HK40081473B - Pharmaceutical composition comprising antibody drug conjugate and use thereof - Google Patents

Description

A pharmaceutical composition containing an antibody-drug conjugate and its use

This application claims priority to Chinese patent application filed on March 25, 2020 (application number CN 202010219601.7) and Chinese patent application filed on March 17, 2021 (application number CN 202110287012.7).

Technical Field

This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition containing an antibody-drug conjugate and its use as an anticancer drug.

Background Technology

The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of cytotoxins, while avoiding the drawbacks of low efficacy of the former and excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329–332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

In 2000, the first antibody-drug conjugate (gemtuzumab-oczogamicin, Wyeth Pharmaceuticals) was approved by the US FDA for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686; US4970198; US5079233; US5585089; US5606040; US5693762; US5739116; US5767285; US5773001).

In August 2011, brentuximab vedotin (Seattle Genetic Engineering) received fast-track approval from the US FDA for the treatment of Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma (Nat. Biotechnol (2003) 21(7):778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). It is a novel targeted ADC drug that allows the drug to directly act on the target CD30 on lymphoma cells, causing endocytosis and inducing apoptosis of tumor cells.

Both are targeted therapies for hematologic malignancies, which have relatively simpler tissue structures compared to solid tumors. In February 2013, ado-trastuzumab emtansine (T-DM1) received FDA approval in the United States for the treatment of HER2-positive patients with advanced or metastatic breast cancer who are resistant to both trastuzumab (trade name: Herceptin) and paclitaxel (WO2005037992; US8088387). It was the first ADC drug approved by the FDA for the treatment of solid tumors.

Several classes of small molecules with cytotoxicity are used in antibody-drug conjugates (ADCs), one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. Reports on the application of the camptothecin derivative eczemacon (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) include WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of more effective ADC drugs is still needed.

However, ADCs have a more complex heterogeneous structure than antibodies, which poses a greater challenge to ADC formulations for therapeutic purposes.

Summary of the Invention

This disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has a structure as shown in the general formula (Pc-L-Y-D):

in:

Y is selected from -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-, -O-CR ¹ R ² -(CR ^a R ^b ) _m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)- and -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclic groups;

Alternatively, ^Ra and ^Rb together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

R ¹ is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

R ² is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups;

Alternatively, ^Ra and ^R2 together with the carbon atom attached to them form cycloalkyl or heterocyclic groups;

m is an integer from 0 to 4;

n is between 1 and 10, and n is a decimal or an integer;

L represents the connector unit;

Pc is an antibody or its antigen-binding fragment;

The pH of the composition is from about 4.5 to about 6.0, preferably from about 4.8 to about 5.3, and more preferably from about 5.0 to about 5.1.

In optional embodiments, the buffer in the pharmaceutical composition has a pH of about 4.5 to about 6.0, with non-limiting examples including about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0, preferably about 4.8 to about 5.3, and more preferably about 5.0 to about 5.1. In some embodiments, the pH of the pharmaceutical composition is 4.5 to 5.2, preferably 4.8 to 5.2, and more preferably 5.0 to 5.1. In some embodiments, the pH of the pharmaceutical composition is 5.0.

In an optional embodiment, the pharmaceutical composition further comprises a surfactant, optionally selected from polysorbate, polysorbate 20, polysorbate 80, polyhydroxyalkanoates, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-saccharide Examples of surfactants include betaine, cetyl-betaine, lauramidopropyl-betaine, cocarbamateopropyl-betaine, linoleamideopropyl-betaine, myristamidopropyl-betaine, palmitoamideopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamideopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In an optional embodiment, the concentration of the surfactant in the pharmaceutical composition is from about 0.01 mg/mL to about 1.0 mg/mL. In an optional embodiment, the concentration of the surfactant in the pharmaceutical composition is from about 0.05 mg/mL to about 0.5 mg/mL, preferably from about 0.1 mg/mL to about 0.3 mg/mL, from about 0.2 mg/mL to about 0.6 mg/mL, from about 0.2 mg/mL to about 0.5 mg/mL, or from about 0.2 mg/mL to about 0.3 mg/mL, more preferably from about 0.2 mg/mL. Non-limiting examples include 0.1 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL, 0.45 mg/mL, 0.5 mg/mL, and 0.6 mg/mL.

In an optional embodiment, the aforementioned pharmaceutical composition further comprises a sugar. The "sugar" disclosed herein includes conventional compositions ( _CH₂O ) _ₙ and their derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and so on. The sugar may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, sylitol, sorbitol, mannitol, melibiose, pinetriose, melitriose, mannitol, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, isomaltulose, and so on. Preferred sugars are non-reducing disaccharides, more preferably trehalose or sucrose, and most preferably sucrose.

In optional embodiments, the sugar concentration in the aforementioned pharmaceutical composition is from about 60 mg/mL to about 90 mg/mL, with non-limiting examples including 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, and 90 mg/mL, preferably 80 mg/mL. In some embodiments, the sugar concentration is from 70 mg/mL to 90 mg/mL.

In optional embodiments, the concentration of the antibody-drug conjugate in the pharmaceutical composition is from about 1 mg/mL to about 100 mg/mL. Non-limiting examples include 1 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, 24 mg/mL, 25 mg/mL, 26 mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, and 100 mg/mL, preferably from about 10 mg/mL to about 30 mg/mL; more preferably from about 20 mg/mL to about 22 mg/mL. Specifically, non-limiting examples include 20.1 mg/mL, 20.2 mg/mL, 20.3 mg/mL, 20.4 mg/mL, 20.5 mg/mL, 20.6 mg/mL, 20.7 mg/mL, 20.8 mg/mL, 20.81 mg/mL, 20.82 mg/mL, 20.83 mg/mL, 20.84 mg/mL, 20.85 mg/mL, and 20.86 mg/mL. 20.87 mg/mL, 20.88 mg/mL, 20.89 mg/mL, 20.9 mg/mL, 20.9 mg/mL, 20.91 mg/mL, 20.92 mg/mL, 20.93 mg/mL, 20.94 mg/mL, 20.95 mg/mL, 20.96 mg/mL, 20.97 mg/mL, 20.98 mg/mL, 20.99 mg/mL, 21 mg/mL. In an optional embodiment, the concentration of the antibody-drug conjugate in the pharmaceutical composition is approximately 10 mg/mL to approximately 30 mg/mL, preferably approximately 20 mg/mL, based on the naked antibody (i.e., the antibody portion in the ADC).

In an optional embodiment, the buffer in the aforementioned pharmaceutical composition is selected from histidine buffers, succinate buffers, and citrate buffers, preferably succinate buffers, and more preferably succinic acid-sodium succinate buffers.

In an optional embodiment, the concentration of the buffer in the pharmaceutical composition is from about 5 mM to about 50 mM, and non-limiting examples include 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, and 50 mM, preferably from about 5 mM to about 20 mM; most preferably from about 10 mM.

In an optional embodiment, the drug load (n) can range from 3 to 8, 4 to 8, 5 to 7, more preferably 5.3 to 6.1, or 5.7 cytotoxic drugs bound to each antibody or its antigen-binding fragment (Pc). n is a decimal or an integer.

In an optional embodiment, the pharmaceutical composition comprises:

(a) the antibody-drug conjugate at a concentration of about 10 mg/mL to about 30 mg/mL, (b) polysorbate at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, (c) sugar at a concentration of about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to about 20 mM; the pH of the pharmaceutical composition is about 4.8 to about 5.3.

In an optional embodiment, the pharmaceutical composition comprises:

(a) the antibody-drug conjugate at a concentration of about 10 mg/mL to about 30 mg/mL, (b) polysorbate at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, (c) sugar at a concentration of about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to about 20 mM; the pH of the pharmaceutical composition is from 4.8 to 5.2.

In an optional embodiment, the pharmaceutical composition comprises:

(a) the antibody-drug conjugate at a concentration of about 20 mg/mL to about 22 mg/mL, (b) polysorbate 80 at a concentration of about 0.2 mg/mL, (c) sucrose at a concentration of 80 mg/mL, and (d) a 10 mM succinate buffer, wherein the pH of the pharmaceutical composition is about 5.0 to about 5.1. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.1.

In an optional implementation, in the aforementioned antibody-drug conjugate,

-Y- is -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens and alkyl groups;

^R1 is a _C3-7 cycloalkyl or _C3-7 cycloalkyl;

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

m is 0 or 1.

In an optional implementation, in the aforementioned antibody-drug conjugate,

-Y- is -O-(CH ₂ )m-CR ¹ R ² -C(O)-;

^R1 is a _C3-7 cycloalkyl or _C3-7 cycloalkyl;

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

m is 0 or 1.

In an optional implementation, in the aforementioned antibody-drug conjugate,

-Y- is -O-(CH ₂ )m-CR ¹ R ² -C(O)-;

^R1 is a _C3-7 cycloalkyl or _C3-7 cycloalkyl;

^R2 is a hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

m is 0 or 1.

In an optional implementation, in the aforementioned antibody-drug conjugate,

-Y- is -O-(CH ₂ )m-CR ¹ R ² -C(O)-;

^R1 is a _C3-7 cycloalkyl or _C3-7 cycloalkyl;

^R2 is a hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

m is 0.

In an optional implementation, in the aforementioned antibody-drug conjugate,

-Y- is selected from:

In an optional implementation, in the aforementioned antibody-drug conjugate,

The O end of the -Y- is connected to the connector unit L.

In an optional implementation, in the aforementioned antibody-drug conjugate,

-Y- is selected from:

In an optional embodiment, the aforementioned antibody-drug conjugate has a structure as shown in general formula (Pc- _LD1 ):

in:

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-7 cycloalkyl or _C3-7 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

m is 0 or 1;

n can be 1 to 10, and can be an integer or a decimal;

Pc represents the antibody or its antigen-binding fragment; and L represents the adapter unit.

In an optional implementation, in the aforementioned antibody-drug conjugate, n is 2 to 8, which can be an integer or a decimal; preferably 3 to 8, which can be an integer or a decimal.

In an optional embodiment, in the aforementioned antibody-drug conjugate, the linker unit -L- is ^{-L1 -L2 -L3 -L4-} , wherein:

^L1 is selected from -(succinimide-3-yl-N)-WC(O)-, _-CH2 -C(O) ^-NR3- WC(O)- and -C(O)-WC(O)-, wherein W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl and straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein the _C1-8 alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _CH2CH2O ) _p1CH2C ( _O )-, ^-S ( _CH2 ) ^p1C (O)- and chemical bonds, where ^p1 is an integer from ¹ _to 20 _; ^L2 is preferably a chemical bond;

^L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids may optionally be further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl.

^L4 is selected from ^-NR5 ( ^CR6R7 ) _t- , -C(O) ^NR5 , -C(O) ^NR5 ( _CH2 ) _t- and chemical bonds, where t is an integer ^from 1 to 6; ^L4 is preferably ^-NR5 ( ^{CR6R7 )} t-.

^R3 , ^R4 and ^R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

In an optional embodiment, in the aforementioned antibody-drug conjugate, the linker unit ^L1 is selected from -(succinimide-3-yl-N)-( _CH2 ) ^s1 -C(O)-, -(succinimide- ₃ -yl-N)-CH2-cyclohexyl-C(O)-, -(succinimide-3- _yl -N)-( _CH2CH2O ) ^s2- _{CH2CH2 -} C(O)-, _-CH2 -C(O) ^-NR3- ( _CH2 ) ^s3 -C(O)- and -C(O)-( _CH2 ) ^s4C (O)-, wherein ^s1 is an integer from 2 to 8, ^s2 is an integer from 1 to 3, ^s3 is an integer from 1 to 8, and ^s4 is an integer from 1 to 8; ^s1 is preferably 5.

In _an optional embodiment, in the aforementioned antibody-drug conjugate, the linker unit ^L2 is ^-NR4 ( _CH2CH2O ) ^p1CH2C (O)- or a chemical bond, _where ^p1 is an integer from 6 to 12.

In an optional embodiment, in the aforementioned antibody-drug conjugate, ^L4 is ^-NR5 ( ^CR6R7 )t- ^{, R5} is a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different, and each is independently a hydrogen atom or an alkyl group, t is 1 or 2, preferably 2; ^L4 is preferably ^-NR5CR6R7- , more _preferably ^{-NHCH2- .}

In an optional embodiment, in the aforementioned antibody-drug conjugate, the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is an integer from 2 to 8 for ^s1 ;

^L2 is a chemical bond;

^L3 is a tetrapeptide residue;

^L4 is ^-NR5 ( ^CR6R7 )t-, where ^R5 is a hydrogen atom or an alkyl group, ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group, ^and t is 1 or 2.

In an optional embodiment, in the aforementioned antibody-drug conjugate, the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is -(succinimide-3-yl-N) _-CH2 -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-;

^L3 is a tetrapeptide residue;

^L4 is ^-NR5 ( ^CR6R7 )t-, where ^R5 is a hydrogen atom or an alkyl group, ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group, ^and t is 1 or 2.

In an optional embodiment, in the aforementioned antibody-drug conjugate, the peptide residue of ^L3 is an amino acid residue formed from one, two, or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (N); preferably, it is an amino acid residue formed from one, two, or more amino acids selected from phenylalanine and glycine; more preferably, it is a tetrapeptide residue; and most preferably, it is a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).

In an optional embodiment, in the aforementioned antibody-drug conjugate, the connector unit -L- is ^{-L1 -L2} -L3 ^{-L4 -} , with its ^L1 end connected to the antibody or its antigen-binding fragment, and its ^L4 end connected to Y.

In an optional implementation, in the aforementioned antibody-drug conjugate, the -L-Y- is:

^L1 is -(succinimide-3-yl-N)-( _CH2 ) ^s1 -C(O)- or -(succinimide-3-yl-N) _-CH2 -cyclohexyl-C(O)-;

^L2 is ^-NR4 ( _CH2CH2O ) _p1CH2C (O)- or a chemical bond, ^{where p1} is an integer from 6 to 12; ^R4 is selected from hydrogen atoms, _alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-7 cycloalkyl or _C3-7 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8; preferably 5;

m is an integer from 0 to 4.

In an optional implementation, in the aforementioned antibody-drug conjugate, the -L-Y- is:

Preferred options are:

^L2 is ^-NR4 ( _CH2CH2O ) _9CH2C (O)-; ^R4 is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuteralkyl groups _, and hydroxyalkyl _groups ;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-7 cycloalkyl or _C3-7 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

m is an integer from 0 to 4.

In an optional implementation, in the aforementioned antibody-drug conjugate, the -L-Y- is:

^L2 is a chemical bond;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-7 cycloalkyl or _C3-7 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8; preferably 5;

m is an integer from 0 to 4.

In an optional embodiment, in the aforementioned antibody-drug conjugate, wherein -L-Y- is:

in:

^L1 is -(succinimide-3-yl-N)-( _CH2 ) ^s1 -C(O)- or -(succinimide-3-yl-N) _-CH2 -cyclohexyl-C(O)-;

^L2 is ^-NR4 ( _CH2CH2O ) _p1CH2C (O)- or a chemical bond, ^{where p1} is an integer from 1 to 20; ^R4 is selected from hydrogen atoms, _alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-7 cycloalkyl or _C3-7 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

^R5 , ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer from 0 to 4.

In an optional implementation, in the aforementioned antibody-drug conjugate, the -L-Y- is:

in:

^L2 is a chemical bond;

^L3 is a tetrapeptide residue of GGFG;

^R1 is a cycloalkyl or cycloalkyl group; preferably a _C3-7 cycloalkyl or _C3-7 cycloalkyl group.

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-7 cycloalkyl groups; preferably hydrogen atoms;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

^R5 is selected from hydrogen atoms or alkyl groups, and ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer from 0 to 4.

In an optional embodiment, the antibody-drug conjugate has a structure as shown in the general formula (Pc- _La -YD):

in,

W is selected from _C1-8 alkyl, _C1-8 alkyl- _C3-7 cycloalkyl, and straight-chain heteroalkyl with 1 to 8 atoms, wherein the straight-chain heteroalkyl contains 1 to 3 heteroatoms selected from N, O, or S, wherein each of the _C1-8 alkyl, _C3-7 cycloalkyl, and straight-chain heteroalkyl is independently and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, _C1-6 alkyl, _chloroC1-6 alkyl, _{deuteratedC1-6} alkyl, _C1-6 alkoxy, and _C3-7 cycloalkyl;

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _CH2CH2O ) _p1CH2C ( _O )-, ^{-S (} _CH2 ) ^p1C (O)- and chemical bonds, where ^p1 is an integer from ¹ _to 20; _R4 is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuteralkyl groups and hydroxyalkyl groups;

^L3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, _C1-6 alkyl, _chloroC1-6 alkyl, _{deuteratedC1-6} alkyl, _C1-6 alkoxy, and _C3-7 cycloalkyl;

^R1 is a halogenated _C1-6 alkyl or _C3-7 cycloalkyl;

^R2 is selected from hydrogen atoms, halogenated _C1-6 alkyl groups, and _C3-7 cycloalkyl groups;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group;

R ⁵ is selected from hydrogen atom, _C1-6 alkyl, halo- _C1-6 alkyl, deuterated _C1-6 alkyl and hydroxy- _C1-6 alkyl;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, _C1-6 alkyl, halo- _C1-6 alkyl, deuterated- _C1-6 alkyl and hydroxy- _C1-6 alkyl;

m is 0 or 1;

n is a decimal or integer between 3 and 8;

Pc represents an antibody or its antigen-binding fragment.

