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WO2025031305A1 - 双联烯连接子、连接子与药物缀合物、抗体偶联药物及其用途 - Google Patents

双联烯连接子、连接子与药物缀合物、抗体偶联药物及其用途 Download PDF

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WO2025031305A1
WO2025031305A1 PCT/CN2024/109676 CN2024109676W WO2025031305A1 WO 2025031305 A1 WO2025031305 A1 WO 2025031305A1 CN 2024109676 W CN2024109676 W CN 2024109676W WO 2025031305 A1 WO2025031305 A1 WO 2025031305A1
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integer
antibody
same
cancer
linker
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French (fr)
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邹斌
刘绍军
杜平
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Taichengsi Shanghai Biomedical Co Ltd
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Taichengsi Shanghai Biomedical Co Ltd
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    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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    • C07C233/20—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
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    • C07C233/22—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
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    • C07C237/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
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Definitions

  • the present invention relates to a biolefin linker, a linker-drug conjugate, an antibody-drug conjugate and uses thereof.
  • ADCs antibody-drug conjugates
  • ADCs antibody-drug conjugates
  • Many widely used antibody conjugation methods still have great limitations.
  • the preparation of ADCs with uniform composition is a challenge that still needs to be overcome.
  • Common conjugation methods use chemical reactions between lysine residues or cysteine moieties (through the reduction of interchain disulfide bonds) and electrophilic reagents. Since the above chemical reactions are currently not selective, the number and sites of conjugation are uncertain, so the resulting conjugates are not uniform products, but contain multiple components.
  • Each ADC with a different DAR value has different pharmacokinetics, toxicology, and efficacy. This diverse ADC mixture is not only difficult to develop, but may also be accompanied by an undesirable therapeutic window.
  • Disulfide bridging is a strategy to form uniform ADCs.
  • Different reagents have been used for disulfide bridging, including dithiones, next-generation maleimides (NGMs, a class of maleimide reagents modified with two halide or thiophenol leaving groups), pyridazinediones such as dibromomethylquinoxaline, arylidene dipropionitrile, divinylpyrimidine (DVP) (Chem. Soc.
  • DAR4's ADC is a mixture of "half antibody” and "full antibody”. There is no way to make the main component "half antibody” or “full antibody”, or the ADC content of DAR4 is relatively low.
  • the purpose of the present invention is to provide a novel bivalent olefin linker that can be covalently bridged with most antibodies.
  • the present invention also provides an antibody/active agent coupling method and its application, such as application in antibody drug conjugates.
  • a biolefin linker characterized in that it comprises a compound or an isotope-labeled substance as shown in the general formula (I):
  • the L 1 is selected from
  • the L1 is selected from
  • the L1 is selected from
  • the L1 is selected from
  • n1 is the same as n2
  • n3 is the same as n4
  • n1 an integer of 0-8,
  • n2 an integer of 0-8,
  • n3 an integer of 0-8,
  • n4 an integer of 0-8;
  • the biolefin linker in the general formula (I) is selected from at least one of the following compounds:
  • Another aspect of the present invention is to provide a linker-drug conjugate, characterized in that the carboxyl group in the aforementioned biolefin linker is A compound formed by bonding the amino group of the amino acid residue in L 3 via an amide bond;
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • D is a fragment formed by the loss of one or more atoms or groups from a cytotoxic small molecule drug or active agent, and D is covalently bonded to L3 via sulfur, hydrogen or oxygen in D.
  • the cytotoxic small molecule drug is a drug for treating tumor diseases;
  • the active agent is a microtubule inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalating agent, a DNA-reactive agent, a DNA alkylating agent or an immunosuppressant.
  • the tubulin inhibitors include but are not limited to monomethyl auristatin, maytansinoid, maytansinol or DM1 maytansine.
  • the monomethyl auristatin may be monomethyl auristatin E or monomethyl auristatin F, etc.
  • the RNA polymerase inhibitors include but are not limited to ⁇ -amanitin, etc.
  • the topoisomerase inhibitors include but are not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan, etc.
  • the intercalating agents include but are not limited to pyrrolobenzodiazepine (PBD), etc.
  • the DNA-reactants include but are not limited to calicheamicin, tiancimycins or other enediynes, etc.
  • the DNA alkylating agents include but are not limited to trabectedin or rubectin and other ecteinascin derivatives.
  • Another aspect of the present invention is to provide a linker-drug conjugate, characterized in that it comprises a compound having a structure as shown in general formula (II):
  • the L 1 is selected from
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent.
