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧)-7-乙氧基-1,10,11,11-四氢-3H,5H-螺并[e]吡咯并[1,2-a][1,4]氮杂-2,1’-环丙烷]-5-酮(化合物7-5)(20mg,29.42μmol)溶于甲醇(5mL)和四氢呋喃(5mL)混合溶剂中，加入10％钯碳(2mg)，氢气置换并保护下搅拌反应2小时。反应液垫硅藻土过滤，滤液减压浓缩后经制备高效液相色谱纯化，冷冻干燥得到化合物7-34(2.77mg,3.13μmol)和化合物7-35(5.66mg,6.69μmol)。(S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-ethoxy-1,10,11,11-tetrahydro-3H,5H-spiro[e]pyrrolo[1,2-a][1,4]aza-2,1’-cyclopropane]-5-one (compound 7-5) (20 mg, 29.42 μmol) was dissolved in a mixed solvent of methanol (5 mL) and tetrahydrofuran (5 mL), and 10% palladium on carbon (2 mg) was added. The mixture was stirred for 2 hours under hydrogen purging and protection. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and purified by preparative high performance liquid chromatography. After freeze drying, compounds 7-34 (2.77 mg, 3.13 μmol) and 7-35 (5.66 mg, 6.69 μmol) were obtained.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):双峰682.5[M+H]+ MS m/z (ESI): Bimodal 682.5 [M+H] +

化合物7-34：1H NMR(400MHz,DMSO)δ7.40-7.24(m,4H),7.10(d,J＝8.3Hz,2H),6.31(t,J＝13.0Hz,2H),3.97(s,1H),3.92(t,J＝6.2Hz,4H),3.83(d,J＝7.6Hz,2H),3.65(d,J＝5.1Hz,6H),3.55-3.47(m,4H),3.30(dd,J＝19.7,10.5Hz,2H),3.26-3.11(m,2H),2.31(dd,J＝12.5,7.5Hz,1H),2.17-2.09(m,1H),1.96(dd,J＝12.4,7.2Hz,1H),1.86-1.72(m,5H),1.60-1.51(m,2H),0.68-0.48(m,4H).Compound 7-34: ¹H NMR (400MHz, DMSO) δ 7.40–7.24 (m, 4H), 7.10 (d, J = 8.3 Hz, 2H), 6.31 (t, J = 13.0 Hz, 2H), 3.97 (s, 1H), 3.92 (t, J = 6.2 Hz, 4H), 3.83 (d, J = 7.6 Hz, 2H), 3.65 (d, J = 5.1 Hz, 6H), 3.55–3.47 (m, 4H). 3.30(dd,J＝19.7,10.5Hz,2H),3.26-3.11(m,2H),2.31(dd,J＝12.5,7.5Hz,1H),2.17-2.09(m ,1H),1.96(dd,J＝12.4,7.2Hz,1H),1.86-1.72(m,5H),1.60-1.51(m,2H),0.68-0.48(m,4H).

化合物7-35：1H NMR(400MHz,DMSO)δ7.35(dd,J＝18.7,10.3Hz,4H),7.11(d,J＝8.2Hz,2H),6.29(d,J＝15.3Hz,2H),4.05(d,J＝3.6Hz,1H),3.92(t,J＝6.4Hz,4H),3.83(s,2H),3.65(t,J＝2.7Hz,6H),3.53(d,J＝11.8Hz,4H),3.33(d,J＝11.7Hz,2H),3.12(ddd,J＝34.2,13.2,8.2Hz,2H),2.46(d,J＝5.1Hz,1H),2.00-1.93(m,1H),1.87-1.70(m,6H),1.56(d,J＝6.6Hz,2H),0.58(ddt,J＝15.7,12.7,6.9Hz,4H).Compound 7-35: ¹H NMR (400MHz, DMSO) δ 7.35 (dd, J = 18.7, 10.3Hz, 4H), 7.11 (d, J = 8.2Hz, 2H), 6.29 (d, J = 15.3Hz, 2H), 4.05 (d, J = 3.6Hz, 1H), 3.92 (t, J = 6.4Hz, 4H), 3.83 (s, 2H), 3.65 (t, J = 2.7Hz, 6H), 3.53 (d, J = 11... .8Hz,4H),3.33(d,J＝11.7Hz,2H),3.12(ddd,J＝34.2,13.2,8.2Hz,2H),2.46(d,J＝5.1Hz,1H),2 .00-1.93(m,1H),1.87-1.70(m,6H),1.56(d,J＝6.6Hz,2H),0.58(ddt,J＝15.7,12.7,6.9Hz,4H).

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

制备例二十:(S)-8-((5-(((S)-2-(呋喃-3-基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-36)的制备
Preparation Example 20: Preparation of (S)-8-((5-(((S)-2-(furan-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-36)

步骤一:(S)-8-((5-溴戊基)氧基)-2-(呋喃-3-基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-36-1)的制备Step 1: Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-1)

将呋喃-3-基硼酸(40.15mg,358.85μmol)，(S)-8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基三氟甲磺酸酯(100mg,179.43μmol)，四(三苯基膦)钯(20.73mg,17.94μmol)和碳酸钠(76.07mg,717.70μmol)混合一起，加入水(0.5mL)和甲苯(2mL)以及乙醇(0.5mL)，然后抽换氮气三次，升温80℃加热反应3小时。反应完成后，反应液加水10mL，乙酸乙酯5mL X 3萃取，有机相饱和食盐水10mL洗涤，无水硫酸钠干燥后减压浓缩。粗品经硅胶柱色谱纯化(乙酸乙酯/石油醚＝0％～70％)后再次减压浓缩得标题化合物(60mg,126.23μmol)。Furan-3-ylboronic acid (40.15 mg, 358.85 μmol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (100 mg, 179.43 μmol), tetra(triphenylphosphine)palladium (20.73 mg, 17.94 μmol), and sodium carbonate (76.07 mg, 717.70 μmol) were mixed together, and water (0.5 mL), toluene (2 mL), and ethanol (0.5 mL) were added. The mixture was then purged with nitrogen three times and heated to 80 °C for 3 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and then concentrated again under reduced pressure to obtain the title compound (60 mg, 126.23 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):475.1[M+H]+ ESI-MS (m/z): 475.1 [M+H] +

步骤二:(S)-2-(呋喃-3-基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-36-2)的制备Step 2: Preparation of (S)-2-(furan-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-2)

将(S)-8-((5-溴戊基)氧基)-2-(呋喃-3-基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(60mg,126.23μmol)和(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(34.63mg,126.23μmol)溶于DMF(1mL)中，加入碳酸钾(52.34mg,378.68μmol)，室温搅拌反应20小时。反应完成后，反应液加水10mL，有大量固体析出，乙酸乙酯：甲醇＝10：1的混合溶剂10mL X 3萃取， 有机相盐水10mL洗涤，无水硫酸钠干燥后减压浓缩。粗品制备薄层色谱纯化(DCM：MeOH＝8:1)后再次减压浓缩得标题化合物(43mg,50.80μmol)。(S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (60 mg, 126.23 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (34.63 mg, 126.23 μmol) were dissolved in DMF (1 mL), and potassium carbonate (52.34 mg, 378.68 μmol) was added. The mixture was stirred at room temperature for 20 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and a large amount of solid precipitated out. The solid was then extracted with 10 mL of a 10:1 mixed solvent of ethyl acetate and methanol three times. The sample was washed with 10 mL of organic brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by thin-layer chromatography (DCM:MeOH = 8:1) and concentrated again under reduced pressure to give the title compound (43 mg, 50.80 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):669.3[M+H]+ ESI-MS (m/z): 669.3 [M+H] +

步骤三:(S)-8-((5-(((S)-2-(呋喃-3-基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-36)的制备Step 3: Preparation of (S)-8-((5-(((S)-2-(furan-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-36)

将(S)-2-(呋喃-3-基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(24mg,35.89μmol)溶于THF(2mL)中，加入NaBH4(12.22mg,323.00μmol)，然后滴入TFA(24.55mg,215.33μmol)，体系有大量气泡产生。待气泡消失后，转移至油浴锅加热至75℃反应22小时。反应完成后，反应液加水5mL，乙酸乙酯5mL X 3萃取三次，有机相饱和食盐水5mL洗涤，无水硫酸钠干燥，减压浓缩。粗品经制备高效液相色谱纯化后冷冻干燥得标题化合物(6.56mg,9.77μmol)。(S)-2-(furan-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (24 mg, 35.89 μmol) was dissolved in THF (2 mL), NaBH4 (12.22 mg, 323.00 μmol) was added, and then TFA (24.55 mg, 215.33 μmol) was added dropwise. A large number of bubbles were generated in the system. After the bubbles disappeared, the mixture was transferred to an oil bath and heated to 75°C for 22 hours. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted three times with 5 mL of ethyl acetate (3 times). The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography and then freeze-dried to obtain the title compound (6.56 mg, 9.77 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):655.4[M+H]+ ESI-MS (m/z): 655.4 [M+H] +

1H NMR(400MHz,DMSO-d6)δ7.66(d,J＝10.0Hz,2H),7.32(s,2H),7.28(s,1H),6.85(s,1H),6.53(d,J＝6.4Hz,1H),6.31-6.26(m,3H),4.20-4.13(m,1H),3.98-3.88(m,4H),3.88-3.70(m,1H),3.66(s,3H),3.64(s,3H),3.58-3.43(m,4H),3.25-3.11(m,4H),2.62(dd,J＝16.8,4.4Hz,1H),1.95(dd,J＝12.4,7.2Hz,1H),1.79(dd,J＝12.4,7.2Hz,5H),1.61-1.49(m,2H),0.68-0.61(m,1H),0.60-0.48(m,3H). 1 H NMR (400MHz, DMSO-d 6 )δ7.66(d,J＝10.0Hz,2H),7.32(s,2H),7.28(s,1H),6.85(s,1H),6.53(d,J＝6.4Hz,1H),6.31 -6.26(m,3H),4.20-4.13(m,1H),3.98-3.88(m,4H),3.88-3.70(m,1H),3.66(s,3H),3.64(s,3 H),3.58-3.43(m,4H),3.25-3.11(m,4H),2.62(dd,J＝16.8,4.4Hz,1H),1.95(dd,J＝12.4,7.2H z,1H),1.79(dd,J=12.4,7.2Hz,5H),1.61-1.49(m,2H),0.68-0.61(m,1H),0.60-0.48(m,3H).

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十一:(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2-(噻吩-3-基)-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-37)的制备
Preparation Example 21: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-37)

步骤一:(S)-8-((5-溴戊基)氧基)-2-(呋喃-3-基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-37-1)的制备Step 1: Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-1)

将噻吩-3-基硼酸(91.84mg,717.70μmol)，(S)-8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基三氟甲磺酸酯(200mg,358.85μmol)，四(三苯基膦)钯(41.47mg,35.89μmol)以及碳酸钠(152.14mg,1.44mmol)混合一起，加入水(1mL)，甲苯(4mL)和乙醇(1mL)，然后抽换氮气三次，升温80℃加热反应2小时。反应完成后，反应液加水10mL，乙酸乙酯5mL X 3萃取三次，有机相饱和食盐水10mL洗涤，无水硫酸钠干燥后减压浓缩。粗品经硅胶柱色谱纯化(乙酸乙酯/石油醚＝0％～70％)后再次减压浓缩得标题化合物(58mg,118.03μmol)。Thiophene-3-ylboronic acid (91.84 mg, 717.70 μmol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (200 mg, 358.85 μmol), tetra(triphenylphosphine)palladium (41.47 mg, 35.89 μmol), and sodium carbonate (152.14 mg, 1.44 mmol) were mixed together, and water (1 mL), toluene (4 mL), and ethanol (1 mL) were added. The mixture was then purged with nitrogen three times and heated to 80 °C for 2 hours. After the reaction was complete, the reaction solution was extracted three times with 10 mL of water and 5 mL of ethyl acetate (3 times each). The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–70%) and then concentrated under reduced pressure to obtain the title compound (58 mg, 118.03 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):491.1[M+H]+ ESI-MS (m/z): 491.1 [M+H] +

步骤二:(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯)[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-2-(噻吩-3-基)-1,11a-二氢-5H-苯并[e] 吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-37-2)的制备Step 2: (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrole)[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-(thiophene-3-yl)-1,11a-dihydro-5H-benzo[e] Preparation of pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-2)

将(S)-8-((5-溴戊基)氧基)-2-(呋喃-3-基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(55mg,111.93μmol)和(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(30.70mg,111.93μmol)溶于DMF(2mL)中，加入碳酸钾(46.41mg,335.78μmol)，室温搅拌反应40小时。反应完成后，反应液过滤除去不溶物，滤液经制备高效液相色谱纯化后冷冻干燥得标题化合物(15mg,21.90μmol)。(S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (55 mg, 111.93 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (30.70 mg, 111.93 μmol) were dissolved in DMF (2 mL), and potassium carbonate (46.41 mg, 335.78 μmol) was added. The mixture was stirred at room temperature for 40 hours. After the reaction was completed, the reaction solution was filtered to remove insoluble matter, and the filtrate was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (15 mg, 21.90 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):685.3[M+H]+ ESI-MS (m/z): 685.3 [M+H] +

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤三:(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2-(噻吩-3-基)-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-37)的制备Step 3: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-37)

将(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-2-(噻吩-3-基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(15mg,21.90μmol)溶于THF(2mL)中，加入硼氢化钠(8.29mg,219.04μmol)，有少许气泡产生。滴入TFA(14.99mg,131.43μmol)，有大量气泡产生。升温至70℃反应24小时。反应完成后，反应液减压浓缩，进一步经制备高效液相色谱纯化后冷冻干燥得标题化合物(2.83mg,4.01μmol)。(S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-(thiophen-3-yl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (15 mg, 21.90 μmol) was dissolved in THF (2 mL), and sodium borohydride (8.29 mg, 219.04 μmol) was added, producing a small amount of bubbles. TFA (14.99 mg, 131.43 μmol) was added dropwise, producing a large amount of bubbles. The reaction was heated to 70 °C and carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, further purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (2.83 mg, 4.01 μmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):671.3[M+H]+ ESI-MS (m/z): 671.3 [M+H] +

1H NMR(400MHz,DMSO-d6)δ7.53(dd,J＝4.8,2.8Hz,1H),7.45(dd,J＝3.6,1.2Hz,2H),7.32(s,1H),7.29-7.28(m,2H),6.55(d,J＝6.0Hz,1H),6.31(s,1H),6.30-6.26(m,2H),4.21-4.15(m,1H),3.93(dd,J＝13.2,6.6Hz,4H),3.83(dd,J＝15.2,7.6Hz,1H),3.66(s,3H),3.64(s,3H),3.55-3.50(m,2H),3.50-3.44(m,1H),3.24(dd,J＝13.2,10.0Hz,2H),3.14(dd,J＝12.0,8.8Hz,1H),2.74(dd,J＝16.4,4.0Hz,1H),2.03-1.90(m,2H),1.79(dd,J＝12.4,7.6Hz,5H),1.61-1.50(m,2H),0.68-0.61(m,1H),0.61-0.48(m,3H). 1 H NMR (400MHz, DMSO-d 6 )δ7.53(dd,J＝4.8,2.8Hz,1H),7.45(dd,J＝3.6,1.2Hz,2H),7.32(s,1H),7.29-7.28(m,2H),6.55(d,J＝6.0Hz,1H),6.31(s ,1H),6.30-6.26(m,2H),4.21-4.15(m,1H),3.93(dd,J＝13.2,6.6Hz,4H),3.83(dd,J＝15.2,7.6Hz,1H),3.66(s,3H),3.64 (s,3H),3.55-3.50(m,2H),3.50-3.44(m,1H),3.24(dd,J＝13.2,10.0Hz,2H),3.14(dd,J＝12.0,8.8Hz,1H),2.74(dd,J＝16 .4,4.0Hz,1H),2.03-1.90(m,2H),1.79(dd,J＝12.4,7.6Hz,5H),1.61-1.50(m,2H),0.68-0.61(m,1H),0.61-0.48(m,3H).

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十二：(S)-8-((5-(((S)-2-(5-(羟甲基)噻吩-3-基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺环[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-38)的制备
Preparation Example 22: Preparation of (S)-8-((5-(((S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spirocyclic[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-5-one (7-38)

步骤一:(S)-8-((5-溴戊基)氧基)-2-(5-(羟甲基)噻吩-3-基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-38-1)的制备Step 1: Preparation of (S)-8-((5-bromopentyl)oxy)-2-(5-(hydroxymethyl)thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-1)

将(5-(羟甲基)噻吩-3-基)硼酸(113.38mg,717.70μmol),(S)-8-((5-溴戊基)氧基)-7-甲氧基-5,11-二氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基三氟甲磺酸酯(200mg,358.85μmol)，四(三苯基膦)钯(41.47mg,35.89μmol)，碳酸钠(152.14mg,1.44mmol)混合一起，加入水(1mL)，甲苯(4mL)以及乙醇(1mL)，然后抽换氮气三次，升温80℃加热反应2小时。反应完成后，向反应液中加水10mL，乙酸乙酯5mL X 3萃取三次，有机相饱和食盐水10mL洗涤，无水硫酸钠干燥后减压浓缩。粗品经硅胶柱色谱纯化(乙酸乙酯/石油醚＝0％～100％)后再次减压浓缩得标题化合物(30mg,50.06μmol)。(5-(hydroxymethyl)thiophen-3-yl)boronic acid (113.38 mg, 717.70 μmol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (200 mg, 358.85 μmol), tetra(triphenylphosphine)palladium (41.47 mg, 35.89 μmol), and sodium carbonate (152.14 mg, 1.44 mmol) were mixed together, and water (1 mL), toluene (4 mL), and ethanol (1 mL) were added. The mixture was then purged with nitrogen three times and heated to 80 °C for 2 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, and the mixture was extracted three times with 5 mL of ethyl acetate (3 times). The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0%–100%) and then concentrated again under reduced pressure to obtain the title compound (30 mg, 50.06 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):521.1[M+H]+ ESI-MS (m/z): 521.1 [M+H] +

步骤二:(S)-2-(5-(羟甲基)噻吩-3-基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-38-2)的制备Step 2: Preparation of (S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-2)

将(S)-8-((5-溴戊基)氧基)-2-(5-(羟甲基)噻吩-3-基)-7-甲氧基-1,11a-二氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5,11(10H)-二酮(30mg,57.53μmol)和(S)-8-羟基-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(15.78mg,57.53μmol)溶于DMF(1mL)中，加入碳酸钾(23.85mg,172.60μmol)，室温搅拌40小时。反应完成后，反应液过滤除去不溶物，滤液经制备高效液相色谱纯化后冷冻干燥得标题化合物(10mg,13.99μmol)。(S)-8-((5-bromopentyl)oxy)-2-(5-(hydroxymethyl)thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (30 mg, 57.53 μmol) and (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (15.78 mg, 57.53 μmol) were dissolved in DMF (1 mL), potassium carbonate (23.85 mg, 172.60 μmol) was added, and the mixture was stirred at room temperature for 40 hours. After the reaction was completed, the reaction solution was filtered to remove insoluble matter, and the filtrate was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (10 mg, 13.99 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):715.3[M+H]+ ESI-MS (m/z): 715.3 [M+H] +

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤三:(S)-8-((5-(((S)-2-(5-(羟甲基)噻吩-3-基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺环[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(7-38)的制备Step 3: Preparation of (S)-8-((5-(((S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spirocyclic[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-38)

将(S)-2-(5-(羟甲基)噻吩-3-基)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a)-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙烷]-8-基)氧基)戊基)氧基)-1,11a-二氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(10mg,13.99μmol)溶于THF(2mL)中，加入硼氢化钠(5.15mg,139.89μmol)，有少许气泡产生。滴入TFA(9.57mg,83.94μmol)。升温至70℃反应24小时。反应完成后，反应液减压浓缩，残余物甲醇溶解后经制备高效液相色谱纯化后冷冻干燥得标题化合物(2.18mg,2.96μmol)。(S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a)-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (10 mg, 13.99 μmol) was dissolved in THF (2 mL), and sodium borohydride (5.15 mg, 139.89 μmol) was added, producing a few bubbles. TFA (9.57 mg, 83.94 μmol) was added dropwise. The temperature was raised to 70℃ and the reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in methanol, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (2.18 mg, 2.96 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):701.3[M+H]+ ESI-MS (m/z): 701.3 [M+H] +

1H NMR(400MHz,DMSO-d6)δ7.35(s,1H),7.32(s,1H),7.28(s,1H),7.24(s,1H),7.15(d,J＝1.2Hz,1H),6.55(d,J＝6.4Hz,1H),6.30(d,J＝10.8Hz,3H),5.48(s,1H),4.60(d,J＝3.6Hz,2H),4.17(dd,J＝15.2,10.0Hz,1H),3.93(dd,J＝12.8,6.4Hz,4H),3.83(dd,J＝14.8,7.2Hz,1H),3.65(d,J＝8.4Hz,6H),3.55-3.46(m,4H),3.29-3.21(m,2H),3.21-3.09(m,2H),2.71(dd,J＝16.8,4.0Hz,1H),2.03-1.91(m,2H),1.81-1.75(m,,5H),1.59-1.51(m,2H),0.68-0.61(m,1H),0.60-0.48(m,3H). 1 H NMR (400MHz, DMSO-d 6 )δ7.35(s,1H),7.32(s,1H),7.28(s,1H),7.24(s,1H),7.15(d,J＝1.2Hz,1H),6.55(d,J＝6.4Hz,1H),6.30(d,J＝10 .8Hz,3H),5.48(s,1H),4.60(d,J＝3.6Hz,2H),4.17(dd,J＝15.2,10.0Hz,1H),3.93(dd,J＝12.8,6.4Hz,4H),3.83(d d,J＝14.8,7.2Hz,1H),3.65(d,J＝8.4Hz,6H),3.55-3.46(m,4H),3.29-3.21(m,2H),3.21-3.09(m,2H),2.71(dd,J＝ 16.8,4.0Hz,1H),2.03-1.91(m,2H),1.81-1.75(m,,5H),1.59-1.51(m,2H),0.68-0.61(m,1H),0.60-0.48(m,3H).

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十三：(S)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-39)的制备
Preparation Example 23: Preparation of (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-39)

步骤一:烯丙基(S)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮卓-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸酯(7-39-1)的制备Step 1: Preparation of allyl (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid ester (7-39-1)

将(S)-2,2-二氟-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(19mg,66.84μmol)和(S)-8-((5-溴戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(39.13mg,66.84μmol)溶于DMF(0.5mL)中，加入碳酸钾(27.71mg,200.52μmol)，室温搅拌24小时。加入碘化钾(11.10mg,66.84μmol)，继续室温搅拌48小时。反应完成后，反应液加水5mL，乙酸乙酯5mL X 3萃取，有机相饱和食盐水5mL洗涤，无水硫酸钠干燥后减压浓缩得标题化合物的粗品(70mg,64.51μmol)，未经纯化直接用于下一步反应。(S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (19 mg, 66.84 μmol) and (S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (39.13 mg, 66.84 μmol) were dissolved in DMF (0.5 mL), and potassium carbonate (27.71 mg, 200.52 μmol) was added. The mixture was stirred at room temperature for 24 hours. Potassium iodide (11.10 mg, 66.84 μmol) was added, and the mixture was stirred at room temperature for 48 hours. After the reaction was complete, 5 mL of water was added to the reaction solution, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic phase was washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (70 mg, 64.51 μmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):790.4[M+H]+ ESI-MS (m/z): 790.4 [M+H] +

步骤二：(S)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-39)的制备 Step 2: Preparation of (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-39)

将(S)-8-((5-(((S)-2,2-二氟-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(70mg,88.74μmol)溶于DMF(1mL)中，加入四氢吡咯(12.62mg,177.48μmol)和四三苯基磷钯(10.25mg,8.87μmol)，抽换氮气三次，在氮气保护氛围下室温搅拌反应3小时。反应完成后，反应液过滤，滤液经制备高效液相色谱纯化后冷冻干燥得标题化合物(9.01mg,12.66μmol)。(S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (70 mg, 88.74 μmol) was dissolved in DMF (1 mL), and tetrahydropyrrole (12.62 mg, 177.48 μmol) and tetra-triphenylphosphine palladium (10.25 mg, 8.87 μmol) were added. The mixture was purged with nitrogen three times and stirred at room temperature for 3 hours under a nitrogen protective atmosphere. After the reaction was complete, the reaction solution was filtered, and the filtrate was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (9.01 mg, 12.66 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):705.3[M+H]+ ESI-MS (m/z): 705.3 [M+H] +

1H NMR(400MHz,DMSO-d6)δ7.45(s,1H),7.39(s,1H),7.37(s,1H),7.34(s,1H),7.29(s,1H),6.91(d,J＝8.8Hz,2H),6.56(d,J＝6.0Hz,1H),6.49(d,J＝6.0Hz,1H),6.31(d,J＝4.8Hz,1H),4.22-4.16(m,1H),3.98-3.86(m,7H),3.75(s,3H),3.66(s,3H),3.64(s,3H),3.57-3.52(m,2H),3.31-3.14(m,3H),2.78-2.68(m,2H),2.43-2.29(m,1H),1.82-1.77(m,4H),1.59 -1.52(m,2H). 1 H NMR (400MHz, DMSO-d 6 )δ7.45(s,1H),7.39(s,1H),7.37(s,1H),7.34(s,1H),7.29(s,1H),6.91(d,J＝8.8H z,2H),6.56(d,J＝6.0Hz,1H),6.49(d,J＝6.0Hz,1H),6.31(d,J＝4.8Hz,1H),4.22-4.1 6(m,1H),3.98-3.86(m,7H),3.75(s,3H),3.66(s,3H),3.64(s,3H),3.57-3.52(m,2 H),3.31-3.14(m,3H),2.78-2.68(m,2H),2.43-2.29(m,1H),1.82-1.77(m,4H),1.59 -1.52(m,2H).