In an optional embodiment, the antibody-drug conjugate has a structure as shown in the general formula (Pc- _Lb -YD):

in:

^s1 is an integer from 2 to 8;

Pc, ^R1 , ^R2 , ^R5 , ^R6 , ^R7 , m and n are defined as in the general formula (Pc-L _a -YD).

In an optional implementation, in the aforementioned antibody-drug conjugate, the -L-Y- includes, but is not limited to:

In an optional embodiment, the aforementioned antibody-drug conjugate has the following structure:

Where Pc and n are defined as in the general formula (Pc-La-Y-D).

In an optional embodiment, the antibody-drug conjugate has the structure shown in the following formula:

in:

n is between 3 and 8, and n is a decimal or an integer;

Pc represents an antibody or its antigen-binding fragment.

In an optional embodiment, Pc is an antibody or its antigen-binding fragment, wherein the antibody is selected from chimeric antibodies, humanized antibodies or fully human antibodies; preferably a monoclonal antibody.

In an optional embodiment, the Pc is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, and anti-Mesothelin antibody, or antigen-binding fragments thereof.

In an optional embodiment, the antibody or its antigen-binding fragment in the antibody-drug conjugate is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab, or their antigen-binding fragments.

In an optional embodiment, the antibody-drug conjugate has the structure shown in the following formula:

Wherein, n is a non-zero integer or decimal from 0 to 10, preferably an integer or decimal between 1 and 10; more preferably 2 to 8, which can be an integer or a decimal; most preferably 3 to 8, which can be an integer or a decimal.

In an optional embodiment, the antibody-drug conjugate in the pharmaceutical composition has a structure as shown in the following formula:

Where n is between 3 and 8, and n is a decimal or an integer.

This disclosure provides a pharmaceutical composition comprising: (a) the antibody-drug conjugate at a concentration of about 10 mg/mL to about 30 mg/mL, (b) polysorbate at a concentration of about 0.05 mg/mL to about 0.5 mg/mL, (c) a sugar at a concentration of about 60 mg/mL to about 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to about 20 mM; the composition having a pH of 4.8 to 5.2;

The antibody-drug conjugate has the structure shown in the following formula:

Where n is between 3 and 8, and n is a decimal or an integer.

This disclosure provides a pharmaceutical composition comprising: (a) an antibody-drug conjugate at about 20 mg/mL to about 22 mg/mL, (b) polysorbate 80 at about 0.2 mg/mL, (c) sucrose at about 80 mg/mL, and (d) about 10 mM succinate buffer, wherein the pH of the pharmaceutical composition is from 5.0 to 5.1.

The antibody-drug conjugate has the structure shown in the following formula:

Where n is between 3 and 8, and n is a decimal or an integer.

This disclosure also provides a lyophilized formulation containing an antibody-drug conjugate, characterized in that the formulation, upon reconstitution, can form the pharmaceutical composition described above.

This disclosure also provides a method for preparing a lyophilized formulation containing an antibody-drug conjugate, including the step of freeze-drying the pharmaceutical composition as described above.

In an optional embodiment, the freeze-drying process in the method for preparing a lyophilized formulation comprising an antibody-drug conjugate sequentially includes pre-freezing, primary drying, and secondary drying steps. Freeze-drying is performed by freezing the formulation and subsequently sublimating water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or collapse temperature of the formulation. Typically, the primary drying temperature ranges from about -30 to 25°C (assuming the product remains frozen during the primary drying process). The size and type of the formulation, the container holding the sample (e.g., a glass vial), and the volume of liquid determine the required drying time, which can range from several hours to several days (e.g., 40-60 hours). The secondary drying stage can be performed at about 0-40°C, depending primarily on the type and size of the container and the type of protein used. The secondary drying time is determined by the desired residual moisture level in the product and typically requires at least about 5 hours. Typically, the water content of the low-pressure freeze-dried formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as the pressure applied in the primary drying step; preferably, the pressure for secondary drying is lower than that for primary drying. Freeze-drying conditions can vary depending on the formulation and vial size.

In one optional embodiment of this disclosure, 5 mL of the pharmaceutical composition stock solution is lyophilized. The lyophilization procedure is as follows: the pre-freezing temperature is -5°C or -45°C, the first drying temperature is -20°C and the vacuum degree is 10 Pa, and the second drying temperature is 25°C and the vacuum degree is 1 Pa.

In some embodiments, the lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

This disclosure also provides a lyophilized formulation comprising an antibody-drug conjugate, said formulation being obtained by lyophilizing a pharmaceutical composition comprising an anti-HER2 antibody-drug conjugate as described above.

This disclosure also provides a reconstitution solution containing an antibody-drug conjugate, characterized in that the reconstitution solution is obtained by reconstitution of the lyophilized formulation as described above.

This disclosure also provides a method for preparing the above-mentioned reconstituted solution, including a step of reconstituted the aforementioned lyophilized preparation, wherein the solution used for reconstitution is selected from, but is not limited to, water for injection, physiological saline or glucose solution.

In an optional embodiment, the reconstituted solution comprises the following components:

(a) the antibody-drug conjugate at a concentration of about 10 mg/mL to 30 mg/mL, (b) polysorbate at a concentration of about 0.05 mg/mL to 0.5 mg/mL, (c) sugar at a concentration of about 60 mg/mL to 90 mg/mL, and (d) a buffer at a concentration of about 5 mM to 20 mM; the reconstituted solution has a pH of 4.8-5.2.

In an optional embodiment, the reconstituted solution comprises the following components:

(a) an antibody-drug conjugate at a concentration of about 20 mg/mL to 22 mg/mL, (b) polysorbate 80 at a concentration of about 0.2 mg/mL, (c) sucrose at a concentration of about 80 mg/mL, and (d) a succinate buffer at a concentration of about 10 mM, wherein the reconstituted solution has a pH of 5.0-5.1.

This disclosure also provides an article of manufacture comprising a container containing the pharmaceutical composition, lyophilized formulation, or reconstituted solution as described above. In some embodiments, the container is a neutral borosilicate glass vial for injection.

This disclosure also provides the use of the aforementioned pharmaceutical compositions or lyophilized formulations or reconstituted solutions or products in the preparation of medicaments for the treatment or prevention of tumors.

This disclosure also provides methods for treating diseases, including providing the aforementioned pharmaceutical compositions or lyophilized preparations or reconstituted solutions or products.

This disclosure also provides the aforementioned pharmaceutical compositions, or lyophilized preparations, or reconstituted solutions, or articles as medicines, preferably medicines used to treat or prevent tumor diseases.

In an optional implementation, the disease or tumor is a cancer associated with the expression of HER2, HER3, B7H3, or EGFR.

In an optional implementation, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

As is well known to those skilled in the art, one, some, or all of the features of the various embodiments described in this disclosure can be further combined to form other embodiments of this disclosure. The above embodiments of this disclosure and other embodiments obtained by combination are further described in detail below.

This disclosure provides a pharmaceutical composition that is easier to manufacture and administer, and has stable performance. Specifically, the pharmaceutical composition described in this disclosure comprises an antibody-drug conjugate and a buffer.

the term

To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

This disclosure incorporates the entire contents of application PCT/CN2019/107873 (WO2020/063676).

Antibody-drug conjugates (ADCs) are drugs that link antibodies or antibody fragments to biologically active cytotoxic drugs or small molecule drugs with cytotoxic activity via stable chemical linker compounds. They fully utilize the specificity of antibodies in binding to tumor cell-specific or highly expressed antigens and the high efficiency of cytotoxic agents, while avoiding toxic side effects on normal cells. Compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

"Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetates, succinates, gluconates, histidines, oxalates, lactates, phosphates, citrates, tartrates, fumarates, glycylglycine, and other organic acid buffers.

"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., with histidine-acetate buffer being preferred. Histidine-acetate buffer is prepared by reacting histidine with acetic acid, and histidine-hydrochloride buffer is prepared by reacting histidine with hydrochloric acid.

"Citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc. A preferred citrate buffer is sodium citrate.

"Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, etc. A preferred succinate buffer is sodium succinate-succinate. Exemplarily, the sodium succinate-succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

A "phosphate buffer" is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

"Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc. Sodium acetate is a preferred acetate buffer.

"Pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates or their physiologically/pharmacologically acceptable salts or prodrugs described herein, along with other chemical components, such as physiologically/pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the antibody active ingredient, facilitate administration to the organism, and enhance the absorption of the active ingredient to exert its biological activity.

In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

The pharmaceutical compositions described in this disclosure are in solution form, and unless otherwise specified, the solvent is water.

"Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by a vacuum freeze-drying step after the liquid or solution form has been processed.

As used herein, the terms “about” and “approximately” mean a numerical value within the acceptable margin of error of a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within the acceptable margin of error of that specific value.

The pharmaceutical compositions disclosed herein achieve a stable effect: the antibody-drug conjugates therein substantially retain their physical and/or chemical stability and/or biological activity after storage; preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

A stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, and 6 months. Typical examples of stability include: aggregation or degradation of antibody monomers typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. A reduction of not more than about 10%, preferably not more than about 5%, is typically observed. Aggregation typically forms at a rate of not more than about 10%, preferably not more than about 5%.

If, after visual inspection of color and/or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody-drug conjugate does not show significant increase in aggregation, precipitation, and/or denaturation, then the antibody-drug conjugate "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

If an antibody-drug conjugate does not exhibit significant chemical changes, then the antibody "retains its chemical stability" in the drug formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

If the biological activity of an antibody-drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the drug formulation is prepared, then the antibody-drug conjugate "retains its biological activity" in the drug formulation.

The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

The "antibody" mentioned in this disclosure refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain can further divide it into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain. The antibodies disclosed herein are preferably specific antibodies against cell surface antigens on target cells. Non-limiting examples include the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, and anti-Integr antibody. The antibody is selected from one or more of the following: anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody; preferably trastuzumab (trade name Herceptin), pertuzumab (also known as 2C4, trade name Perjeta), nimotuzumab (trade name Taixinsheng), enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96, and glematumamab.

The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragments disclosed herein conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.

In this disclosure, the antibody light chain may further include a light chain constant region, which includes a human or mouse κ, λ chain or a variant thereof.

In this disclosure, the antibody heavy chain may further include a heavy chain constant region, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

The antibodies disclosed herein include murine antibodies, chimeric antibodies, and humanized antibodies, with humanized antibodies being preferred.

The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from mouse hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system.

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. This overcomes the strong heterologous response induced by chimeric antibodies due to carrying a large number of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the VBase human germline sequence database and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR.

The term "naked antibody" refers to an antibody that is not conjugated to a heterologous module (such as a cytotoxic module) or a radiolabel.

The "antigen-binding fragment of an antibody" mentioned in this disclosure can refer to Fab fragments, Fab' fragments, F(ab') ₂ fragments, and Fv fragments (scFv fragments) that bind to antigens. Fv fragments contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain using different linkers, called a single-chain antibody or single-chain Fv (sFv).

The term “antigen binding site” in this disclosure refers to a continuous or discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment disclosed herein.

The “ADCC” mentioned in this disclosure, or antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc fragment of an antibody. The ADCC effector function of antibodies can be reduced or eliminated by modifying the Fc fragment of IgG. This modification refers to mutations in the constant region of the antibody's heavy chain, such as N297A, L234A, and L235A selected from IgG1; IgG2/4 chimeras; and F234A/L235A mutations in IgG4.

The term "mutation" in the mutated sequences described in this disclosure includes, but is not limited to, "reverse mutations," "conservative modifications," or "conservative substitutions or replacements." "Conservative modifications" or "conservative substitutions or replacements" as described in this disclosure refer to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

The “mutated sequence” as described in this disclosure refers to a nucleotide and/or amino acid sequence that, when modified by appropriate substitutions, insertions, or deletions, exhibits a different percentage of sequence identity with the nucleotide and/or amino acid sequence disclosed herein. The sequence identity described in this disclosure may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Sequence comparisons and identity percentage determinations between two sequences can be performed using the default settings of the BLASTN/BLASTP algorithm available on the National Center for Biotechnology Institute website.

The term "linker unit,""connectorfragment," or "connector cell" refers to a chemical structural fragment or bond that is connected at one end to an antibody or its antigen-binding fragment and at the other end to a drug. It may also be connected to other linkers before being linked to an antibody or drug. The preferred embodiment disclosed herein is represented by L and ^L1 to ^L4 , wherein the ^L1 end is connected to an antibody, and the ^L4 end is connected to structural unit Y, which in turn connects to a compound or toxin.

The linker, including extensions, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), light-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to antibody glycosylation, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting antibodies can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any usable connection point. The substituents are preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene ( _-CH2- ), 1,1-ethylene (-CH( _{CH3 )} -), 1,2-ethylene ( _{-CH2CH2 )} -, 1,1-propylene (-CH( _CH2CH3 )-), 1,2-propylene ( _{-CH2CH ( CH3} )-), 1,3-propylene ( _{-CH2CH2CH2- )} , _1,4 -butylene _{( -CH2CH2CH2CH2-} ) _, and _{1,5 - butylene ( -CH2CH2CH2CH2CH2-} ). The alkylene group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring comprises 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 cyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 cyclic, more preferably 7 to 10 cyclic. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups according to the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospirocyclic group. Non-limiting examples of spiroheterocyclic groups include:

The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

wait.

The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrrole, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

The term "amino protecting group" is used to protect the amino group by a group that is easily removed, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenemethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be replaced by 1-3 substituents selected from halogens, alkoxy groups, or nitro groups. The amino protecting group is preferably 9-fluorenemethoxycarbonyl.

The term "cycloalkylalkyl" refers to an alkyl group that is substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein the alkyl group is as defined above, and the cycloalkyl group is as defined above.

The term "haloalkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

The term "hydroxyl group" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

The term "amino" refers to _-NH2 .

The term "nitro" refers to _-NO2 .

"Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the possibility that the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

"Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

The term "drug loading" refers to the average number of cytotoxic drugs loaded onto each antibody or its antigen-binding fragment in an antibody-drug conjugate molecule. It can also be expressed as the ratio of drug amount to antibody amount. The drug loading range can be 0-12 cytotoxic drugs attached to each antibody or its antigen-binding fragment (Pc), preferably 1-10, more preferably 3-8, and most preferably 5.3-6.1 cytotoxic drugs. In the embodiments disclosed herein, the drug loading is expressed as n, also known as the DAR (Drug-antibody Ratio) value, and can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The average number of drugs per ADC molecule after the conjugation reaction can be identified using conventional methods such as ultraviolet-visible spectroscopy (UV-Vis), hydrophobic chromatography (HIC) mass spectrometry, ELISA assays, and HPLC characterization.

The loading of cytotoxic drugs can be controlled using the following non-limiting methods, including:

(1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

(2) Control the reaction time and temperature.

(3) Choose different reaction reagents.

For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

The term "carrier" used in the context of this disclosure refers to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferably such as micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "giving" and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid. "Giving" and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

"Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

An "effective amount" includes the amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

"Displacement" refers to the replacement of the solvent system in which the antibody protein is dissolved. For example, using a buffer system of a stabilizing formulation, a high-salt or hypertonic solvent system containing the antibody protein is replaced by a physical manipulation, thereby ensuring the antibody protein remains in the stabilizing formulation. The physical manipulation methods include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.

Attached Figure Description

Figure 1A: Results of plasma stability experiments for ADC-19 disclosed herein.

Figure 1B: Plasma stability test results of the disclosed ADC-18.

Figure 1C: Plasma stability test results of the disclosed ADC-20.

Figure 2: Evaluation of the efficacy of ADC-21 and ADC-24 in JIMT-1 tumor-bearing mice disclosed in this paper.

Figure 3: Evaluation of the efficacy of the ADC disclosed in this study on nude mice with human breast cancer cell SK-BR-3 xenograft tumors.

Figure 4: Plasma stability test results of the disclosed ADC-25.

Figure 5: The efficacy of this disclosed ADC on human astrocytoblastoma U87MG xenografts in nude mice.

Figure 6: The efficacy of this disclosed ADC on Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases.

Figure 7: The efficacy of this disclosed ADC in human glioma U87MG xenografts in nude mice.

Figure 8: Trend chart of prescription screening experiment fitting, where the unit of PS80 is ^10⁻⁴ g/mL; the unit of ADC-32 protein concentration (calculated as naked antibody concentration) is mg/mL.

Detailed Implementation

The following description, in conjunction with examples, further illustrates this disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor Laboratory's *Antibody Technology Laboratory Manual* and *Molecular Cloning Handbook*; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

I. Antibody-Drug Conjugates

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ), and deuterated methanol ( _CD3OD ). The internal standard was tetramethylsilane (TMS). Chemical shifts are given in units of ^10⁻⁶ (ppm).

MS measurements were performed using a Finnigan LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

UPLC measurements were performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

The UV-HPLC determination was performed using a Thermo nanodrop2000 UV spectrophotometer.

The proliferation inhibition rate and _IC50 value were determined using a Phera starFS microplate reader (BMG GmbH, Germany).

Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC are 0.15mm to 0.2mm in diameter, while those used for TLC separation and purification are 0.4mm to 0.5mm in diameter.

Column chromatography typically uses 200-300 mesh silica gel from Yantai Huanghai as the carrier.

The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co.KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Darui Chemicals.

Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere.

Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature.

Room temperature is the optimal reaction temperature, with a range of 20℃ to 30℃.

Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g _{of KH2PO4} , 8.56g _{of K2HPO4 ·} 3H2O, 5.85g of NaCl and 1.5g of EDTA and place them in a bottle, make up to 2L, sonicate to dissolve completely, and shake well to obtain the solution.

The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and small amounts of triethylamine and acidic or basic reagents can also be added for adjustment.