  • D is covalently bonded to L 3 via the sulfur, nitrogen or oxygen in D.
  • the L1 is selected from
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent.
  • D is covalently bonded to L 3 via the sulfur, nitrogen or oxygen in D.
  • the L1 is selected from
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or other active agent.
  • D is covalently bonded to L 3 via the sulfur, nitrogen or oxygen in D.
  • the L1 is selected from
  • n1 is the same as n2
  • n3 is the same as n4
  • n1 an integer of 0-8,
  • n2 an integer of 0-8,
  • n3 an integer of 0-8,
  • n4 an integer of 0-8;
  • D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or other active agent.
  • D is covalently bonded to L 3 via the sulfur, nitrogen or oxygen in D.
  • the L1 is selected from
  • n1 is the same as n2
  • n3 is the same as n4
  • n1 an integer of 0-8,
  • n2 an integer of 0-8,
  • n3 an integer of 0-8,
  • n4 an integer of 0-8;
  • the cytotoxic small molecule drug is a drug for treating tumor diseases;
  • the active agent is a microtubule inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalating agent, a DNA-reactive agent, a DNA alkylating agent or an immunosuppressant.
  • the tubulin inhibitor includes but is not limited to monomethyl auristatin, maytansinoid, maytansinol or DM1 maytansine.
  • the monomethyl auristatin includes but is not limited to monomethyl auristatin E or monomethyl auristatin F, etc.
  • the RNA polymerase inhibitor includes but is not limited to ⁇ -amanitin, etc.
  • the topoisomerase inhibitor includes but is not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan, etc.
  • the intercalating agent includes but is not limited to pyrrolobenzodiazepine (PBD), etc.
  • the DNA-reactant includes but is not limited to calicheamicin, tiancimycins or other enediynes, etc.
  • the DNA alkylating agent includes but is not limited to trabectedin or rubectin and other ecteinascin derivatives.
  • the compound of general formula (II) is selected from at least one of the compounds in the following table:
  • Another aspect of the present invention is to provide an antibody-drug conjugate, characterized in that the conjugate is formed by coupling the aforementioned linker with the bifenal group in the drug conjugate and the thiol group after reduction of Ab;
  • the Ab is an antibody, an antibody fragment or a protein.
  • the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, antigen-binding fragments thereof, immunologically active portions or mutants of the above antibodies.
  • Another aspect of the present invention is to provide an antibody-drug conjugate, characterized in that it comprises a structure as shown in general formula (III):
  • the L1 is selected from
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent.
  • D is covalently bonded to L 3 via the sulfur, nitrogen or oxygen in D.
  • n 16 is selected from any value between 1 and 20;
  • the Ab is an antibody, an antibody fragment or a protein.
  • the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, antigen-binding fragments thereof, immunologically active portions or mutants of the above antibodies.
  • the L1 is selected from
  • n 10 an integer from 1 to 20
  • n 11 an integer from 1 to 20
  • n 12 an integer from 1 to 20
  • n 13 an integer from 1 to 20;
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • the cytotoxic small molecule drug or active agent is a fragment formed by the cytotoxic small molecule drug or other active agent losing one or more atoms or groups;
  • D is covalently bonded to L 3 via the sulfur, nitrogen or oxygen in D.
  • n 16 is selected from any value between 1 and 20;
  • the Ab is an antibody, an antibody fragment or a protein.
  • the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, antigen-binding fragments thereof, immunologically active portions or mutants of the above antibodies.
  • the L1 is selected from
  • the M is selected from The n1 is different from n2 , n3 is the same as or different from n4 , and n5 is the same as or different from n6 .
  • n 1 an integer of 0-10
  • n 2 an integer of 0-10
  • n 3 an integer of 0-10
  • n 4 an integer of 0-10
  • n 5 an integer of 1-10
  • n 6 an integer of 1-10;
  • n 14 an integer from 1 to 20
  • n 15 an integer from 1 to 20
  • D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent
  • D is covalently bonded to L 3 through the sulfur, nitrogen or oxygen in D;
  • n 16 is selected from any value between 1 and 20;
  • the Ab is an antibody, an antibody fragment or a protein.
  • the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, antigen-binding fragments thereof, immunologically active portions or mutants of the above antibodies.