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十四：(S)-8-((5-(((2R,11aS)-2-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-40)的制备
Preparation Example 24: Preparation of (S)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40)

步骤一:(S)-8-((5-(((2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(7-40-1)的制备Step 1: Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-40-1)

将(S)-8-((5-溴戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(45mg,76.86μmol)，(2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-8-羟基-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(34.91mg,92.23μmol)溶于DMF(1mL)，加入碳酸钾(21.24mg,153.72μmol)，搅拌反应16小时。加水析出固体，过滤，固体水洗后干燥得粗品(65mg,73.60μmol)，未经纯化直接用于下一步。(S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (45 mg, 76.86 μmol) and (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (34.91 mg, 92.23 μmol) were dissolved in DMF (1 mL), and potassium carbonate (21.24 mg, 153.72 μmol) was added. The mixture was stirred and reacted for 16 hours. Water was added to precipitate the solid, which was then filtered, washed with water, and dried to obtain the crude product (65 mg, 73.60 μmol), which was used directly in the next step without purification.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):883.1[M+H]+ MS m/z(ESI): 883.1 [M+H] +

步骤二:(S)-8-((5-(((2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-40-2)的制备Step 2: Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40-2)

将(S)-8-((5-(((2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(65mg,73.60μmol)溶于DMF(1mL)，加入四(三苯基膦)钯(8.51mg,7.36μmol)和四氢吡 咯(5.23mg,73.60μmol)，氮气置换并保护下搅拌反应12小时。加入乙酸乙酯和水，搅拌，过滤，静置分液，有机相用饱和食盐水洗涤3次后干燥，减压浓缩得粗品(58mg,73.60μmol)，未经纯化直接用于下一步。(S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (65 mg, 73.60 μmol) was dissolved in DMF (1 mL), and tetra(triphenylphosphine)palladium (8.51 mg, 7.36 μmol) and tetrahydropyrrolo[5H]carboxylic acid were added. The reaction was carried out under nitrogen purging and protection with 5.23 mg (73.60 μmol) and stirred for 12 hours. Ethyl acetate and water were added, the mixture was stirred, filtered, allowed to stand, and separated. The organic phase was washed three times with saturated brine, dried, and concentrated under reduced pressure to obtain the crude product (58 mg, 73.60 μmol), which was used directly in the next step without purification.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):800.5[M+H]+ MS m/z(ESI): 800.5 [M+H] +

步骤三:(S)-8-((5-(((2R,11aS)-2-羟基-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-40)的制备Step 3: Preparation of (S)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40)

将(S)-8-((5-(((2R,11aS)-2-((叔丁基二甲基甲硅烷基)氧基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(58mg,73.60μmol)溶于四氢呋喃(1mL)，滴入四丁基氟化铵溶液(1M,145.17μL)，搅拌反应1小时。加入乙酸乙酯稀释，饱和氯化铵水溶液洗涤2次，饱和食盐水洗涤2次，减压浓缩。经制备高效液相色谱纯化后冷冻干燥得到标题化合物((17.11mg,24.24μmol)。(S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (58 mg, 73.60 μmol) was dissolved in tetrahydrofuran (1 mL), and tetrabutylammonium fluoride solution (1 M, 145.17 μL) was added dropwise. The mixture was stirred for 1 hour. The solution was diluted with ethyl acetate, washed twice with saturated ammonium chloride aqueous solution, and twice with saturated brine. The solution was then concentrated under reduced pressure. The title compound (17.11 mg, 24.24 μmol) was obtained by preparative high performance liquid chromatography purification followed by freeze drying.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):685.3[M+H]+ MS m/z(ESI): 685.3 [M+H] +

1H NMR(400MHz,DMSO)δ7.45(s,1H),7.42-7.36(m,3H),7.29(s,1H),6.91(d,J＝8.9Hz,2H),6.55(d,J＝5.7Hz,1H),6.36(d,J＝5.9Hz,1H),6.31(s,1H),6.26(s,1H),4.92(d,J＝3.2Hz,1H),4.19(s,2H),3.93(dd,J＝13.7,6.7Hz,4H),3.80(d,J＝9.1Hz,1H),3.76(s,3H),3.65(d,J＝9.5Hz,6H),3.51(dt,J＝12.1,9.0Hz,4H),3.28-3.19(m,2H),2.99-2.93(m,1H),2.79-2.74(m,1H),2.08(dd,J＝13.0,6.7Hz,1H),1.80(s,4H),1.73-1.67(m,1H),1.56(d,J＝6.4Hz,2H). 1 H NMR (400MHz, DMSO) δ7.45 (s, 1H), 7.42-7.36 (m, 3H), 7.29 (s, 1H), 6.91 (d, J = 8.9Hz, 2H), 6.55 (d, J = 5.7Hz, 1H), 6. 36(d,J＝5.9Hz,1H),6.31(s,1H),6.26(s,1H),4.92(d,J＝3.2Hz,1H),4.19(s,2H),3.93(dd,J＝13.7,6.7Hz,4H),3. 80(d,J＝9.1Hz,1H),3.76(s,3H),3.65(d,J＝9.5Hz,6H),3.51(dt,J＝12.1,9.0Hz,4H),3.28-3.19(m,2H),2.99-2. 93(m,1H),2.79-2.74(m,1H),2.08(dd,J＝13.0,6.7Hz,1H),1.80(s,4H),1.73-1.67(m,1H),1.56(d,J＝6.4Hz,2H).

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十五：(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-2-亚甲基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-41)的制备
Preparation Example 25: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41)

步骤一:(S)-4-亚甲基吡咯烷-2-甲酸甲酯(7-41-2)的制备Step 1: Preparation of (S)-4-methylenepyrrolidine-2-carboxylate (7-41-2)

将(S)-1-(叔丁氧基羰基)-4-亚甲基吡咯烷-2-羧酸(941.87mg,4.14mmol)溶于MeOH(7mL)，滴入HCl/dioxane(4M,7mL)，室温搅拌反应24小时。反应完成后，反应液直接减压浓缩得标题化合物的盐酸盐粗品(794mg,4.02mmol)，未经纯化直接用于下一步反应。(S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid (941.87 mg, 4.14 mmol) was dissolved in MeOH (7 mL), and HCl/dioxane (4 M, 7 mL) was added dropwise. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the reaction solution was directly concentrated under reduced pressure to obtain crude hydrochloride of the title compound (794 mg, 4.02 mmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):142.2[M+H]+ ESI-MS (m/z): 142.2 [M+H] +

步骤二:(S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-亚甲基吡咯烷-2-甲酸甲酯(7-41- 3)的制备Step 2: (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylic acid methyl ester (7-41- 3) Preparation

将4-(苄氧基)-5-甲氧基-2-硝基苯甲酸(1.35g,4.45mmol)溶于DMF(15mL)中加入HATU(2.03g,5.34mmol)和DIPEA(1.72g,13.34mmol,2.32mL)，室温搅拌10分钟。然后加入(S)-4-亚甲基吡咯烷-2-甲酸甲酯的盐酸盐(790mg,4.45mmol)和DIPEA(574.80mg,4.45mmol)的DMF(15mL)混合溶液。加完后，室温搅拌反应2小时。反应完成后，反应液加水100mL，乙酸乙酯50mL X 3萃取，有机相食盐水洗涤，无水硫酸钠干燥后减压浓缩。粗品经硅胶柱色谱纯化(乙酸乙酯/石油醚＝0～60％)后再次减压浓缩得标题化合物(1.53g,3.58mmol)。4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.35 g, 4.45 mmol) was dissolved in DMF (15 mL), and HATU (2.03 g, 5.34 mmol) and DIPEA (1.72 g, 13.34 mmol, 2.32 mL) were added. The mixture was stirred at room temperature for 10 minutes. Then, a mixture of (S)-4-methylenepyrrolidine-2-carboxylate hydrochloride (790 mg, 4.45 mmol) and DIPEA (574.80 mg, 4.45 mmol) in DMF (15 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, 100 mL of water was added to the reaction mixture, and the mixture was extracted with 50 mL of ethyl acetate three times. The organic phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0–60%) and concentrated again under reduced pressure to give the title compound (1.53 g, 3.58 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):427.4[M+H]+ ESI-MS (m/z): 427.4 [M+H] +

步骤三:(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-41-4)的制备Step 3: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-41-4)

将(S)-1-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-4-亚甲基吡咯烷-2-甲酸甲酯(1.53g,3.59mmol)溶于饱和氯化铵水溶液(8mL)和甲醇(80mL)中，加入锌粉(2.35g,35.88mmol)，升温至75℃搅拌反应4小时。反应完成后，反应液冷却至室温过滤，滤液减压浓缩，残余物然后加水50ml，乙酸乙酯:甲醇＝4：1的混合溶剂50mL X 3萃取，有机相饱和食盐水洗涤，无水硫酸钠干燥后减压浓缩得标题化合物的粗品(1.3g,3.57mmol)，未经纯化直接用于下一步反应。Methyl (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-methylenepyrrolidine-2-carboxylate (1.53 g, 3.59 mmol) was dissolved in saturated ammonium chloride aqueous solution (8 mL) and methanol (80 mL). Zinc powder (2.35 g, 35.88 mmol) was added, and the mixture was heated to 75 °C and stirred for 4 hours. After the reaction was complete, the reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the residue was then extracted with 50 mL of water and 3 times of a mixed solvent of ethyl acetate:methanol (4:1). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (1.3 g, 3.57 mmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):365.2[M+H]+ ESI-MS (m/z): 365.2 [M+H] +

步骤四:(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-10-((2-(三甲基甲硅烷基)乙氧基)甲基)-1,2,3,11a-四氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-41-5)的制备Step 4: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5,11(10H)-dione (7-41-5)

将(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5,11(10H)-二酮(0.2g,548.86μmol)溶于THF(3mL)中，氮气保护下降温至-40℃，然后逐滴加入n-BuLi(2.5M,230.52μL)。加完后，保温反应30分钟。然后-40℃滴加2-(三甲基硅烷基)乙氧甲基氯(100.66mg,603.74μmol)，加毕，自然升温反应过夜。反应完成后，反应液加氯化铵水溶液5mL淬灭，然后加水10mL和乙酸乙酯10mL X 3萃取，有机相饱和食盐水洗涤后用无水硫酸钠干燥，然后减压浓缩得标题化合物的粗品(286mg, 520.36μmol)，未经纯化直接用于下一步反应。(S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-5,11(10H)-dione (0.2 g, 548.86 μmol) was dissolved in THF (3 mL), and the mixture was cooled to -40 °C under nitrogen protection. Then, n-BuLi (2.5 M, 230.52 μL) was added dropwise. After the addition was complete, the reaction was maintained at this temperature for 30 minutes. Then, 2-(trimethylsilyl)ethoxymethyl chloride (100.66 mg, 603.74 μmol) was added dropwise at -40 °C. After the addition was complete, the mixture was allowed to rise naturally overnight. After the reaction was complete, the reaction solution was quenched with 5 mL of ammonium chloride aqueous solution, then extracted with 10 mL of water and 10 mL of ethyl acetate (3 times). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, then concentrated under reduced pressure to obtain the crude product of the title compound (286 mg). 520.36 μmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):496.3[M+H]+ ESI-MS (m/z): 496.3 [M+H] +

步骤五:(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-41-6)的制备Step 5: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41-6)

将(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5-酮(180mg,363.89μmol)溶于THF(10mL)中，氮气保护下降温至-78℃。然后滴加三乙基硼氢化锂(1M,727.78μL)，加毕保温反应2小时。补加三乙基硼氢化锂(1M,727.78μL)三次，每次间隔1小时。反应完成后，反应液加入水15mL中，乙酸乙酯萃取三次10mL X 3,有机相饱和食盐水洗涤，无水硫酸钠干燥后减压浓缩得标题化合物的粗品(200mg,344.44μmol)，未经纯化直接用于下一步反应。(S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-5-one (180 mg, 363.89 μmol) was dissolved in THF (10 mL) and cooled to -78 °C under nitrogen protection. Then, triethyllithium borohydride (1 M, 727.78 μL) was added dropwise, and the reaction was maintained at this temperature for 2 hours. Triethyllithium borohydride (1 M, 727.78 μL) was added three times, each time at 1-hour intervals. After the reaction was complete, the reaction solution was added to 15 mL of water, extracted three times with ethyl acetate (10 mL x 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (200 mg, 344.44 μmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):367.3[M+H+H2O]+；697.4[2M+H]+ ESI-MS(m/z):367.3[M+H+H 2 O] + ; 697.4[2M+H] +

步骤六:(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-41-7)的制备Step Six: Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41-7)

将(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-5-酮(80mg,229.62μmol)溶于二氯甲烷(1mL)和甲醇(1mL)中，加入三乙酰氧基硼氢化钠(97.33mg,459.25μmol)，室温搅拌过夜16小时。补加NaBH4(17.37mg,459.25μmol)，反应2小时后再次补加NaBH4(17.37mg,459.25μmol)反应2小时。反应完成后，将反应液减压浓缩，甲醇溶解后反相柱色谱纯化(乙腈/0.05％甲酸水溶液＝0～50％)后冷冻干燥得标题化合物(15mg,42.81μmol)。(S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-5-one (80 mg, 229.62 μmol) was dissolved in dichloromethane (1 mL) and methanol (1 mL), and sodium triacetoxyborohydride (97.33 mg, 459.25 μmol) was added. The mixture was stirred overnight at room temperature for 16 hours. NaBH4 (17.37 mg, 459.25 μmol) was added, and after reacting for 2 hours, NaBH4 (17.37 mg, 459.25 μmol) was added again, and the reaction was continued for another 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, dissolved in methanol, purified by reversed-phase column chromatography (acetonitrile/0.05% formic acid aqueous solution = 0–50%), and then freeze-dried to obtain the title compound (15 mg, 42.81 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):351.3[M+H]+ ESI-MS (m/z): 351.3 [M+H] +

步骤七:(S)-8-羟基-7-甲氧基-2-亚甲基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-41-8)的制备Step 7: Preparation of (S)-8-hydroxy-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41-8)

将(S)-8-(苄氧基)-7-甲氧基-2-亚甲基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4] 二氮杂-5-酮(15mg,42.81μmol)中加入二氯甲烷(1mL)，然后滴加甲磺酸(0.1mL)，室温搅拌反应1小时。反应完成后，反应液加水2mL，饱和碳酸氢钠水溶液调节pH＝8～9，二氯甲烷3mL X 3萃取，有机相饱和食盐水3mL洗涤，无水硫酸钠干燥后减压浓缩得标题化合物的粗品(11mg,42.26μmol)，未经纯化直接用于下一步反应。(S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4] Dichloromethane (1 mL) was added to diaza-5-one (15 mg, 42.81 μmol), followed by the dropwise addition of methanesulfonic acid (0.1 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 2 mL of water was added to the reaction solution, the pH was adjusted to 8–9 with saturated sodium bicarbonate solution, and the mixture was extracted with 3 mL of dichloromethane three times. The organic phase was washed with 3 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (11 mg, 42.26 μmol), which was used directly in the next reaction without purification.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):261.1[M+H]+ ESI-MS (m/z): 261.1 [M+H] +

步骤八:(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-2-亚甲基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并)[e]吡咯并[1,2-a][1,4]二氮卓-8-基)氧基)戊基)氧基)-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(7-41-9)的制备Step 8: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo)[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-41-9)

将(S)-8-((5-溴戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(24.74mg,42.26μmol)和(S)-8-羟基-7-甲氧基-2-亚甲基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(11mg,42.26μmol)溶于DMF(1mL)中加入碳酸钾(17.52mg,126.78μmol)，室温搅拌反应16小时。反应完成后，反应液直接经制备高效液相色谱纯化后冷冻干燥得标题化合物(5mg,6.54μmol)。(S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (24.74 mg, 42.26 μmol) and (S)-8-hydroxy-7-methoxy-2-methylene-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (11 mg, 42.26 μmol) were dissolved in DMF (1 mL) and potassium carbonate (17.52 mg, 126.78 μmol) was added. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (5 mg, 6.54 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):766.3[M+H]+ ESI-MS (m/z): 766.3 [M+H] +

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤九:(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-2-亚甲基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-2-(4-甲氧基苯基)-1,10,11,11a-四氢- 5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-41)的制备Step Nine: (S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro- Preparation of 5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-41)

将(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-2-亚甲基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并)[e]吡咯并[1,2-a][1,4]二氮卓-8-基)氧基)戊基)氧基)-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(5mg,6.54μmol)溶于DMF(0.5mL)中，加入四氢吡咯(464.92μg,6.54μmol)和四三苯基膦钯(755.41μg,6.54e-1μmol)，置换氮气三次，然后再氮气保护下室温搅拌反应2小时。反应完成后，反应液直接经制备高效液相色谱纯化后冷冻干燥得标题化合物(1.89mg,2.47μmol)。(S)-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo)[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (5 mg, 6.54 μmol) was dissolved in DMF (0.5 mL), and tetrahydropyrrole (464.92 μg, 6.54 μmol) and tetratriphenylphosphine palladium (755.41 μg, 6.54e-1 μmol) were added. Nitrogen gas was purged three times, and then the reaction was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (1.89 mg, 2.47 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):681.4[M+H]+ ESI-MS (m/z): 681.4 [M+H] +

其制备方法如下：Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: Waters SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十六：(1aR,9aS,10aR)-5-甲氧基-6-((5-(((S)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1a,8,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-3(1H)-酮(7-43)的制备
Preparation Example 26: Preparation of (1aR,9aS,10aR)-5-methoxy-6-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1a,8,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3(1H)-one (7-43)

步骤一:(1R,3S,5R)-2-氮杂双环[3.1.0]己烷-3-甲酸甲酯盐酸盐(7-43-2)的制备Step 1: Preparation of (1R,3S,5R)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid methyl ester hydrochloride (7-43-2)

将(1R,3S,5R)-2-氮杂双环[3.1.0]己烷-3-甲酸(2.00g,8.80mmol)溶于甲醇(10mL)，滴入4M盐酸二氧六环溶液(10mL)后搅拌反应4小时。减压浓缩后直接进行下一步反应。(1R,3S,5R)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid (2.00 g, 8.80 mmol) was dissolved in methanol (10 mL), and 4 M dioxane hydrochloride solution (10 mL) was added dropwise, followed by stirring for 4 hours. After concentration under reduced pressure, the reaction proceeded directly to the next step.

步骤二:(1R,3S,5R)-2-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-2-氮杂双环[3.1.0]己烷-3-甲酸甲酯(7-43-3)的制备Step 2: Preparation of (1R,3S,5R)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid methyl ester (7-43-3)

将4-苄氧基-5-甲氧基-2-硝基-苯甲酸(1.18g,3.89mmol)，(1R,3S,5R)-2-氮杂双环[3.1.0]己烷-3-甲酸甲酯盐酸盐(760.26mg,4.28mmol)，HATU(2.22g,5.84mmol)溶于DMF(20mL)，滴入DIPEA(1.51g,11.67mmol)，搅拌反应2小时。加水和EA搅拌，分液，水相用EA萃取2次，合并有机相，饱和食盐水洗涤2次，干燥，浓缩。硅胶柱纯化(洗脱剂30-65％乙酸乙酯/石油醚)后再次浓缩得标题化合物(1.64g,3.85mmol)。4-Benzyloxy-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.89 mmol), (1R,3S,5R)-2-azabicyclo[3.1.0]hexane-3-carboxylic acid methyl ester hydrochloride (760.26 mg, 4.28 mmol), and HATU (2.22 g, 5.84 mmol) were dissolved in DMF (20 mL), and DIPEA (1.51 g, 11.67 mmol) was added dropwise. The mixture was stirred for 2 hours. Water and EA were added and stirred. The mixture was separated, and the aqueous phase was extracted twice with EA. The organic phases were combined, washed twice with saturated brine, dried, and concentrated. After purification by silica gel column chromatography (eluent 30-65% ethyl acetate/petroleum ether), the mixture was concentrated again to give the title compound (1.64 g, 3.85 mmol).

步骤三:(1aR,9aS,10aR)-6-(苄氧基)-5-甲氧基-1a,9a,10,10a-四氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-3,9(1H,8H)-二酮(7-43-4)的制备Step 3: Preparation of (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (7-43-4)

将(1R,3S,5R)-2-(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-2-氮杂双环[3.1.0]己烷-3-甲酸甲酯(870mg,2.04mmol)溶于甲醇(10mL)，加入饱和氯化铵水溶液(2mL)，再分批加入锌粉(1.33g,20.40mmol)，升温至80℃回流反应16小时。冷却至室温，过滤，滤液减压浓缩后硅胶柱纯化(洗脱剂：5％甲醇/二氯甲烷)后再次浓缩得标题化合物(682mg,1.87mmol)。 Methyl (1R,3S,5R)-2-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-2-azabicyclo[3.1.0]hexane-3-carboxylate (870 mg, 2.04 mmol) was dissolved in methanol (10 mL), and saturated ammonium chloride aqueous solution (2 mL) was added. Zinc powder (1.33 g, 20.40 mmol) was then added in portions, and the mixture was refluxed at 80 °C for 16 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 5% methanol/dichloromethane). The filtrate was then concentrated again to give the title compound (682 mg, 1.87 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):365.1[M+H]+ MS m/z(ESI): 365.1 [M+H] +

步骤四:(1aR,9aS,10aR)-6-(苄氧基)-5-甲氧基-1a,9a,10,10a-四氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-3,9(1H,8H)-二酮(7-43-5)的制备Step 4: Preparation of (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (7-43-5)

将(1aR,9aS,10aR)-6-(苄氧基)-5-甲氧基-1a,9a,10,10a-四氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-3,9(1H,8H)-二酮(180mg,493.97μmol)溶于干燥THF(10mL)，搅拌下加入硼氢化钠(56.06mg,1.48mmol)，再滴入三氟乙酸(112.65mg,987.94μmol,75.65μL)的四氢呋喃溶液(100μL)，大量气泡产生。升温至75℃搅拌反应4小时。减压浓缩，加水和乙酸乙酯搅拌，分液，有机相减压浓缩，硅胶柱纯化(洗脱剂：5％甲醇/二氯甲烷)后再次浓缩得标题化合物(142mg,405.24μmol)。(1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (180 mg, 493.97 μmol) was dissolved in dry THF (10 mL). Sodium borohydride (56.06 mg, 1.48 mmol) was added with stirring, followed by a 100 μL solution of trifluoroacetic acid (112.65 mg, 987.94 μmol, 75.65 μL) in tetrahydrofuran. A large number of bubbles were generated. The mixture was heated to 75 °C and stirred for 4 hours. The mixture was concentrated under reduced pressure, then water and ethyl acetate were added and stirred. The mixture was separated, and the organic phase was concentrated under reduced pressure. After purification by silica gel column chromatography (eluent: 5% methanol/dichloromethane), the mixture was concentrated again to give the title compound (142 mg, 405.24 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):367.3[M+H2O]+ MS m/z (ESI): 367.3 [M+H2O] +

步骤五:(1aR,9aS,10aR)-6-(苄氧基)-5-甲氧基-3-氧代-1,1a,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-8(3H)-羧酸烯丙酯(7-43-6)的制备Step 5: Preparation of (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropane[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (7-43-6)

将(1aR,9aS,10aR)-6-(苄氧基)-5-甲氧基-1a,9a,10,10a-四氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-3,9(1H,8H)-二酮(142mg,405.24μmol)溶于干燥二氯甲烷(5mL)，滴入吡啶(48.08mg,607.86μmol)，冷却搅拌至0℃，滴入氯酸烯丙酯(195.38mg,1.62mmol,172.29μL)，恢复至室温搅拌反应1小时。加二氯甲烷稀释，加10％柠檬酸水搅拌，静置分液，有机相水洗后干燥浓缩。硅胶柱纯化(洗脱剂：80％乙酸乙酯/石油醚)后再次浓缩得标题化合物(153mg,352.14μmol)。(1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-1a,9a,10,10a-tetrahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3,9(1H,8H)-dione (142 mg, 405.24 μmol) was dissolved in dry dichloromethane (5 mL), and pyridine (48.08 mg, 607.86 μmol) was added dropwise. The mixture was cooled and stirred to 0 °C, and allyl chlorate (195.38 mg, 1.62 mmol, 172.29 μL) was added dropwise. The mixture was then brought back to room temperature and stirred for 1 hour. The solution was diluted with dichloromethane, and 10% citric acid solution was added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water and then dried and concentrated. After silica gel column purification (eluent: 80% ethyl acetate/petroleum ether), the compound was concentrated again to give the title compound (153 mg, 352.14 μmol).