Some of the compounds disclosed herein were characterized by Q-TOF LC/MS. The Q-TOF LC/MS was performed using an Agilent 6530 Precision Mass Number Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Chromatography System (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm column).

Example 1-1

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

Add 1 mL of N,N-dimethylformamide to eczema sulfonate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopropylformic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (3.8 mg, 13.7 μmol) to the reaction solution in sequence. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 1 (1.6 mg, yield: 82.1%).

MS m/z (ESI): 520.2 [M+1]

¹ H NMR (400MHz, CDCl ₃ ): δ7.90-7.84(m,1H),7.80-7.68(m,1H),5.80-5.70(m,1H),5.62-5.54(m,2H),5.44-5.32(m,2H),5.28-5.10(m,2H),3.40-3. 15(m,3H),2.44(s,3H),2.23(t,1H),2.06-1.75(m,2H),1.68-1.56(m,1H),1.22-1.18(m,2H),1.04-0.98(m,2H),0.89(t,3H).

Examples 1-2

(S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide 2-A

(R)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide 2-B

To compound 1b (4 mg, 7.53 μmol), add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, purge three times with argon, cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared using the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) sequentially to the reaction solution. After the additions are complete, stir the reaction mixture at 0–5 °C for 1 hour. Remove the ice-water bath, heat to 30 °C, and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ), B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

MS m/z(ESI):534.0[M+1].

Single-configuration compound 2-B (shorter retention time):

UPLC analysis: retention time 1.06 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1 H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,2H),3.60(t,1H),3.19-3.1 3(m,1H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H ),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H).

Compound 2-A with a single configuration (longer retention time):

UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H),5.58-5.53(m, 1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3.20-3.15(m ,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m,1H), 1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H).

Examples 1-3

(S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-3,3,3-trifluoro-2-hydroxypropionyl

Amine 3-A

(R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-B

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to compound 1b (5.0 mg, 9.41 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction mixture at 0–5 °C for 10 minutes. Remove the ice-water bath, heat to 30 °C, and stir for 8 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 3 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (1.5mg, 1.5mg).

MS m/z(ESI): 561.9 [M+1].

Single-configuration compounds (shorter retention time):

UPLC analysis: retention time 1.11 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H),6.53(s,1H),5. 61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66-4.57(m,1H ),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08(m,1H) ,2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H).

Single-configuration compounds (longer retention time):

UPLC analysis: retention time 1.19 min, purity: 90% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,1H),4.66-4 .57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.22-2.14(m,1H),2.04-1.95(m,2H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H).

Examples 1-4

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide 4

Add 1 mL of N,N-dimethylformamide to compound 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared using the method disclosed in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction mixture at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 4 (2.5 mg, yield: 80.9%).

MS m/z(ESI):548.0[M+1].

¹ H NMR (400MHz, CDCl ₃ ): δ7.73-7.62(m,2H),5.75-5.62(m,1H),5.46-5.32(m,2H),5.26-5.10(m,1H),3.30-3.10(m,1H),2 .43(s,3H),2.28-2.20(m,2H),2.08-1.84(m,8H),1.69-1.58(m,2H),1.04-1.00(m,2H),0.89(t,3H).

Examples 1-5

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

Add 1 mL of N,N-dimethylformamide to compound 1b (2.0 mg, 3.76 μmol), cool in an ice-water bath to 0–5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared using the method disclosed in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2 mg, 7.24 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 5 (1.0 mg, yield: 50%).

MS m/z(ESI): 533.9 [M+1].

¹ H NMR (400MHz, CDCl ₃ ): δ8.07(s,1H),7.23-7.18(m,2H),6.71-6.64(m,1H),6.55-6.51(m,1H),5.36-5.27(m,2H),4.67-4.61(m,2H),3.53-3.48(m,1H),3 .30-3.22(m,2H),3.18-3.13(m,1H),2.71-2.61(m,2H),2.35-2.28(m,1H),2.04-1.91(m,4H),1.53-1.40(m,3H),0.91-0.75(m,4H).

Examples 1-6

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

Add 1 mL of N,N-dimethylformamide to compound 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol; prepared according to the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 6 (2.1 mg, yield: 67.9%).

MS m/z(ESI):548.0[M+1].

¹ H NMR (400MHz, DMSO-d ₆ ): δ7.85-7.62(m,1H),6.88(br,1H),5.87-5.48(m,2H),5.47-5.33(m,1H),5.31-5.06(m,1H),4.25-3.91(m,2H),3.25(br,1 H),2.60-2.32(m,3H),2.23(t,1H),2.15-1.95(m,3H),1.70-1.56(m,2H),1.41-1.17(m,9H),1.03(s,1H),0.95-0.80(m,2H).

Examples 1-7

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclobutane-1-carboxamide 7

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to compound 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, from the supplier), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 10 minutes. Remove the ice-water bath and stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, and purify the residue by thin-layer chromatography using solvent system B to give title product 7 (2.5 mg, yield: 83.1%).

MS m/z(ESI):534.0[M+1].

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H),6.12(s,1H),5. 59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17(m,1H),3.1 5-3.05(m,1H),2.71-2.63(m,1H),2.37(s,3H),2.12-2.05(m,1H),2.03 -1.94(m,2H),1.92-1.78(m,4H),1.50-1.42(m,1H),0.90-0.83(m,4H).

Examples 1-8

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

first step

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid benzyl ester 8c

1-Hydroxycyclopropane-1-carboxylic acid benzyl ester 8a (104 mg, 0.54 mmol; prepared by the method disclosed in patent application "US2005/20645") and 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in patent application "CN105829346A") were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon three times, the temperature was lowered to 0-5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice bath was removed, the mixture was heated to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 3 mL of 1,4-dioxane, and 0.6 mL of water was added. Sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to give the title product 8c (100 mg, yield: 73.6%).

MS m/z(ESI):501.0[M+1].

Step 2

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d

Compound 8c (50 mg, 0.10 mmol) was dissolved in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (25 mg, 10%) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated to give the title product 8d (41 mg, yield: 100%).

MS m/z(ESI):411.0[M+1].

Step 3

(9H-fluorene-9-yl)methyl(2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)2-oxoethyl)carbamate 8e

Compound 1b (7 mg, 0.013 mmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Then, 0.5 mL of N,N-dimethylformamide solution of compound 8d (7 mg, 0.017 mmol) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (7 mg, 0.026 mmol). The mixture was stirred in an ice bath for 35 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 8e (8.5 mg, 78.0% yield).

MS m/z(ESI):828.0[M+1].

Step 4

1-((2-aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8f

Compound 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, and 0.1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred. The upper n-hexane layer was decanted. This process was repeated three times. The crude product 8f (2.9 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

MS m/z(ESI): 606.0 [M+1].

Step 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

Crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, and cooled to 0-5 °C in an ice-water bath. 0.3 mL of N,N-dimethylformamide solution containing 8 g (2.7 mg, 5.80 μmol, prepared using the method disclosed in patent application "EP2907824") of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrolo-1-yl)hexamylamino)acetamido)acetamido)-3-phenylpropionic acid was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7 mg, 9.67 μmol). The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was heated to room temperature and stirred for 15 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 8 (2mg, yield: 39.0%).

MS m/z(ESI):1060.0[M+1].

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H),7.24-7.07(m,4 H),6.98(s,1H),6.50(s,1H),5.61(q,1H),5.40(s,2H),5.32(t,1H),5.12(q,2H),4.62(t,1H),4.52(t,1 H),4.40-4.32(m,1H),3.73-3.47(m,8H),3.16-3.04(m,2H),2.89(dd,1H),2.69-2.55(m,2H),2.37-2.2 3(m,4H),2.12-1.93(m,4H),1.90-1.74(m,2H),1.52-1.38(m,4H),1.33-1.11(m,5H),0.91-0.81(m,4H).

Examples 1-9

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-A

N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

first step

2-Cyclopropyl-2-hydroxyacetic acid benzyl ester 9a

Compound 2a (1.3 g, 11.2 mmol; prepared by the method disclosed in patent application "WO2013/106717") was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent system C to give the title product 9a (2 g, yield: 86.9%).

Step 2

10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 9b

Compounds 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, followed by the addition of 4 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0–5 °C in an ice-water bath, and then potassium tert-butoxide (109 mg, 0.98 mmol) was added. The ice bath was removed, and the mixture was brought to room temperature and stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 4 mL of dioxane, and 2 mL of water was added. Sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorene methyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system C to give the title product 9b (48 mg, yield: 19%).

MS m/z(ESI):515.0[M+1].

Step 3

10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid 9c

Compound 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1). Palladium on carbon (12 mg, 10% purity, dry form) was added, and the mixture was purged with hydrogen three times. The mixture was stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to give crude product 9c (13 mg). This product was used directly in the next reaction without further purification.

MS m/z(ESI):424.9[M+1].

Step 4

(9H-fluorene-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

Compound 1b (10 mg, 18.8 μmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Crude product 9c (13 mg, 30.6 μmol) was then added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (16.9 mg, 61.2 μmol). The mixture was stirred in an ice bath for 40 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phases were washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 9d (19 mg, yield: 73.6%).

MS m/z(ESI):842.1[M+1].

Step 5

2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide 9e

Compound 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude title product 9e (17 mg). The product was used directly in the next reaction without purification.

MS m/z(ESI):638.0[M+18].

Step 6

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-A

N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

Crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (21.2 mg, 44.8 μmol) of 0.3 mL of N,N-dimethylformamide solution was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

MS m/z(ESI):1074.4[M+1].

Compound 9-A with a single configuration (shorter retention time):

UPLC analysis: retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7 .26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1H),4.54-4 .40(m,2H),3.76-3.64(m,4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m,3H), 2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H).

Single-configuration compound 9-B (longer retention time):

UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.68-8.60(m,1H),8.58-8.50(m,1H), 8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6. 55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H),5.35-5.15(m,2H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H) ,3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2. 04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H).

Examples 1-10

N-((2S,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 10-A

N-((2R,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 10-B

first step

Benzyl 3,3,3-trifluoro-2-hydroxypropionate 10a

Compound 3a (1.80 g, 12.5 mmol) was dissolved in 100 mL of acetonitrile, and potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 mL, 37.5 mmol), and tetrabutylammonium iodide (231 mg, 0.63 mmol) were added sequentially. The reaction mixture was heated to 60 °C and stirred for 5 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent C as the developing solvent to give the title product 10a (980 mg, yield: 33.5%).

¹ H NMR (400MHz, CDCl ₃ ): δ7.43-7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H).

Step 2

1-(9H-fluorene-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 10b

Compounds 8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to a reaction flask, followed by the addition of 3 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then potassium tert-butoxide (38 mg, 0.34 mmol) was added. The ice bath was removed, and the mixture was brought to room temperature and stirred for 20 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 2 mL of dioxane, and 0.4 mL of water was added. Sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorene methyl chloroformate (49 mg, 0.19 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system C to give the title product 10b (51 mg, yield: 55.3%).

MS m/z(ESI):559.9[M+18].

Step 3

1-(9H-fluorene-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazabutane-11-acid 10c

Compound 10b (15 mg, 0.28 mmol) was dissolved in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (15 mg, 10%) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated to give the crude product 10c (13 mg).

MS m/z(ESI):452.9[M+1].

Step 4

(9H-fluorene-9-yl)methyl(2-((((3-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,1,1-trifluoro-3-oxopropyl-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 10d

Compound 1b (10 mg, 18.8 μmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Then, 0.5 mL of N,N-dimethylformamide solution containing 10c (13 mg, 28.7 μmol) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (11 mg, 39.7 μmol). The mixture was stirred in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 10d (16 mg, 97.8% yield).

MS m/z(ESI):870.0[M+1].

Step 5

2-((2-aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-3,3,3-trifluoropropionamide 10e

Compound 10d (16 mg, 18.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. This process was repeated three times. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 10e (12 mg). The product was used directly in the next reaction without purification.

MS m/z(ESI): 647.9 [M+1].

Step 6

N-((2S,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 10-A

N-((2R,10S)-10-benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 10-B

Crude product 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, 8 g (14 mg, 29.6 μmol) of 0.3 mL of N,N-dimethylformamide solution was added, followed by 15 mg (54.2 μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride. The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ) B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (2.7mg, 2.6mg).

MS m/z(ESI):1102.0[M+1].

Single-configuration compounds (shorter retention time):

UPLC analysis: retention time 1.18 min, purity: 91% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.97(d,1H),8.85-8.76(m,1H),8.37-8.27(m,1H),8.12-8.02(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.31(s,1H), 7.26-7.10(m,4H),6.99(s,1H),6.66(br,1H),6.52(s,1H),5.65-5.54(m,1H),5.41(s,1H),5.37-5.25(m,3H),5.23-5. 13(m,1H),4.81-4.68(m,2H),4.51-4.41(m,1H),3.78-3.45(m,6H),3.21-3.13(m,1H),3.02-2.93(m,1H),2.77-2.63(m ,2H),2.45-2.29(m,3H),2.24-2.05(m,3H),2.04-1.93(m,5H),1.90-1.75(m,2H),1.52-1.38(m,4H),0.90-0.78(m,5H).

Single-configuration compounds (longer retention time):

UPLC analysis: retention time 1.23 min, purity: 90% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.05(d,1H),8.97-8.88(m,1H),8.35-8.27(m,1H),8.11-8.03(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.34(s,1H), 7.29-7.13(m,4H),6.99(s,1H),6.66(br,1H),6.54(s,1H),5.64-5.55(m,1H),5.43(s,1H),5.36-5.20(m,3H),4.92-4. 85(m,1H),4.82-4.72(m,2H),4.52-4.42(m,1H),3.77-3.48(m,6H),3.21-3.14(m,1H),3.03-2.95(m,1H),2.79-2.65(m ,2H),2.47-2.28(m,3H),2.25-2.05(m,3H),2.05-1.94(m,5H),1.91-1.76(m,2H),1.52-1.37(m,4H),0.92-0.77(m,5H).

Examples 1-11

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 11

first step

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclobutane-1-carboxylic acid benzyl ester 11b

1-Hydroxycyclobutane-benzyl carboxylate 11a (167 mg, 0.81 mmol, prepared according to the method disclosed in the literature "Journal of Medicinal Chemistry, 2013, vol. 56, #13, p. 5541-5552") and 8b (150 mg, 0.41 mmol) were added to a reaction flask, followed by the addition of 5 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then potassium tert-butoxide (92 mg, 0.82 mmol) was added. The ice bath was removed, and the mixture was heated to room temperature and stirred for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 3 mL of dioxane, and 0.6 mL of water was added. Sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorene methyl chloroformate (105 mg, 0.41 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (8 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system C to give title product 11b (37 mg, yield: 17.6%).

MS m/z(ESI): 514.6 [M+1].

Step 2

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclobutane-1-carboxylic acid 11c

Compound 11b (37 mg, 71.9 μmol) was dissolved in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (15 mg, 10%) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 11c (35 mg, yield: 82%), which was then directly proceeded to the next step.

Step 3

(9H-fluorene-9-yl)methyl(2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclobutoxy)methyl)amino)2-oxoethyl)carbamate 11d

Compound 1b (10 mg, 0.018 mmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Then, 0.5 mL of an N,N-dimethylformamide solution of compound 11c (13 mg, 0.031 mmol) was added, followed by 25 mg of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (0.091 mmol). The mixture was stirred in an ice bath for 40 minutes. 8 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (8 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system A to give the title product 11d (19 mg, 73.9% yield).

MS m/z(ESI):842.3[M+1].

Step 4

1-((2-aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 11e

Compound 11d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 4 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product, 11e (15 mg), was concentrated under reduced pressure and used directly in the next reaction step without purification.

Step 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 11

Crude product 11e (2 mg, 3.22 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (1.5 mg, 3.17 μmol) of 0.3 mL of N,N-dimethylformamide solution was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7 mg, 9.67 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was evaporated to dryness using an oil pump to remove DMF. The residue was dissolved in DCM and purified twice by thin-layer chromatography (evolving solvent polarity: DCM/MeOH = 10/1) to obtain the title product 11 (1 mg, yield: 28.8%).

MS m/z(ESI): 1073.6 [M+1].

¹ H NMR (400MHz, CDCl ₃ ): δ8.70-8.60(m,1H),8.28-8.19(m,1H),8.13-7.91(m,3H),7.79-7.71(d,1H),7.29(s,1H),7.25-7.09(m,4H),6.9 8(s,1H),6.71-6.62(m,1H),6.55-6.47(m,1H),5.64-5.54(m,2H),5.40(s,1H),5.35-5.27(t,2H),5.17-5.10(m,2H ),4.60-4.51(m,1H),4.51-4.35(m,2H),3.93-3.78(m,3H),3.71-3.59(m,3H),3.01-2.88(m,3H),2.70-2.64(m,2H) ,2.44-2.30(m,3H),2.28-2.14(m,3H),2.11-1.92(m,6H),1.90-1.76(m,3H),1.51-1.39(m,4H),0.92-0.75(m,6H).

Examples 1-12

(S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxypropionamide 12-A

(R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxypropionamide 12-B

first step

12b of 3-cyclopropyl-2-hydroxypropionic acid

Compound 12a (0.5 g, 3.87 mmol, supplier Adamas) was dissolved in 35 mL of a mixed solvent of water and acetic acid (V:V = 4:1). The solution was cooled to 0–5 °C in an ice-water bath, and a 2 M aqueous solution of sodium nitrite (0.53 g, 7.74 mmol) was added dropwise. The mixture was then heated to room temperature and stirred for 3 hours. Solid sodium chloride was added to the reaction solution to saturate the aqueous phase. The solution was extracted with ethyl acetate (8 mL × 8), dried over anhydrous sodium sulfate, filtered, and concentrated to give the title product 12b (0.45 g, yield: 89.3%).