  • the L1 is selected from
  • n1 is the same as n2
  • n3 is the same as n4
  • n1 an integer of 0-8,
  • n2 an integer of 0-8,
  • n3 an integer of 0-8,
  • n4 an integer of 0-8;
  • the cytotoxic small molecule drug is a drug for treating tumor diseases;
  • the active agent is a tubulin inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalating agent, a DNA-reactive agent, a DNA alkylating agent or an immunosuppressant;
  • n 16 is selected from any value between 1 and 20;
  • the Ab is an antibody, an antibody fragment or a protein.
  • the tubulin inhibitors include but are not limited to monomethyl auristatin, maytansinoid, maytansinol or DM1 maytansine.
  • the monomethyl auristatin may be monomethyl auristatin E or monomethyl auristatin F, etc.
  • the RNA polymerase inhibitors include but are not limited to ⁇ -amanitin, etc.
  • the topoisomerase inhibitors include but are not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan, etc.
  • the intercalating agents include but are not limited to pyrrolobenzodiazepine (PBD), etc.
  • the DNA-reactants include but are not limited to calicheamicin, tiancimycins or other enediynes, etc.
  • the DNA alkylating agents include but are not limited to trabectedin or rubectin and other ecteinascin derivatives.
  • the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies, antigen-binding fragments thereof, immunologically active portions or mutants of the above antibodies.
  • the L1 is selected from
  • n1 is the same as n2
  • n3 is the same as n4
  • n1 an integer of 0-8,
  • n2 an integer of 0-8,
  • n3 an integer of 0-8,
  • n4 an integer of 0-8;
  • n 16 is selected from any value between 1 and 20;
  • the cytotoxic small molecule drug is an inflammatory disease drug, a nervous system disease drug or a cardiovascular disease drug;
  • the active agent is a tubulin inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalating agent, a DNA-reactive agent, a DNA alkylating agent or an immunosuppressant;
  • the Ab is abciximab, alemtuzumab, anetuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatumomab, brentuximab, catumaxomab, cetuximab, cetuximab, cotuximab, daclizumab, daratumumab, deninutuzumab, denosumab, depatuxizumab, denutuximab, durvalumab, elotuzumab, infortumumab, gebatumumab, gemtuzumab, ibritumomab tiuxetan, indalatuzumab, indoximab, inotuzumab, ipilimumab, labetuzumab, latuzumab, latuximab, rifatuzumab,
  • the L1 is selected from
  • n1 is the same as n2
  • n3 is the same as n4
  • n1 an integer of 0-8
  • n2 an integer of 0-8,
  • n3 an integer of 0-8,
  • n4 an integer of 0-8;
  • the cytotoxic small molecule drug is a drug for treating tumor diseases;
  • the active agent is a tubulin inhibitor, an RNA polymerase inhibitor, a topoisomerase inhibitor, an intercalating agent, a DNA-reactive agent, a DNA alkylating agent or an immunosuppressant;
  • n 16 is selected from any value between 2 and 6;
  • the Ab is selected from pertrastuzumab, saccituzumab, metuximab, cetuximab, trastuzumab, anti-B7-H3 monoclonal antibody, cetuximab or mutants of the above antibodies at position 226 or position 229 of the heavy chain Eu numbering.
  • the mutant is a mutant in which the cysteine that forms a disulfide bond between heavy chains is mutated to serine.
  • the antibody-drug conjugate in the general formula (III) is selected from at least one of the compounds of the following structural formulas:
  • n 16 is selected from any value between 2 and 6.
  • S is the sulfur atom contained in the antibody Ab.
  • the microtubule inhibitors of the present invention include, but are not limited to, monomethyl auristatin, maytansinoid, maytansinol or DM1 maytansine.
  • the monomethyl auristatin can be It is monomethyl auristatin E or monomethyl auristatin F, etc.
  • the RNA polymerase inhibitor includes but is not limited to ⁇ -amanitin, etc.
  • the topoisomerase inhibitor includes but is not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan, etc.
  • the inserter includes but is not limited to pyrrolobenzodiazepine (English name: pyrrolobenzodiazepine) (PBD), etc.
  • the DNA-reactant includes but is not limited to calicheamicin, tiancimycins (English name: tiancimycins) or other enediynes, etc.