步骤六:(1aR,9aS,10aR)-6-羟基-5-甲氧基-3-氧代-1,1a,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-8(3H)-羧酸烯丙酯(7-43-7)的制备Step Six: Preparation of (1aR,9aS,10aR)-6-hydroxy-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropane[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (7-43-7)

将(1aR,9aS,10aR)-6-(苄氧基)-5-甲氧基-3-氧代-1,1a,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-8(3H)-羧酸烯丙酯(130mg,299.21μmol)溶于二氯甲烷(1mL)，搅拌下滴入甲磺酸(0.5mL)，继续反应1小时。加入二氯甲烷稀释，加入碳酸氢钠水溶液搅拌，静置分液，有机相干燥浓缩。制备高效液相色谱纯化，冷冻干燥得到标题化合物(57mg,165.52μmol)。 (1aR,9aS,10aR)-6-(benzyloxy)-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (130 mg, 299.21 μmol) was dissolved in dichloromethane (1 mL), and methanesulfonic acid (0.5 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After dilution with dichloromethane, sodium bicarbonate aqueous solution was added and stirred. The mixture was allowed to stand and separated, and the organic phase was dried and concentrated. The solution was purified by preparative high-performance liquid chromatography and freeze-dried to give the title compound (57 mg, 165.52 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):345.1[M+H]+ MS m/z(ESI): 345.1 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤七:(1aR,9aS,10aR)-6-((5-(((S)-10-((烯丙氧基)羰基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-5-甲氧基-3-氧代-1,1a,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-8(3H)-羧酸烯丙酯(7-43-8)的制备Step 7: Preparation of (1aR,9aS,10aR)-6-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (7-43-8)

将(S)-8-((5-溴戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(10.20mg,17.42μmol)，(1aR,9aS,10aR)-6-羟基-5-甲氧基-3-氧代-1,1a,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-8(3H)-羧酸烯丙酯(5mg,14.52μmol)，碳酸钾(3.01mg,21.78μmol)溶于DMF(1mL)，搅拌反应16小时。加水析出固体，过滤，固体水洗后真空干燥得标题化合物(12mg,14.14μmol)。(S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (10.20 mg, 17.42 μmol), (1aR,9aS,10aR)-6-hydroxy-5-methoxy-3-oxo-1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (5 mg, 14.52 μmol), and potassium carbonate (3.01 mg, 21.78 μmol) were dissolved in DMF (1 mL), and the reaction was stirred for 16 hours. Water was added to precipitate the solid, which was then filtered, washed with water, and dried under vacuum to obtain the title compound (12 mg, 14.14 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):849.4[M+H]+ MS m/z(ESI): 849.4 [M+H] +

步骤八:(1aR,9aS,10aR)-5-甲氧基-6-((5-(((S)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-1a,8,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-3(1H)-酮(7-43)的制备Step 8: Preparation of (1aR,9aS,10aR)-5-methoxy-6-((5-(((S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1a,8,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-3(1H)-one (7-43)

将(1aR,9aS,10aR)-6-((5-(((S)-10-((烯丙氧基)羰基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-5-甲氧基-3-氧 代-1,1a,9,9a,10,10a-六氢苯并[e]环丙[4,5]吡咯并[1,2-a][1,4]二氮杂-8(3H)-羧酸烯丙酯(12mg,14.14μmol)溶于DMF(1mL)，滴入四氢吡咯(1.01mg,14.14μmol)，加入四(三苯基膦)钯(1.63mg,1.41μmol)，氮气置换并保护下反应16小时。经制备高效液相色谱纯化，冷冻干燥得到标题化合物(6.18mg,8.99μmol)。The (1aR,9aS,10aR)-6-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-5-methoxy-3-oxo 1,1a,9,9a,10,10a-hexahydrobenzo[e]cyclopropyl[4,5]pyrrolo[1,2-a][1,4]diaza-8(3H)-carboxylic acid allyl ester (12 mg, 14.14 μmol) was dissolved in DMF (1 mL), tetrahydropyrrole (1.01 mg, 14.14 μmol) was added dropwise, followed by tetra(triphenylphosphine)palladium (1.63 mg, 1.41 μmol). The reaction was carried out under nitrogen purging and protection for 16 hours. After purification by preparative high-performance liquid chromatography, the compound was lyophilized to give the title compound (6.18 mg, 8.99 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):681.3[M+H]+ MS m/z(ESI): 681.3 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十七：(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮(7-44)的制备
Preparation Example 27: Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-44)

步骤一:(S)-8-(苄氧基)-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(7-44-1)的制备Step 1: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-44-1)

将(4-(苄氧基)-5-甲氧基-2-硝基苯甲酰基)-L-脯氨酸甲酯(870mg,2.04mmol)溶于甲醇(100mL)，加入饱和氯化铵水溶液(10mL)，再分批加入锌粉(2.84g,43.44mmol)，升温至80℃回流反应16小时。冷却至室温，过滤，滤液减压浓缩后硅胶柱纯化(洗脱剂：5％甲醇/二氯甲烷)后再次浓缩得标题化合物(1.15g,3.26mmol)。(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-L-proline methyl ester (870 mg, 2.04 mmol) was dissolved in methanol (100 mL), and saturated ammonium chloride aqueous solution (10 mL) was added. Zinc powder (2.84 g, 43.44 mmol) was then added in portions, and the mixture was refluxed at 80 °C for 16 hours. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: 5% methanol/dichloromethane). The filtrate was then concentrated again to give the title compound (1.15 g, 3.26 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):353.1[M+H]+ MS m/z(ESI): 353.1 [M+H] +

步骤二:(S)-8-(苄氧基)-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(7-44-2)的制备Step 2: Preparation of (S)-8-(benzyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-44-2)

将(S)-8-(苄氧基)-7-甲氧基-1,2,3,11a-四氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5,11(10H)-二酮(1.1g,3.12mmol)溶于干燥THF(10mL)，搅拌下加入硼氢化钠(354.29mg,9.36mmol)，再滴入三氟乙酸(711.85mg,6.24mmol,478.07μL)的四氢呋喃溶液(100μL)，大量气泡产生。升温至75℃搅拌反应4小时。减压浓缩，加水和乙酸乙酯搅拌，分液，有机相减压浓缩，硅胶柱纯化(洗脱剂：5％甲醇/二氯甲烷)后再次浓缩得标题化合物(1.01g,2.98mmol)。 (S)-8-(benzyloxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (1.1 g, 3.12 mmol) was dissolved in dry THF (10 mL), and sodium borohydride (354.29 mg, 9.36 mmol) was added with stirring. Then, a tetrahydrofuran solution (100 μL) of trifluoroacetic acid (711.85 mg, 6.24 mmol, 478.07 μL) was added dropwise, resulting in the generation of numerous bubbles. The mixture was heated to 75 °C and stirred for 4 hours. The solution was concentrated under reduced pressure, and water and ethyl acetate were added with stirring. The mixture was separated, and the organic phase was concentrated under reduced pressure. After purification by silica gel column chromatography (eluent: 5% methanol/dichloromethane), the solution was concentrated again to give the title compound (1.01 g, 2.98 mmol).

步骤三:(S)-8-(苄氧基)-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(7-44-3)的制备Step 3: Preparation of (S)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-44-3)

将(S)-8-(苄氧基)-7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-5-酮(1g,2.96mmol)溶于干燥二氯甲烷(50mL)，滴入吡啶(350.62mg,4.43mmol,358.51μL)，冷却搅拌至0℃，滴入氯酸烯丙酯(1.07g,8.87mmol,942.30μL)，恢复至室温搅拌反应1小时。加二氯甲烷稀释，加10％柠檬酸水搅拌，静置分液，有机相水洗后干燥浓缩。硅胶柱纯化(洗脱剂：80％乙酸乙酯/石油醚)后再次浓缩得标题化合物(530mg,1.25mmol)。(S)-8-(benzyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (1 g, 2.96 mmol) was dissolved in dry dichloromethane (50 mL), and pyridine (350.62 mg, 4.43 mmol, 358.51 μL) was added dropwise. The mixture was cooled and stirred to 0 °C, and allyl chlorate (1.07 g, 8.87 mmol, 942.30 μL) was added dropwise. The mixture was then brought back to room temperature and stirred for 1 hour. The solution was diluted with dichloromethane, and 10% citric acid solution was added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water and dried and concentrated. The solution was purified by silica gel column chromatography (eluent: 80% ethyl acetate/petroleum ether) and then concentrated again to give the title compound (530 mg, 1.25 mmol).

步骤四:(S)-8-羟基-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(7-44-4)的制备Step 4: Preparation of (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-44-4)

将(S)-8-(苄氧基)-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(530mg,1.25mmol)溶于二氯甲烷(4mL)，搅拌下滴入甲磺酸(2mL)，继续反应1小时。加入二氯甲烷稀释，加入碳酸氢钠水溶液搅拌，静置分液，有机相干燥浓缩得到标题化合物(410mg,1.23mmol)。(S)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (530 mg, 1.25 mmol) was dissolved in dichloromethane (4 mL), and methanesulfonic acid (2 mL) was added dropwise with stirring. The reaction was continued for 1 hour. After dilution with dichloromethane, sodium bicarbonate aqueous solution was added and stirred. The mixture was allowed to stand and separated. The organic phase was dried and concentrated to give the title compound (410 mg, 1.23 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):333.2[M+H]+ MS m/z(ESI): 333.2 [M+H] +

步骤五:(S)-8-((5-(((S)-10-((烯丙氧基)羰基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(7-44-5)的制备Step 5: Preparation of (S)-8-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-44-5)

将(S)-8-((5-溴戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(40mg,68.32μmol)，(S)-8-羟基-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(22.71mg,68.32μmol)，碳酸钾(14.16mg,102.48μmol)溶于DMF(1mL)，搅拌反应16小时。加水析出固体，过滤，固体水洗后真空干燥得标题化合物(55mg,65.72μmol)。(S)-8-((5-bromopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (40 mg, 68.32 μmol), (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (22.71 mg, 68.32 μmol), and potassium carbonate (14.16 mg, 102.48 μmol) were dissolved in DMF (1 mL), and the mixture was stirred for 16 hours. Water was added to precipitate the solid, which was then filtered, washed with water, and dried under vacuum to obtain the title compound (55 mg, 65.72 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):838.4[M+H]+ MS m/z(ESI): 838.4 [M+H] +

步骤六:(S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-2-(4-甲氧基苯基)-1,10,11,11a-四氢-5H-苯并[e]吡 咯[1,2-a][1,4]二氮杂-5-酮(7-44)的制备Step Six: (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrole Preparation of [1,2-a][1,4]diaza-5-one (7-44)

将(S)-8-((5-(((S)-10-((烯丙氧基)羰基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-2-(4-甲氧基苯基)-5-氧代-11,11a-二氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(55mg,65.72μmol)溶于DMF(1mL)，滴入四氢吡咯(4.67mg,65.72μmol)，加入四(三苯基膦)钯(7.59mg,6.57μmol)，氮气置换并保护下反应16小时。经制备高效液相色谱纯化，冷冻干燥得到标题化合物(11.71mg,17.33μmol)。Allyl (55 mg, 65.72 μmol) of (S)-8-((5-(((S)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid (1 mL) was dissolved in DMF, and tetrahydropyrrole (4.67 mg, 65.72 μmol) was added dropwise. Tetra(triphenylphosphine)palladium (7.59 mg, 6.57 μmol) was added, and the reaction was carried out under nitrogen purging and protection for 16 hours. The compound was purified by preparative high-performance liquid chromatography and freeze-dried to obtain the title compound (11.71 mg, 17.33 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):669.3[M+H]+ MS m/z(ESI): 669.3 [M+H] +

1H NMR(400MHz,CDCl3)δ7.49(s,2H),7.31(d,J＝8.7Hz,2H),7.26(s,3H),6.89(d,J＝8.8Hz,2H),4.29(s,1H),3.99(s,4H),3.87(s,5H),3.81(d,J＝11.6Hz,5H),3.70(s,1H),3.58(d,J＝10.2Hz,4H),3.32(s,3H),2.74(dd,J＝16.1,3.6Hz,1H),2.21(dd,J＝12.8,6.3Hz,1H),1.90(m,6H),1.65(m,4H). 1 H NMR (400MHz, CDCl 3 )δ7.49(s,2H),7.31(d,J＝8.7Hz,2H),7.26(s,3H),6.89(d,J＝8.8Hz,2H),4.29(s,1H),3.99(s,4H),3.87(s,5H),3.81(d,J＝11.6Hz,5H) ,3.70(s,1H),3.58(d,J＝10.2Hz,4H),3.32(s,3H),2.74(dd,J＝16.1,3.6Hz,1H),2.21(dd,J＝12.8,6.3Hz,1H),1.90(m,6H),1.65(m,4H).

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

制备例二十八：(11aS,11a’S)-8,8’-(戊烷-1,5-二基双(氧基))双(7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮)(7-45)的制备
Preparation Example 28: Preparation of (11aS,11a'S)-8,8'-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one) (7-45)

步骤一:(S)-8-((5-(((R)-10-((烯丙氧基)羰基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H- 苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(7-45-1)的制备Step 1: (S)-8-((5-(((R)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H- Preparation of benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-45-1)

将1,5-二溴戊烷(12.45mg,54.16μmol)，(S)-8-羟基-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(40mg,120.35μmol)，碳酸钾(49.90mg,361.06μmol)溶于DMF(1mL)，搅拌反应16小时。加水析出固体，过滤，固体水洗后真空干燥得标题化合物(40mg,54.58μmol)。1,5-Dibromopentane (12.45 mg, 54.16 μmol), (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (40 mg, 120.35 μmol), and potassium carbonate (49.90 mg, 361.06 μmol) were dissolved in DMF (1 mL), and the mixture was stirred for 16 hours. Water was added to precipitate the solid, which was filtered, washed with water, and dried under vacuum to give the title compound (40 mg, 54.58 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):733.4[M+H]+ MS m/z(ESI): 733.4 [M+H] +

步骤二:(11aS,11a’S)-8,8’-(戊烷-1,5-二基双(氧基))双(7-甲氧基-1,2,3,10,11,11a-六氢-5H-苯并[e]吡咯[1,2-a][1,4]二氮杂-5-酮)(7-45)的制备Step 2: Preparation of (11aS,11a’S)-8,8’-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one) (7-45)

将(S)-8-((5-(((R)-10-((烯丙氧基)羰基)-7-甲氧基-5-氧代-2,3,5,10,11,11a-六氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-5-氧代-2,3,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-10(5H)-羧酸烯丙酯(40mg,54.58μmol)溶于DMF(1mL)，滴入四氢吡咯(3.88mg,54.58μmol)，加入四(三苯基膦)钯(6.31mg,5.46μmol)，氮气置换并保护下反应16小时。经制备高效液相色谱纯化，冷冻干燥得到标题化合物(4.96mg,8.70μmol)。(S)-8-((5-(((R)-10-((allyloxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (40 mg, 54.58 μmol) was dissolved in DMF (1 mL), tetrahydropyrrole (3.88 mg, 54.58 μmol) was added dropwise, and tetra(triphenylphosphine)palladium (6.31 mg, 5.46 μmol) was added. The reaction was carried out under nitrogen purging and protection for 16 hours. The compound was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (4.96 mg, 8.70 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):565.3[M+H]+ MS m/z(ESI): 565.3 [M+H] +

1H NMR(400MHz,CDCl3)δ7.52(s,2H),6.13(s,2H),3.99(s,4H),3.95-3.73(m,9H),3.70(s,2H),3.57(d,J＝11.7Hz,2H),3.29(s,3H),2.22(dd,J＝12.6,6.1Hz,2H),1.90(dt,J＝13.4,6.5Hz,8H),1.68(d,J＝20.9Hz,6H). 1 H NMR (400MHz, CDCl 3 )δ7.52(s,2H),6.13(s,2H),3.99(s,4H),3.95-3.73(m,9H),3.70(s,2H),3.57(d,J＝11.7Hz,2H) ,3.29(s,3H),2.22(dd,J＝12.6,6.1Hz,2H),1.90(dt,J＝13.4,6.5Hz,8H),1.68(d,J＝20.9Hz,6H).

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

中间体制备例一：1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(INT-1)的制备：
Example 1 of intermediate preparation: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (INT-1):

步骤一：1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(INT-1-2)的制备Step 1: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrica-31-carboxylic acid (INT-1-2)

使用标准的固相合成方法：Using standard solid-phase synthesis methods:

1)树脂制备:将2-CTC树脂(3.00mmol,3.70g,0.81mmol/g)和N-(((9H-芴-9-基)甲氧基)羰基)-N-甲基甘氨酸(3.00mol,933mg,3.00当量)、DIPEA(4.00当量)加入到二氯甲烷(10.0mL)中，在氮气氛围下反应2小时。然后在30分钟内氮气鼓泡氛围下将MeOH(1.0mL)加入到树脂中，过滤获得树脂。1) Resin preparation: 2-CTC resin (3.00 mmol, 3.70 g, 0.81 mmol/g), N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (3.00 mol, 933 mg, 3.00 equivalents), and DIPEA (4.00 equivalents) were added to dichloromethane (10.0 mL) and reacted under a nitrogen atmosphere for 2 hours. Then, MeOH (1.0 mL) was added to the resin over a nitrogen bubbling atmosphere over 30 minutes, and the resin was obtained by filtration.

2)偶联：在氮气鼓泡的情况下，将N-(((9H-芴-9-基)甲氧基)羰基)-N-甲基甘氨酸(5.60g，6.00当量)、HATU(6.58g，5.70当量)的DMF(10.0mL)溶液加入到树脂中。滴加DIPEA(6.00当量)后，在20℃下氮气鼓泡30分钟。在进行下一步骤之前用DMF(30.0mL X 5)洗涤树脂。2) Coupling: Under nitrogen bubbling, a DMF solution (10.0 mL) of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (5.60 g, 6.00 equivalent) and HATU (6.58 g, 5.70 equivalent) was added to the resin. After adding DIPEA (6.00 equivalent), the mixture was bubbled under nitrogen at 20°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) before proceeding to the next step.

3)脱保护：将20％的哌啶DMF溶液(30.0mL)加入到树脂中，并在20℃下用氮气鼓泡30分钟。然后用DMF(30.0mL)×5洗涤树脂。 3) Deprotection: Add 30.0 mL of 20% piperidine DMF solution to the resin and bubble with nitrogen at 20°C for 30 minutes. Then wash the resin with 5 x 30.0 mL DMF solutions.

4)使用表1中的氨基酸重复步骤2和3：表1中的编号2-10。4) Repeat steps 2 and 3 using the amino acids in Table 1: numbers 2-10 in Table 1.

5)然后用DMF(30.0mL X 5)、MeOH(30.0mL X 5)洗涤树脂,然后在真空下干燥。5) Then wash the resin with DMF (30.0 mL x 5) and MeOH (30.0 mL x 5), and then dry it under vacuum.

表1:
Table 1:

多肽的切割和纯化：Peptide cleavage and purification:

1)在室温下将裂解溶液(TFA/DCM，1/100，v/v，200.0mL)加入到含有侧链保护肽的烧瓶中，并搅拌3分钟两次。 1) Add the lysis solution (TFA/DCM, 1/100, v/v, 200.0 mL) to a flask containing the side-chain protected peptide at room temperature and stir twice for 3 minutes.

2)过滤后，合并滤液并浓缩。2) After filtration, combine the filtrates and concentrate them.

3)粗品经高效液相色谱制备纯化后，冷冻干燥得标题化合物(1117.1mg，TFA盐)。3) The crude product was prepared and purified by high performance liquid chromatography and then freeze-dried to obtain the title compound (1117.1 mg, TFA salt).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):1010.4(M+H)+。ESI-MS(m/z): 1010.4(M+H) + .

其纯化方法如下：
The purification method is as follows:

步骤二：1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(INT-1)的制备Step 2: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (INT-1)

将1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(665mg,615.04μmol)加到水(7.5mL)和乙腈(15mL)中，加入高碘酸钠(1.32g,6.15mmol)和三氯化钌水合物(51.03mg,246.02μmol)后在25℃反应1小时。反应液直接经反相纯化(乙腈/水(0.05％甲酸)＝0-25％)，冷冻干燥得标题化合物(515mg,449.69μmol)。1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatricarne-31-carboxylic acid (665 mg, 615.04 μmol) was added to water (7.5 mL) and acetonitrile (15 mL), followed by the addition of sodium periodate (1.32 g, 6.15 mmol) and ruthenium trichloride hydrate (51.03 mg, 246.02 μmol), and the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by reverse-phase reaction (acetonitrile/water (0.05% formic acid) = 0-25%) and freeze-dried to give the title compound (515 mg, 449.69 μmol).

其结构表征数据如下： Its structural characterization data are as follows:

ESI-MS(m/z):1074.4(M+H)+。ESI-MS(m/z):1074.4(M+H) + .

中间体制备例二:1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂壬烷-29-酸(INT-2)的制备
Example 2 of intermediate preparation: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-acid (INT-2)

步骤一：3,5-二(2-(甲硫基)嘧啶-5-基)苯甲酸甲酯(INT-2-2)的制备Step 1: Preparation of methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (INT-2-2)

将原料3,5-二溴苯甲酸甲酯(720mg，2.45mmol)、2-甲硫基嘧啶-5-硼酸(874mg，5.14mmol)、XPhosPd G3(207mg,245μmol)、K3PO4(1.56g,7.35mmol)加入到二噁烷(12mL)和水(4mL)中，反应体系在氮气氛围下于90℃搅拌反应3小时，LC-MS监控反应，垫硅藻土过滤，往滤液中加水和乙酸乙酯，萃取，浓缩，得到粗品，经柱层析纯化(EA/PE＝0-25％)得到3,5-二(2-(甲硫基)嘧啶-5-基)苯甲酸甲酯710mg。The raw materials methyl 3,5-dibromobenzoate (720 mg, 2.45 mmol), 2-methylthiopyrimidin-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and water (4 mL). The reaction system was stirred at 90 °C for 3 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was filtered through diatomaceous earth, and water and ethyl acetate were added to the filtrate for extraction and concentration to obtain the crude product. The crude product was purified by column chromatography (EA/PE = 0-25%) to obtain 710 mg of methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):385.1[M+H]+。ESI-MS(m/z): 385.1 [M+H] + .

步骤二：3,5-二(2-(甲硫基)嘧啶-5-基)苯甲酸(INT-2-3)的制备Step 2: Preparation of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (INT-2-3)

将化合物3,5-二(2-(甲硫基)嘧啶-5-基)苯甲酸甲酯(650mg，1.69mol)、氢氧化锂(121mg,5.07mmol)溶于THF(2mL)、MeOH(2mL)和H2O(2mL)中。25℃搅拌反应2小时，LC-MS监控反应，用1N HCl调节体系pH约为2，析出大量固体，过滤收集滤饼，干燥得3,5-二(2-(甲硫基)嘧啶-5-基)苯甲酸560mg。 Methyl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and H₂O ( 2 mL). The reaction was stirred at 25 °C for 2 hours, and the reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1 N HCl. A large amount of solid precipitated, and the filter cake was collected by filtration and dried to obtain 560 mg of 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid.