Step 2

(S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxypropionamide 12-A

(R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxypropionamide 12-B

To compound 1b (45 mg, 0.085 mmol), 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide were added, and the mixture was purged three times with argon gas. 0.1 mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction solution became clear. Compound 12b (90 mg, 0.691 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.256 mmol) were added sequentially to the reaction solution. After the additions were complete, the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 12 was purified by high-performance liquid chromatography (HPLC) (separation conditions: column: Sharpsil-T C18 5 μm 21.2*250 mm; mobile phase: A-water (10 mmol _NH₄OAc ), B-acetonitrile, gradient elution, flow rate: 18 mL/min) to obtain the title product (7 mg, 15 mg).

MS m/z (ESI): 547.9 [M+1].

Single-configuration compounds (shorter retention time):

UPLC analysis: retention time 1.345 min, purity: 72% (column: ZORBAX Ecliphase Plus C18 1.8um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.42(d,1H),7.78(d,1H),7.30(s,1H),6.51(s,1H),5.60-5.50(m ,2H),5.42(s,1H),5.19(q,2H),4.02-4.00(m,1H),3.21-3.11(m,2H), 2.39(s,3H),2.21-2.07(m,2H),2.05-1.95(m,1H),1.92-1.68(m,4H), 1.53-1.41(m,1H),0.87(t,3H),0.48-0.34(m,2H),0.14-0.01(m,2H).

Single-configuration compounds (longer retention time):

UPLC analysis: retention time 1.399 min, purity: 88% (column: ZORBAX Ecliphase Plus C18 1.8um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

^1H NMR (400MHz, DMSO-d6 ₎ ): δ8.36(d,1H),7.77(d,1H),7.31(s,1H),6.51(s,1H),5.58-5.51(m,1H),5. 48(d,1H),5.42(s,1H),5.20(q,2H),4.09-4.02(m,1H),3.22-3.11(m,2H),2.3 9(s,3H),2.27-2.06(m,2H),2.05-1.95(m,1H),1.93-1.81(m,2H),1.65-1.43 (m,2H),1.32-1.21(m,1H),0.87(t,3H),0.48-0.33(m,2H),0.14-0.01(m,2H).

Examples 1-13 (Reference Examples)

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide

Title compound 13 was prepared according to the method disclosed in Example 76 on page 147 of the specification of patent “EP2907824A1”.

Examples 1-14

N-((2R,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide 14-A

N-((2S,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide14-B

first step

3-Cyclopropyl-2-hydroxypropanoate benzyl ester 14a

Compound 12b (200 mg, 1.54 mmol) was dissolved in 20 mL of acetonitrile, and potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 mL, 1.34 mmol), and tetrabutylammonium iodide (28 mg, 0.07 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent C as the developing solvent to give the title product 14a (140 mg, yield: 41.3%).

Step 2

10-(cyclopropylmethyl)-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 14b

Compounds 14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) were added to a reaction flask, followed by the addition of 10 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0–5 °C in an ice-water bath, and then potassium tert-butoxide (79 mg, 0.704 mmol) was added. The ice bath was removed, and the mixture was brought to room temperature and stirred for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (10 mL × 4). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system C to give the title product 14b (50 mg, yield: 26.8%).

MS m/z(ESI): 529.2 [M+1].

Step 3

10-(cyclopropylmethyl)-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid 14c

Compound 14b (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate, and palladium on carbon (7 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to obtain the crude product 14c (23 mg). The product was directly used in the next step of the reaction without further purification.

MS m/z(ESI):439.1[M+1].

Step 4

(9H-fluorene-9-yl)methyl(2-((((3-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropyl-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 14d

Compound 1b (22 mg, 42.38 μmol) was added to a reaction flask, followed by 3 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then triethylamine (4.3 mg, 42.49 μmol) was added dropwise. Crude product 14c (23 mg, 51.1 μmol) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (17.6 mg, 63.6 μmol). The mixture was stirred in an ice bath for 40 minutes. 15 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phases were combined. The organic phases were washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 14d (29 mg, yield: 79.9%).

MS m/z(ESI): 856.1 [M+1].

Step 5

2-((2-aminoacetamido)methoxy)-3-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)propionamide 14e

Compound 14d (29 mg, 33.9 μmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted. This process was repeated three times. The residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 14e (22 mg). The product was used directly in the next reaction without purification.

MS m/z(ESI): 634.1 [M+1].

Step 6

N-((2R,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide 14-A

N-((2S,10S)-10-benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide14-B

Crude product 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide, purged three times with argon, and cooled to 0-5 °C in an ice-water bath. Then, 8 g (24 mg, 50.8 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (14 mg, 50.6 μmol) were added sequentially. The ice bath was removed, and the mixture was brought to room temperature and stirred for 1 hour to produce compound 14. The reaction solution was purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH₄OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min) to obtain the title product (2 mg, 2 mg).

MS m/z(ESI):1088.4[M+1].

Single-configuration compounds (shorter retention time):

UPLC analysis: retention time 1.18 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

Single-configuration compounds (longer retention time):

UPLC analysis: retention time 1.23 min, purity: 96% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

Examples 1-15

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 15

first step

1-(10-(9H-fluorene-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid benzyl ester 15b

Compound 8b (500 mg, 1.35 mmol) was added to a reaction flask, followed by 6 mL of tetrahydrofuran. 1-hydroxymethylcyclopropane-1-carboxylic acid benzyl ester 15a (233 mg, 1.13 mmol; prepared using the method disclosed in Example 22-2 on page 262 of patent application EP2862856A1) was added to the flask. The mixture was purged three times with argon gas, cooled to 0-5°C in an ice-water bath, and then sodium hydride (54 mg, 1.35 mmol) was added. The ice bath was removed, and the mixture was brought to room temperature and stirred for 40 minutes. The mixture was then cooled to 0°C and 20 mL of ice water was added. The mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to obtain the title product 15b (15 mg, yield: 2.5%).

MS m/z(ESI): 515.2 [M+1].

Step 2

1-(10-(9H-fluorene-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15c

Compound 15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate, and palladium on carbon (3 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 4.5 hours. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the filtrate was concentrated to give the title product 15c (11 mg, yield: 89%).

MS m/z(ESI):425.2[M+1].

Step 3

(9H-fluorene-9-yl)methyl(2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropyl)methoxy)methyl)amino)2-oxoethyl)carbamate 15d

Compound 1b (10 mg, 0.021 mmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Compound 15c (11 mg, 0.026 mmol) was then added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (10.7 mg, 0.039 mmol). After the addition was complete, the mixture was stirred at room temperature for 60 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 15d (19 mg, 87.0% yield).

MS m/z(ESI):842.2[M+1].

Step 4

1-(((2-aminoacetamido)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 15e

Compound 15d (19 mg, 22.56 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure at 0 °C, and 1 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product 15e (13.9 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

MS m/z(ESI): 620.1 [M+1].

Step 5

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 15

Crude product 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (15.8 mg, 33.4 μmol) was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (9.3 mg, 33.6 μmol) was added, and the mixture was stirred at room temperature for 60 minutes. The reaction solution was purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product 15 (2.5 mg, yield: 10.3%).

MS m/z(ESI):1074.2[M+1].

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.51-8.37(m,1H),8.22(t,1H),8.14-8.02(m,2H),8.011-7.94(m,1H),7.82-7.73(m,1H),7.29(s,1H),7.26-7.10(m,3H),6.98( s,1H),6.53-6.47(m,1H),5.62-5.50(m,1H),5.45-5.36(m,1H),5.35-5.23(m,2H),5.13-5.02(m,2H),4.61-4.50(m,2H),4.42-4.28( m,2H),3.76-3.61(m,3H),3.60-3.45(m,3H),3.27-3.23(m,1H),3.20-2.81(m,7H),2.75-2.61(m,3H),241-2.28(m,3H),2.23-2.13(m ,2H),2.11-2.01(m,1H),2.03-1.94(m,1H),1.90(s,1H),1.87-1.74(m,2H),1.53-1.36(m,3H),1.29-1.08(m,4H),0.90-0.68(m,4H).

Examples 1-16

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 16

first step

1-(hydroxymethyl)cyclobutane-1-carboxylic acid 16b

Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplier Alfa) was dissolved in methanol (2 mL) and water (1 mL), and sodium hydroxide (126 mg, 3.15 mmol) was added. The mixture was heated to 40 °C and stirred for 3 hours. After cooling to room temperature, the organic solvent was removed by concentration under reduced pressure, and the mixture was back-extracted with diethyl ether (10 mL), collecting the aqueous phase. The aqueous phase was adjusted to pH 3-4 with 6N hydrochloric acid aqueous solution and concentrated under reduced pressure to obtain a solid. 3 mL of toluene was added, and the mixture was concentrated under reduced pressure and evaporated to dryness. This process was repeated three times. The product was then dried using an oil pump to obtain the crude product 16b (206 mg), which was used directly in the next reaction without purification.

MS m/z(ESI, NEG):129.2[M-1].

Step 2

1-(hydroxymethyl)cyclobutane-1-carboxylic acid benzyl ester 16c

Crude product 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 mL), anhydrous potassium carbonate (1.09 g, 7.90 mmol) and tetrabutylammonium iodide (29 mg, 78.51 μmol) were added, followed by benzyl bromide (216 mg, 1.26 mmol). The mixture was stirred overnight at room temperature. The solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system C to give the title product 16c (112 mg, yield: 32.1%).

MS m/z(ESI):221.1[M+1].

Step 3

1-(10-(9H-fluorene-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid benzyl ester 16d

Compounds 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to a reaction flask, followed by the addition of 3 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0–5 °C in an ice-water bath, and then potassium tert-butoxide (61 mg, 0.54 mmol) was added. The mixture was stirred in an ice-water bath for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 3 mL of 1,4-dioxane, and 0.5 mL of water was added. Sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorene methyl chloroformate (71 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system C to give the title product 16d (24 mg, yield: 16.7%).

MS m/z(ESI):551.3[M+23].

Step 4

1-(10-(9H-fluorene-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid 16e

Compound 16d (12 mg, 22.7 μmol) was dissolved in 1.5 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (5 mg, 10%) was added. The mixture was purged three times with hydrogen and stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to obtain the crude product 16e (10 mg), which was used directly in the next reaction without further purification.

Step 5

(9H-fluorene-9-yl)methyl(2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclobutyl)methoxy)methyl)amino)2-oxoethyl)carbamate 16f

Compound 1b (7.5 mg, 0.014 mmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. Then, a 0.5 mL solution of crude 16e (10 mg) in N,N-dimethylformamide was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (6 mg, 0.026 mmol). The mixture was stirred in an ice bath for 30 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 16f (10.6 mg, 87.8% yield).

MS m/z (ESI): 856.2 [M+1].

Step 6

1-(((2-aminoacetamido)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 16g

Compound 16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product (16 g, 8 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

MS m/z(ESI): 634.1 [M+1].

Step 7

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-5,8,11,14,17-pentoxo-2-oxa-4,7,10,13,16-pentazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclobutane-1-carboxamide 16

16 g (8 mg) of the crude product was dissolved in 1 mL of N,N-dimethylformamide, and 8 g (8.8 mg, 18.6 μmol) was added. Then, 5.2 mg (18.8 μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH₄OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min) to give the title product 16 (1.0 mg, yield: 7.2%).

MS m/z(ESI):1088.0[M+1].

Examples 1-17

(1r,4r)-N-((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12,15-pentoxo-17,20,23,26,29,32,35,38,41-nonoxa-2,5,8,11,14-pentazatritoxy-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 17

first step

1-Phenylacetic-2,5,8,11,14,17,20,23,26,29-decaoxane-31-tert-butyl ester 17b

1-Phenyl-2,5,8,11,14,17,20,23,26-nonoxataocta-28-ol 17a (0.34 g, 0.67 mmol, supplier: Bidet) was dissolved in 10 mL of dichloromethane, and silver oxide (0.24 g, 1.01 mmol), tert-butyl bromoacetate (0.16 g, 0.81 mmol), and potassium iodide (0.07 g, 0.40 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to give the title product 17b (0.42 g, yield: 100%).

MS m/z(ESI): 636.3 [M+18].

Step 2

29-Hydroxy-3,6,9,12,15,18,21,24,27-Nineoxa-2,9-nona-1-octa-tert-butyl ester 17c

Compound 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran, and palladium on carbon (110 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times, and the temperature was raised to 60 °C with stirring for 3 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to obtain the crude product 17c (357 mg). The product was directly used in the next reaction without purification.

MS m/z(ESI): 546.2 [M+18].

Step 3

29-Azide-3,6,9,12,15,18,21,24,27-nonazo-1-octanoic acid tert-butyl ester 17d

Compound 17c (357 mg, 0.675 mmol) was dissolved in 10 mL of toluene, and diphenyl azidophosphate (279 mg, 1.014 mmol) and 1,8-diazabicycloundec-7-ene (206 mg, 1.353 mmol) were added. The mixture was purged with argon three times, stirred at room temperature for 2 hours, and then heated to 105 °C for 19 hours. The reaction solution was cooled to room temperature, concentrated, and extracted with ethyl acetate (10 mL × 4). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to give the crude title product 17d (412 mg).

MS m/z(ESI):571.3[M+18].

Step 4

29-Amino-3,6,9,12,15,18,21,24,27-nonoxa-2,9-dioxo-1-acid tert-butyl ester 17e

Compound 17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran, and palladium on carbon (58 mg, 10% purity, dry form) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 2 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to obtain the crude product 17e (220 mg). The product was directly used in the next step of the reaction without further purification.

MS m/z(ESI): 528.2 [M+1].

Step 5

1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonoxa-2-aza-tetrazol-31-oic acid tert-butyl ester 17f

(1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) was dissolved in 10 mL of dichloromethane. 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol) were added. The mixture was purged three times with argon gas. Crude 17e (220 mg, 0.417 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 15 mL of water was added, and the mixture was extracted with dichloromethane (8 mL × 3). The organic phases were combined. The organic phases were washed with saturated sodium chloride solution (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to give the title product 17f (122 mg, yield: 39.2%).

MS m/z(ESI):747.2[M+1].

Step 6

1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa-2-azatritria-31-acid 17g

Compound 17f (122 mg, 0.163 mmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 1 hour. The solution was diluted with 15 mL of dichloromethane and concentrated under reduced pressure. Then, 10 mL of n-hexane was added, and the solution was concentrated under reduced pressure, repeated twice. Finally, 10 mL of toluene was added and the solution was concentrated under reduced pressure. The mixture was then slurried three times with a 5:1 mixture of n-hexane and diethyl ether until the pH was close to 7. The solution was concentrated and dried using an oil pump to give 17 g (98 mg, yield: 86.8%) of the title product.

MS m/z(ESI): 691.2 [M+1].

Step 7

2,4-Dimethoxybenzyl 1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropyl-1-carboxylic acid ester 17h

Compound 8d (164 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL), and 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.60 mmol), and 4-dimethylaminopyridine (5 mg, 0.041 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was shaken to separate the layers. The aqueous phase was extracted with dichloromethane (8 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent system C to give the title product 17h (124 mg, yield: 55.4%).

MS m/z(ESI):583.1[M+23].

Step 8

2,4-Dimethoxybenzyl(S)-1-((11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazahepta-17-yl)oxy)cyclopropyl-1-carboxylic acid ester 17j

Compound 17h (39 mg, 69.6 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the upper n-hexane layer was decanted. This process was repeated three times, and the mixture was concentrated under reduced pressure. The crude product was dissolved in 2 mL of N,N-dimethylformamide, and (((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared using the method disclosed in Examples 7-12 on page 13 of patent application CN108853514A) was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (23 mg, 83.1 μmol) was added, and the mixture was stirred at room temperature for 1 hour. Add 10 mL of water and extract with ethyl acetate (10 mL × 3). Combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by thin-layer chromatography with developing solvent system B to give the title product 17j (48 mg, yield: 83.9%).

MS m/z(ESI):822.0[M+1].

Step 9

(S)-1-((11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2-oxa-4,7,10,13,16-pentazahepta-17-yl)oxy)cyclopropane-1-carboxylic acid 17k

Compound 17j (48 mg, 58.4 μmol) was dissolved in 1.4 mL of a 3% (v/v) dichloroacetic acid solution in dichloromethane. The solution was cooled to 0–5 °C in an ice-water bath, and triethylsilane (21 mg, 180.6 μmol) was added. The mixture was stirred in an ice bath for 3 hours. The solution was concentrated under reduced pressure in an ice bath to remove half of the organic solvent. 5 mL of diethyl ether was added, and the mixture was allowed to rise naturally to room temperature and stirred until a white solid precipitated. The solid was filtered, the filter cake was collected, and the mixture was dried using an oil pump to give the title product 17k (33 mg, yield: 84.1%).

Step 10

(9H-fluorene-9-yl)methyl((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl-13-yl)carbamate 17l

Compound 1b (20 mg, 42.4 μmol) was added to a reaction flask, followed by 1 mL of a 10% (v/v) methanol-dichloromethane solution. The mixture was purged three times with argon gas, cooled to 0–5 °C in an ice-water bath, and one drop of triethylamine was added. The mixture was stirred until compound 1b dissolved. Compound 17k (33 mg, 49.1 μmol) was dissolved in 1 mL of a 10% (v/v) methanol-dichloromethane solution and then added dropwise to the above reaction solution. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (17.6 mg, 63.6 μmol) was then added. The mixture was brought to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, and the mixture was stirred for 5 minutes. The mixture was allowed to stand and separate into layers. The organic phase was collected. The aqueous phase was extracted with dichloromethane (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 17l (37 mg, yield: 80.2%).