  • the DNA alkylating agent includes but is not limited to
  • the English name of the abciximab described in the present invention is Abciximab; the English name of alemtuzumab is Alemtuzumab; the English name of anetuzumab is Anetumab; the English name of atezolizumab is Atezolizumab; the English name of avelumab is Avelumab; the English name of basiliximab is Basiliximab; the English name of bevacizumab is Bevacizumab; the English name of blinatomumab is Brentuximab; the English name of catumaxomab is Catumaxomab; the English name of cetuximab is Cetuximab; the English name of cirmtuzumab is Cirmtuzumab; the English name of coltuximab is Coltuximab; the English name of daclizumab is Daclizumab; the English name of daratumumab is Daratumuma
  • the cancers include lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancies, squamous cell carcinoma (such as epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung), peritoneal cancer, liver cancer, stomach cancer, stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioma, cervical cancer, ovarian cancer, oral cancer, bladder cancer, urinary tract cancer, breast cancer, HER2-positive breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer and related metastatic tumors.
  • squamous cell carcinoma such as epithelial squamous cell carcinoma
  • lung cancer including
  • the present invention provides a new double ene linker, which can be simply used for site-specific coupling of antibodies based on covalent thiol bridging, and can be applied to most antibodies. Compared with the cumbersome recombinant modification of antibodies, the method of site-specific coupling sites using the double ene linker in the present invention does not require recombinant modification of antibodies, so it can save a lot of time, cost and resources, and has a broader application prospect.
  • the novel bivalent olefin linker provided by the present invention can be covalently bridged and coupled with an antibody by a simple chemical method.
  • the DAR value distribution of the conjugate obtained by using the linker of the present invention is narrower, the generated product has higher uniformity, and the proportion of the single distributed component (DAR4) of the obtained conjugate can exceed 80%, and some can even exceed 95%, and the "half-antibody" (DAR4) component is the main component.
  • novel biolefin linker provided by the present invention can also be covalently bridged and coupled with most antibody mutants, for example, with mutants at position 226 (Eu numbering) or 229 (Eu numbering) of the heavy chain. Since the cysteine that forms the disulfide bond between the heavy chains is mutated to other amino acids, a uniform DAR3 antibody-drug conjugate can be produced.
  • Figure 1 is a HIC diagram of ADC-1 prepared in an embodiment of the present invention. As can be seen from Figure 1, the proportion of a single distributed component (DAR4) of the obtained conjugate can exceed 80%.
  • DAR4 distributed component
  • FIG. 2 is a SEC diagram of ADC-1 prepared in an embodiment of the present invention.
  • FIG. 3 is a MS graph of ADC-1 prepared in an embodiment of the present invention.
  • FIG. 4 is an SDS-PAGE image of ADC-1 prepared in an example of the present invention.
  • FIG. 5 is a diagram of ADC-2HIC prepared in an embodiment of the present invention.
  • FIG. 6 is a SEC diagram of ADC-2 prepared in an example of the present invention.
  • FIG. 7 is a diagram of ADC-3HIC prepared in an embodiment of the present invention.
  • FIG8 is a graph of ADC-3SEC prepared in an embodiment of the present invention.
  • FIG. 9 is an SDS-PAGE image of ADC-3 prepared in an embodiment of the present invention.
  • FIG. 10 is a diagram of ADC-5HIC prepared in an embodiment of the present invention.
  • FIG. 11 is a SEC graph of ADC-5 prepared in an embodiment of the present invention.
  • FIG. 12 is an SDS-PAGE image of ADC-5 prepared in an embodiment of the present invention.
  • Figure 13 is a HIC graph of ADC-10 prepared in an embodiment of the present invention. As can be seen from Figure 13, the proportion of the single distributed component (DAR4) of the obtained conjugate can exceed 95%.
  • DAR4 single distributed component
  • FIG. 14 is a graph of ADC-10SEC prepared in an embodiment of the present invention.
  • FIG. 15 is an SDS-PAGE image of ADC-10 prepared in an embodiment of the present invention.
  • FIG. 16 is a diagram of ADC-25HIC prepared in an embodiment of the present invention.
  • FIG. 17 is a graph of ADC-25SEC prepared in an embodiment of the present invention.
  • FIG. 18 is an SDS-PAGE image of ADC-25 prepared in an embodiment of the present invention.
  • FIG. 19 is a HIC diagram of ADC-26 prepared in an embodiment of the present invention.
  • FIG. 20 is a diagram of ADC-26SEC prepared in an embodiment of the present invention.
  • FIG. 21 is an SDS-PAGE image of ADC-26 prepared in an embodiment of the present invention.