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):371.1[M+H]+。ESI-MS(m/z): 371.1[M+H] + .

步骤三：1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸叔丁酯(INT-2-4)的制备Step 3: Preparation of tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecan-29-olate (INT-2-4)

将3,5-二(2-(甲硫基)嘧啶-5-基)苯甲酸(3.00g,8.10mmol)和1-氨基-3,6,9,12,15,18,21,24-八氧杂庚烷-27-酸叔丁酯(4.03g,8.10mmol)加入到DMF(40mL)中，依次加入HOBt(3.28g,24.3mmol)、EDCI(4.66g,24.3mmol)和DIPEA(4.19g,32.4mmol,5.64mL)，反应体系于60℃搅拌2小时。向反应液中加入水(100mL)和乙酸乙酯萃取(60mL x 3)，有机相合并后用无水硫酸钠干燥,过滤，浓缩得1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸叔丁酯(4.20g,4.14mmol)，未经纯化直接用于下一步。3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-oate (4.03 g, 8.10 mmol) were added to DMF (40 mL), followed by HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL). The reaction mixture was stirred at 60 °C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain tert-butyl 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-oic acid (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

步骤四：1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸(INT-2-5)的制备Step 4: Preparation of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecan-29-acid (INT-2-5)

将1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸叔丁酯(3.60g,4.24mmol)溶于二氯甲烷(30mL)中，加入TFA(15.3g,134mmol,10mL)，反应体系于25℃搅拌6小时。向反应液加入水(60mL)和乙酸乙酯萃取(40mL x 3)。有机相合并后用无水硫酸钠干燥,过滤，浓缩得粗品，粗品经制备高效液相色谱纯化后冷冻干燥得1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸(2.93g,3.63mmol)。3.60 g (4.24 mmol) of 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-oic acid tert-butyl ester was dissolved in dichloromethane (30 mL), and TFA (15.3 g, 134 mmol, 10 mL) was added. The reaction mixture was stirred at 25 °C for 6 hours. Water (60 mL) and ethyl acetate (40 mL x 3) were added to the reaction mixture for extraction. After the organic phases were combined, they were dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-acid (2.93 g, 3.63 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):794.3[M+H]+。ESI-MS(m/z): 794.3[M+H] + .

其纯化方法如下：The purification method is as follows:

色谱柱：Phenomenex luna C18(250mm*70mm*10μm)Column: Phenomenex Luna C18 (250mm*70mm*10μm)

流动相A：乙腈；流动相B：水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤五：1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸(INT-2)的制备Step 5: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecano-29-acid (INT-2)

将1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸(148mg，0.186mmol)加到乙腈(15mL)和水(7.5mL)中，再将高碘酸钠(398.71mg，1.86mmol)和三氯化钌水合物(15.47mg，74.56μmol)加到反应体系中，25℃搅拌反应30分钟，反应体系经水和乙酸乙酯萃取，浓缩得1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂二十九烷-29-酸(155mg)。1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-acid (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), followed by sodium periodate (398.71 mg, 1.86 mmol) and ruthenium trichloride. The hydrate (15.47 mg, 74.56 μmol) was added to the reaction system, and the mixture was stirred at 25 °C for 30 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to obtain 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonadecan-29-acid (155 mg).

其结构表征数据如下：Its structural characterization data are as follows:

ESI-MS(m/z):858.3[M+H]+。ESI-MS(m/z): 858.3[M+H] + .

中间体制备例三:1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸(INT-5)的制备
Example 3 of intermediate preparation: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (INT-5)

步骤一：2,5-二氧代吡咯烷-1-基3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酸酯(INT-5-1)的制备Step 1: Preparation of 2,5-dioxopyrrolidine-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (INT-5-1)

将3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酸(0.6g,1.62mmol)溶于THF(15mL)中，加入N-羟基丁二酰亚胺(278.89mg,2.43mmol)，然后加入二环己基碳二亚胺(0.4g,1.94mmol)，室温搅拌2小时。反应完毕后，抽滤，收集滤液，减压浓缩得标题化合物的粗品(1.5g,3.42mmol)，未经纯化直接用于下一步反应。3,5-Bis(2-(methylthio)pyrimidin-5-yl)benzoic acid (0.6 g, 1.62 mmol) was dissolved in THF (15 mL), and N-hydroxysuccinimide (278.89 mg, 2.43 mmol) was added, followed by dicyclohexylcarbodiimide (0.4 g, 1.94 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product of the title compound (1.5 g, 3.42 mmol), which was used directly in the next reaction without purification.

步骤二：1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷 -20-羧酸(INT-5-2)的制备Step 2: 1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane Preparation of -20-carboxylic acid (INT-5-2)

将2,5-二氧代吡咯烷-1-基3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酸酯(331.5mg,0.99mmol)溶于DMF(4mL)中，加入1-氨基-3,6,9,12,15-五氧杂十八烷-18-羧酸(0.3g,0.67mmol)，DIPEA(588.24mg,4.56mol)，室温搅拌2小时。反应完毕后，经快速柱层析(C18,水/乙腈＝0.5)得标题化合物(315.50mg,0.44mmol)。2,5-Dioxopyrrolidone-1-yl 3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoate (331.5 mg, 0.99 mmol) was dissolved in DMF (4 mL), and 1-amino-3,6,9,12,15-pentaoctadecane-18-carboxylic acid (0.3 g, 0.67 mmol) and DIPEA (588.24 mg, 4.56 mol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solution was obtained by rapid column chromatography (C18, water/acetonitrile = 0.5) to give the title compound (315.50 mg, 0.44 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI):662.2[M+H]+MS m/z (ESI): 662.2 [M+H]+

步骤三：1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸(INT-5)的制备Step 3: Preparation of 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid (INT-5)

将1-(3,5-双(2-(甲硫基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-油酸(315.50mg,0.44mmol)溶于乙腈(3mL)，水(1.5mL)中，加入高碘酸钠(470.50mg,2.20mmol)，一水合三氯化钌(9.11mg,0.04mmol)，室温搅拌0.5小时。反应完毕后，经快速柱层析(C18,水/乙腈＝2/1)后冷冻干燥得标题化合物(115.50mg,0.16mmol)。1-(3,5-bis(2-(methylthio)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosano-20-oleic acid (315.50 mg, 0.44 mmol) was dissolved in acetonitrile (3 mL) and water (1.5 mL), and sodium periodate (470.50 mg, 2.20 mmol) and ruthenium trichloride monohydrate (9.11 mg, 0.04 mmol) were added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the solution was subjected to rapid column chromatography (C18, water/acetonitrile = 2/1) and then freeze-dried to give the title compound (115.50 mg, 0.16 mmol).

其结构表征数据如下：Its structural characterization data are as follows:

MS m/z(ESI):726.1[M+H]+ MS m/z(ESI): 726.1 [M+H] +

中间体制备例四:N6-(二苯基(对甲苯基)甲基)-N2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰基)-L-赖氨酸(INT-6)的制备
Example 4 of intermediate preparation: Preparation of N, 6- (diphenyl(p-tolyl)methyl)-N, 2- (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-6)

步骤一：N6-(二苯基(对甲苯基)甲基)-L-赖氨酸(INT-6-2)的制备Step 1: Preparation of N, 6- (diphenyl(p-tolyl)methyl)-L-lysine (INT-6-2)

将N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(二苯基(对甲苯基)甲基)-L-赖氨酸(150.0mg,0.24mmol)溶于DMF(2mL)中，加入二乙胺(182.3mg,2.5mmol)，室温搅拌1小时。反应完毕后，加入(EA/PE＝1/3)混合溶剂10mL重结晶1h，过滤得标题化合物(90.1mg,0.22mmol)。 N₂ -(((9H-fluorene-9-yl)methoxy)carbonyl) -N₆- (diphenyl(p-tolyl)methyl)-L-lysine (150.0 mg, 0.24 mmol) was dissolved in DMF (2 mL), and diethylamine (182.3 mg, 2.5 mmol) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, 10 mL of a mixed solvent (EA/PE = 1/3) was added, and the mixture was recrystallized for 1 hour. The solution was filtered to obtain the title compound (90.1 mg, 0.22 mmol).

步骤二：N6-(二苯基(对甲苯基)甲基)-N2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰基)-L- 赖氨酸(INT-6)的制备Step 2: N 6- (diphenyl(p-tolyl)methyl)-N 2- (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L- Preparation of Lysine (INT-6)

将N6-(二苯基(对甲苯基)甲基)-L-赖氨酸(90.1mg,0.22mmol)溶于DMF(3mL)中，加入DIPEA(86.3mg,0.66mmol)，加入2,5-二氧吡咯烷-1-基-6-(2-(甲磺酰基)嘧啶-5-基)己基-5-炔酸酯(160.6mg,0.44mmol)，反应完毕后，经快速柱层析(C18,水/乙腈＝0.5)纯化后冷冻干燥得标题化合物(75.2mg,0.11mmol)。N, 6- (diphenyl(p-tolyl)methyl)-L-lysine (90.1 mg, 0.22 mmol) was dissolved in DMF (3 mL), DIPEA (86.3 mg, 0.66 mmol) was added, followed by 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexyl-5-acetylacetate (160.6 mg, 0.44 mmol). After the reaction was complete, the mixture was purified by rapid column chromatography (C18, water/acetonitrile = 0.5) and then freeze-dried to obtain the title compound (75.2 mg, 0.11 mmol).

中间体制备例五：(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7)的制备
Example 5 of intermediate preparation: Preparation of (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (INT-7)

步骤一：(9H-芴-9-基)甲基((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酸酯(INT-7-1)的制备Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (INT-7-1)

将(((9H-芴-9-基)甲氧基)羰基)-L-缬氨酰-L-丙氨酸(13.28mg,32.36μmol)和(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧)-7-乙氧基-1,10,11,11-四氢-3H,5H-螺并[e]吡咯并[1,2-a][1,4]氮杂-2,1’-环丙烷]-5-酮(20mg,29.42μmol)溶于DMF(1mL)，搅拌下加入HATU(16.77mg,44.13μmol)，再滴入二异丙基乙胺(11.41mg,88.26μmol)，搅拌反应1小时。加水和二氯甲烷搅拌，静置分液，有机相水洗后干燥浓缩得粗品，经硅胶柱纯化(0-5％甲醇/二氯甲烷)后再次浓缩得标题化合物(10mg,9.33μmol)。The (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine (13.28 mg, 32.36 μmol) and (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-ethoxy -1,10,11,11-tetrahydro-3H,5H-spiro[e]pyrrolo[1,2-a][1,4]aza-2,1’-cyclopropane]-5-one (20 mg, 29.42 μmol) was dissolved in DMF (1 mL), and HATU (16.77 mg, 44.13 μmol) was added with stirring, followed by the dropwise addition of diisopropylethylamine (11.41 mg, 88.26 μmol). The reaction was stirred for 1 hour. Water and dichloromethane were added and stirred. The mixture was allowed to stand and separated. The organic phase was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (0-5% methanol/dichloromethane) and then concentrated again to obtain the title compound (10 mg, 9.33 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1072.6[M+H]+MS m/z (ESI): 1072.6 [M+H]+

步骤二：(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a- 四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7)的制备Step 2: (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a- Preparation of tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (INT-7)

将(9H-芴-9-基)甲基((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酸酯(10mg,9.33μmol)溶于DMF(1mL)，搅拌下滴入二乙胺(0.5mL)，继续反应0.5小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(3mg,3.53μmol)。The (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo -5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (10 mg, 9.33 μmol) was dissolved in DMF (1 mL), and diethylamine (0.5 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (3 mg, 3.53 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):850.3[M+H]+MS m/z (ESI): 850.3 [M+H]+

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

中间体制备例六：N2-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰基)-N6-(叔丁氧基羰基)-L-赖氨酸(INT-8)的制备
Example 6: Preparation of intermediate N2- (3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl) -N6- (tert-butoxycarbonyl)-L-lysine (INT-8)

步骤一：2,5-二氧吡咯烷-1-基3,5-双(2-(甲硫基)嘧啶-4-基)苯甲酸酯(INT-8-1)的制备Step 1: Preparation of 2,5-dioxopyrrolidine-1-yl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (INT-8-1)

将3,5-双(2-(甲硫基)嘧啶-4-基)苯甲酸(5g,13.50mmol)，1-羟基吡咯烷-2,5-二酮(1.55g,13.50mmol)，加到乙腈(75mL)和DMF(75mL)的混合溶剂中，再加入EDCI(5.17g,26.99mmol)，在25℃反应1.5小试。将反应液过滤，收集滤饼，干燥得标题化合物(2.41g,5.15mmol)。3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoic acid (5 g, 13.50 mmol) and 1-hydroxypyrrolidine-2,5-dione (1.55 g, 13.50 mmol) were added to a mixed solvent of acetonitrile (75 mL) and DMF (75 mL), followed by the addition of EDCI (5.17 g, 26.99 mmol). The reaction mixture was reacted at 25 °C for 1.5 minutes. The reaction solution was filtered, the filter cake was collected, and dried to give the title compound (2.41 g, 5.15 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):468.3[M+H]+ MS m/z(ESI): 468.3 [M+H] +

步骤二：N2-(3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酰基)-N6-(叔丁氧基羰基)-L-赖氨酸(INT-8-2)的制备Step 2: Preparation of N2- (3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl) -N6- (tert-butoxycarbonyl)-L-lysine (INT-8-2)

将N6-(叔丁氧基羰基)-L-赖氨酸(895.61mg,3.64mmol)和2,5-二氧吡咯烷-1-基3,5-双(2-(甲硫基)嘧啶-4-基)苯甲酸酯(1.7g,3.64mmol)加入到DMSO(50mL)中，升温至60℃反应14小时。将反应液倒入水中，析出大量白色固体，用1N稀盐酸调节体系pH约为2，过滤，收集滤饼，干燥得标题化合物(2.16g,3.61mmol)。N, 6- (tert-butoxycarbonyl)-L-lysine (895.61 mg, 3.64 mmol) and 2,5-dioxopyrrolidone-1-yl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (1.7 g, 3.64 mmol) were added to DMSO (50 mL), and the mixture was heated to 60 °C and reacted for 14 hours. The reaction solution was poured into water, and a large amount of white solid precipitated. The pH of the system was adjusted to approximately 2 with 1N dilute hydrochloric acid, filtered, and the filter cake was collected and dried to obtain the title compound (2.16 g, 3.61 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):526.2[M+H-56]+ MS m/z(ESI): 526.2 [M+H-56] +

步骤三：N2-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰基)-N6-(叔丁氧基羰基)-L-赖氨酸(INT-8)的制备Step 3: Preparation of N2- (3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl) -N6- (tert-butoxycarbonyl)-L-lysine (INT-8)

将N2-(3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酰基)-N6-(叔丁氧基羰基)-L-赖氨酸(1.06g,1.77 mmol)溶于水(25mL)和乙腈(50mL)的混合溶液中，再加入高碘酸钠(2.46g,11.51mmol)和水合三氯化钌(36.72mg,177.04mol)，在25℃反应20分钟。相反应体系中加入水，用1N稀盐酸调节体系pH约为2，EA萃取，浓缩，乙腈溶解粗品，过反相纯化(ACN/H2O＝0-45％,0.05％甲酸)，冻干得标题化合物(825mg,1.24mmol)。 N2- (3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl) -N6- (tert-butoxycarbonyl)-L-lysine (1.06 g, 1.77 g) The compound (11.51 mmol) was dissolved in a mixture of water (25 mL) and acetonitrile (50 mL), and sodium periodate (2.46 g, 11.51 mmol) and ruthenium trichloride hydrate (36.72 mg, 177.04 mol) were added. The mixture was reacted at 25 °C for 20 minutes. Water was then added to the reaction system, and the pH was adjusted to approximately 2 with 1 N dilute hydrochloric acid. The mixture was extracted with EA, concentrated, dissolved in acetonitrile, purified by reverse-phase chromatography (ACN/ H₂O = 0.45%, 0.05% formic acid), and lyophilized to give the title compound (825 mg, 1.24 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):680.2[M+H2O]+ MS m/z (ESI): 680.2 [M+ H₂O ] ⁺

中间体制备例七：N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰基)-L-赖氨酸(INT-9)的制备
Example 7 of intermediate preparation: Preparation of N6 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N2- (3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (INT-9)

步骤一：N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酰基)-L-赖氨酸(INT-9-1)的制备Step 1: Preparation of N6 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N2- (3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl)-L-lysine (INT-9-1)

向DMSO(20mL)中加入N6-(((9H-芴-9-基)甲氧基)羰基)-L-赖氨酸三氟乙酸盐(2.8g,5.80mmol)和DIPEA(1.50g,11.61mmol)，室温搅拌反应10分钟，体系澄清。再加入2,5-二氧吡咯烷-1-基3,5-双(2-(甲硫基)嘧啶-4-基)苯甲酸酯(2.71g,5.80mmol)，加毕，室温搅拌反应，反应过程中逐渐析出固体，升温至37℃反应。将反应液过反相柱(ACN/H2O＝0-45％,0.05％甲酸)纯化后冷冻干燥得标题化合物(2.8g,3.88mmol)。 N6 -(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine trifluoroacetate (2.8 g, 5.80 mmol) and DIPEA (1.50 g, 11.61 mmol) were added to DMSO (20 mL), and the mixture was stirred at room temperature for 10 minutes until the system became clear. Then, 2,5-dioxopyrrolidone-1-yl 3,5-bis(2-(methylthio)pyrimidin-4-yl)benzoate (2.71 g, 5.80 mmol) was added. After the addition was complete, the mixture was stirred at room temperature, and a solid gradually precipitated during the reaction. The temperature was then increased to 37 °C. The reaction solution was purified by passing it through a reverse-phase column (ACN/H2O = 0-45%, 0.05% formic acid) and then freeze-dried to obtain the title compound (2.8 g, 3.88 mmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):721.9[M+H]+ MS m/z(ESI): 721.9 [M+H] +

步骤二：N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰基)-L-赖氨酸(INT-9)的制备Step 2: Preparation of N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-N2-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzoyl)-L-lysine (INT-9)

向乙腈(80mL)和水(40mL)的混合溶剂中加入N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(3,5-双(2-(甲硫基)嘧啶-5-基)苯甲酰基)-L-赖氨酸(580mg,804.60μmol)、高碘酸钠(1.38g,6.44mmol)和RuCl3·H2O(36.28mg,160.92μmol),加毕,室温搅拌反应40分钟。向反应液中加水(150mL)后，用乙酸乙酯(100mL*3)萃取三次，硅藻土过滤后分液，有机相用水(50mL)洗涤 一次，饱和氯化钠水溶液(50mL)洗涤一次，无水硫酸钠干燥后过滤浓缩，残留物用乙腈和水溶解,冷冻干燥得标题化合物(590mg,676.56μmol)。Add N6 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N2- (3,5-bis(2-(methylthio)pyrimidin-5-yl)benzoyl)-L-lysine (580 mg, 804.60 μmol), sodium periodate (1.38 g, 6.44 mmol), and RuCl3 · H2O (36.28 mg, 160.92 μmol) to a mixed solvent of acetonitrile (80 mL) and water (40 mL). After the addition is complete, stir the mixture at room temperature for 40 minutes. Add water (150 mL) to the reaction solution, and extract three times with ethyl acetate (100 mL * 3). After filtration through diatomaceous earth, separate the liquid and wash the organic phase with water (50 mL). The compound was washed once with saturated sodium chloride aqueous solution (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was dissolved in acetonitrile and water and freeze-dried to give the title compound (590 mg, 676.56 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):785.9[M+H]+。MS m/z(ESI):785.9[M+H] + .

实施例一:N-(2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-2)的制备
Example 1: N-(2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy Preparation of (B-2)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-2)

步骤一：甘氨酰甘氨酸(B-2-2)的制备Step 1: Preparation of glycylglycine (B-2-2)

将(((9H-芴-9-基)甲氧基)羰基)甘氨酰甘氨酸(50mg,141.2μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(15mg,113.6μmol)，未经纯化直接用于下一步。(((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycine (50 mg, 141.2 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (15 mg, 113.6 μmol), which was used directly in the next step without purification.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):133.2[M+H]+ MS m/z(ESI): 133.2 [M+H] +

步骤二：(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰基)甘氨酰甘氨酸(B-2-3)的制备Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycine (B-2-3)

将甘氨酰甘氨酸(B-2-2,15mg,113.6μmol)溶于DMF(1mL)中，依次加入DIPEA(16mg)和2,5-二氧代吡咯烷-1-基6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸酯(50mg,136.32μmol)，加毕在25℃反应2小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(32mg,83.8μmol)。Glycylglycine (B-2-2, 15 mg, 113.6 μmol) was dissolved in DMF (1 mL), and DIPEA (16 mg) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (50 mg, 136.32 μmol) were added sequentially. The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (32 mg, 83.8 μmol).

其结构表征数据如下: Its structural characterization data are as follows:

MS m/z(ESI):383.1[M+H]+ MS m/z(ESI): 383.1 [M+H] +

步骤三：N-(2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-2)的制备Step 3: N-(2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy Preparation of 5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-2)

将(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰基)甘氨酰甘氨酸(B-2-3,15mg,39.3μmol)溶于DMF(1mL)中，依次加入DIPEA(16mg)、(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(Int-7,37.9mg,39.3μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(4.08mg,3.33μmol)。Dissolve (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycine (B-2-3, 15 mg, 39.3 μmol) in DMF (1 mL), then add DIPEA (16 mg) and (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-) in sequence. Spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutyramide (Int-7, 37.9 mg, 39.3 μmol) was added and reacted at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (4.08 mg, 3.33 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1214.4[M+H]+ MS m/z(ESI): 1214.4 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例二:N-((S)-1-(((S)-6-氨基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-10)的制备
Example 2: Preparation of N-((S)-1-(((S)-6-amino-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-10)

步骤一：(9H-芴-9-基)甲基叔丁基((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-6-氧代己烷-1,5-二基)二氨基甲酸酯(B-10-1)的制备Step 1: Preparation of (9H-fluorene-9-yl)methyl tert-butyl((S)-6-((4-(((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (B-10-1)

将(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙]-5-酮(7-5,50mg,73.55μmol)溶于DMF(1mL)中，依次加入DIPEA(28.52mg)、HATU(33.54mg)和N2-(((9H-芴-9-基)甲氧基)羰基)-N6-(叔丁氧羰基)-L-赖氨酸(41.35mg,88.26μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(76mg,67.24μmol)。Dissolve (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1'-cyclopropyl]-5-one (7-5,50 mg, 73.55 μmol) in DMF (1 mL), then add DIPEA (28.52 mg), HATU (33.54 mg), and N2 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N6 -(tert-Butoxycarbonyl)-L-lysine (41.35 mg, 88.26 μmol) was added and the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (76 mg, 67.24 μmol).

其结构表征数据如下: Its structural characterization data are as follows:

MS m/z(ESI):1331.2[M+H]+ MS m/z(ESI): 1331.2 [M+H] +

步骤二：((S)-5-氨基-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-6-氧代己基)氨基甲酸叔丁酯(B-10-2)的制备Step 2: Preparation of ((S)-5-amino-6-((4-(((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-6-oxohexyl)tert-butyl carbamate (B-10-2)

将(9H-芴-9-基)甲基叔丁基((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-6-氧代己烷-1,5-二基)二氨基甲酸酯(76mg,67.24μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(61.06mg,67.24μmol)，未经纯化直接用于下一步。(9H-fluorene-9-yl)methyl tert-butyl((S)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (76 mg, 67.24 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (61.06 mg, 67.24 μmol), which was used directly in the next step without purification.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):909.1[M+H]+ MS m/z(ESI): 909.1 [M+H] +

步骤三：(9H-芴-9-基)甲基((S)-1-(((S)-6-((叔丁氧羰基)氨基)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酸酯(B-10-3)的制备Step 3: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-6-((tert-butoxycarbonyl)amino)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (B-10-3)

将((S)-5-氨基-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-6-氧代己基)氨基甲酸叔丁酯(30mg,33.04μmol)溶于DMF(1mL)中，依次加入DIPEA(12.81mg)、HATU(15.06mg)和(((9H-芴-9-基)甲氧基)羰基)-L-缬氨酸(13.45mg,39.64μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝2/1)纯化后冷冻干燥得标题化合物(26mg,21.15μmol)。The ((S)-5-amino-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazaphen-2-yl)phenyl)amino)-6-oxohexyl)carbamate tert-butyl ester (30 mg, 33.04 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (12.81 mg), HATU (15.06 mg), and (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (13.45 mg, 39.64 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 2/1) and then freeze-dried to give the title compound (26 mg, 21.15 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1230.2[M+H]+ MS m/z(ESI): 1230.2 [M+H] +

步骤四：((S)-5-((S)-2-氨基-3-甲基丁酰胺基)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊 基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-6-氧代己基)氨基甲酸叔丁酯(B-10-4)的制备Step 4: ((S)-5-((S)-2-amino-3-methylbutamido)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl Preparation of tert-butyl carbamate (B-10-4) with α-(α)-(β ...