MS m/z(ESI):1090.1[M+1].

Step 11

(1r,4r)-N-((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12,15-pentoxo-17,20,23,26,29,32,35,38,41-nonoxa-2,5,8,11,14-pentazatritoxy-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 17

Compound 17l (15.5 mg, 14.23 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant was decanted. This process was repeated three times. The mixture was concentrated under reduced pressure and then dried using an oil pump. The crude product was dissolved in 1 mL of N,N-dimethylformamide, and compound 17g (11 mg, 15.92 μmol) was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (6.0 mg, 21.68 μmol) was added. The mixture was purged with argon three times, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 17 (6mg, yield: 27.4%).

MS m/z (ESI): 1556.4 [M+18].

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.98(d,1H),8.76(s,1H),8.20(br,1H),8.12-7.95(m,3H),7.93-7.76(m,2H),7.75-7.66(m,2H),7.24( s,1H),7.20-7.05(m,6H),6.97(s,1H),6.64(br,1H),6.55(d,1H),6.47(s,1H),5.61-5.52(m,2H),5.37(s, 1H),5.33-5.23(m,2H),5.18(s,1H),5.13(s,1H),5.05(s,1H),5.00(s,1H),4.65-4 .55(m,2H),4.53-4.45(m,1H),4.38-4.28(m,2H),3.84(s,2H),3.67(d,3H),3.60-3 .40(m,33H),3.18(d,1H),3.15-3.08(m,3H),2.28(s,3H),2.00-1.92(m,3H),1.85( s,2H),1.82-1.73(m,2H),1.68-1.52(m,4H),1.29-1.15(m,3H),0.86-0.76(m,5H).

Examples 1-18

(1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline -1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaza-hexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 18

first step

(R)-2-Cyclopropyl-2-hydroxyacetic acid benzyl ester 18a

(S)-2-Cyclopropyl-2-hydroxyacetic acid benzyl ester 18b

Compound 2a (7.4 g, 63.7 mmol) was dissolved in 200 mL of acetonitrile, and potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol), and tetrabutylammonium iodide (500 mg, 1.36 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 16 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue (4.1 g) was purified by silica gel column chromatography with solvent C as the developing solvent. Further chiral resolution and purification yielded the title products 18a (1.1 g) and 18b (1.2 g).

Step 2

(R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid benzyl ester 18c

Compound 8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL), and compound 18a (2.0 g, 9.70 mmol) was added. The mixture was cooled to 0–5 °C in an ice-water bath, and potassium tert-butoxide (1.89 g, 16.84 mmol) was added. The mixture was stirred in an ice-water bath for 10 minutes. Ethyl acetate (30 mL) and water (20 mL) were added, and the mixture was allowed to stand for separation. The aqueous phase was extracted with chloroform (30 mL × 5), and the organic phases were combined. The organic phase was concentrated under reduced pressure, and the residue was dissolved in 1,4-dioxane (32 mL) and water (8 mL). Sodium carbonate (1.78 g, 16.79 mmol) and fluorenyl chloroformate (2.18 g, 8.42 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with developing solvent system C to give the title product 18c (1.3 g, yield: 30.0%).

MS m/z(ESI): 515.2 [M+1].

Step 3

(R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid 18d

Compound 18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL), and palladium on carbon (260 mg, 10% purity, dry type) was added. The mixture was purged three times with hydrogen and stirred at room temperature for 5 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (20 mL) and methanol (20 mL). The filtrate was concentrated to give crude product 18d (980 mg). The product was directly used in the next reaction without purification.

MS m/z(ESI):425.1[M+1].

Step 4

2,4-Dimethoxybenzyl(R)-10-cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-ester 18e

Crude product 18d (980 mg, 2.31 mmol) was dissolved in dichloromethane (15 mL), and 2,4-dimethoxybenzyl alcohol (777 mg, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (664 mg, 3.46 mmol), and 4-dimethylaminopyridine (28 mg, 0.23 mmol) were added. The mixture was stirred at room temperature for one hour. The organic solvent was removed by concentration under reduced pressure, and 20 mL of water was added. The mixture was extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The organic phases were washed with saturated sodium chloride solution (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with solvent system C to give the title product 18e (810 mg, yield: 61.1%).

MS m/z(ESI): 575.0 [M+1].

Step 5

2,4-Dimethoxybenzyl(R)-2-((2-aminoacetamido)methoxy)-2-cyclopropylacetate 18f

Compound 18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product, 18f (21 mg), was concentrated under reduced pressure and used directly in the next reaction step without purification.

Step 6

2,4-Dimethoxybenzyl(11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentazaeicosoe-20-ester 18g

Crude product 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, compound 17i (29 mg, 57.8 μmol) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (19 mg, 68.7 μmol). The mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phases were washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give 18 g (37 mg, yield: 77.1%) of the title product.

MS m/z(ESI):853.0[M+18].

Step 7

(11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2,18-dioxa-4,7,10,13,16-pentazaeicosoe-20-acid 18h

18 g (37 mg, 44.3 μmol) of the compound was dissolved in 1.4 mL of a 3% (v/v) dichloroacetic acid solution in dichloromethane. The solution was cooled to 0–5 °C in an ice-water bath, and triethylsilane (15.4 mg, 132.4 μmol) was added. The mixture was stirred in an ice bath for 3 hours. Half of the organic solvent was removed by vacuum concentration in an ice bath. 5 mL of diethyl ether was added, and the mixture was allowed to rise naturally to room temperature and stirred until a white solid precipitated. The solid was filtered, the filter cake was collected, and the mixture was dried by an oil pump to give the title product 18h (24 mg, yield: 79.1%).

MS m/z(ESI):708.2[M+23].

Step 8

(9H-fluorene-9-yl)methyl((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexa-16-yl)carbamate 18i

Compound 1b (30 mg, 63.6 μmol) was added to a reaction flask, followed by 1 mL of a 10% (v/v) methanol-dichloromethane solution. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. The mixture was stirred until compound 1b dissolved. Compound 18h (65 mg, 94.8 μmol) was dissolved in 1 mL of a 10% (v/v) methanol-dichloromethane solution and then added dropwise to the above reaction solution. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (27 mg, 97.6 μmol) was then added. The mixture was brought to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, and the mixture was stirred for 5 minutes. The mixture was allowed to stand and separate into layers. The organic phase was collected. The aqueous phase was extracted with dichloromethane (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 18i (25 mg, yield: 35.6%).

MS m/z(ESI):1104.4[M+1].

Step 9

(S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((R)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropionamide18j

Compound 18i (12 mg, 10.9 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product, titled 18j (10 mg), was concentrated under reduced pressure and used directly in the next reaction step without purification.

MS m/z(ESI):881.0[M+1].

Step 10

(1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline lin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentazatetrahexocetrimi-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 18

Crude product 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 17 g of compound (8.5 mg, 12.3 μmol) was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.6 mg, 16.6 μmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was filtered and purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge PrepC18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain title product 18 (9.5 mg, yield: 56.2%).

MS m/z (ESI): 1570.2 [M+18].

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.77(d,1H),8.59-8.55(m,1H),8.42(d,1H),8.37-8.28(m,1H),8.25-8.06(m,2H),7.96-7.86(m,1H),7.86-7. 70(m,2H),7.32-7.28(m,1H),7.25-7.14(m,3H),6.67(m,1H),5.96(s,1H),5.80-5.72(m,1H),5.62-5.52(m,2H),5. 43-5.30(m,3H),5.28-5.17(m,2H),5.12-5.08(m,1H),4.72-4.35(m,8H),3.95-3.70(m,13H),3.35-3.22(m,14H),2 .42-2.32(m,3H),2.05-1.98(m,4H),1.88-1.82(m,12H),1.47-1.39(m,3H),1.32-1.18(m,11H),0.90-0.80(m,4H), 0.52-0.37(m,3H),0.32-0.18(m,2H).

Examples 1-19

(1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline (Lin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaza-hexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 19

first step

(S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid benzyl ester 19a

Compound 18b (252 mg, 1.22 mmol) was added to a reaction flask, followed by 4 mL of dichloromethane. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then lithium tert-butoxide (98 mg, 1.22 mmol) was added. The mixture was stirred in an ice-water bath for 15 minutes until the solution became clear. Then, 8b (300 mg, 814.3 μmol) was added, and the mixture was stirred in an ice-water bath for 2.5 hours. Water (10 mL) was added, and the mixture was separated. The aqueous phase was extracted with dichloromethane (8 mL × 2). The combined organic phases were washed with water (10 mL × 1) and saturated brine (10 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The residue was purified by silica gel column chromatography using solvent system C to give the title product 19a (282 mg, yield: 67.2%).

Step 2

(S)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid 19b

Compound 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate, and palladium on carbon (84 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 3 hours. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to obtain the crude product 19b (230 mg). The product was directly used in the next step of the reaction without further purification.

Step 3

2,4-Dimethoxybenzyl(S)-10-cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabutane-11-ester 19c

Crude product 19b (230 mg, 541.8 μmol) was dissolved in 7 mL of dichloromethane, and 2,4-dimethoxybenzyl alcohol (136.7 mg, 812.7 μmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (155 mg, 808.5 μmol), and 4-dimethylaminopyridine (6.6 mg, 53.5 μmol) were added sequentially. The mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with 10 mL of dichloromethane, washed with water (10 mL × 1), washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, and concentrated by filtration to obtain the crude product. The residue was purified by thin-layer chromatography using solvent system B to give the title product 19c (159 mg, yield: 51.0%).

Step 4

2,4-Dimethoxybenzyl(S)-2-((2-aminoacetamido)methoxy)-2-cyclopropylacetate 19d

Compound 19c (60 mg, 104.4 μmol) was dissolved in 1 mL of dichloromethane, and 0.5 mL of diethylamine was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was discarded. This process was repeated three times. The crude product, 19d (21 mg), was concentrated under reduced pressure and used directly in the next reaction step without purification.

Step 5

2,4-Dimethoxybenzyl(11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentazaeicosoe-20-ester 19e

Crude product 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, compound 17i (52 mg, 103.6 μmol) was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (34.6 mg, 125.0 μmol). The mixture was stirred at room temperature for 1 hour. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phases were washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product 19e (70 mg, yield: 80.2%).

Step 6

(11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentoxo-2,18-dioxa-4,7,10,13,16-pentazaeicosoe-20-acid 19f

Compound 19e (70 mg, 83.7 μmol) was dissolved in 2.5 mL of a 3% (v/v) dichloroacetic acid solution in dichloromethane. The solution was cooled to 0–5 °C in an ice-water bath, and triethylsilane (29 mg, 249.4 μmol) was added. The mixture was stirred in an ice bath for 3 hours. The solution was concentrated under reduced pressure in an ice bath to remove half of the organic solvent. 5 mL of diethyl ether was added, and the mixture was allowed to rise naturally to room temperature and stirred until a white solid precipitated. The solid was filtered, the filter cake was collected, and the mixture was dried using an oil pump to give the title product 19f (57 mg, yield: 99.2%).

Step 7

(9H-fluorene-9-yl)methyl((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexa-16-yl)carbamate 19g

Compound 1b (30 mg, 63.6 μmol) was added to a reaction flask, followed by 1 mL of a 10% (v/v) methanol-dichloromethane solution. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. The mixture was stirred until compound 1b dissolved. Compound 19f (57 mg, 83.1 μmol) was dissolved in 1 mL of a 10% (v/v) methanol-dichloromethane solution and then added dropwise to the above reaction solution. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (26 mg, 93.9 μmol) was then added. The mixture was brought to room temperature and stirred for 1 hour. 10 mL of dichloromethane and 5 mL of water were added, and the mixture was stirred for 5 minutes. The mixture was allowed to stand and separate into layers. The organic phase was collected. The aqueous phase was extracted with dichloromethane (10 mL × 3), and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give 19 g (56 mg, yield: 79.8%) of the title product.

MS m/z(ESI):1103.1[M+1].

Step 8

(S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-((((S)-1-cyclopropyl-2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropionamide 19h

19 g (4.6 mg, 4.16 μmol) of the compound was dissolved in 1.5 mL of dichloromethane, and 0.75 mL of diethylamine was added. The mixture was stirred at room temperature for 1.6 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred until the supernatant n-hexane was decanted. This process was repeated three times. The crude product 19 h (4.0 mg) was concentrated under reduced pressure and used directly in the next reaction without purification.

Step 9

(1r,4r)-N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline (Lin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaza-hexa-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide 19

The crude product 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 17 g (2.9 mg, 4.2 μmol) of compound was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (1.5 mg, 5.4 μmol) was added, and the mixture was stirred at room temperature for 40 minutes. The reaction solution was filtered and purified by high-performance liquid chromatography (HPLC) (separation conditions: column: XBridge PrepC18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 19 (2.1 mg, yield: 32.4%).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.71-8.62(m,1H),8.59-8.51(m,1H),8.34-8.26(m,1H),8.14-8.02 (m,2H),7.95-7.86(m,1H),7.83-7.69(m,2H),7.35-7.31(m,1H),7.29-7 .11(m,3H),7.01(s,1H),6.72-6.50(m,3H),5.59-5.50(m,2H),5.42(s,2 H),5.38-5.18(m,3H),4.79-4.69(m,2H),4.61-4.42(m,3H),3.91(s,2H) ,3.79-3.65(m,4H),3.63-3.44(m,13H),3.41-3.30(m,2H),3.26-3.09( m,5H),3.08-2.84(m,4H),2.81-2.64(m,3H),2.42-2.28(m,3H),2.24-2. 12(m,2H),2.05-1.93(m,4H),1.89-1.77(m,2H),1.72-1.56(m,3H),1.53 -1.38(m,3H),1.34-1.10(m,11H),0.94-0.78(m,5H),0.52-0.35(m,3H).

Examples 1-20 (Reference Examples)

The title compound 20 was synthesized according to the method provided in Example 58 on page 163 of the specification of patent CN104755494A.

The following antibodies were prepared using standard antibody preparation methods, such as vector construction, transfection into eukaryotic cells like HEK293 cells (Life Technologies Cat. No. 11625019), expression, and purification.

The following is the sequence of Trastuzumab:

Light chain

Heavy chain

The following is the sequence of Pertuzumab:

Light chain

Heavy chain

The following is the sequence of B7H3 antibody 1F9DS:

Light chain

Heavy chain

Example 1-21 ADC-1

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 2.5 mL, 9.96 mg/mL, 0.168 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 2.0 mL of the solution was taken out and used for further reaction.

Compound 10—a compound with a shorter retention time (2.1 mg, 2.02 μmol)—was dissolved in 0.10 mL of DMSO and added to the above 2.0 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (5.0 mg/mL, 1.1 mL) of the exemplary product ADC-1 of the general formula FADC-1, which was stored at 4 °C.

UV-HPLC calculated average: n = 5.09.

Example 1-22 ADC-2

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 2.5 mL, 9.96 mg/mL, 0.168 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 2.0 mL of the solution was taken out and used for further reaction.

Compound 10—a compound with a longer retention time (2.1 mg, 2.02 μmol)—was dissolved in 0.10 mL of DMSO and added to the above 2.0 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (4.95 mg/mL, 1.1 mL) of the exemplary product ADC-2 of the general formula FADC-1, which was stored at 4 °C.

UV-HPLC calculated average: n = 7.39.

Example 1-23 ADC-3

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 2.5 mL, 9.96 mg/mL, 0.168 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 2.0 mL of the solution was taken out and used for further reaction.

Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO and added to the above 2.0 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (5.24 mg/mL, 1.1 mL) of the exemplary product ADC-3 of the general formula FADC-3, which was stored at 4 °C.

UV-HPLC calculated average: n = 7.36.

Example 1-24 ADC-4

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 3.74 mL, 13.38 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 6.7 mg/mL, and 1.3 mL of the solution was taken out and used for further reaction.

Compound 9-A (1.0 mg, 0.93 μmol), a compound with a shorter retention time, was dissolved in 0.10 mL of DMSO and added to the above 1.3 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.72 mg/mL, 2.36 mL) of the exemplary product ADC-4 of the general formula FADC-4A, which was stored at 4 °C.

UV-HPLC calculated average: n = 7.39.

Example 1-25 ADC-5

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol) was added to the PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 3.0 mL, 6.70 mg/mL, 0.136 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, and 0.614 mL of the solution was taken out and the reaction was continued.

Compound 9-A (0.5 mg, 0.42 μmol), a compound with a shorter retention time, was dissolved in 0.031 mL of DMSO and added to the above 0.614 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (3.08 mg/mL, 0.82 mL) of the exemplary product ADC-5 of the general formula FADC-4A, which was stored at 4 °C.

UV-HPLC calculated average: n = 3.16.

Example 1-26 ADC-6

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 3.74 mL, 13.38 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 6.7 mg/mL, and 0.75 mL of the solution was taken out and used for further reaction.

Compound 9-B (0.68 mg, 0.63 μmol), a compound with a longer retention time, was dissolved in 0.10 mL of DMSO and added to the above 0.75 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.78 mg/mL, 1.78 mL) of the exemplary product ADC-6 of the general formula FADC-4B, which was stored at 4 °C.

UV-HPLC calculated average: n = 3.94.

Example 1-27 ADC-7

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to the PBS buffered aqueous solution of the antibody Pertuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 5.0 mL, 10 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 1.0 mL of the solution was taken out and used for further reaction.

Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 mL of DMSO and added to the above 1.0 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.42 mg/mL, 2.15 mL) of the exemplary product ADC-7 of the general formula FADC-7, which was stored at 4 °C.