将(9H-芴-9-基)甲基((S)-1-(((S)-6-((叔丁氧羰基)氨基)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酸酯(B-10-3,26mg,21.15μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(21.30mg,21.15μmol)，未经纯化直接用于下一步。The (9H-fluorene-9-yl)methyl((S)-1-(((S)-6-((tert-butoxycarbonyl)amino)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)- 5-O-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (B-10-3, 26 mg, 21.15 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (21.30 mg, 21.15 μmol), which was used directly in the next step without purification.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1008.1[M+H]+ MS m/z(ESI): 1008.1 [M+H] +

步骤五：((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-5-(((S)-3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)-6-氧代己基)氨基甲酸叔丁酯(B-10-5)的制备Step 5: Preparation of ((S)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-5-(((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamide)butamido)-6-oxohexyl)tert-butyl carbamate (B-10-5)

将(9H-芴-9-基)甲基((S)-1-(((S)-6-((叔丁氧羰基)氨基)-1-((4-((S)-7-甲氧基-8-((5-(S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H，3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己烷-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酸酯(B-10-4,21.30mg,21.15μmol)溶于DMF(1mL)中，依次加入DIPEA(27mg)和2,5-二氧代吡咯烷-1-基6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸酯(9.27mg,25.38μmol)，加毕在25℃反应2小时。反应液直接经快速柱层析(C18,水/乙腈＝1/1)纯化后冷冻干燥得标题化合物(22mg,17.50μmol)。The (9H-fluorene-9-yl)methyl((S)-1-(((S)-6-((tert-butoxycarbonyl)amino)-1-((4-((S)-7-methoxy-8-((5-(S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohexane-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate (B-10-4, 21.30 mg, 21.15 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (27 mg) and 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (9.27 mg, 25.38 μmol). The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/1) and then freeze-dried to give the title compound (22 mg, 17.50 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1258.5[M+H]+ MS m/z(ESI): 1258.5 [M+H] +

步骤六：N-((S)-1-(((S)-6-氨基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-10)的制备Step Six: Preparation of N-((S)-1-(((S)-6-amino-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-10)

将((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并 [e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-5-(((S)-3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)-6-氧代己基)氨基甲酸叔丁酯(B-10-5,22mg,17.50μmol)溶于DCM(1mL)中，加入TFA(1mL)，加毕在25℃反应1小时。反应液室温减压蒸干后经制备高效液相色谱纯化后冷冻干燥得到标题化合物(0.57mg,0.49μmol)。((S)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[ [e]pyrrolo[1,2-a][1,4]diazaphen-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-5-(((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)butamido)-6-oxohexyl)tert-butyl carbamate (B-10-5, 22 mg, 17.50 μmol) was dissolved in DCM (1 mL), and TFA (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was evaporated to dryness under reduced pressure at room temperature, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (0.57 mg, 0.49 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1158.4[M+H]+ MS m/z(ESI): 1158.4 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例三:N-(2-(((S)-6-氨基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-2-氧代乙基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-11)的制备
Example 3: Preparation of N-(2-(((S)-6-amino-1-((4-(((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxohexyl-2-yl)amino)-2-oxoethyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-11)

步骤一：(9H-芴-9-基)甲基(2-(((S)-6-((叔丁氧基羰基)氨基)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-2-氧代乙基)氨基甲酸酯(B-11-1)的制备Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-(((S)-6-((tert-butoxycarbonyl)amino)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohex-2-yl)amino)-2-oxoethyl)carbamate (B-11-1)

将((S)-5-氨基-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-6-氧代己基)氨基甲酸叔丁酯 (B-10-2,30mg,33.04μmol)溶于DMF(1mL)中，依次加入DIPEA(12.81mg)、HATU(15.06mg)和(((9H-芴-9-基)甲氧基)羰基)甘氨酸(11.79mg,39.64μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝2/1)纯化后冷冻干燥得标题化合物(31mg,26.11μmol)。The tert-butyl carbamate ((S)-5-amino-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-6-oxohexyl)carbamate (B-10-2, 30 mg, 33.04 μmol) was dissolved in DMF (1 mL), and DIPEA (12.81 mg), HATU (15.06 mg), and (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (11.79 mg, 39.64 μmol) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 2/1) and then freeze-dried to give the title compound (31 mg, 26.11 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1188.4[M+H]+ MS m/z(ESI): 1188.4 [M+H] +

步骤二：((S)-5-(2-氨基乙酰胺基)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代)-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-6(-氧代己基)氨基甲酸叔丁酯(B-11-2)的制备Step 2: Preparation of ((S)-5-(2-aminoacetamido)-6-((4-(((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo)-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-6(-oxohexyl)carbamate tert-butyl ester (B-11-2)

将(9H-芴-9-基)甲基(2-(((S)-6-((叔丁氧基羰基)氨基)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-2-氧代乙基)氨基甲酸酯(B-11-1,31mg,26.11μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(25.20mg,26.11μmol)，未经纯化直接用于下一步。The (9H-fluorene-9-yl)methyl(2-(((S)-6-((tert-butoxycarbonyl)amino)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropyl]oxy)pentyl)oxy 5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxohexyl)amino)-2-oxoethyl)carbamate (B-11-1, 31 mg, 26.11 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (25.20 mg, 26.11 μmol), which was used directly in the next step without purification.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):966.2[M+H]+ MS m/z(ESI): 966.2 [M+H] +

步骤三：((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a)-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-5-(2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)乙酰胺基)-6-氧代己基)氨基甲酸叔丁酯(B-11-3)的制备Step 3: Preparation of ((S)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a)-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-5-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)acetamyl)-6-oxohexyl)tert-butyl carbamate (B-11-3)

将((S)-5-(2-氨基乙酰胺基)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代)-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-6(-氧代己基)氨基甲酸叔丁酯(B-11-2,25.20mg,26.11μmol)溶于DMF(1mL)中，依次加入DIPEA(12mg)和2,5-二氧代吡咯烷-1-基6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸酯(11.36mg,31.08μmol)，加毕在25℃反应2小时。反应液直接经快速柱层析(C18,水/乙腈＝1/1)纯化 后冷冻干燥得标题化合物(26mg,21.39μmol)。The ((S)-5-(2-aminoacetamido)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo)-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1, 2-a][1,4]diaza-2-yl)phenyl)amino)-6(-oxohexyl)carbamate tert-butyl ester (B-11-2, 25.20 mg, 26.11 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (12 mg) and 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (11.36 mg, 31.08 μmol). The reaction mixture was then incubated at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/1). The compound was then freeze-dried to give the title compound (26 mg, 21.39 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1216.4[M+H]+ MS m/z(ESI): 1216.4 [M+H] +

步骤四：N-(2-(((S)-6-氨基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-2-氧代乙基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-11)的制备Step 4: Preparation of N-(2-(((S)-6-amino-1-((4-(((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxohexyl-2-yl)amino)-2-oxoethyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-11)

将((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a)-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-5-(2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)乙酰胺基)-6-氧代己基)氨基甲酸叔丁酯(B-11-3,26mg,21.39μmol)溶于DCM(1mL)中，加入TFA(1mL)，加毕在25℃反应1小时。反应液室温减压蒸干后经制备高效液相色谱纯化后冷冻干燥得到标题化合物(1.04mg,0.923μmol)。The ((S)-6-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a)-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[ [e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-5-(2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)acetamyl)-6-oxohexyl)tert-butyl carbamate (B-11-3, 26 mg, 21.39 μmol) was dissolved in DCM (1 mL), and TFA (1 mL) was added. After the addition was complete, the reaction mixture was reacted at 25 °C for 1 hour. The reaction solution was evaporated to dryness under reduced pressure at room temperature, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (1.04 mg, 0.923 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1116.3[M+H]+ MS m/z(ESI): 1116.3 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例四:2,2’,2”-(10-(2-(((S)-6-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮卓-2-基)苯基)氨基)-1-氧 代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-5-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-6-氧代己基)氨基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-1)的制备
Example 4: 2,2',2”-(10-(2-(((S)-6-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxo Preparation of propan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-5-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

步骤一：N-((S)-6-((二苯基(对甲苯基)甲基)氨基)-1-(((S)-1-(((S)-1-((4-((S)-7)-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-1-氧代己烷-2-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-1-1)的制备Step 1: N-((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((S)-1-(((S)-1-((4-((S)-7)-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl) Preparation of benzo[e]pyrrole[1,2-a][1,4]diazepine-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-1-oxohexane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-1-1)

将N6-(二苯基(对甲苯基)甲基)-N2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰基)-L-赖氨酸(INT-6,31.10mg,47.65μmol)溶于DMF(1mL)中，依次加入DIPEA(12.32mg)、HATU(19.31mg)和(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代- 5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7,27mg,31.76μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(20mg,13.47μmol)。 N6- (diphenyl(p-tolyl)methyl) -N2- (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-lysine (INT-6, 31.10 mg, 47.65 μmol) was dissolved in DMF (1 mL), and DIPEA (12.32 mg), HATU (19.31 mg), and (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo- 5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (INT-7, 27 mg, 31.76 μmol) was added and reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (20 mg, 13.47 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1485.8[M+H]+ MS m/z(ESI): 1485.8 [M+H] +

步骤二：N-((S)-6-氨基-1-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-1-氧代己烷-2-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-1-2)的制备Step 2: N-((S)-6-amino-1-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy) Preparation of -5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-1-oxohexane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-1-2)

将N-((S)-6-((二苯基(对甲苯基)甲基)氨基)-1-(((S)-1-(((S)-1-((4-((S)-7)-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-1-氧代己烷-2-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-1-1,20mg,13.47μmol)溶于DCM(1mL)中，加入甲酸(2mL)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(15mg,11.77μmol)。N-((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-(((S)-1-(((S)-1-((4-((S)-7)-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,1 1a-Tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diazepine-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-1-oxohexane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-1-1, 20 mg, 13.47 μmol) was dissolved in DCM (1 mL), and formic acid (2 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (15 mg, 11.77 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1275.5[M+H]+ MS m/z(ESI): 1275.5 [M+H] +

步骤三：2,2’,2”-(10-(2-(((S)-6-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-5-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-6-氧代己基)氨基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-1)的制备Step 3: 2,2’,2”-(10-(2-(((S)-6-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11, Preparation of 11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diazazo-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-5-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynylamido)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-1)

将N-((S)-6-氨基-1-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-1-氧代己烷-2-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-1-2,15mg,11.77μmol)溶于DMF(1mL)中，依次加入DIPEA(8.42mg)和 2,2’,2”-(10-(2-((2,5-二氧代吡咯烷-1-基)氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(19.59mg,39.07μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(3.32mg,1.88μmol)。The N-((S)-6-amino-1-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1 H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-1-oxohexane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-1-2, 15 mg, 11.77 μmol) was dissolved in DMF (1 mL), and DIPEA (8.42 mg) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (19.59 mg, 39.07 μmol) was added and reacted at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (3.32 mg, 1.88 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1661.9[M+H]+ MS m/z(ESI): 1661.9 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例五:2,2’,2”-(10-((2S,5S,14S)-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-14-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-1,4,7,10,13,20-六氧代-3,6,9,12,19-五氮杂二十一烷-21-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-2)的制备
Example 5: 2,2',2”-(10-((2S,5S,14S)-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a Preparation of 1H-tetrahydro-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamido)-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentazatetracosano-21-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-2)

步骤一：(9H-芴-9-基)甲基(2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酸酯(A-2-1)的制备Step 1: (9H-fluorene-9-yl)methyl(2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8- Preparation of benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (A-2-1)

将(((9H-芴-9-基)甲氧基)羰基)甘氨酰甘氨酸(30.02mg,84.70μmol)溶于DMF(1mL)中， 依次加入DIPEA(27.37mg)、HATU(32.19mg)和(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7,60mg,70.59μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/1)纯化后冷冻干燥得标题化合物(75mg,63.22μmol)。其结构表征数据如下:Dissolve (((9H-fluorene-9-yl)methoxy)carbonyl)glycylglycine (30.02 mg, 84.70 μmol) in DMF (1 mL). DIPEA (27.37 mg), HATU (32.19 mg), and (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (INT-7, 60 mg, 70.59 μmol) were added sequentially, and the reaction was carried out at 25 °C for 1 hour after the addition was complete. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/1) and then freeze-dried to give the title compound (75 mg, 63.22 μmol). Its structural characterization data are as follows:

MS m/z(ESI):1187.4[M+H]+ MS m/z(ESI): 1187.4 [M+H] +

步骤二：(S)-2-(2-(2-氨基乙酰胺基)乙酰胺基)-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(A-2-2)的制备Step 2: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (A-2-2)

将(9H-芴-9-基)甲基(2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酸酯(A-2-1,75mg,63.22μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(60.95mg,63.22μmol)。The (9H-fluorene-9-yl)methyl(2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11, 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (A-2-1, 75 mg, 63.22 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction mixture was reacted at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (60.95 mg, 63.22 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):965.1[M+H]+ MS m/z (ESI): 965.1 [M+H] +

步骤三：N-((7S,16S,19S)-16-异丙基-20-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-19-甲基-8,11,14,17,20-五氧代-1,1-二苯基-1-(对甲苯基)-2,9,12,15,18-五氮杂二十烷-7-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-2-3)的制备Step 3: N-((7S,16S,19S)-16-isopropyl-20-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,1 Preparation of 1,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-19-methyl-8,11,14,17,20-pentoxo-1,1-diphenyl-1-(p-tolyl)-2,9,12,15,18-pentazaeicosaecan-7-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-2-3)

将N6-(二苯基(对甲苯基)甲基)-N2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-L-赖氨酸(INT-6,32.50mg,49.79μmol)溶于DMF(1mL)中，依次加入DIPEA(16.08mg)、HATU(18.92mg)和(S)-2-(2-(2-氨基乙酰胺基)乙酰胺基)-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2- 基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(A-2-2,27mg,31.76μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(40mg,25.02μmol)。N- 6- (diphenyl(p-tolyl)methyl)-N- 2- (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)-L-lysine (INT-6, 32.50 mg, 49.79 μmol) was dissolved in DMF (1 mL), and then DIPEA (16.08 mg), HATU (18.92 mg), and (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((4-((S))) were added sequentially. -7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2- (A-2-2, 27 mg, 31.76 μmol) was added and reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (40 mg, 25.02 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1599.9[M+H]+ MS m/z(ESI): 1599.9 [M+H] +

步骤四：N-((2S,5S,14S)-18-氨基-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-1,4,7,10,13-五氧代-3,6,9,12-四氮杂十八烷-14-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-2-4)的制备Step 4: N-((2S,5S,14S)-18-amino-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy Preparation of 5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-1,4,7,10,13-pentoxo-3,6,9,12-tetraazaoctadecane-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-2-4)

将N-((7S,16S,19S)-16-异丙基-20-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-19-甲基-8,11,14,17,20-五氧代-1,1-二苯基-1-(对甲苯基)-2,9,12,15,18-五氮杂二十烷-7-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-2-3,40mg,25.02μmol)溶于DCM(1mL)中，加入甲酸(2mL)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(15mg,11.17μmol)。N-((7S,16S,19S)-16-isopropyl-20-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2] [1,4]diaza-2-yl)phenyl)amino)-19-methyl-8,11,14,17,20-pentoxo-1,1-diphenyl-1-(p-tolyl)-2,9,12,15,18-pentazaeicosano-7-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-2-3, 40 mg, 25.02 μmol) was dissolved in DCM (1 mL), and formic acid (2 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (15 mg, 11.17 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1343.6[M+H]+ MS m/z(ESI): 1343.6 [M+H] +

步骤五：2,2’,2”-(10-((2S,5S,14S)-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-14-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-1,4,7,10,13,20-六氧代-3,6,9,12,19-五氮杂二十一烷-21-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-2)的制备Step 5: 2,2’,2”-(10-((2S,5S,14S)-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a Preparation of 1H-tetrahydro-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamido)-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentazatetracosano-21-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-2)

将N-((2S,5S,14S)-18-氨基-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-1,4,7,10,13-五氧代-3,6,9,12-四氮杂十八烷-14-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺 (A-2-4,15mg,11.17μmol)溶于DMF(1mL)中，依次加入DIPEA(4.32mg)和2,2’,2”-(10-(2-((2,5-二氧代吡咯烷-1-基)氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(16.79mg,33.51μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(2.16mg,1.24μmol)。N-((2S,5S,14S)-18-amino-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl (I)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-1,4,7,10,13-pentoxo-3,6,9,12-tetraazaoctadecane-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (A-2-4, 15 mg, 11.17 μmol) was dissolved in DMF (1 mL), and DIPEA (4.32 mg) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (16.79 mg, 33.51 μmol) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (2.16 mg, 1.24 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1730.1[M+H]+ MS m/z(ESI): 1730.1 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例六:2,2’,2”-(10-((2S,5S,14S)-23-羧基-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-14-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)-1,4,7,10,13,20-六氧代-3,6,9,12,19-五氮杂二十三烷-23-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-3)的制备
Example 6: 2,2',2”-(10-((2S,5S,14S)-23-carboxy-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11 Preparation of 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-14-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)-1,4,7,10,13,20-hexaoxo-3,6,9,12,19-pentazatoritriane-23-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-3)

将N-((2S,5S,14S)-18-氨基-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-1,4,7,10,13-五氧代-3,6,9,12-四氮杂十八烷-14-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺(A-2-4,15mg,11.17μmol)溶于DMF(1mL)中，依次加入DIPEA(4.32mg)和2,2’,2”-(10-(1-羧基-4-(4-硝基苯氧基)-4-氧代丁基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(20.00mg,33.51μmol)，加毕在25℃反应2小时。反应液直接经快速柱层析(C18,水/乙腈＝1/1)纯化后冷冻干燥得标题化合物(2.98mg,1.64μmol)。N-((2S,5S,14S)-18-amino-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-1,4,7,10,13-pentoxo-3,6,9,12-tetraazaoctadecane-14- A 2-4 compound (15 mg, 11.17 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (4.32 mg) and 2,2',2”-(10-(1-carboxy-4-(4-nitrophenoxy)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (20.00 mg, 33.51 μmol). The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/1) and then freeze-dried to give the title compound (2.98 mg, 1.64 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1802.0[M+H]+ MS m/z(ESI): 1802.0 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例七:2,2’,2”-(10-(2-(((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-5-((S)-3-甲基-2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-酰胺基)丁酰胺基)-6-氧代己基)氨基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-4)的制备
Example 7: 2,2',2”-(10-(2-(((S)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11, Preparation of 11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)butamido)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-4)

将N-((S)-1-(((S)-6-氨基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-10,20mg,17.28μmol)溶于DMF(1mL)中，依次加入DIPEA(6.69mg)和2,2’,2”-(10-(2-((2,5-二氧代吡咯烷-1-基)氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(26.00,51.84μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(7.79mg,5.00μmol)。The N-((S)-1-(((S)-6-amino-1-((4-(((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohexyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-6- (2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide (B-10, 20 mg, 17.28 μmol) was dissolved in DMF (1 mL), and DIPEA (6.69 mg) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (26.00, 51.84 μmol) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (7.79 mg, 5.00 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1544.8[M+H]+ MS m/z(ESI): 1544.8 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm) Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例八:2,2’,2”-(10-(1-羧基-4-(((S)-6-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-5-((S)-3-甲基-2-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)-6-氧代己基)氨基)-4-氧代丁基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(A-5)的制备
Example 8: 2,2',2”-(10-(1-carboxyl-4-(((S)-6-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,1 Preparation of 1,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-5-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)butamido)-6-oxohexyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (A-5)

将N-((S)-1-(((S)-6-氨基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(B-10,20mg,17.28μmol)溶于DMF(1mL)中，依次加入DIPEA(6.70mg)和2,2’,2”-(10-(1-羧基-4-(4-硝基苯氧基)-4-氧代丁基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(10.33mg,17.28μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(11.83mg,7.25μmol)。 The N-((S)-1-(((S)-6-amino-1-((4-(((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxohex-2-yl)amino)-3-methyl-1-oxobut-2-yl)- 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide (B-10, 20 mg, 17.28 μmol) was dissolved in DMF (1 mL), and DIPEA (6.70 mg) and 2,2',2”-(10-(1-carboxy-4-(4-nitrophenoxy)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (10.33 mg, 17.28 μmol) were added sequentially. The reaction mixture was then reacted at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (11.83 mg, 7.25 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1616.8[M+H]+ MS m/z(ESI): 1616.8 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例九：N-((29S,32S,35S)-36-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-29,32,35-三甲基-27,30,33,36-四氧代-3,6,9,12,15,18,21,24-八氧杂-28,31,34-三氮杂三十六烷基)-3,5-双(2-(甲磺酰基)嘧啶-5-基)苯甲酰胺(G-5)的制备
Example 9: N-((29S,32S,35S)-36-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11 Preparation of α-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-29,32,35-trimethyl-27,30,33,36-tetraoxo-3,6,9,12,15,18,21,24-octaoxa-28,31,34-triazahexadecyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-5)

步骤一：(9H-芴-9-基)甲基((S)-1-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)氨基甲酸酯(G-5-1)的制备Step 1: Preparation of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (G-5-1)

将(((9H-芴-9-基)甲氧基)羰基)-L-丙氨酰-L-丙氨酰-L-丙氨酸(19.27mg,38.25μmol)，(S)-8-((5-(((S)-2-(4-氨基苯基)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯[1,2-a][1,4]二氮杂-8-基)氧基)戊基)氧基)-7-甲氧基-1,10,11,11a-四氢-3H,5H-螺[苯并[e]吡咯[1,2-a][1,4]二氮杂-2,1’-环丙烷]-5-酮(20mg,29.42μmol)，HATU(16.78mg,44.13μmol)溶于干燥DMF(0.5mL)，滴入DIPEA(11.41mg,88.26μmol)，搅拌反应2小时。加入乙酸乙酯和水，搅拌，静置分液，有机相用饱和食盐水洗涤后减压浓缩；硅胶柱纯化(洗脱剂:0-5％甲醇/二氯甲烷)后再次浓缩得标题化合物(10mg,8.97μmol)The following ingredients were added: (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanyl-L-alanine (19.27 mg, 38.25 μmol), (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7- Methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (20 mg, 29.42 μmol), HATU (16.78 mg, 44.13 μmol) dissolved in dry DMF (0.5 mL), and DIPEA (11.41 mg, 88.26 μmol) was added dropwise. The mixture was stirred for 2 hours. Ethyl acetate and water were added, the mixture was stirred, allowed to stand, and separated. The organic phase was washed with saturated brine and concentrated under reduced pressure. After purification by silica gel column chromatography (eluent: 0-5% methanol/dichloromethane), the mixture was concentrated again to give the title compound (10 mg, 8.97 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1116.6[M+H]+ MS m/z(ESI): 1116.6 [M+H] +

步骤二：(S)-2-氨基-N-((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代- 5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)丙酰胺(G-5-2)的制备Step 2: (S)-2-amino-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo- Preparation of 5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (G-5-2)

将((9H-芴-9-基)甲基((S)-1-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)氨基甲酸酯(10mg,8.97μmol)溶于DMF(1mL)，搅拌下滴入二乙胺(0.5mL)，继续反应0.5小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(5mg,5.60μmol)。The ((9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo) [e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)carbamate (10 mg, 8.97 μmol) was dissolved in DMF (1 mL), and diethylamine (0.5 mL) was added dropwise with stirring. The reaction was continued for 0.5 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (5 mg, 5.60 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):894.3[M+H]+ MS m/z(ESI): 894.3 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤三：N-((29S,32S,35S)-36-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-29,32,35-三甲基-27,30,33,36-四氧代-3,6,9,12,15,18,21,24-八氧杂-28,31,34-三氮杂三十六烷基)-3,5-双(2-(甲磺酰基)嘧啶-5-基)苯甲酰胺(G-5)的制备Step 3: N-((29S,32S,35S)-36-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a Preparation of tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-29,32,35-trimethyl-27,30,33,36-tetraoxo-3,6,9,12,15,18,21,24-octaoxa-28,31,34-triazahexadecyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-5)