UV-HPLC calculated average: n = 6.91.

Example 1-28 ADC-8

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Pertuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 5.0 mL, 10 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 1.6 mL of the solution was taken out and used for further reaction.

Compound 10—a compound with a shorter retention time (1.04 mg, 1.0 μmol)—was dissolved in 0.1 mL of DMSO and added to the above 1.6 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.14 mg/mL, 2.31 mL) of the exemplary product ADC-8 of the general formula FADC-8, which was stored at 4 °C.

UV-HPLC calculated average: n = 6.58.

Example 1-29 ADC-9

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol) was added to a PBS buffered aqueous solution of the antibody Pertuzumab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 5.0 mL, 10 mg/mL, 0.338 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg/mL, and 0.8 mL of the solution was taken out and used for further reaction.

Compound 9-A (0.55 mg, 0.5 μmol), a compound with a shorter retention time, was dissolved in 0.1 mL of DMSO and added to the above 0.8 mL solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.27 mg/mL, 1.11 mL) of the exemplary product ADC-9 of the general formula FADC-9A, which was stored at 4 °C.

UV-HPLC calculated average: n = 3.16.

Example 1-30 ADC-10

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/mL, 0.574 mL, 38.78 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 14—a compound with a shorter retention time (0.64 mg, 588 nmol)—was dissolved in 40 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (5.48 mg/mL, 1.03 mL) of the exemplary product ADC-10 of the general formula FADC-10, which was stored at 4 °C.

UV-Vis calculated average: n = 6.25.

Example 1-31 ADC-11

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 0.646 mL, 43.64 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 14—a compound with a longer retention time (0.72 mg, 662 nmol)—was dissolved in 40 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.13 mg/mL, 1.87 mL) of the exemplary product ADC-11 of the general formula FADC-10, which was stored at 4 °C.

UV-Vis calculated average: n = 7.03.

Example 1-32 ADC-12

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/mL, 0.726 mL, 49.05 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (3.34 mg/mL, 1.45 mL) of the exemplary product ADC-12 of the general formula FADC-12, which was stored at 4 °C.

UV-Vis calculated average: n = 6.93.

Example 1-33 ADC-13

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/mL, 0.287 mL, 19.39 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.37 mg/mL, 0.88 mL) of the exemplary product ADC-13 of the general formula FADC-13, which was stored at 4 °C.

UV-Vis calculated average: n = 6.53.

Example 1-34 ADC-14

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/mL, 0.592 mL, 40.0 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.30 mg/mL, 12.0 mL) of the exemplary product ADC-14 of the general formula FADC-14, which was stored at 4 °C.

UV-Vis calculated average: n = 7.61.

Example 1-35 ADC-15

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffer; 10.0 mg/mL, 0.592 mL, 40.0 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.32 mg/mL, 11.8 mL) of the exemplary product ADC-15 of the general formula FADC-15, which was stored at 4 °C.

UV-Vis calculated average: n = 7.89.

Example 1-36 ADC-16

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 0.53 mL, 35.8 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.32 mg/mL, 12.0 mL) of the exemplary product ADC-16 of the general formula FADC-16, which was stored at 4 °C.

UV-Vis calculated average: n = 7.43.

Example 1-37 ADC-17

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 2.0 mL, 135.12 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (2.22 mg, 2067 nmol), a compound with a shorter retention time, was dissolved in 175 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.32 mg/mL, 12.0 mL) of the exemplary product ADC-17 of the general formula FADC-4A, which was stored at 4 °C.

UV-Vis calculated average: n = 5.42.

Example 1-38 ADC-18 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 1.5 mL, 101.3 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.79 mg/mL, 13.0 mL) of the exemplary product ADC-18 of the general formula FADC-18, which was stored at 4 °C.

UV-Vis calculated average: n = 7.23.

Example 1-39 ADC-19

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 1.36 mL, 91.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (2.0 mg, 1862 nmol), a compound with a shorter retention time, was dissolved in 100 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.73 mg/mL, 13.0 mL) of the exemplary product ADC-19 of the general formula FADC-4A, which was stored at 4 °C.

UV-Vis calculated average: n = 6.26.

Example 1-40 ADC-20

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/ml, 1.5 mL, 101.3 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 10—a compound with a longer retention time (2.0 mg, 1815 nmol)—was dissolved in 100 μl of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.73 mg/mL, 13.0 mL) of the exemplary product ADC-20 of the general formula FADC-1, which was stored at 4 °C.

UV-Vis calculated average: n = 7.43.

Example 1-41 ADC-21 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 1.86 mL, 125.4 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.91 mg/mL, 4.44 mL) of the exemplary product ADC-21 of the general formula FADC-18, which was stored at 4 °C.

UV-Vis calculated average: n = 7.23.

Example 1-42 ADC-22

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 1.88 mL, 127.2 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (2.1 mg, 1955 nmol), a compound with a shorter retention time, was dissolved in 150 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (3.56 mg/mL, 3.98 mL) of the exemplary product ADC-22 of the general formula FADC-4A, which was stored at 4 °C.

UV-Vis calculated average: n = 6.79.

Examples 1-43 ADC-23 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 345 mL, 23.31 μmol). The solution was placed in a water bath and shaken at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL of MeCN and 3.56 mL of DMSO, and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by ultrafiltration through a membrane membrane using PBS buffer solution (0.05 M PBS buffer solution, pH = 6.5) containing 2% (v/v) MeCN and 1% (v/v) DMSO, and succinate buffer solution (0.01 M succinate buffer solution, pH = 5.3). Sucrose was then added to a concentration of 60 mg/mL and Tween 20 to a concentration of 0.2 mg/mL. The mixture was then lyophilized to obtain an exemplary product of the general formula FADC-18, ADC-23, as a lyophilized powder sample, which was stored at 4 °C.

UV-Vis calculated average: n = 7.05.

Example 1-44 ADC-24

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol) was added to a PBS-buffered solution of the antibody Trastuzumab (pH = 6.5, 0.05 M PBS buffered solution; 10.0 mg/mL, 332 mL, 22.43 μmol). The solution was placed in a water bath and shaken at 37°C for 3.5 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (241 mg, 224 μmol), a compound with a shorter retention time, was dissolved in 13.76 mL of MeCN and 6.88 mL of DMSO. This solution was added to the above reaction mixture, and the mixture was placed in a water bath and shaken at 25 °C for 3 hours. The reaction was then stopped. The reaction mixture was purified by ultrafiltration using a PBS buffer solution containing 4% (v/v) MeCN and 2% (v/v) DMSO (0.05 M PBS buffer solution, pH = 6.5) and a succinate buffer solution (0.01 M succinate buffer solution, pH = 5.3). Sucrose was then added to a concentration of 60 mg/mL, and Tween 20 was added to a concentration of 0.2 mg/mL. The mixture was then lyophilized to obtain an exemplary product of the general formula FADC-4A, ADC-24, as a lyophilized powder sample, which was stored at 4 °C.

UV-Vis calculated average: n = 7.07.

Example 1-45 ADC-25

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol) was added to a PBS buffered aqueous solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.14 mL, 144.60 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (3.0 mg, 2793 nmol), a compound with a shorter retention time, was dissolved in 150 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.28 mg/mL, 13.0 mL) of the exemplary product ADC-25 of the general formula FADC-25, which was stored at 4 °C.

UV-Vis calculated average: n = 6.87.

Examples 1-46 ADC-26 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to a PBS buffer solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffer solution; 10.0 mg/mL, 0.89 mL, 60.14 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.61 mg/mL, 4.0 mL) of the exemplary product ADC-26 of the general formula FADC-26, which was stored at 4 °C.

UV-Vis calculated average: n = 6.15.

Example 1-47 ADC-27

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to a PBS buffer solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffer solution; 10.0 mg/mL, 0.89 mL, 60.14 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (1.02 mg, 950 nmol), a compound with a shorter retention time, was dissolved in 100 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.94 mg/mL, 3.5 mL) of the exemplary product ADC-27 of the general formula FADC-25, which was stored at 4 °C.

UV-Vis calculated average: n = 6.11.

Example 1-48 ADC-28 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol) was added to a PBS buffered aqueous solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.36 mL, 159.47 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.29 mg/mL, 13.0 mL) of the exemplary product ADC-28 of the general formula FADC-26, which was stored at 4 °C.

UV-Vis calculated average: n = 7.46.

Example 1-49 ADC-29

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 290 nmol) was added to a PBS buffer solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffer solution; 10.0 mg/mL, 0.80 mL, 50.06 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (1.29 mg, 1201 nmol), a compound with a shorter retention time, was dissolved in 100 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.63 mg/mL, 2.4 mL) of the exemplary product ADC-29 of the general formula FADC-25, which was stored at 4 °C.

UV-Vis calculated average: n = 7.24.

Example 1-50 ADC-30 (Reference Example)

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol) was added to a PBS-buffered aqueous solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/ml, 0.86 mL, 58.4 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.61 mg/mL, 4.0 mL) of the exemplary product ADC-30 of the general formula FADC-26, which was stored at 4 °C.

UV-Vis calculated average: n = 6.15.

Example 1-51 ADC-31

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol) was added to a PBS buffer solution of antibody B7H3 antibody 1F9DS (pH = 6.5, 0.05 M PBS buffer solution; 10.0 mg/mL, 0.89 mL, 60.14 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.47 mg/mL, 4.5 mL) of the exemplary product ADC-31 of the general formula FADC-31, which was stored at 4 °C.

UV-Vis calculated average: n = 6.33.

Example 1-52 ADC-32

At 25°C, 5.0 g of trastuzumab antibody stock solution (34.44 μmol, diluted to a final antibody concentration of 15 mg/mL with 20 mM histidine-hydrochloric acid buffer) and 34.64 mg of tris(2-carboxyethyl)phosphonic acid hydrochloride (reducing agent TCEP, Sigma, 120.84 μmol) were stirred in a constant temperature water bath for 3 hours to generate intermediate I solution.

Compound 9-A (406.2 mg, 378.17 μmol), with a shorter retention time than compound 9, was dissolved in 9.98 mL of DMSO to generate a DMSO solution of compound 9-A. 23.42 mL of DMSO was pre-added to the above intermediate I solution, and then the DMSO solution of compound 9-A was added to the pre-added DMSO intermediate I solution. The reaction was stirred for 1 hour in a water bath at 25°C, and the reaction was quenched with cysteine. The mixture was then filtered. At 25°C, the reaction solution was passed through an ultrafiltration membrane (30 kDa) and subjected to 10-fold and 16-fold volume changes of ultrafiltration with 20 mM histidine-hydrochloric acid and 2.5 mM EDTA buffer (pH = 6.0) containing 10% (v/v) DMSO, and 10 mM histidine-hydrochloric acid buffer (pH = 5.5) to remove small molecules and residual solvent, yielding the exemplary product ADC-32 of the general formula FADC-4A.

The average value was calculated using the HIC method: n = 6.0.

Example 1-53 ADC-33

At 37°C, 8.0 g of trastuzumab antibody stock solution (55.11 μmol, diluted to a final antibody concentration of 15 mg/mL with 20 mM histidine-hydrochloric acid and 2.5 mM EDTA) and 123.95 mg of tris(2-carboxyethyl)phosphonic acid hydrochloride (reducing agent TCEP, Sigma, 432.41 μmol) were stirred in a constant temperature water bath for 5 hours to generate intermediate I solution.

Compound 9-A (754.7 mg, 702.63 μmol), with a shorter retention time than compound 9, was dissolved in 15.99 mL of DMSO to generate a DMSO solution of compound 9-A. 37.34 mL of DMSO was pre-added to the above intermediate I solution, and then the DMSO solution of compound 9-A was added to the pre-added DMSO intermediate I solution. The reaction was stirred in a water bath at 37°C for 1 hour, then stopped and filtered. At 25°C, the reaction solution was passed through an ultrafiltration membrane (30 kDa) and subjected to 8-fold and 16-fold volume replacements of 20 mM histidine-hydrochloric acid and 2.5 mM EDTA buffer solution (pH = 6.0) containing 10% (v/v) DMSO, and 10 mM histidine-hydrochloric acid buffer solution (pH = 6.0) to remove small molecules and residual solvent, yielding the exemplary product ADC-33 of the general formula FADC-4A.

The average value was calculated using the HIC method: n = 7.15.

ADC drug loading analysis

Experimental Objectives and Principles

Antibody-associated drug (ADC) is a type of antibody-crosslinked drug. Its mechanism of action relies on the targeted delivery of toxin molecules into cells, thereby killing the cells. The drug loading plays a decisive role in efficacy. The drug loading of ADC stock solutions was determined using ultraviolet-visible spectrophotometry (UV-Vis) or hydrophobic chromatography (HIC).

I. Ultraviolet-Visible Spectrophotometry

After placing cuvettes containing sodium succinate buffer solution into the reference absorption cell and the sample absorption cell, respectively, and subtracting the solvent blank, cuvettes containing the test solution were placed into the sample absorption cell, and absorbance was measured at 280 nm and 370 nm.

Results Calculation: The ADC stock solution loading was determined using ultraviolet spectrophotometry (instrument: Thermo Nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody at that wavelength, i.e.:

(1)A _280nm =ε _mab-280 bC _mab +ε _Drug-280 bC _Drug

ε _Drug-280 : The drug has an average molar extinction coefficient of 5100 at 280 nm;

C _Drug : Drug concentration;

ε _mab-280 : Trastuzumab antigen solution or pertuzumab antigen solution has an average molar extinction coefficient of 214600 at 280 nm;

C _mab : Concentration of trastuzumab antigen solution or pertuzumab antigen solution;

b: The optical path length is 1cm.

Similarly, the equation for the total absorbance of the sample at 370 nm can be obtained:

(2)A _370nm =ε _mab-370 bC _mab +ε _Drug-370 bC _Drug

ε _Drug-370 : The drug has an average molar extinction coefficient of 19000 at 370 nm;

C _Drug : Drug concentration;

ε _mab-370 : Trastuzumab antigen solution or pertuzumab antigen solution has an extinction coefficient of 0 at 370nm;

C _mab : Concentration of trastuzumab antigen solution;

b: The optical path length is 1cm.

The drug loading can be calculated by combining equations (1) and (2) with the extinction coefficients and concentration data of the monoclonal antibody and drug at two detection wavelengths.

Drug load n = C_{_Drug} / C _{_mab} .

II. Hydrophobic Chromatography

2.1 Perform HPLC analysis under the following measurement conditions:

HPLC System: Agilent High Performance Liquid Chromatography (HPLC) System

Detector: DAD detector (measurement wavelength: 280nm)

Column: TSKgel Butyl-NPR (4.6mm × 100mm, 2.5μm)

Column temperature: 30℃

Flow rate: 0.5 ml/min

Sample chamber temperature: 4℃

Mobile phase A: pH 7.00 aqueous solution containing 1.5M ammonium sulfate ((NH4)2SO4) and 20mM disodium hydrogen phosphate (Na2HPO4).

Mobile phase B: A mixed solution at pH 7.00 containing 75% 20mM disodium hydrogen phosphate (Na2HPO4) and 25% isopropanol.

Gradient program: 0.0%B - 0.0%B (0 min - 3.0 min), 0.0%B - 100.0%B (3.0 min - 30.0 min), 100.0%B -

100.0%B (30.0min-33.0min), 0.0%B-0.0%B (33.1min-40.0min),

Sample volume injected: 50 μg

2.2 Data Analysis

Based on current data, and due to column characteristics and differences in salt concentration, antibody-drug conjugates are eluted in order of increasing drug-bound number. Therefore, the distribution of drug-bound number is obtained by measuring the peak area. The peaks are eluted in the order of D0 (antibody without drug-bound linker), D2, D4, D6, and D8. The drug loading DAR(n) is calculated using the following formula:

Table 1. Calculation of Drug Loading in Hydrophobic Chromatography

name Number of connected drugs Weighted peak area percentage D0 0 D0 peak area * number of connected drugs D2 2 D2 peak area * number of connected drugs D4 4 D4 peak area * number of drugs connected D6 6 D6 peak area * number of drugs connected D8 8 D8 peak area * number of drugs connected

Drug loading n = Σ(weighted peak area) / 100

Biological evaluation

Test Example 1-1: In vitro inhibition test of the disclosed compound on tumor cell proliferation

I. Purpose of the Test

The purpose of this experiment was to detect the inhibitory activity of the disclosed drug compound on the in vitro proliferation of U87MG cells (Chinese Academy of Sciences Cell Bank, Catalog #TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30). Cells were treated with different concentrations of the compound in vitro, and after 6 days of culture, cell proliferation was detected using the CTG (Cell Titer-Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent. The in vitro activity of the compound was evaluated based on the IC50 value.

II. Experimental Methods

The following example, using the in vitro proliferation inhibition assay method for U87MG cells, illustrates the method for testing the in vitro proliferation inhibition activity of the disclosed compounds against tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, catalog number SH30024.01) with 10% FBS and McCoy's 5A medium (Gibco, catalog number 16600-108) with 10% FBS, respectively.

2. Cell preparation: Take U87MG and SK-BR-3 cells in the logarithmic growth phase, wash them once with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to prepare a single-cell suspension.

3. Cell Plating: Mix U87MG and SK-BR-3 single-cell suspensions thoroughly. Adjust the viable cell density to 2.75 × ^10³ cells/mL and 8.25 × ^10³ cells/mL respectively using cell culture medium. Mix the adjusted cell suspensions thoroughly and add 180 μL/well to a 96-well cell culture plate. Add only 200 μL of culture medium to the outer wells of the 96-well plate. Incubate the plates in an incubator for 24 hours (37℃, 5% _CO₂ ).