将1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17,20,23,26-八氧杂-2-氮杂壬烷-29-酸(Int-2，5.28mg,6.16μmol)和(S)-2-氨基-N-((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环 丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)丙酰胺(5mg,5.60μmol)溶于DMF(1mL)，加入HATU(3.19mg,8.40μmol)，滴入DIPEA(2.17mg,16.80μmol)，搅拌反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(3.53mg,1.94μmol)。1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonane-29-acid (Int-2, 5.28 mg, 6.16 μmol) and (S)-2-amino-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclo) [Propyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxopropyl-2-yl)propionamide (5 mg, 5.60 μmol) was dissolved in DMF (1 mL), HATU (3.19 mg, 8.40 μmol) was added, and DIPEA (2.17 mg, 16.80 μmol) was added dropwise. The mixture was stirred for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (3.53 mg, 1.94 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1733.2[M+H]+ MS m/z(ESI): 1733.2 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例十：N-((31S,34S)-31-异丙基-35-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-3,6,9,12,15,18,21,24,27,34-十甲基-2,5,8,11,14,17,20,23,26,29,32,35-十二氧代-3,6,9,12,15,18,21,24,27,30,33-十一氮杂三十五烷烷基)-N-甲基-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-6)的制备
Example 10: N-((31S,34S)-31-isopropyl-35-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- Preparation of [1,4]diaza-2-yl)phenyl)amino)-3,6,9,12,15,18,21,24,27,34-decamethyl-2,5,8,11,14,17,20,23,26,29,32,35-dodecoxo-3,6,9,12,15,18,21,24,27,30,33-undecazapentadecyl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-6)

将1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(INT-1)(13.47mg,11.76μmol)、(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7)(10mg,11.76μmol)和HATU(6.71mg,17.65μmol)溶于干燥DMF(0.5mL)，滴入DIPEA(4.56mg,35.29μmol)，搅拌反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(13.51mg,6.40μmol)。1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (INT-1) (13.47 mg, 11.76 μmol), (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetra- Hydrogen-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (INT-7) (10 mg, 11.76 μmol) and HATU (6.71 mg, 17.65 μmol) were dissolved in dry DMF (0.5 mL), and DIPEA (4.56 mg, 35.29 μmol) was added dropwise. The mixture was stirred for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (13.51 mg, 6.40 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1977.3[M+H]+ MS m/z(ESI):1977.3[M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

实施例十一：N-((31S,34S,37S)-38-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-3,6,9,12,15,18,21,24,27,31,34,37-十二甲基-2,5,8,11,14,17,20,23,26,29,32,35,38-十三氧代-3,6,9,12,15,18,21,24,27,30,33,36-十二氮杂三十八烷基)-N-甲基-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-7)的制备
Example 11: N-((31S,34S,37S)-38-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4] Preparation of diaza-2-yl)phenyl)amino)-3,6,9,12,15,18,21,24,27,31,34,37-dodecanoyl-2,5,8,11,14,17,20,23,26,29,32,35,38-tetraoxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecanoyl)octadecyl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-7)

将1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(INT-1)(12.82mg,11.20μmol)、(S)-2-氨基-N-((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-1-氧代丙-2-基)丙酰胺(10mg,11.20μmol)和HATU(6.38mg,16.80μmol)溶于干燥DMF(0.5mL)，滴入DIPEA(4.56mg,35.29μmol)，搅拌反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(5.5mg,2.30μmol)。1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (INT-1) (12.82 mg, 11.20 μmol), (S)-2-amino-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11) α-Tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxopropane-2-yl)propionamide (10 mg, 11.20 μmol) and HATU (6.38 mg, 16.80 μmol) were dissolved in dry DMF (0.5 mL), and DIPEA (4.56 mg, 35.29 μmol) was added dropwise. The reaction mixture was stirred for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (5.5 mg, 2.30 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):2020.3[M+H]+ MS m/z(ESI):2020.3[M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

实施例十二：N-((37S,40S)-37-异丙基-41-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-3,6,9,12,15,18,21,24,27,40-十甲基-2,5,8,11,14,17,20,23,26,29,32,35,38,41-十四氧代-3,6,9,12,15,18,21,24,27,30,33,36,39-十三氮杂四十一烷基)-N-甲基-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-8)的制备
Example 12: N-((37S,40S)-37-isopropyl-41-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1, Preparation of [4]diaza-2-yl)phenyl)amino)-3,6,9,12,15,18,21,24,27,40-decamethyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecano-3,6,9,12,15,18,21,24,27,30,33,36,39-tetrazata-tetradecanoyl)-N-methyl-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-8)

步骤一：(9H-芴-9-基)甲基(2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5- 氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酸酯(G-8-1)的制备Step 1: (9H-fluorene-9-yl)methyl(2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5- Preparation of oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (G-8-1)

将(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7)(10mg,11.76μmol)溶于DMF(1mL)，加入HATU(6.71mg,17.65μmol)，滴入DIPEA(4.56mg,35.29μmol)，搅拌反应1小时。反应液直接进行下一步反应。The (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1 H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (INT-7) (10 mg, 11.76 μmol) was dissolved in DMF (1 mL), HATU (6.71 mg, 17.65 μmol) was added, and DIPEA (4.56 mg, 35.29 μmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was then directly used for the next step of the reaction.

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1186.3[M+H]+ MS m/z(ESI): 1186.3 [M+H] +

步骤二：(S)-2-(2-(2-氨基乙酰胺基)乙酰胺基)-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S))-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(G-8-2)的制备Step 2: Preparation of (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S))-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (G-8-2)

将(9H-芴-9-基)甲基(2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酸酯的DMF反应液中滴入二乙胺(0.5mL)，继续搅拌反应0.5小时。将反应液减压浓缩后经制备高效液相色谱纯化后冷冻干燥得到标题化合物(5mg,5.19μmol)。(9H-fluorene-9-yl)methyl(2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5 Diethylamine (0.5 mL) was added dropwise to the DMF reaction solution of -oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate, and the reaction was stirred for 0.5 hours. The reaction solution was concentrated under reduced pressure, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (5 mg, 5.19 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):964.2[M+H]+ MS m/z(ESI): 964.2 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤三：N-((37S,40S)-37-异丙基-41-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-3,6,9,12,15,18,21,24,27,40-十甲基-2,5,8,11,14,17,20,23,26,29,32,35,38,41-十四氧代-3,6,9,12,15,18,21,24,27,30,33,36,39-十三氮杂四十一烷基)-N-甲基-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-8)的制备Step 3: N-((37S,40S)-37-isopropyl-41-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4 Preparation of diaza-2-yl)phenyl)amino)-3,6,9,12,15,18,21,24,27,40-decamethyl-2,5,8,11,14,17,20,23,26,29,32,35,38,41-tetradecano-3,6,9,12,15,18,21,24,27,30,33,36,39-tetrazata-tetradecanoyl)-N-methyl-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-8)

将1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-2,5,8,11,14,17,20,23,26,29-十甲基-1,4,7,10,13,16,19,22,25,28-十氧代-2,5,8,11,14,17,20,23,26,29-十氮杂三十一烷-31-羧酸(INT-1)(5.94mg,5.19μmol)和(S)-2-(2-(2-氨基乙酰胺基)乙酰胺基)-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S))-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(5mg,5.19μmol)溶于DMF(0.5mL)，加入HATU(2.96mg,7.78μmol)，滴入DIPEA(2.01mg,15.56μmol)，搅拌反应1小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(1.58mg,0.75μmol)。1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-2,5,8,11,14,17,20,23,26,29-decamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-2,5,8,11,14,17,20,23,26,29-decaazatrione-31-carboxylic acid (INT-1) (5.94 mg, 5.19 μmol) and (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S))-7-methoxy-5-oxo- 5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (5 mg, 5.19 μmol) was dissolved in DMF (0.5 mL), HATU (2.96 mg, 7.78 μmol) was added, and DIPEA (2.01 mg, 15.56 μmol) was added dropwise. The mixture was stirred for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (1.58 mg, 0.75 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):2091.3[M+H]+ MS m/z(ESI): 2091.3 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例十三:2,2’,2”-(10-(2-(((S)-5-(2,4-双(2-(甲磺酰基)嘧啶-5-基)苯甲酰氨基)-6-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-6-氧代己基)氨基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(G-9)的制备
Example 13: 2,2',2”-(10-(2-(((S)-5-(2,4-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoylamino)-6-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1' Preparation of [-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-9)

步骤一:((S)-5-(2,4-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺基)-6-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4] 二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-6-氧代己基)氨基甲酸叔丁酯(G-9-1)的制备Step 1: ((S)-5-(2,4-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]) Preparation of tert-butyl carbamate (G-9-1) of diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-6-oxohexyl)carbamate

将((S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(30.0mg,35.3μmol)，N2-(2,4-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰基)-N6-(叔丁氧基羰基)-L-赖氨酸(25.8mg,38.9μmol)，溶于DMF(2mL)中，依次加入DIPEA(15mg)、HATU(20.2mg)，加毕在25℃反应1小时。反应完毕后，经快速柱层析(C18,水/乙腈＝0.75)纯化后冷冻干燥得标题化合物(37.1mg,24.7μmol)。The following ingredients were added: ((S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (30.0 mg, 35.3 μmol), N2- (2,4-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoyl) -N6 -(tert-butoxycarbonyl)-L-lysine (25.8 mg, 38.9 μmol) was dissolved in DMF (2 mL), and DIPEA (15 mg) and HATU (20.2 mg) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 1 hour. After the reaction was completed, the product was purified by rapid column chromatography (C18, water/acetonitrile = 0.75) and then freeze-dried to give the title compound (37.1 mg, 24.7 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1494.8[M+H]+ MS m/z(ESI): 1494.8 [M+H] +

步骤二:N-((S)-6-氨基-1-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-1-氧代己烷-2-基)-2,4-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-9-2)的制备Step 2: N-((S)-6-amino-1-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy) Preparation of -5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-1-oxohexane-2-yl)-2,4-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-9-2)

将((S)-5-(2,4-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺基)-6-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-6-氧代己基)氨基甲酸叔丁酯(37.1mg,24.7μmol)，溶于DCM(2mL)中，加入三氟乙酸(1mL)，加毕在25℃反应0.5小时。反应完毕后，经快速柱层析(C18,水/乙腈＝0.6)纯化后冷冻干燥得标题化合物(30.9mg,22.2μmol)。The radical is ((S)-5-(2,4-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)-6-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy 5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-6-oxohexyl)tert-butyl carbamate (37.1 mg, 24.7 μmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (1 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 0.5 h. After the reaction was completed, the mixture was purified by rapid column chromatography (C18, water/acetonitrile = 0.6) and then freeze-dried to give the title compound (30.9 mg, 22.2 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1394.8[M+H]+ MS m/z(ESI): 1394.8 [M+H] +

步骤三:2,2’,2”-(10-(2-(((S)-5-(2,4-双(2-(甲磺酰基)嘧啶-5-基)苯甲酰氨基)-6-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡 咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-6-氧代己基)氨基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(G-9)的制备Step 3: 2,2',2”-(10-(2-(((S)-5-(2,4-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzoylamino)-6-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrene]) Preparation of pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-6-oxohexyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-9)

将N-((S)-6-氨基-1-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂卓-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-1-氧代己烷-2-基)-2,4-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(30.9mg,22.2μmol)溶于DMF(2mL)中，加入DIPEA(15mg)，加入2,2’,2”-(10-(2-((2,5-二氧代吡咯烷-1-基)氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(33.4mg,66.6μmol)，反应完毕后，反应液直接经制备高效液相色谱纯化后冷冻干燥得标题化合物(12.0mg,6.7μmol)。N-((S)-6-amino-1-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropan]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl) (Amino)-1-oxohexane-2-yl)-2,4-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (30.9 mg, 22.2 μmol) was dissolved in DMF (2 mL), DIPEA (15 mg) was added, and 2,2’,2”-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (33.4 mg, 66.6 μmol) was added. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (12.0 mg, 6.7 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):[M/2+H]+＝1780.9MS m/z(ESI):[M/2+H] + =1780.9

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

实施例十四:2,2’,2”-(10-((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S))-7-甲氧基-5-氧代-5,10,11,11a-四氢)-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酰基)-1,20,28-三氧代-5,8,11,14,17-五氧杂-2,21,27-三氮杂二十九烷-29-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(G-10)的制备
Example 14: 2,2',2”-(10-((S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S))-7-methoxy-5-oxo-5,10,11,11a-tetrahydro)-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5 Preparation of -oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamoyl)-1,20,28-trioxo-5,8,11,14,17-pentaoxa-2,21,27-triazanonadecan-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-10)

步骤一：2,5-二氧代吡咯烷-1-基1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸酯(G-10-1)的制备Step 1: Preparation of 2,5-dioxopyrrolidine-1-yl 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosane-20-carboxylic acid ester (G-10-1)

将1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸(INT-5,30mg,41.33μmol)溶于DMF(2mL)中，依次加入DIPEA(8.04mg)、EDCI(23.81mg)和吡咯烷-2,5-二酮(4.91mg,49.60μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(25mg,30.41μmol)。1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicoseoane-20-carboxylic acid (INT-5, 30 mg, 41.33 μmol) was dissolved in DMF (2 mL), followed by the addition of DIPEA (8.04 mg), EDCI (23.81 mg), and pyrrolidine-2,5-dione (4.91 mg, 49.60 μmol). The reaction mixture was then reacted at 25 °C for 1 hour. The reaction solution was purified directly by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (25 mg, 30.41 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):823.2[M+H]+ MS m/z(ESI): 823.2 [M+H] +

步骤二：N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-酰基)-L-赖氨酸(G-10-2)的制备 Step 2: Preparation of N6 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N2- (1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosano-20-acyl)-L-lysine (G-10-2)

将2,5-二氧代吡咯烷-1-基1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸酯(G-10-1,25mg,30.41μmol)溶于DMF(1mL)中，加入DIPEA(11.77mg)和N6-(((9H-芴-9-基)甲氧基)羰基)-L-赖氨酸(13.43mg,36.49μmol)，加毕在25℃反应1小时。反应液直接冷冻干燥得到标题化合物(22mg,20.45μmol)。2,5-Dioxopyrrolidone-1-yl 1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosaecan-20-carboxylic acid ester (G-10-1, 25 mg, 30.41 μmol) was dissolved in DMF (1 mL), and DIPEA (11.77 mg) and N6-(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine (13.43 mg, 36.49 μmol) were added. After the addition was complete, the reaction mixture was reacted at 25 °C for 1 hour. The reaction solution was directly freeze-dried to give the title compound (22 mg, 20.45 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1077.2[M+H]+ MS m/z(ESI): 1077.2 [M+H] +

步骤三：(9H-芴-9-基)甲基((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H),3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-1-氧代丁-2-基)氨基甲酰基)-1,20-二氧代-5,8,11,14,17-五氧杂-2,21-二氮杂二十六烷-26-基)氨基甲酸甲酯(G-10-3)的制备Step 3: (9H-fluorene-9-yl)methyl((S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H),3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8 Preparation of methyl carbamate (G-10-3)

将N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-酰基)-L-赖氨酸(G-10-2,22mg,20.45μmol)溶于DMF(1mL)中，依次加入DIPEA(7.91mg)、HATU(9.32mg)和(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7,17.38mg,20.45μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(30mg,15.72μmol)。 N6 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N2- (1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-penta-2-azaeicosano-20-acyl)-L-lysine (G-10-2, 22 mg, 20.45 μmol) was dissolved in DMF (1 mL), and DIPEA (7.91 mg), HATU (9.32 mg), and (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy)) were added sequentially. -5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (INT-7, 17.38 mg, 20.45 μmol), after addition, reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (30 mg, 15.72 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1909.2[M+H]+ MS m/z(ESI): 1909.2 [M+H] +

步骤四：N-((20S,23S,26S)-20-(4-氨基丁基)-23-异丙基-27-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-26-甲基-18,21,24,27-四氧代-3,6,9,12,15-五氧杂-19,22,25-三氮杂二十七烷基)-3,5-双(2-(甲磺酰)嘧啶-5-基)苯甲酰胺(G-10-4)的制备Step 4: N-((20S,23S,26S)-20-(4-aminobutyl)-23-isopropyl-27-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy) Preparation of -5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-26-methyl-18,21,24,27-tetraoxo-3,6,9,12,15-pentaoxa-19,22,25-triazaheptadecyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-10-4)

将(9H-芴-9-基)甲基((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-(((S)-1-(((S)-1-((4- ((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H),3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-1-氧代丁-2-基)氨基甲酰基)-1,20-二氧代-5,8,11,14,17-五氧杂-2,21-二氮杂二十六烷-26-基)氨基甲酸甲酯(G-10-3,30mg,15.72μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应20分钟。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(6mg,3.56μmol)。(9H-fluorene-9-yl)methyl((S)-1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-22-(((S)-1-(((S)-1-((4-) ((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H),3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza- Methyl 2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-1-oxobut-2-yl)carbamoyl)-1,20-dioxo-5,8,11,14,17-pentaoxa-2,21-diazahexacosane-26-yl)carbamate (G-10-3, 30 mg, 15.72 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. After the addition was complete, the reaction mixture was reacted at 25 °C for 20 min. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (6 mg, 3.56 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1687.0[M+H]+ MS m/z(ESI): 1687.0 [M+H] +

步骤五：2,2’,2”-(10-((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S))-7-甲氧基-5-氧代-5,10,11,11a-四氢)-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基甲酰基)-1,20,28-三氧代-5,8,11,14,17-五氧杂-2,21,27-三氮杂二十九烷-29-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(G-10)的制备Step 5: 2,2’,2”-(10-((S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S))-7-methoxy-5-oxo-5,10,11,11a-tetrahydro)-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5- Preparation of oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamoyl)-1,20,28-trioxo-5,8,11,14,17-pentaza-2,21,27-triazanonadecan-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-10)

将N-((20S,23S,26S)-20-(4-氨基丁基)-23-异丙基-27-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-26-甲基-18,21,24,27-四氧代-3,6,9,12,15-五氧杂-19,22,25-三氮杂二十七烷基)-3,5-双(2-(甲磺酰)嘧啶-5-基)苯甲酰胺(G-10-4,6mg,3.56μmol)溶于DMF(1mL)中，依次加入DIPEA(1.41mg)和2,2’,2”-(10-(2-((2,5-二氧代吡咯烷-1-基)氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(5.49mg,10.95μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(0.98mg,0.47μmol)。N-((20S,23S,26S)-20-(4-aminobutyl)-23-isopropyl-27-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-26-methyl-18,21,24,27-tetraoxo-3,6,9,12,15-penta- 19,22,25-Triazaheptadecyl)-3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)benzamide (G-10-4, 6 mg, 3.56 μmol) was dissolved in DMF (1 mL), and DIPEA (1.41 mg) and 2,2’,2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (5.49 mg, 10.95 μmol) were added sequentially. After the addition was complete, the reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (0.98 mg, 0.47 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):2073.4[M+H]+ MS m/z(ESI): 2073.4 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例十五:2,2’,2”-(10-((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酰基)-1,20,28-三氧代-5,8,11,14,17-五氧杂-2,21,27-三氮杂二十九烷-29-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(G-11)的制备
Example 15: 2,2',2”-(10-((S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10 Preparation of ,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamoyl)-1,20,28-trioxo-5,8,11,14,17-pentaoxa-2,21,27-triazanonadecan-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-11)

步骤一：(9H-芴-9-基)甲基((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酰基)-1,20-二氧代-5,8,11,14,17-五氧杂-2,21-二氮杂二十六烷-26-基)氨基甲酸酯(G-11-1)的制备Step 1: (9H-fluorene-9-yl)methyl((S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl Preparation of 5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamoyl)-1,20-dioxo-5,8,11,14,17-pentaoxa-2,21-diazahexacosane-26-yl)carbamate (G-11-1)

将N6-(((9H-芴-9-基)甲氧基)羰基)-N2-(1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-酰基)-L-赖氨酸(G-10-2,33.49mg,31.12μmol)溶于DMF(1mL)中，依次加入DIPEA(10.05mg)、HATU(11.82mg)和(S)-2-(2-(2-氨基乙酰胺基)乙酰胺基)-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四 氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(A-2-2,25mg,25.93μmol)，加毕在25℃反应1小时。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(25mg,12.36μmol)。 N6 -(((9H-fluorene-9-yl)methoxy)carbonyl) -N2- (1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-penta-2-azaeicosano-20-acyl)-L-lysine (G-10-2, 33.49 mg, 31.12 μmol) was dissolved in DMF (1 mL), and DIPEA (10.05 mg), HATU (11.82 mg), and (S)-2-(2 -(2-Aminoacetamido)acetamido)-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro) Hydrogen-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (A-2-2, 25 mg, 25.93 μmol) was added and reacted at 25 °C for 1 hour. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (25 mg, 12.36 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):2023.3[M+H]+ MS m/z(ESI):2023.3[M+H] +

步骤二：N-((2S,5S,14S)-14-(4-氨基丁基)-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-1,4,7,10,13,16-六氧代-19,22,25,28,31-五氧杂-3,6,9,12,15-五氮杂三十三烷-33-基)-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-11-2)的制备Step 2: N-((2S,5S,14S)-14-(4-aminobutyl)-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,1 Preparation of 0,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-1,4,7,10,13,16-hexaoxo-19,22,25,28,31-pentaoxa-3,6,9,12,15-pentazatritane-33-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-11-2)

将(9H-芴-9-基)甲基((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酰基)-1,20-二氧代-5,8,11,14,17-五氧杂-2,21-二氮杂二十六烷-26-基)氨基甲酸酯(G-11-1,25mg,12.36μmol)溶于DMF(1mL)中，加入二乙胺(1mL)，加毕在25℃反应20分钟。反应液直接经快速柱层析(C18,水/乙腈＝1/2)纯化后冷冻干燥得标题化合物(8.90mg,4.94μmol)。(9H-fluorene-9-yl)methyl((S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-((((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1 H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamoyl)-1,20-dioxo-5,8,11,14,17-pentaoxa-2,21-diazahexacosane-26-yl)carbamate (G-11-1, 25 mg, 12.36 μmol) was dissolved in DMF (1 mL), and diethylamine (1 mL) was added. The reaction mixture was then reacted at 25 °C for 20 min. The reaction solution was directly purified by rapid column chromatography (C18, water/acetonitrile = 1/2) and then freeze-dried to give the title compound (8.90 mg, 4.94 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1801.1[M+H]+ MS m/z(ESI): 1801.1 [M+H] +

步骤三：2,2’,2”-(10-((S)-1-(3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯基)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-2-氧代乙基)氨基)-2-氧代乙基)氨基甲酰基)-1,20,28-三氧代-5,8,11,14,17-五氧杂-2,21,27-三氮杂二十九烷-29-基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(G-11)的制备Step 3: 2,2’,2”-(10-((S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)phenyl)-22-((2-((2-(((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10, Preparation of 11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamoyl)-1,20,28-trioxo-5,8,11,14,17-pentaoxa-2,21,27-triazanonadecan-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (G-11)

将N-((2S,5S,14S)-14-(4-氨基丁基)-5-异丙基-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊 基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-2-甲基-1,4,7,10,13,16-六氧代-19,22,25,28,31-五氧杂-3,6,9,12,15-五氮杂三十三烷-33-基)-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-11-2,8.90mg,4.94μmol)溶于DMF(1mL)中，依次加入DIPEA(1.91mg)和2,2’,2”-(10-(2-((2,5-二氧代吡咯烷-1-基)氧基)-2-氧代乙基)-1,4,7,10-四氮杂环十二烷-1,4,7-三基)三乙酸(7.42mg,14.82μmol)，加毕在25℃反应2小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(4.32mg,1.96μmol)。N-((2S,5S,14S)-14-(4-aminobutyl)-5-isopropyl-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl 5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-2-methyl-1,4,7,10,13,16-hexaoxo-19,22,25,28,31-pentaoxa-3,6,9,12,15-pentazatritane-33-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-11-2, 8.90 mg, 4.94 μmol) l) The compound was dissolved in DMF (1 mL), and DIPEA (1.91 mg) and 2,2',2”-(10-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (7.42 mg, 14.82 μmol) were added sequentially. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (4.32 mg, 1.96 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):2187.4[M+H]+ MS m/z(ESI): 2187.4 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例十六:N-((20S,23S)-20-异丙基-24-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-23-甲基-18,21,24-三氧代-3,6,9,12,15-五氧杂-19,22-二氮杂二十四烷基)-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-1)的制备
Example 16: N-((20S,23S)-20-isopropyl-24-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo Preparation of 5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-23-methyl-18,21,24-trioxo-3,6,9,12,15-pentaoxa-19,22-diazatetracosyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-1)

将1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸(INT-5,20.49mg,28.23μmol)溶于DMF(1mL)中，依次加入DIPEA(9.12mg)、HATU(10.73mg)和(S)-2-氨基-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-)5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(INT-7,20mg,23.53μmol)，加毕在25℃反应1小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(17.5mg,11.12μmol)。1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicosano-20-carboxylic acid (INT-5, 20.49 mg, 28.23 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (9.12 mg), HATU (10.73 mg), and (S)-2-amino-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5) -O-)5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (INT-7, 20 mg, 23.53 μmol), added and reacted at 25 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (17.5 mg, 11.12 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1558.8[M+H]+ MS m/z(ESI): 1558.8 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例十七:N-((26S,29S)-26-异丙基-30-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧 基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-29-甲基-18,21,24,27,30-五氧代-3,6,9,12,15-五氧代-19,22,25,28-四氮杂三十烷基)-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(G-12)的制备
Example 17: N-((26S,29S)-26-isopropyl-30-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropyl]-8-yl)oxy)pentyl)oxy Preparation of 2,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-12)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-29-methyl-18,21,24,27,30-pentoxo-3,6,9,12,15-pentoxo-19,22,25,28-tetraazatrianealkyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (G-12)

将1-(3,5-双(2-(甲磺酰基)嘧啶-5-基)苯基)-1-氧代-5,8,11,14,17-五氧杂-2-氮杂二十烷-20-羧酸(INT-5,18.07mg,24.89μmol)溶于DMF(1mL)中，依次加入DIPEA(8.04mg)、HATU(9.46mg)和(S)-2-(2-(2-氨基乙酰胺基)乙酰胺基)-N-((S)-1-((4-((S)-7-甲氧基-8-((5-(((S)-7-甲氧基-5-氧代-5,10,11,11a-四氢-1H,3H-螺[苯并[e]吡咯并[1,2-a][1,4]二氮杂-2,1’-环丙]-8-基)氧基)戊基)氧基)-5-氧代-5,10,11,11a-四氢-1H-苯并[e]吡咯并[1,2-a][1,4]二氮杂-2-基)苯基)氨基)-1-氧代丙-2-基)-3-甲基丁酰胺(A-2-2,20mg,23.53μmol)，加毕在25℃反应1小时。反应液直接经制备高效液相色谱纯化后冷冻干燥得到标题化合物(11.86mg,7.02μmol)。1-(3,5-bis(2-(methanesulfonyl)pyrimidin-5-yl)phenyl)-1-oxo-5,8,11,14,17-pentaoxa-2-azaeicoseoane-20-carboxylic acid (INT-5, 18.07 mg, 24.89 μmol) was dissolved in DMF (1 mL), followed by the addition of DIPEA (8.04 mg), HATU (9.46 mg), and (S)-2-(2-(2-aminoacetamido)acetamido)-N-((S)-1-((4-((S)-7-methoxy-8-((5-(((S))) -7-Methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (A-2-2, 20 mg, 23.53 μmol), added and reacted at 25 °C for 1 hour. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to give the title compound (11.86 mg, 7.02 μmol).

其结构表征数据如下:Its structural characterization data are as follows:

MS m/z(ESI):1672.9[M+H]+ MS m/z(ESI): 1672.9 [M+H] +

其制备方法如下:Its preparation method is as follows:

色谱柱:Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A:乙腈；流动相B:水(0.05％甲酸)

Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

本申请所涉及的药物-连接体化合物均可以参照上述实施例的制备用类似的方法获得。The drug-linker compounds involved in this application can all be obtained using similar methods to those described in the above embodiments.

二、抗体的制备及结合活性测定II. Antibody Preparation and Binding Activity Assay

1、抗体的获得及纯化1. Antibody acquisition and purification

根据IMGT数据库中Trastuzumab氨基酸序列(IMGT/mAb-DB ID：97)，Pertuzumab氨基酸序列(IMGT/mAb-DB ID：80)，进行密码子优化并合成编码基因，构建至表达载体中，转染CHO细胞并加压筛选构建稳定表达细胞株。表达并收集上清，通过Protein A亲和填料纯化得到相应抗体。Based on the Trastuzumab amino acid sequence (IMGT/mAb-DB ID: 97) and Pertuzumab amino acid sequence (IMGT/mAb-DB ID: 80) from the IMGT database, codon optimization was performed, and the encoding gene was synthesized. This gene was then constructed into an expression vector, transfected into CHO cells, and subjected to pressure selection to establish a stable expression cell line. The expression was performed, and the supernatant was collected. The supernatant was then purified using Protein A affinity reagent to obtain the corresponding antibody.

三、包含细胞生物活性分子和连接体的化合物与抗体的偶联III. Conjugation of antibodies with compounds containing cellular bioactive molecules and linkers

以下实施例所制备得到的抗体药物偶联物中所涉及的抗体Trastuzumab和Pertuzumab即为上述第二部分中所述的Trastuzumab和Pertuzumab。The antibodies Trastuzumab and Pertuzumab involved in the antibody-drug conjugates prepared in the following examples are the same as those described in Part II above.

1、Trastuzumab-G-6的制备
1. Preparation of Trastuzumab-G-6

取1.235mL Trastuzumab抗体(16.2mg/mL)，用61.73uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至8.0，加入10mM TCEP(三(2-羧乙基)膦，75.8uL，pH 7.60)溶液混匀，室温放置1.5h。再依次加入相对于抗体的7倍物质的量的溶解在二甲基亚砜的G-6(97.44uL，10mM)溶液混匀，室温静置18h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物(即Trastuzumab-G-6)。质谱法测定DAR值为4.51。 1.235 mL of Trastuzumab antibody (16.2 mg/mL) was diluted with 61.73 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), and then the pH was adjusted to 8.0 with 1 M Na₂HPO₄ solution . 10 mM TCEP (tris(2-carboxyethyl)phosphine, 75.8 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 h. Then, 7 times the amount of G-6 dissolved in dimethyl sulfoxide (97.44 μL, 10 mM) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 h. Finally, the buffer solution was replaced with 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-G-6). The DAR value was 4.51 as determined by mass spectrometry.

2、Trastuzumab-G-8的制备
2. Preparation of Trastuzumab-G-8

取0.309mL Trastuzumab抗体(16.2mg/mL)，用35.4uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.6，加入10mM TCEP(三(2-羧乙基)膦，19uL，pH 7.60)溶液混匀，室温放置1.5h。再依次加入相对于抗体的6倍物质的量的溶解在二甲基亚砜的G-8(20.82uL，10mM)溶液混匀，室温静置18h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物(即Trastuzumab-G-8)。质谱法测定DAR值为4.46。Take 0.309 mL of Trastuzumab antibody (16.2 mg/mL), dilute with 35.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na₂HPO₄ solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 19 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 6 times the amount of G-8 solution (20.82 μL, 10 mM) dissolved in dimethyl sulfoxide and mix well. Incubate at room temperature for 18 h. After that, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-G-8). The DAR value was determined by mass spectrometry to be 4.46.

3、Trastuzumab-A-3的制备
3. Preparation of Trastuzumab-A-3

取1.601mL Trastuzumab抗体(14.8mg/mL)，用80uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至8.0，加入10mM TCEP(三(2-羧乙基)膦，49.0uL，pH 7.60)溶液混匀，室温放置1.5h。再加入相对于抗体的6.8倍物质的量的溶解在二甲基亚砜的A-3(112uL，10mM)溶液混匀，室温静置18h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物(即Trastuzumab-A-3)。质谱法测定DAR值为4.07。Take 1.601 mL of Trastuzumab antibody (14.8 mg/mL), dilute with 80 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 8.0 with 1 M Na₂HPO₄ solution . Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 49.0 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 6.8 times the amount of antibody dissolved in dimethyl sulfoxide (112 μL, 10 mM) A-3 solution and mix well. Incubate at room temperature for 18 h. After that, replace the buffer with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-A-3). The DAR value was 4.07 by mass spectrometry.

4、Trastuzumab-A-1的制备
4. Preparation of Trastuzumab-A-1

取1.857mL Trastuzumab抗体(14.7mg/mL)，加入93.0uL 20mM PB+0.1M EDTA(pH 7.60)，然后用1M Na2HPO4溶液调pH至7.6，加入10mM TCEP(三(2-羧乙基)膦，103.4uL，pH 7.60)溶液混匀，室温放置1.5h。再加入相对于抗体的10倍物质的量的溶解在二甲基亚砜的A-1(200.0uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物缀合物(即Trastuzumab-A-1)。质谱法测定DAR值为6.88。Take 1.857 mL of Trastuzumab antibody (14.7 mg/mL), add 93.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na₂HPO₄ solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 103.4 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 10 times the amount of antibody dissolved in dimethyl sulfoxide (200.0 μL, 10 mM) solution of A-1 and mix well. Incubate at room temperature for 2 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-A-1). The DAR value was determined by mass spectrometry to be 6.88.

5、Trastuzumab-A-2的制备
5. Preparation of Trastuzumab-A-2

取2.118mL Trastuzumab抗体(14.8mg/mL)，加入106.0uL 20mM PB+0.1M EDTA(pH 7.60)，然后用1M Na2HPO4溶液调pH至7.6，加入10mM TCEP(三(2-羧乙基)膦，49.0uL，pH 7.60)溶液混匀，室温放置1.5h。再加入相对于抗体的6.8倍物质的量的溶解在二甲基亚砜的A-2(112.0uL，10mM)溶液混匀，室温静置18h，完毕后采用NAP凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物缀合物(即Trastuzumab-A-2)。质谱法测定DAR值为4.07。Take 2.118 mL of Trastuzumab antibody (14.8 mg/mL), add 106.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na₂HPO₄ solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 49.0 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 6.8 times the amount of antibody dissolved in dimethyl sulfoxide A-2 (112.0 μL, 10 mM) solution and mix well. Incubate at room temperature for 18 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 6.0 using a NAP gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-A-2). The DAR value was determined by mass spectrometry to be 4.07.

本申请所涉及的ADC均可以参照上述偶联实施例的类似的方法通过将相应的药物-连接体化合物与抗体(例如Trastuzumab或Pertuzumab)获得。All ADCs involved in this application can be obtained by referring to the similar methods described in the above-described conjugation embodiments by combining the corresponding drug-linker compound with an antibody (e.g., Trastuzumab or Pertuzumab).

四、ADC的生物学评价IV. Biological Evaluation of ADCs

1、本发明ADC在NCI-N87胃癌移植瘤模型上的药效检测 1. Efficacy detection of the ADC of the present invention in the NCI-N87 gastric cancer xenograft model

(1)检测采用的实验动物及细胞系：(1) Experimental animals and cell lines used in the detection:

Balb/c Nude小鼠(成都药康生物科技有限公司,生产许可证号:SCXK(川)2020-0034)Balb/c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd., Production License No.: SCXK(川)2020-0034)

人胃癌细胞NCI-N87(ATCC)Human gastric cancer cells NCI-N87 (ATCC)

(2)检测步骤：(2) Detection steps:

用含10％胎牛血清的RPMI 1640培养液，在37℃、5％ CO2的条件下培养NCI-N87细胞。收集指数生长期的NCI-N87细胞,用PBS重悬至适合浓度,接种于雌性Balb/c Nude小鼠皮下建立胃癌模型。待肿瘤平均体积约150mm3左右时,根据肿瘤大小随机分组,依次为:溶媒对照组(即阴性对照或Vehicle组，采用0.9％ NaCl注射液)、Trastuzumab-G-6组(给药剂量2mg/kg)，各组均采用尾静脉注射(i.v.),在Day0、Day7、Day14给药,共给药3次。NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2 . NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female Balb/c Nude mice to establish a gastric cancer model. When the average tumor volume reached approximately 150 mm³ , the cells were randomly divided into two groups according to tumor size: a solvent control group (i.e., negative control or Vehicle group, treated with 0.9% NaCl injection) and a Trastuzumab-G-6 group (dose 2 mg/kg). All groups received intravenous injection (iv) via tail vein on Day 0, Day 7, and Day 14, for a total of three administrations.

给药后每周1次用游标卡尺测量肿瘤直径，并按如下计算公式计算肿瘤体积:V＝0.5a×b2,其中a和b分别表示肿瘤的长径和短径。每天观察记录动物死亡情况。After administration, the tumor diameter was measured weekly using calipers, and the tumor volume was calculated using the following formula: V = 0.5a × b² , where a and b represent the major and minor diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

采用以下公式计算肿瘤生长抑制率TGI(％),用于评价ADC的抑瘤疗效:The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

VT末>VT0,TGI(％)＝[1-(VT末-VT0)/(VC末-VC0)]*100％或VT末≤VT0,TGI(％)＝[1-(VT末-VT0)/VT0]*100％。If VT end > VT0 , TGI(%) = [1 - (VT end - VT0 ) / (VT end - VT0 )] * 100% or if VT end ≤ VT0 , TGI(%) = [1 - (VT end - VT0 ) / VT0 ] * 100%.

其中VT末:治疗组实验结束时肿瘤体积均值；Where V T end : mean tumor volume at the end of the experiment in the treatment group;

VT0:治疗组给药开始时肿瘤体积均值；V T0 : Mean tumor volume at the start of treatment in the treatment group;

VC末:阴性对照组实验结束时肿瘤体积均值；V C end : Mean tumor volume at the end of the experiment in the negative control group;

VC0:阴性对照组给药开始时肿瘤体积均值；V C0 : Mean tumor volume at the start of drug administration in the negative control group;

采用以下公式计算肿瘤相对增殖率T/C(％),用于评价ADC的抑瘤疗效:The tumor relative proliferation rate (T/C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

T/C(％)＝(VT末/VT0)/(VC末/VC0)*100％。T/C (%) = (V <sub>T</sub> final /V<sub> T0 </sub>) / (V <sub>C</sub> final /V <sub>C0</sub> ) * 100%.

具体结果见表2、图1及图2：The specific results are shown in Table 2, Figure 1, and Figure 2:

表2本发明ADC在人胃癌细胞NCI-N87 CDX模型上的药效
Table 2. Efficacy of the ADC of the present invention in the human gastric cancer cell NCI-N87 CDX model.

由表2可见,与Vehicle组相比，本发明Trastuzumab-G-6组的肿瘤生长抑制率(TGI)为72.11％，具有显著的肿瘤生长抑制活性。在第35天时治疗组无动物死亡及无显著地动物体重降低，未见明显的药物毒性反应，治疗期间小鼠对本发明ADC耐受性良好。本发明ADC(例如ADC A-1～A-9、ADC B-1～B-19、ADC G-1～G-16)均对NCI-N87胃癌移植瘤模型的肿瘤生长具有明确的抑制作用。这说明，本发明ADC具有显著的肿瘤生长抑制活性，并且安全性及耐受性良好。As shown in Table 2, compared with the Vehicle group, the Trastuzumab-G-6 group of this invention exhibited a tumor growth inhibition rate (TGI) of 72.11%, demonstrating significant tumor growth inhibitory activity. On day 35, no animals in the treatment group died or experienced significant weight loss, and no obvious drug toxicity was observed. Mice tolerated the ADC of this invention well during treatment. The ADCs of this invention (e.g., ADC A-1 to A-9, ADC B-1 to B-19, ADC G-1 to G-16) all showed clear inhibitory effects on tumor growth in the NCI-N87 gastric cancer xenograft model. This indicates that the ADCs of this invention have significant tumor growth inhibitory activity and good safety and tolerability.

2、本发明ADC在JIMT-1人乳腺癌移植瘤模型上的药效检测2. Efficacy detection of the ADC of the present invention in the JIMT-1 human breast cancer xenograft model

(1)检测采用的实验动物及细胞系：(1) Experimental animals and cell lines used in the detection:

NOD SCID小鼠(江苏集萃药康生物科技股份有限公司,生产许可证号:SCXK(苏)2023-0009)NOD SCID mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Production License No.: SCXK(Su)2023-0009)

人乳腺癌细胞JIMT-1(南京科佰)Human breast cancer cell line JIMT-1 (Nanjing Kebai)

(2)检测步骤：(2) Detection steps:

用含10％胎牛血清的DMEM培养液，在37℃、5％ CO2的条件下培养JIMT-1细胞。收集指数生长期的JIMT-1细胞,用含50％基质胶的PBS重悬至适合浓度,接种于雌性NOD SCID小鼠皮下建立乳腺癌模型。待肿瘤平均体积约150mm3左右时,根据肿瘤大小随机分组,依次为:溶媒对照组(即阴性对照或Vehicle组，采用0.9％ NaCl注射液)、Trastuzumab-A-1组(给药剂量3mg/kg)，各组均采用尾静脉注射(i.v.),在Day0、Day7、Day14给药,共给药3次。JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 . JIMT-1 cells in the exponential growth phase were collected, resuspended in PBS containing 50% matrix gel to a suitable concentration, and subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. When the average tumor volume reached approximately 150 mm³ , the cells were randomly divided into two groups according to tumor size: a solvent control group (i.e., negative control or Vehicle group, treated with 0.9% NaCl injection) and a Trastuzumab-A-1 group (dose 3 mg/kg). All groups received intravenous injection (iv) via tail vein on Day 0, Day 7, and Day 14, for a total of three administrations.

给药后每周1次用游标卡尺测量肿瘤直径，并按如下计算公式计算肿瘤体积:V＝0.5a×b2,其中a和b分别表示肿瘤的长径和短径。每天观察记录动物死亡情况。After administration, the tumor diameter was measured weekly using calipers, and the tumor volume was calculated using the following formula: V = 0.5a × b² , where a and b represent the major and minor diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

采用以下公式计算肿瘤生长抑制率TGI(％),用于评价ADC的抑瘤疗效:The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

VT末>VT0,TGI(％)＝[1-(VT末-VT0)/(VC末-VC0)]*100％或VT末≤VT0,TGI(％)＝[1-(VT末- VT0)/VT0]*100％。If VT<sub> end </sub> >VT<sub>0</sub>, TGI(%) = [1 - (VT<sub>end</sub> - VT <sub> 0 </sub> ) / (VC<sub> end </sub> - VC<sub>0</sub>)] * 100% or VT <sub>end </sub> ≤ VT<sub>0</sub> , TGI(%) = [1 - (VT <sub>end</sub> - VC<sub>0</sub>)] * 100%. V <sub>T0 </sub>)/V <sub>T0 </sub>]*100%.

其中VT末:治疗组实验结束时肿瘤体积均值；Where V T end : mean tumor volume at the end of the experiment in the treatment group;

VT0:治疗组给药开始时肿瘤体积均值；V T0 : Mean tumor volume at the start of treatment in the treatment group;

VC末:阴性对照组实验结束时肿瘤体积均值；V C end : Mean tumor volume at the end of the experiment in the negative control group;

VC0:阴性对照组给药开始时肿瘤体积均值；V C0 : Mean tumor volume at the start of drug administration in the negative control group;

采用以下公式计算肿瘤相对增殖率T/C(％),用于评价ADC的抑瘤疗效:The tumor relative proliferation rate (T/C%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

T/C(％)＝(VT末/VT0)/(VC末/VC0)*100％。T/C (%) = (V <sub>T</sub> final /V<sub> T0 </sub>) / (V <sub>C</sub> final /V <sub>C0</sub> ) * 100%.

具体结果见表3、图3及图4：The specific results are shown in Table 3, Figure 3, and Figure 4:

表3本发明ADC在人乳腺癌细胞JIMT-1CDX模型上的药效
Table 3. Efficacy of the ADC of the present invention in the human breast cancer cell JIMT-1CDX model.

由表3可见,与Vehicle组相比，本发明Trastuzumab-A-1组的肿瘤生长抑制率(TGI)为56.61％，具有显著的肿瘤生长抑制活性。在第25天时治疗组无动物死亡及无显著地动物体重降低，未见明显的药物毒性反应，治疗期间小鼠对本发明ADC耐受性良好。本发明ADC(例如ADC A-1～A-9、ADC B-1～B-19、ADC G-1～G-16)均对人乳腺癌细胞JIMT-1模型的肿瘤生长具有明确的抑制作用。这说明，本发明ADC具有显著的肿瘤生长抑制活性，并且安全性及耐受性良好。As shown in Table 3, compared with the Vehicle group, the tumor growth inhibition rate (TGI) of the Trastuzumab-A-1 group of the present invention was 56.61%, demonstrating significant tumor growth inhibitory activity. On day 25, no animal deaths or significant weight loss were observed in the treatment group, and no obvious drug toxicity was observed. Mice tolerated the ADC of the present invention well during treatment. The ADCs of the present invention (e.g., ADC A-1 to A-9, ADC B-1 to B-19, ADC G-1 to G-16) all showed a clear inhibitory effect on tumor growth in the JIMT-1 human breast cancer cell model. This indicates that the ADCs of the present invention have significant tumor growth inhibitory activity and good safety and tolerability.

3、本发明ADC在JIMT-1人乳腺癌移植瘤模型上的药效检测3. Efficacy detection of the ADC of the present invention in the JIMT-1 human breast cancer xenograft model

(1)检测采用的实验动物及细胞系：(1) Experimental animals and cell lines used in the detection:

NOD SCID小鼠(江苏集萃药康生物科技股份有限公司,生产许可证号:SCXK(苏)2023-0009)NOD SCID mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Production License No.: SCXK(Su)2023-0009)

人乳腺癌细胞JIMT-1(南京科佰) Human breast cancer cell line JIMT-1 (Nanjing Kebai)

(2)检测步骤：(2) Detection steps:

用含10％胎牛血清的DMEM培养液，在37℃、5％ CO2的条件下培养JIMT-1细胞。收集指数生长期的JIMT-1细胞,用含50％基质胶的PBS重悬至适合浓度,接种于雌性NOD SCID小鼠皮下建立乳腺癌模型。待肿瘤平均体积约150mm3左右时,根据肿瘤大小随机分组,依次为:溶媒对照组(即阴性对照或Vehicle组，采用0.9％ NaCl注射液)、Trastuzumab-A-2。Trastuzumab-A-2在Day0给药3mg/kg，在Day14给药6mg/kg，共给药2次。各组均采用尾静脉注射(i.v.)JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 . JIMT-1 cells in the exponential growth phase were collected, resuspended in PBS containing 50% matrix gel to a suitable concentration, and subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. When the average tumor volume reached approximately 150 mm³ , the cells were randomly assigned to groups based on tumor size: a solvent control group (i.e., negative control or Vehicle group, using 0.9% NaCl injection) and Trastuzumab-A-2. Trastuzumab-A-2 was administered twice, at 3 mg/kg on Day 0 and 6 mg/kg on Day 14. All groups received intravenous (iv) injection via tail vein.

给药后每周1次用游标卡尺测量肿瘤直径，并按如下计算公式计算肿瘤体积:V＝0.5a×b2,其中a和b分别表示肿瘤的长径和短径。每天观察记录动物死亡情况。After administration, the tumor diameter was measured weekly using calipers, and the tumor volume was calculated using the following formula: V = 0.5a × b² , where a and b represent the major and minor diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

采用以下公式计算肿瘤生长抑制率TGI(％),用于评价ADC的抑瘤疗效:The tumor growth inhibition rate (TGI%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs:

VT末>VT0,TGI(％)＝[1-(VT末-VT0)/(VC末-VC0)]*100％或VT末≤VT0,TGI(％)＝[1-(VT末-VT0)/VT0]*100％。If VT end > VT0 , TGI(%) = [1 - (VT end - VT0 ) / (VT end - VT0 )] * 100% or if VT end ≤ VT0 , TGI(%) = [1 - (VT end - VT0 ) / VT0 ] * 100%.

其中VT末:治疗组实验结束时肿瘤体积均值；Where V T end : mean tumor volume at the end of the experiment in the treatment group;

VT0:治疗组给药开始时肿瘤体积均值；V T0 : Mean tumor volume at the start of treatment in the treatment group;

VC末:阴性对照组实验结束时肿瘤体积均值；V C end : Mean tumor volume at the end of the experiment in the negative control group;

VC0:阴性对照组给药开始时肿瘤体积均值；V C0 : Mean tumor volume at the start of drug administration in the negative control group;

采用以下公式计算肿瘤相对增殖率T/C(％),用于评价ADC的抑瘤疗效:T/C(％)＝(VT 末/VT0)/(VC末/VC0)*100％。The tumor relative proliferation rate T/C (%) was calculated using the following formula to evaluate the tumor-suppressive efficacy of ADC: T/C (%) = (V<sub> T </sub> end /V <sub>T0 </sub>) / (V <sub>C</sub> end /V <sub>C0</sub> ) * 100%.