4. Compound preparation: Dissolve the compound in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.

The initial concentration of the small molecule compound is 500 nM, and the preparation method is as follows.

Add 30 μL of each different test sample (100 μM) to the first well of a 96-well U-bottom preparation plate. Add 20 μL of DMSO to each well in columns 2 through 11. Take 10 μL of the sample from the first column and add it to the 20 μL of DMSO in the second column, mix well, and then add 10 μL to the third column, and so on up to the tenth column. Transfer 5 μL of the drug from each well of the preparation plate to 95 μL of EMEM medium, mix well, and set aside for later use.

The initial concentration of the ADC is 10 nM or 500 nM, and the preparation method is as follows.

Add 100 μL of different test samples to the first column of a 96-well plate, with a sample concentration of 100 nM or 5 μM. Add 100 μL of PBS to each well in columns 2 through 11. Take 50 μL of the sample from the first column and add it to the 100 μL of PBS in the second column, mix well, take 50 μL and add it to the third column, and so on, diluting 3-fold up to the 10th column.

5. Sample addition procedure: Add 20 μL of the prepared test sample at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% _CO2 ).

6. Color development procedure: Take out the 96-well cell culture plate, add 90 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

7. Plate reading procedure: Take out the 96-well cell culture plate and place it in a microplate reader (BMG Labtech, PHERAstar FS) to measure chemiluminescence.

III. Data Analysis

The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in Table 2 below.

Table 2. _IC50 values of the small molecule fragments disclosed herein on the in vitro proliferation inhibition of SK-BR-3 and U87 cells.

Conclusion: The small molecule fragment disclosed in this study has significant inhibitory activity against the proliferation of SK-BR-3 and U87 cells, and the chiral center has a certain influence on the inhibitory activity of the compound.

Test Examples 1-2: In vitro proliferation inhibition test of the antibody-drug conjugates disclosed herein on HER2-targeted tumor cells

The purpose of this experiment was to detect the inhibitory activity of the disclosed antibody-drug conjugate targeting HER2 on the in vitro proliferation of SK-BR-3 (human breast cancer cells, ATCC, catalog number HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, catalog number HTB-132). Cells were treated with different concentrations of the compound in vitro, and after 6 days of culture, cell proliferation was detected using CTG reagent. The in vitro activity of the compound was evaluated based on the _IC50 value.

Following the test method in Test Example 1, the test cells were SK-BR-3 and MDA-MB-468. The cell culture media were McCoy's 5A medium (Gibco, catalog number 16600-108) containing 10% FBS, EMEM medium (GE, catalog number SH30024.01) containing 10% FBS, and L-15 medium (ThermoFisher, catalog number 11415-114) containing 10% FBS, respectively. The viable cell densities of the three cell lines were adjusted to 8.33 × ^10³ cells/mL, ^{8.33 × 10³} cells/mL, and 1.39 × ^10⁴ cells/mL using the cell culture media. The density-adjusted cell suspensions were mixed thoroughly, and 180 μL/well was added to a 96-well cell culture plate. The relevant compounds were tested, and the results are shown in Table 3 below.

Table 3. _IC50 values of the antibody-drug conjugate disclosed herein for in vitro inhibition of HER2-targeted tumor cell proliferation.

Conclusion: The antibody-drug conjugates targeting HER2 disclosed in this study have significant inhibitory activity against the proliferation of HER2-positive SK-BR-3 cells; at the same time, they have weak inhibitory activity against the proliferation of HER2-negative MDA-MB-468 cells, demonstrating good selectivity.

Test Examples 1-3: Her2-ADC Plasma Stability Experiment

ADC-19, ADC-18, ADC-20 samples, human plasma, monkey plasma (Shanghai Medicilon Biopharmaceutical Co., Ltd.), and 1% BSA (Sigma) PBS solution (Shanghai Sangon Biotech) were sterilized by filtration through a 0.22 μm filter. ADC-19, ADC-18, and ADC-20 were added to the sterile plasma or 1% BSA PBS solution at a final concentration of 200 μg/mL and incubated at 37°C in a cell culture incubator. The day of incubation was designated as day 0. Samples were then collected on days 7, 14, and 21 for free toxin detection.

Transfer 25 μL of sample to a 96-well plate; add 50 μL of internal standard working solution (100 ng/mL camptothecin acetonitrile solution) and 150 μL of acetonitrile; vortex for 5 minutes, centrifuge for 10 minutes (4000 rpm), and analyze 5 μL by LC/MS/MS (Applied Biosystems, Inc.).

The results showed that ADC-19 was quite stable in human and monkey plasma, as well as in 1% BSA PBS solution, with the release rate of free toxin not exceeding 2.1% at most, and tending to stabilize on day 14 (Figure 1A).

ADC-18 exhibits poor stability in human and monkey plasma, with the highest release rates of free toxins reaching 14.5% and 8.10%, respectively. It is relatively stable in 1% BSA PBS solution (see Figure 1B).

ADC-20 exhibited poor stability in human plasma, monkey plasma, and 1% BSA PBS solution, with the highest release rates of free toxins at 21.7%, 29.7%, and 21.7%, respectively. Furthermore, it remained in a degrading state in 1% BSA PBS solution (see Figure 1C).

Test Examples 1-4: Evaluation of JIMT-1 Efficacy in Tumor-Bearing Mice

I. Experimental Objective

Nunu mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies T-DM1, ADC-21, and ADC-24 administered intraperitoneally in nunu mice with trastuzumab-resistant human breast cancer cell line JIMT-1 xenografts.

II. Test Drugs and Materials

1. Test drug

T-DM1 (prepared according to US20050169933)

ADC-21: 3 mg/kg

ADC-21: 10 mg/kg

ADC-24: 3 mg/kg

ADC-24: 10 mg/kg

Blank control: PBS

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

nunu nude mouse, purchased from Beijing Vital River.

III. Test Methods

JIMT-1 cells (Nanjing Kebai) (5× ^10⁶ /mouse, with 50% artificial basement membrane) were subcutaneously injected into the right rib area of mice. After 8 days of tumor growth, when the tumor reached 203.09±11.94 mm³, ^the animals were randomly divided into 6 groups (d1), with 8 mice per group.

The medication was administered via intraperitoneal injection, twice in total. Tumor volume and body weight were measured twice weekly, and the data were recorded.

Data were statistically analyzed using Excel 2003 software: mean values were calculated as averages; SD values were calculated as standard deviations (STDEV); SEM values were calculated as STDEV/SQRT; and p-values for intergroup differences were calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × _{L_length} ^× _{L_short²}

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control group (Vehicle, PBS) and the experimental group at the end of the experiment, respectively.

IV. Test Results

The experimental results are shown in Figure 2. The drug was administered intraperitoneally twice, and the experiment ended on day 34. T-DM1 (10 mg/kg) had no inhibitory effect on tumors; ADC-21, 3 mg/kg, showed a tumor inhibition rate of 46.22% (P < 0.01); ADC-21, 10 mg/kg, showed a tumor inhibition rate of 56.77% (P < 0.001); ADC-24, 3 mg/kg, showed a tumor inhibition rate of 62.77% (P < 0.001); ADC-24, 10 mg/kg, showed a tumor inhibition rate of 76.32% (P < 0.001). At the same dosage, ADC-24 showed significantly better tumor inhibition than ADC-21.

Test Examples 1-5: Efficacy Evaluation of SK-BR-3 Tumor-Bearing Mice

I. Experimental Objective

Nunu mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies ADC-21 and ADC-22 administered intraperitoneally on nunu mice with human breast cancer cell xenograft SK-BR-3.

II. Test Drugs and Materials

1. Test drug

ADC-21: 1 mg/kg

ADC-21: 6 mg/kg

ADC-22: 1 mg/kg

ADC-22: 6 mg/kg

Blank control: PBS.

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

nunu nude mouse, purchased from Beijing Vital River.

III. Test Methods

SK-BR-3 cells (ATCC) (5× ^10⁶ /mouse, with 50% artificial basement membrane) were subcutaneously injected into the right rib area of mice. After 20 days of tumor growth, when the tumor reached 153.34±11.73 mm³, the animals were randomly divided into 5 groups (d0), with 8 mice per group.

The medication was administered once via intraperitoneal injection. Tumor volume and body weight were measured twice weekly, and the data were recorded.

Data were statistically analyzed using Excel 2003 software: mean values were calculated as averages; SD values were calculated as standard deviations (STDEV); SEM values were calculated as STDEV/SQRT; and p-values for intergroup differences were calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × _{L_length} ^× _{L_short²}

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control and experimental group at the end of the experiment, respectively.

IV. Test Results

The experimental results are shown in Figure 3. After a single intraperitoneal injection, the experiment ended on day 28. The tumor inhibition rate of ADC-21 (1 mg/kg) was 15.01%, and that of ADC-21 (6 mg/kg) was 77.4%, showing a highly significant difference compared to the control group (P < 0.001). The tumor inhibition rate of ADC-22 (1 mg/kg) was 19.82%, and that of ADC-22 (6 mg/kg) was 98.38% (P < 0.001). At the same dose of 6 mg/kg, ADC-22 showed significantly better tumor inhibition than ADC-21.

Test Examples 1-6: Plasma Stability

The ADC-25 sample was mixed with human plasma, monkey plasma and 1% BSA PBS solution at a final concentration of 100 μg/mL. After being filtered and sterilized, the mixture was incubated in a 37°C water bath. The day of incubation was marked as day 0. The samples were then taken out on days 7, 14 and 21 for free toxin detection.

After the samples were taken out at different time points, they were placed at room temperature and vortexed to mix. 25 μL of the sample was transferred to a 96-well plate. 50 μL of internal standard working solution (100 ng/mL camptothecin acetonitrile solution) and 150 μL of acetonitrile were added. The mixture was vortexed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 5 μL of the supernatant was taken for LC/MS/MS analysis.

As shown in Figure 4, ADC-25 was quite stable in human and monkey plasma, as well as in 1% BSA PBS solution, with the release rate of free toxin not exceeding 2% at most, and tending to stabilize on day 14.

Test Examples 1-7: Evaluation of the efficacy of ADC in human astrocytoma U87MG xenografts in nude mice

I. Experimental Objective

This experiment used BALB/cA-nude nude mice as test animals to evaluate the efficacy of the disclosed ADC compound on human astrocytoma U87MG nude mouse xenografts.

II. Test Drugs and Materials

1. Test drug

ADC-27 (3 mg/kg)

ADC-26 (3 mg/kg)

Blank control: PBS buffer at pH 7.4.

2. Preparation method: PBS buffer at pH 7.4.

3. Experimental animals

BALB/cA-nude nude mice: purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.

III. Test Methods

Female BALB/cA-nude nude mice, 6-7 weeks old, were subcutaneously inoculated with human astrocytoma U87MG cells (human astrocytoma, Chinese Academy of Sciences Cell Bank, Catalog#TCHu138). Ten days after cell inoculation, the animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injections of the drug were administered once weekly for a total of 3 times. Tumor volume and body weight were measured 2-3 times per week, and data were recorded. The formula for calculating tumor volume (V) is:

V = 1/2 × a × b ²

Where a and b represent length and width, respectively.

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.

IV. Test Results

Intraperitoneal injection (i.p.) was administered once weekly for a total of three times. By day 22, the tumor inhibition rate of ADC-27 3 mg/kg reached 63.3% (P<0.0001), while the inhibition rate of ADC-26 3 mg/kg reached 49.1%. ADC-27 showed stronger antitumor efficacy than ADC-26.

During the administration process, the weight of animals in all groups remained normal, indicating that the ADC had no obvious toxic side effects. The test results are shown in Table 4 and Figure 5. The tested antibody effectively inhibited the growth of U87MG xenografts in tumor-bearing nude mice in a dose-dependent manner.

Table 4. Efficacy of antibody administration on human astroblastoma U87MG xenografts in nude mice (D22)

*** indicates P < 0.001

Test Examples 1-8: Evaluation of the efficacy of ADCs on Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases.

I. Experimental Objective

This experiment used BALB/cA-nude nude mice as test animals to evaluate the efficacy of the disclosed ADC compound on Detroit 562 nude mouse xenografts of human pharyngeal carcinoma pleural effusion metastases.

II. Test Drugs and Materials

1. Test drug

ADC-29 (3 mg/kg)

ADC-28 (3mg/kg)

Negative control ADC (3 mg/kg): Antibody-drug conjugate formed by conjugating a non-B7H3 target antibody with compound 20.

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

BALB/cA-nude nude mice: purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

III. Test Methods

Female BALB/cA-nude nude mice, 6-7 weeks old, were subcutaneously inoculated with Detroit 562 cells (ATCC, Catalog CCL-138 ^™ ) that had metastasized to the pleural effusion of human pharyngeal carcinoma. On day 10 post-inoculation, the animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injections of the drug were administered once weekly for a total of 3 times. Tumor volume and body weight were measured 2-3 times weekly, and data were recorded. The formula for calculating tumor volume (V) is:

V = 1/2 × a × b ²

Where a and b represent length and width, respectively.

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.

IV. Test Results

The drugs were administered intraperitoneally once a week for a total of three times. By day 28, the tumor inhibition rates of the tested ADCs were as follows: ADC-29 3 mg/kg (3 MPk) achieved a tumor inhibition rate of 72.27% (P<0.001); ADC-28 3 mg/kg (3 MPk) achieved a tumor inhibition rate of 56.2% (P<0.001). ADC-29 showed stronger antitumor efficacy than ADC-28 in both cases.

During the administration process, the weight of animals in all groups remained normal, indicating that the ADC had no obvious toxic side effects. The test results are shown in Table 5 and Figure 6. The tested antibody effectively inhibited the growth of Detroit 562 xenografts in tumor-bearing nude mice in a dose-dependent manner.

Table 5. Efficacy of antibody administration on Detroit 562 xenografts in tumor-bearing nude mice (D28)

*** indicates P < 0.001

Test Examples 1-9: Efficacy Evaluation of U87-MG Tumor-Bearing Mice

I. Experimental Objective

Using Balb/c nude mice as test animals, the efficacy of intraperitoneal injection of B7H3-antibody-drug conjugates was evaluated in a human glioma cell U87MG xenograft model.

II. Test Drugs and Materials

1. Test drug

ADC-30 1mg/kg

ADC-30 3mg/kg

ADC-31 1mg/kg

ADC-31 3mg/kg

Blank control: PBS

2. Preparation method: All preparations are made by diluting with PBS.

3. Experimental animals

BALB/cA-nude nude mice: purchased from Shanghai Slack Laboratory Animal Co., Ltd.

III. Test Methods

U87MG cells (human astroblastoma, Chinese Academy of Sciences Cell Bank, Catalog#TCHu138) (2.5× ^10⁶ /mouse) were subcutaneously injected into the right rib area of mice. After the tumor grew for 14 days and reached 167.49 mm³, the animals were randomly divided into 5 groups of 8 mice per group (d1).

The medication was administered via intraperitoneal injection once a week for a total of three times. Tumor volume and body weight were measured twice a week, and the data were recorded.

Data were statistically analyzed using Excel 2003 software: mean values were calculated as averages; SD values were calculated as standard deviations (STDEV); SEM values were calculated as STDEV/SQRT; and p-values for intergroup differences were calculated as TTEST.

The formula for calculating tumor volume (V) is: V = 1/2 × _{L_length} ^× _{L_short²}

Relative volume (RTV) = V _{_T} / V _₀

Tumor inhibition rate (%) = ( _CRTV - _TRTV ) / _CRTV (%)

Where V _₀ and _VT are the tumor volumes at the beginning and end of the experiment, respectively. _CRTV and _TRTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively.

IV. Test Results

The experimental results are shown in Figure 7. After intraperitoneal injection once a week for a total of three administrations, the tumor inhibition rates of the tested ADCs were as follows by day 18: ADC-30 1 mg/kg: 0.31%; ADC-30 3 mg/kg: 45.23% (P<0.0001); ADC-31 1 mg/kg: 39.22% (P<0.01); ADC-31 3 mg/kg: 80.24% (P<0.0001). At the same dosage, ADC-31 showed significantly better tumor inhibition than ADC-30.

II. Formulation

The equipment used in the preparation and testing of the formulation and the calculation methods for the results are as follows:

SEC size exclusion chromatography:

An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

SEC% (SEC monomer content percentage) = A monomer / A total * 100% (A monomer is the peak area of the main peak monomer in the sample, and A total is the sum of the peak areas of all peaks.)

SEC measurement instrument: Agilent 1260; column: Waters, XBrige SEC (300×7.8mm 3.5μm)

CE capillary gel electrophoresis:

An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

The percentage of CE purity is calculated as follows: A_main/A_total * 100% (A_main is the peak area of the light chain main peak + the heavy chain main peak in the sample, and A_total is the sum of the peak areas of all peaks).

CE testing instrument: Beckman Plus 800

Turbidity measurement:

The degree to which light is obstructed when passing through a water layer indicates the water layer's ability to scatter and absorb light. This is related not only to the content of suspended solids but also to the particle composition, size, shape, and surface reflectivity. By comparing the absorbance values of the same protein sample at the same concentration and wavelength (near-ultraviolet and visible wavelength regions), a higher absorbance value indicates greater turbidity and a more pronounced tendency for protein molecules to aggregate in the sample. The instrument used for the measurement was a multi-functional microplate reader (Molecular Devices M5), with the same volume of sample added to a 96-well plate and the absorbance values read.

Osmotic pressure measurement:

The freezing point method for determining osmotic pressure is based on the principle that the freezing point depression is directly proportional to the molar concentration of the solution. It employs a highly sensitive temperature-sensing element to measure the freezing point of the solution and converts the electrical charge into osmotic pressure. The instrument is manufactured by Loser, model OM815.

Protein concentration determination:

Because the toxins in antibody-drug conjugates absorb at 280 nm, the protein concentration is corrected using the following formula.

A280＝Cd*ε280d+Cmab*ε280mab

A370＝Cd*ε370d

Cd represents the concentration of the toxin, Cmab represents the concentration of the protein, ε280d represents the extinction coefficient of the toxin at 280nm, ε280mab represents the extinction coefficient of the protein at 280nm, ε370d represents the extinction coefficient of the toxin at 370nm, ε280mab = 1.49mg-1*cm-1*ml, ε280d = 5000 (molar extinction coefficient of toxin at 280nm)/1074.13 (molecular weight of toxin) = 4.65mg-1*cm-1*mL, ε370d = 19000 (molar extinction coefficient of toxin at 370nm)/1074.13 (molecular weight of toxin) = 17.69mg-1*cm-1*mL. The above extinction coefficients are mass extinction coefficients.

Protein concentration measurement instrument: UV-Vis spectrophotometer, model: Nano Drop oneC, optical path length: 1mm.

For example, the molecular weight of the naked antibody is 145.181 kDa, and the molecular weight of the toxin is 1074 Da. If calculated based on an average DAR value of 5.7, the molecular weight of the ADC is 151.317 kDa, which is 1.042 times that of the naked antibody. For example, if the ADC DAR value is 5.7 and the concentration of the naked antibody protein is 20.00 mg/mL, then the concentration of the ADC is 20.84 mg/mL.

Example 2-1. Screening of formulation buffer system and pH value

The following different buffer systems were used to prepare a formulation with an ADC-33 protein concentration of 20 mg/mL (based on naked antibody concentration) (this formulation does not contain sugar, surfactants, or other buffers except for the buffer systems described below):

1) 10mM histidine-sodium acetate, pH 5.0

2) 10mM histidine-sodium acetate, pH 5.5

3) 10mM histidine-sodium acetate, pH 6.0

4) 10mM histidine-sodium acetate, pH 6.5

5) 10mM histidine-histidine hydrochloride, pH 5.5

6) 10mM succinic acid-sodium succinate, pH 5.5

7) 10mM citric acid-sodium citrate, pH 5.5

Samples were subjected to high-temperature stability studies (40°C) and shaking studies (25°C, 300 rpm). The shaken samples contained 0.1 mg/mL polysorbate 80 (PS80), while the other samples contained 0.4 mg/mL PS80. Each formulation was filtered, filled, capped, and crimped. The samples were then subjected to the forced degradation experiment described above, and their appearance, SEC (secret chemical reaction), and turbidity were assessed.

The results are shown in Table 6. The results indicate that, using histidine-sodium acetate as a buffer, within the pH range of 5.0-6.5, the appearance of the samples gradually deteriorated, turbidity increased, and the SEC monomer ratio decreased with increasing pH. At pH 5.5, the appearance of samples using 10 mM histidine-sodium acetate and 10 mM histidine hydrochloride was similar to the SEC percentage, and subsequently superior to 10 mM succinic acid-sodium succinate and 10 mM citrate-sodium citrate. Surprisingly, the formulation containing 10 mM succinic acid-sodium succinate showed significantly better turbidity than the other formulations after shaking.

Table 6. Results of pH and buffer system stability

Note: In the table, "D" represents days, for example, D12 means 12 days; D0 means the start of the experiment, and the same applies below.

Example 2-2 Screening of Surfactant Types

Formulations containing different polysorbate surfactants, all containing 10 mM succinate-sodium succinate (pH 5.0) buffer, 80 mg/mL sucrose, and 20 mg/mL protein (based on naked antibody concentration) of ADC-32, were prepared. Each formulation was filtered, filled, capped, and crimped. High-temperature stability studies (40 °C) and shaking studies (25 °C, 300 rpm) were conducted on the samples, examining appearance, SEC, and reduction CE.

The results are shown in Table 7. The formulations containing PS20 (polysorbate 20) and PS80 showed no significant differences in appearance, SEC, and reduced CE under different conditions, indicating that both PS20 and PS80 can effectively stabilize ADC-32.

Table 7. Screening results of polysorbates

Example 2-3 Surfactant Concentration Screening

Samples with PS80 concentrations of 0, 0.1, 0.2, 0.4, and 0.6 mg/mL were prepared. Each sample also contained 10 mM succinic acid-sodium succinate (pH 5.0), 80 mg/mL sucrose, and ADC-32 with a protein concentration of 20 mg/mL (based on naked antibody concentration). The samples were filtered to remove bacteria and particles, and diluted with 0.9% NaCl. The diluted ADC-32 concentration was 0.2 mg/mL. The appearance and insoluble particles of the diluted solution were examined.

The results are shown in Table 8. When the concentration of PS80 in the formulation is 0-0.2 mg/mL, the increase in insoluble particles in the diluent decreases and the number of visible particles decreases with increasing concentration. When the concentration is 0.2-0.6 mg/mL, there is no significant difference in the number of insoluble particles in the diluent and the appearance is good.

Table 8. Screening Results of PS80 Concentration

Example 2-4 Sugar type screening

Different samples containing 80 mg/mL sucrose, trehalose, and mannitol were prepared. Each sample also contained 10 mM succinate-sodium succinate (pH 5.0), 0.2 mg/mL PS80, and ADC-32 with a protein concentration of 20 mg/mL (based on naked antibody concentration). After filtration to remove bacteria and particles, each formulation was filtered, filled, capped, and crimped. High-temperature stability studies (40℃) and freeze-thaw cycles (-35℃/4℃) were conducted to examine appearance, SEC, and reduction CE.

The results are shown in Table 9. Under freeze-thaw conditions, sucrose was superior to trehalose and mannitol in appearance. SEC results showed that sucrose and trehalose were superior to mannitol. CE results under 40℃ reduction conditions showed that sucrose was slightly superior to trehalose.

Table 9. Results of Sugar Type Screening

Examples 2-5: pH, antibody-drug conjugate concentration, and polysorbate screening experiments

Using 10 mM succinic acid-sodium succinate as buffer and 80 mg/mL sucrose as stabilizer, formulations were designed targeting pH, ADC-32 protein concentration (based on naked antibody) and polysorbate concentration, as shown in Table 10. High-temperature stability studies (40 °C), light exposure studies (4 °C, 4500 Lx), and freeze-thaw cycles at -35 °C/4 °C were conducted. SEC and reduction CE were used as evaluation indicators, and the results were statistically analyzed using the least squares method.

The results are shown in Table 11 and Figure 8. The disclosed formulation exhibits high pH dependence. Under light, shaking, and 40°C conditions, the decrease in SEC value increases with increasing pH. Similarly, under light conditions, the decrease in monomer peak value of reduced CE increases with increasing pH, suggesting that the disclosed formulation should be used under low pH conditions to improve stability. Under shaking conditions, the decrease in SEC value tends to decrease with increasing PS80 concentration; under other conditions, PS80 has no significant effect on SEC or reduced CE. With increasing ADC-32 protein concentration, the decrease in SEC monomer peak value increases under 40°C conditions; under other conditions, ADC-32 protein concentration has no significant trend in its effect on SEC or reduced CE.

Table 10. Screening Experimental Recipe Design

Table 11. Results of the prescription screening experiment

Examples 2-6: Freeze-drying process development experiments

The stock solution was prepared according to 10 mM succinate-sodium succinate pH 5.0, 80 mg/mL sucrose, 0.2 mg/mL PS80, and 20 mg/mL (protein concentration, calculated as naked antibody) ADC-33. After filtration and filling, the sample was lyophilized according to the lyophilization process parameters 1 (see Table 12). The lyophilized sample was a smooth white powder cake with a slight collapse in the middle of the bottom. The moisture content was 1.05%. After reconstitution with water for injection, the pH was measured. The results are shown in Table 13. The ionic strength ≥10 mM can meet the buffering requirements. The sucrose concentration of 80 mg/mL in this formulation can meet the isotonic requirements.

Table 12. Freeze-drying process parameters 1

Freeze-drying process parameters Set temperature (°C) Set time (min) Duration (h) Vacuum level (Pa) Pre-freezing 5 10 1 N/A Pre-freezing -45 50 2 N/A One-time drying -25 120 40 10 Secondary drying 25 60 8 1

Table 13. Final formulation pH and osmotic pressure results

Buffer pH Original solution osmotic pressure stock solution pH pH after reconstitution 5.00 298mosm 5.06 5.06

The stock solution was prepared according to 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose, 0.2 mg/mL PS80 and 20 mg/mL (protein concentration, calculated as naked antibody) ADC-33. After filtration and filling, it was freeze-dried according to the freeze-drying process parameters 2 (see Table 14). The powder cake surface was flat without wrinkles or collapse, and the moisture content was 0.89%. This freeze-drying process can meet the product quality requirements.

Table 14. Freeze-drying process parameters 2

Freeze-drying process parameters Set temperature (°C) Set time (min) Duration (h) Vacuum level (Pa) Pre-freezing -5 10 1 N/A Pre-freezing -45 40 2 N/A

One-time drying -20 120 35 10 Secondary drying 25 60 8 1

The stock solution was prepared according to 10mM succinic acid-sodium succinate pH5.0, 80mg/mL sucrose, 0.2mg/mL PS80 and 20mg/mL (protein concentration, calculated as naked antibody) ADC-32. After filtration and filling, it was freeze-dried according to the freeze-drying process parameters 3 (see Table 15). The surface of the powder cake was flat without collapse, the bottom edge of the powder cake was slightly reduced, and the moisture content was 1.17%. This freeze-drying process basically meets the product quality requirements.

Table 15. Freeze-drying process parameters 3

Long-term stability data from Examples 2-7

The stock solution was prepared according to 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose, 0.2 mg/mL PS80 and protein concentration, and 20 mg/mL (calculated as naked antibody) ADC-32. After filtration and filling, the stock solution was prepared according to the freeze-drying process parameter 2 to prepare the pilot batch of finished product. It was stored at 2-8℃ for a long time, and after reconstitution, it was tested. The results are shown in Table 16. Compared with D0, M3 at 2-8℃ had SEC, reduced CE and free toxins within acceptable ranges (SEC% ≥ 93%; reduced CE% ≥ 95%; free toxins ≤ 330 ppm). Except for a slight decrease of 0.5% in SEC, there was no change in reduced CE and free toxins.

Table 16. Long-term stability data results

Note: In the table, "M" represents month, for example, M3 represents 3 months.

Claims (29)

1. A pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the buffer is selected from histidine buffers, succinate buffers, and citrate buffers, the concentration of the buffer being from 5 mM to 50 mM, and wherein the concentration of the antibody-drug conjugate is from 1 mg/mL to 100 mg/mL; the antibody-drug conjugate having a structure as shown in the general formula (Pc-L-Y-D): in: Y is selected from -O-( _CH2 )m- ^CR1R2 - ^C (O)-; ^R1 is a _C3-7 cycloalkyl or a _C3-7 cycloalkyl- _C1-6 alkyl; ^R2 is a hydrogen atom; Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-7 cycloalkyl group; m is 0 or 1; n is between 1 and 10, and n is a decimal or an integer; L represents the connector unit; The connector unit -L- is ^{-L1 -L2 -L3 -L4} -, ^L1 is an integer from 2 to 8 for ^s1 ; ^L2 is a chemical bond; ^L3 is a tetrapeptide residue of GGFG; ^L4 is ^-NR5 ( ^CR6R7 )t-, ^{where R5} is a hydrogen atom or a _C1-6 alkyl group, ^R6 and ^R7 may be the same or different, and each is independently a hydrogen atom or a _C1-6 alkyl group, and t is 1 or 2; Pc stands for Trastuzumab; The pH of the pharmaceutical composition is 4.5 to 5.2. The pharmaceutical composition further comprises a surfactant, wherein the surfactant is polysorbate and the concentration is from 0.01 mg/mL to 1.0 mg/mL; The pharmaceutical composition also contains sugar at a concentration of 60 mg/mL to 90 mg/mL. 2. The pharmaceutical composition according to claim 1, wherein the pH is 4.8 to 5.2. 3. The pharmaceutical composition according to claim 2, wherein the pH is 5.0 to 5.1. 4. The pharmaceutical composition according to claim 1, wherein the surfactant is polysorbate 80 or polysorbate 20. 5. The pharmaceutical composition according to claim 4, wherein the surfactant is polysorbate 80. 6. The pharmaceutical composition according to claim 1, wherein the concentration of the surfactant is from 0.05 mg/mL to 0.5 mg/mL. 7. The pharmaceutical composition according to claim 6, wherein the concentration of the surfactant is 0.2 mg/mL. 8. The pharmaceutical composition according to claim 1, wherein the sugar is selected from sucrose, mannitol, sorbitol and trehalose. 9. The pharmaceutical composition according to claim 8, wherein the sugar is sucrose. 10. The pharmaceutical composition according to claim 1, wherein the concentration of the sugar is 80 mg/mL. 11. The pharmaceutical composition according to claim 1, wherein the concentration of the antibody-drug conjugate is from 10 mg/mL to 30 mg/mL. 12. The pharmaceutical composition according to claim 11, wherein the concentration of the antibody-drug conjugate is from 20 mg/mL to 22 mg/mL. 13. The pharmaceutical composition according to claim 1, wherein the buffer is a succinate buffer. 14. The pharmaceutical composition according to claim 13, wherein the buffer is a succinate-sodium succinate buffer. 15. The pharmaceutical composition according to claim 1, wherein the concentration of the buffer is from 5 mM to 20 mM. 16. The pharmaceutical composition according to claim 15, wherein the concentration of the buffer is 10 mM. 17. The pharmaceutical composition according to claim 1, comprising the following components: (a) the antibody-drug conjugate at a concentration of 10 mg/mL to 30 mg/mL, (b) polysorbate at a concentration of 0.05 mg/mL to 0.5 mg/mL, (c) sugar at a concentration of 60 mg/mL to 90 mg/mL, and (d) a buffer at a concentration of 5 mM to 20 mM, wherein the buffer is selected from histidine buffers, succinate buffers, and citrate buffers; the pH of the pharmaceutical composition is 4.8-5.2. 18. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises the following components: (a) 20 mg/mL to 22 mg/mL of the antibody-drug conjugate, (b) 0.2 mg/mL of polysorbate 80, (c) 80 mg/mL of sucrose, and (d) 10 mM succinate buffer, wherein the composition has a pH of 5.0-5.1. 19. The pharmaceutical composition according to claim 1, wherein the antibody conjugate has the structure shown in the following formula: Where n is between 3 and 8, and n is a decimal or an integer. 20. A pharmaceutical composition comprising an antibody-drug conjugate having the structure shown in the following formula: Where n is between 3 and 8, and n is a decimal or an integer; Furthermore, the pharmaceutical composition comprises: (a) the antibody-drug conjugate at a concentration of 10 mg/mL to 30 mg/mL, (b) polysorbate at a concentration of 0.05 mg/mL to 0.5 mg/mL, (c) sugar at a concentration of 60 mg/mL to 90 mg/mL, and (d) a buffer at a concentration of 5 mM to 20 mM, wherein the buffer is selected from histidine buffers, succinate buffers, and citrate buffers; the pH of the pharmaceutical composition is 4.8 to 5.2. 21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition comprises: (a) 20 mg/mL to 22 mg/mL of the antibody-drug conjugate, (b) 0.2 mg/mL of polysorbate 80, (c) 80 mg/mL of sucrose, and (d) 10 mM succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 5.1. 22. A lyophilized formulation comprising an antibody-drug conjugate, characterized in that the formulation, upon reconstitution, can form the pharmaceutical composition according to any one of claims 1 to 21. 23. A method for preparing a lyophilized formulation comprising an antibody-drug conjugate, comprising the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 21. 24. A lyophilized formulation comprising an antibody-drug conjugate, said formulation being obtained by freeze-drying a pharmaceutical composition according to any one of claims 1 to 21. 25. A reconstituted solution comprising an antibody-drug conjugate, characterized in that the reconstituted solution is obtained by reconstituted the lyophilized formulation of claim 22 or 24; The reconstituted solution contains the following components: (a) the antibody-drug conjugate at a concentration of 10 mg/mL to 30 mg/mL, (b) polysorbate at a concentration of 0.05 mg/mL to 0.5 mg/mL, (c) sugar at a concentration of 60 mg/mL to 90 mg/mL, and (d) a buffer at a concentration of 5 mM to 20 mM, wherein the buffer is selected from histidine buffers, succinate buffers, and citrate buffers; the pH of the reconstituted solution is 4.8 to 5.2. 26. The reconstituted solution according to claim 25, wherein the reconstituted solution comprises the following components: (a) 20 mg/mL to 22 mg/mL of antibody-drug conjugate, (b) 0.2 mg/mL of polysorbate 80, (c) 80 mg/mL of sucrose, and (d) 10 mM succinate buffer, wherein the pH of the reconstituted solution is 5.0 to 5.1. 27. An article comprising a container containing a pharmaceutical composition as claimed in any one of claims 1 to 21, a lyophilized formulation as claimed in claim 22 or 24, or a reconstituted solution as claimed in claim 25 or 26. 28. Use of the pharmaceutical composition of any one of claims 1 to 21, the lyophilized formulation of claim 22 or 24, the reconstituted solution of claim 25 or 26, or the article of claim 27 in the preparation of a medicament for treating cancer, wherein the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma. 29. The use according to claim 28, wherein the cancer is selected from endometrial cancer, colon cancer, and rectal cancer.