具体结果见表4、图5及图6：The specific results are shown in Table 4, Figure 5, and Figure 6:

表4本发明ADC在人乳腺癌细胞JIMT-1CDX模型上的药效
Table 4. Efficacy of the ADC of the present invention in the human breast cancer cell JIMT-1CDX model.

由表4可见,与Vehicle组相比，本发明Trastuzumab-A-2组的肿瘤生长抑制率(TGI)为56.20％，具有显著的肿瘤生长抑制活性。在第26天时治疗组无动物死亡及无显著地动物体重降低，未见明显的药物毒性反应，治疗期间小鼠对本发明ADC耐受性良好。本发明ADC(例如ADC A-1～A-9、ADC B-1～B-19、ADC G-1～G-16)均对人乳腺癌细胞JIMT-1模型的肿瘤生长具有明确的抑制作用。这说明，本发明ADC具有显著的肿瘤生长抑制活性，并且安全性及耐受性良好。As shown in Table 4, compared with the Vehicle group, the tumor growth inhibition rate (TGI) of the Trastuzumab-A-2 group of the present invention was 56.20%, demonstrating significant tumor growth inhibitory activity. On day 26, no animal deaths or significant weight loss were observed in the treatment group, and no obvious drug toxicity was observed. Mice tolerated the ADC of the present invention well during treatment. The ADCs of the present invention (e.g., ADC A-1 to A-9, ADC B-1 to B-19, ADC G-1 to G-16) all showed a clear inhibitory effect on tumor growth in the JIMT-1 human breast cancer cell model. This indicates that the ADCs of the present invention have significant tumor growth inhibitory activity and good safety and tolerability.

最后应当说明的是：以上实施例仅用以说明本发明的技术方案而非对其限制；尽管参照较佳实施例对本发明进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本发明的具体实施方式进行修改或者对部分技术特征进行等同替换；而不脱离本发明技术方案的精神，其均应涵盖在本发明请求保护的技术方案范围当中。 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims (20)

一种化合物或其药学上可接受的盐，其具有式D-E-L-M’所示结构，其中：A compound or a pharmaceutically acceptable salt thereof having the structure shown in formula D-E-L-M’, wherein: M’为-M-Lg，其中M是和靶向部位结合的结构片段，Lg为亲核取代反应的离去基团；M’ is -M-Lg, where M is the structural fragment that binds to the target site, and Lg is the leaving group for the nucleophilic substitution reaction; L是连接M和E之间的结构片段；L is the structural segment connecting M and E; E是连接L和D的结构片段；E is a structural segment connecting L and D; D是细胞毒性药物片段；D is a cytotoxic drug fragment; 优选地，M选自以下取代或未取代的结构片段：
Preferably, M is selected from the following substituted or unsubstituted structural segments:
优选地，所述Lg选自卤素(例如F、Cl、Br、I)、卤代C1-6烷基、C1-6烷基磺酰基、卤代C1-6烷基磺酰基、卤代磺酰基、C1-6烷基磺酸酯基、卤代C1-6烷基磺酸酯基、C1-6烷基亚磺酸酯基、C1-6烷基亚砜基、卤代苯氧基、羟基(-OH)、巯基(-SH)、氨基(-NH2)、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段，所述的卤代C1-6烷基、C1-6烷基磺酰基、卤代C1-6烷基磺酰基、卤代磺酰基、C1-6烷基磺酸酯基、卤代C1-6烷基磺酸酯基、C1-6烷基亚磺酸酯基、C1-6烷基亚砜基、卤代苯氧基、烯基、炔基及含炔基的结构片段任选地被一个或多个合适的取代基所取代。Preferably, the Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxy (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments. The halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfonate, C1-6 alkyl sulfinate, C1-6 alkyl sulfinate, C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide ...oxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl sulfoxide, C1-6 alkyl The 1-6 alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments may optionally be replaced by one or more suitable substituents. 权利要求1所述的化合物或其药学上可接受的盐，其中，The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, 所述L选自由下述的一个或多个组成的取代或未取代的结构片段或其立体异构体：C1- 6亚烷基、6-10元芳基、5-6元杂芳基、9-12元含氮杂环基、-N(R’)-、-NH(R’)、-N(R’)2、羰基、-O-、天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH2CH2(OCH2CH2)rOCH3))、Lys(R’)、以及氨基酸组成的短肽(比如Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Gly-Lys、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Cit、Val-Lys、Val-Lys(Ac)、Ala-Ala-Ala、Ala-D-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Gly、D-Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-D-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly)、 其中R’由下述一个或多个基团组成，包括但不限于氢、C1-6烷基、C1-6亚烷基、胺基、羟基、羧基、酰基、-O-、-C1-6亚烷基CO2H、-C1-6亚烷基SO3H、-SO3H、-PO3H2、-C1-6亚烷基-NHC1-6烷基、-C1-6亚烷基-N(C1-6烷基)2、-CH2N(C1- 6烷基)-C(＝O)C1-6亚烷基-杂环、-CH2NH-SO3H、-CH2N(C1-6烷基)-SO3H、-CH2NHC1-6亚烷基-SO3H、-CH2N(C1-6烷基)C1-6亚烷基-SO3H、-CH2N(C1-6亚烷基-SO3H)2、-CH2N+(C1-6亚烷基-SO3H)3、-CH2N+(C1-6烷基)2-C1-6亚烷基-SO3H、-CH2N(C1-6烷基)-C(＝O)C1-6亚烷基-N+(C1-6亚烷基-SO3H)3、-CH2NH-C(＝O)C1-6亚烷基-N+(C1-6亚烷基-SO3H)3、-CH2N(C1-6烷基)-C(＝O)C1-6亚烷基-N+(C1-6烷基)3、-CH2NH-C(＝O)C1-6亚烷基-N+(C1-6烷基)3、-CH2N(C1- 6烷基)-C(＝O)OC2-6亚烷基-N+(C1-6烷基)3、-CH2N(C1-6烷基)-C(＝O)OC2-6亚烷基-N+(C1-6烷基)2-CH2CO2H、-CH2N(C1-6烷基)-C1-6亚烷基-CO2H、-CH2N+(C1-6烷基)2-C1-6亚烷基-CO2H、葡萄糖基、半乳糖基、葡萄糖醛酸基、半乳糖醛酸基、-CH2N(C1-6烷基)-C(＝O)-(CH2CH2O)r-C1-6烷基、-CH2N(C1-6烷基)-C(＝O)-(OCH2CH2)r-OC1-6烷基、-(CH2N(Me)-C(＝O))r-C1-6烷基、或含1-10个EO单元的聚乙二醇片段(即-(CH2CH2O)r-C1-6烷基)、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸残基)、DOTAGA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸,α-丙酰基)、NOTA(1,4,7-三氮杂环壬烷-N,N’,N”-三乙酸残基)、 其中r选自1-20的整数；s选自1-20的整数。 The L is selected from one or more of the following substituted or unsubstituted structural segments or stereoisomers thereof: C 1- 6 -alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic groups, -N(R')-, -NH(R'), -N(R') 2 , carbonyl, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( COCH2CH2 ( OCH2CH2 ) rOCH3 ) (), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Gly-Lys, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Ala-Glu, G ly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Ly s-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, C1-6 alkyl, C1-6 alkylene, amino, hydroxyl, carboxyl, acyl, -O-, -C1-6 alkylene CO2H, -C1-6 alkylene SO3H , -SO3H , -PO3H2, -C1-6 alkylene -NHC1-6 alkyl , -C1-6 alkylene -N( C1-6 alkyl) 2 , -CH2N ( C1-6 alkyl)-C(=O) C1-6 alkylene-heterocyclic, -CH2NH -SO3H, -CH2N (C1-6 alkyl ) -SO3H , -CH2NHC1-6 alkylene- SO3H , -CH2N ( C1-6 alkyl) C1-6 alkylene- SO3H , -CH2N ( C1-6 alkyl) C1-6 alkylene-SO3H, -CH2N ( C1-6 alkyl- SO3 ) H) 2 、-CH 2 N + (C 1-6 alkylene-SO 3 H) 3 、-CH 2 N + (C 1-6 alkyl) 2 -C 1-6 alkylene-SO 3 H、-CH 2 N(C 1-6 alkyl)-C(＝O)C 1-6 alkylene-N + (C 1-6 alkylene-SO 3 H) 3 、-CH 2 NH-C(＝O)C 1-6 alkylene-N + (C 1-6 alkylene-SO 3 H) 3 、-CH 2 N(C 1-6 alkyl)-C(＝O)C 1-6 alkylene-N + (C 1-6 alkyl) 3 、-CH 2 NH-C(＝O)C 1-6 alkylene-N + (C 1-6 alkyl) 3 、-CH 2 N(C 1-6 alkyl)-C(＝O) OC 2-6 alkylene-N + (C 1-6 alkyl) 3 , -CH 2 N(C 1-6 alkyl)-C(＝O)OC 2-6 alkylene-N + (C 1-6 alkyl) 2 -CH 2 CO 2 H, -CH 2 N(C 1-6 alkyl)-C 1-6 alkylene-CO 2 H, -CH 2 N + (C 1-6 alkyl) 2 -C 1-6 alkylene-CO 2 H, glucosyl, galactosyl, glucuronic acid, galacturonic acid, -CH 2 N(C 1-6 alkyl)-C(＝O)-(CH 2 CH 2 O) r -C 1-6 alkyl, -CH 2 N(C 1-6 alkyl)-C(＝O)-(OCH 2 CH 2 ) r -OC 1-6 alkyl, -(CH 2 N(Me)-C(＝O)) r -C 1-6 alkyl, or polyethylene glycol fragments containing 1-10 EO units (i.e., -( CH₂CH₂O ) r - C₁ -6 alkyl), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), Where r is selected from integers from 1 to 20; s is selected from integers from 1 to 20. 如权利要求1或2所述的化合物或其药学上可接受的盐，其中：所述E为单键，取代或未取代的-NH-CH2-或 The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein: E is a single bond, substituted or unsubstituted -NH- CH2- or 优选地，所述E为单键，取代或未取代的-NH-CH2-；Preferably, E is a single bond, substituted or unsubstituted -NH- CH2- ; 进一步优选地，E为单键或-NH-CH2-。More preferably, E is a single bond or -NH- CH2- . 如权利要求1～3任意一项所述的化合物或其药学上可接受的盐，其中：M选自以下取代或未取代的结构片段：
The compound as claimed in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein: M is selected from the following substituted or unsubstituted structural segments:
如权利要求1～4任意一项所述的化合物或其药学上可接受的盐，其中：Lg选自卤素(例如F、Cl、Br、I)、卤代C1-6烷基、C1-6烷基磺酰基、卤代C1-6烷基磺酰基、卤代磺酰基、C1-6烷基磺酸酯基、卤代C1-6烷基磺酸酯基、C1-6烷基亚磺酸酯基、C1-6烷基亚砜基、卤代苯氧基、羟基(-OH)、巯基(-SH)、氨基(-NH2)、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段；The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein: Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkyl sulfonyl, halogenated C1-6 alkyl sulfonyl, halogenated sulfonyl, C1-6 alkyl sulfonate, halogenated C1-6 alkyl sulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxyl (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments; 优选地，Lg选自卤素、取代或未取代的C1-6烷基磺酰基、卤代苯氧基、羟基(-OH)、巯基(-SH)或氨基(-NH2)。Preferably, Lg is selected from halogens, substituted or unsubstituted C1-6 alkylsulfonyl groups, halophenoxy groups, hydroxyl groups (-OH), mercapto groups (-SH), or amino groups ( -NH2 ). 进一步优选地，Lg选自甲基磺酰基。More preferably, Lg is selected from methanesulfonyl. 如权利要求1～5任意一项所述的化合物或其药学上可接受的盐，其中：L选自由下述的一个或多个组成的取代或未取代的结构片段：天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH2CH2(OCH2CH2)rOCH3))、Lys(R’)、以及氨基酸组成的短肽(比如Gly-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala)、其中R’由下述一个或多个基团组成，包括但不限于氢、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸残基)、DOTAGA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸,α-丙酰基)、NOTA(1,4,7-三氮杂环壬烷-N,N’,N”-三乙酸残基)、其中s选自1-20的整数，例如1、 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；优选地，s为3-10，例如，s为5、8、或10。The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, wherein: L is selected from one or more of the following substituted or unsubstituted structural fragments: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D- Leu , D - Ala, Lys(COCH2CH2(OCH2CH2)rOCH3 ) ) , Lys(R'), and short peptides composed of amino acids (e.g., Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). Wherein R' is composed of one or more of the following groups, including but not limited to hydrogen, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-propionyl), and NOTA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residue). Where s is selected from integers from 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, or 10. 优选地，L选自由下述的一个或多个组成的取代或未取代的结构片段：天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、D-Val、D-Leu、D-Ala、Lys(COCH2CH2(OCH2CH2)rOCH3))、以及氨基酸组成的短肽(比如Gly-Lys、Val-Ala、Val-Cit、Val-Lys、Ala-Ala-Ala、Ala-D-Ala-Ala、D-Leu-Ala-Glu、Val-Lys-Gly、Gly-Gly-Val-Ala)、 其中s选自1-20的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、或20；优选地，s为3-10，例如，s为5、8、9或10。Preferably, L is selected from one or more of the following substituted or unsubstituted structural fragments: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), and short peptides composed of amino acids (e.g., Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala). Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10. 如权利要求1～6任意一项所述的化合物或其药学上可接受的盐，其中：选自以下取代或未取代的结构：


The compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 6, wherein: Selected from the following substituted or unsubstituted structures:


其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、 9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10；Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10; 优选地，选自以下取代或未取代的结构：

Preferably, Selected from the following substituted or unsubstituted structures:

如权利要求1～7任意一项所述的化合物或其药学上可接受的盐，其中：所述细胞毒性药物选自下式(I)所示结构的化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：

The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein: the cytotoxic drug is selected from compounds of the structure shown in formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs thereof:

其中，R1和R2各自独立地选自氢、-CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1- 6烷基)2、C1-6烷基、C2-6烯基、C2-6炔基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C2-6烯基、C2-6炔基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代； R1 and R2 are each independently selected from hydrogen, -CN, halogen, -ORα, -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl; wherein the alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl groups; R4和R4’各自独立地选自氢、卤素、-OH、-NH2、C1-6烷基、卤代C1-6烷基和C1-6烷氧基，R5和R5’均为氢； R4 and R4 ' are each independently selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halo- C1-6 alkyl and C1-6 alkoxy, and R5 and R5 ' are both hydrogen; 或者，R4和R4’一起与连接R4和R4’的碳原子成碳碳双键或者3-6元碳环，R5和R5’均为氢；Alternatively, R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms; 或者，R4和R5连接成键或一起与连接R4和R5的碳原子成3-6元碳环，R5’和R4’各自独立地选自氢、卤素、-NH2、C1-6烷基、卤代C1-6烷基和C1-6烷氧基；Alternatively, R4 and R5 may be bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5 , and R5 ' and R4 ' may be independently selected from hydrogen, halogen, -NH2, C1-6 alkyl, halo -C1-6 alkyl and C1-6 alkoxy; R6为氢、C1-6烷基或 R6 is hydrogen, C1-6 alkyl, or... R6’、R7和R7’均为氢；或者，R6’与R7连接成键，R7’为氢；R6 ' , R7 , and R7 ' are all hydrogen; or, R6 ' is bonded to R7 , and R7 ' is hydrogen. Ra选自H、C1-6烷基或C3-6环烷基； Ra is selected from H, C1-6 alkyl, or C3-6 cycloalkyl; 环A选自3-6元环烷基、C6-10芳基、5-10元杂芳基、3-6元杂环基；所述3-6元环烷基、C6-10芳基、5-10元杂芳基、3-6元杂环基任选被以下一个或多个取代基所取代：氢、-CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、C1-6烷基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代；Ring A is selected from 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups; the 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups are optionally substituted by one or more of the following substituents: hydrogen, -CN, halogen, -ORα , -NH2 , -NH( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6-membered heterocyclic group, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more of the following substituents: -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic group, C6-10 aryl ... Substituents of 6-10 aryl and 5-10 heteroaryl groups; R3选自氢、-CN、卤素、-ORa、-NH2、-NH(C1-6烷基)、-N(C1-6烷基)2、C1-6烷基、C3- 6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基；所述烷基、烯基、炔基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自-CN、卤素、-OH、-NH2、C1-6烷基、C1-6烷氧基、C3-6环烷基、3-6元杂环基、C6-10芳基和5-10元杂芳基的取代基所取代； R3 is selected from hydrogen, -CN, halogen, -ORα, -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups; X和Y各自独立地为-CH2-、-CD2-、和-C(O)-； X and Y are independently -CH 2- and -CD 2- , respectively. and -C(O)-; t选自1-10，例如1、2、3、4、5、6、7、8、9或10；t is selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 优选地，所述细胞毒性药物选自以下化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：


Preferably, the cytotoxic drug is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:


进一步优选地，所述细胞毒性药物选自以下化合物或其可药用盐、酯、立体异构体、多晶型物、溶剂合物、氮氧化物、同位素标记物、代谢物或前药：
More preferably, the cytotoxic drug is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
如权利要求1～8任意一项所述的化合物或其药学上可接受的盐，其中：所述化合物结构如下：


The compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 8, wherein the compound has the following structure:


其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10. 如式(II)所示的偶联物，其中：
A-[M-L-E-D]x
式(II)The coupling shown in formula (II) is as follows:
A-[MLED]x
Equation (II) M、L、E和D如权利要求1-9任意一项所述；x为1-10；M, L, E, and D are as described in any one of claims 1-9; x is 1-10; A是靶向部分，A的靶标选自表皮生长因子、Trop-2、CD37、HER2、CD70、EGFRvIII、Mesothelin、Folate eceoptor1、Mucin 1、CD138、CD20、CD19、CD30、SLTRK6、Nectin 4、Tissue factor、Mucin16、Endothelinreceoptor、STEAP1、SLC39A6、Guanylylcyclase C、PSMA、CCD79b、CD22、Sodium phosphate cotransporter 2B、GPNMB、Trophoblast glycoprotein、AGS-16、EGFR、CD33、CD66e、CD74、CD56、PD-L1、TACSTD2、DR5、E16、STEAP1、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、BAFF-R、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2、TENB2、整合素α5β6，α4β7、FGF2、FGFR2、Her3、CD70、CA6、DLL3、DLL4、P-cadherin、EpCAM、pCAD、CD223、LYPD3、LY6E、EFNA4、ROR1、SLITRK6、5T4、ENPP3、SLC39A6、Claudin18.2、BMPR1B、E16、STEAP1、Tyro7、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2，c-Met,ApoE、CD1lc、CD40、CD45(PTPRC)、CD49D(ITGA4)、CD80、CSF1R、CTSD、GZMB、Ly86、MS4A7、PIK3AP1、PIK3CD、CCR5、IFNG、IL10RA1、IL-6、ACTA2、COL7A1、LOX、LRRC15、MCPT8、MMP10、NOG、SERPINEl、STAT1、TGFBR1、CTSS、PGF、VEGFA、C1QA、C1QB、ANGPTL4、EGLN、ANGPTL4、EGLN3、BNIP3、AIF1、CCL5、CXCL10、CXCL11、IFI6、PLOD2、KISS1R、STC2、DDIT4、PFKFB3、PGK1、PDK1、AKR1C1、AKR1C2、CADM1、CDH11、COL6A3、CTGF、HMOX1、KRT33A、LUM、WNT5A、IGFBP3、MMP14、CDCP1、PDGFRA、TCF4、TGF、TGFB1、TGFB2、CDl lb、ADGRE1、EMR2、TNFRSF21、UPK1B、TNFSF9、MMP16、MFI2、IGF-1R、RNF43、NaPi2b和TENB2。 A represents the target group, whose targets are selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, O772P, MPF, Napi3b, Sema 5b, and PSCA. hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASL G659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin α5β6 , α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, R OR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b. PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LO X, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KIS S1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and TENB2. 权利要求10所述的偶联物，其中所述A为抗体或其抗原结合片段，例如单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、非人抗体、人源化抗体、嵌合抗体、全人抗体、前抗(Probody)、双特异性抗体或多特异性抗体；The conjugate of claim 10, wherein A is an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody, or multispecific antibody; 优选地，A是与ErbB家族受体酪氨酸激酶成员表皮生长因子受体2(Her2)特异性结合的抗体或其抗原结合片段Preferably, A is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases. 权利要求10或11所述的抗体药物偶联物，其选自：



The antibody-drug conjugate of claim 10 or 11 is selected from:



其中，各抗体药物偶联物中的HA-(S-或代表抗体或其抗原结合片段；Among them, HA-(S- or) in each antibody-drug conjugate This represents an antibody or its antigen-binding fragment; 其中，-(S-表示抗体或其抗原结合片段中的巯基与M片段的具体连接方式；Wherein, -(S-) represents the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the M fragment; 其中n为1～20的整数；优选为3～15的整数，例如，n为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20；优选地，n为5、8、9或10。Where n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10. 组合物，所述组合物包含一种或多种权利要求10-12任一项所述的抗体药物偶联物，所述组合物的DAR值(药物抗体偶联比)为1-10，例如：1～2，1～3，1～4，1～5，1～6，1～7，1～8，1～9，1～10，2～3，2～4，2～5，2～6，2～7，2～8，2～9，2～10，3～4，3～5，3～6，3～7，3～8，3～9，3～10，4～5，4～6，4～7，4～8，4～9，4～10，5～6，5～7，5～8，5～9，5～10，6～7，6～8，6～9，6～10，7～8，7～9，7～10，8～9，8～10，或9～10，优选为3～8，例如，3.0～3.5，3.0～4.0，3.0～4.5，3.0～5.0，6.0～6.5，6.0～7.0，6.0～7.5，6.0～8.0，6.0～8.5，6.5～7.0，6.5～7.5，6.5～8.0，6.5～8.5，7.0～7.5，7.0～8.0或7.5～8.0。The composition comprises one or more antibody-drug conjugates according to any one of claims 10-12, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5- 7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.0, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.0, 6.5-8.5, 7.0-7.5, 7.0-8.0 or 7.5-8.0. 药物组合物，其包含权利要求10-12任一项所述的偶联物、权利要求17所述的组合物，以及一种或多种药用辅料。A pharmaceutical composition comprising the conjugate of any one of claims 10-12, the composition of claim 17, and one or more pharmaceutical excipients. 权利要求10-12任一项所述的偶联物、权利要求13所述的组合物、权利要求14所述的药物组合物在制备治疗Her2表达癌症的药物中的用途。 Use of the conjugate of any one of claims 10-12, the composition of claim 13, and the pharmaceutical composition of claim 14 in the preparation of a medicament for treating Her2-expressing cancer. 权利要求15所述的用途，其中所述癌症选自实体瘤或血液系统恶性肿瘤；例如选自胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)和尿路上皮癌。The use according to claim 15, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma. 权利要求10-12任一项所述的偶联物、权利要求13所述的组合物、权利要求14所述的药物组合物，其用于治疗Her2表达癌症。The conjugate of any one of claims 10-12, the composition of claim 13, and the pharmaceutical composition of claim 14 are used to treat Her2-expressing cancers. 权利要求10-12任一项所述的偶联物、权利要求13所述的组合物、权利要求18所述的药物组合物，其用于治疗实体瘤或血液系统恶性肿瘤；例如胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)或尿路上皮癌。The conjugate of any one of claims 10-12, the composition of claim 13, and the pharmaceutical composition of claim 18 are for the treatment of solid tumors or hematologic malignancies, such as gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) or urothelial carcinoma. 一种Her2表达癌症的治疗方法，所述方法包括基于有需要的个体治疗有效量的权利要求10-12任一项所述的偶联物、权利要求13所述的组合物、权利要求14所述的药物组合物。A treatment method for Her2-expressing cancer, the method comprising, based on an individually therapeutically effective amount of the conjugate of any one of claims 10-12, the composition of claim 13, or the pharmaceutical composition of claim 14. 权利要求19所述的治疗方法，其中所述癌症选自实体瘤或血液系统恶性肿瘤；例如选自胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)和尿路上皮癌。 The treatment method of claim 19, wherein the cancer is selected from solid tumors or hematologic malignancies; for example, selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.
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