  • Figure 22 is a HIC graph of ADC-27 prepared in an embodiment of the present invention. As can be seen from Figure 22, the proportion of the single distribution component (DAR4) of the obtained conjugate can exceed 95%.
  • DAR4 single distribution component
  • FIG. 23 is a SEC graph of ADC-27 prepared in an embodiment of the present invention.
  • FIG. 24 is a MS graph of ADC-27 prepared in an example of the present invention.
  • FIG. 25 is an SDS-PAGE image of ADC-27 prepared in an example of the present invention.
  • FIG26 is a graph showing the in vitro plasma stability experiment of ADCs based on different connectors.
  • the “whole antibody” is shown in the figure on the right, which is formed by the bridging between the light chain and the heavy chain and the bridging between the heavy chain and the heavy chain:
  • half-antibody is formed by the bridging between the light chain and the heavy chain and the bridging within the heavy chain:
  • Step 1 Synthesis of 2-(trimethylsilyl)ethyl 6-(((benzyloxy)carbonyl)amino)hexanoate.
  • Step 3 Synthesis of methyl 3-((tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropanoate
  • Step 4 Synthesis of 3-((tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropanoic acid
  • Step 5 Synthesis of 2-(trimethylsilyl)ethyl-6-(3-(tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropylamino)hexanoate
  • Step 6 Synthesis of 2-(trimethylsilyl)ethyl-6-(3-amino-2-(aminomethyl)propylamino)hexanoic acid dihydrochloride
  • Step 7 Synthesis of 2-(trimethylsilyl)ethyl-6-(3-(butyl-3-amino)-2-(butyl-3-amino)propylamino)hexanoate
  • Oxalyl chloride (939.1 mg, 7.4 mmol) was added to a suspension of butane-3-acetic acid (623.6 mg, 7.4 mmol) in dichloromethane (10 mL). After stirring at room temperature for 15 min, the reaction solution was cooled to 0°C and 0.15 mL DMF was added. The reaction mixture was stirred at 0°C and then stirred at room temperature for another 90 min.
  • Step 8 Synthesis of 2-(trimethylsilyl)ethyl-6-(3-(butyl-2,3-diamino)-2-(butyl-2,3-diaminomethyl)propylamino)hexanoate
  • Step 9 Synthesize LK1
  • Step 1 Synthesis of methyl 2,3-bis((tert-butyloxycarbonyl)amino)propionate
  • Step 3 Synthesis of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-bis((tert-butoxycarbonyl)amino)propionamido)hexanoate
  • EDCI (658 mg, 3.45 mmol) and HOBt (463.3 mg, 3.45 mmol) were added to a solution of 2,3-bis(tert-butyloxycarbonyl)amino)propionic acid (1.0 g, 3.14 mmol) and LK1-3 (1.46 g, 31.19 mmol) in dichloromethane (20 mL). Stir overnight at room temperature.
  • Step 5 Synthesis of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-di(but-3-ynylamido)propionamido)hexanoate
  • the mixture was extracted with dichloromethane (50 mL ⁇ 3), the organic phases were combined, dried, and concentrated.
  • Step 6 Synthesis of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-di(buta-2,3-dienamido)propionamido)hexanoate
  • Step 7 6-(2,3-di(butane-2,3-dienamido)propionamido)hexanoic acid
  • Oxalyl chloride (972 mg, 7.66 mmol) was added to a suspension of but-3-ynoic acid (644 mg, 7.66 mmol) in dichloromethane (30 mL). After stirring at room temperature for 15 minutes, the reaction solution was cooled to 0 ° C and DMF (0.15 mL) was added. The reaction solution was gradually warmed to room temperature and continued to stir for 90 minutes. The solution was added dropwise to a solution of 6-(3,4-diaminobenzamido)hexanoic acid 2-(trimethylsilyl)ethyl ester (280 mg, 0.766 mmol) in saturated sodium bicarbonate (10 mL) and stirred at room temperature for 2 hours. Extracted with dichloromethane (50 mL ⁇ 3), dried, filtered and concentrated. Purified by silica gel flash column chromatography to obtain the target compound (270 mg, yield 71%) as a yellow solid.
  • Step 4 and Step 5 Synthesis of 6-(3,4-di(butane-2,3-dienylamido)benzamido)hexanoic acid
  • Step 2 Synthesis of 2-(trimethylsilyl)ethyl-6-(3,5-di((tert-butylcarbonyl)amino)methyl)benzylamine)hexanoate
  • Step 6 6-(3,5-di(butane-2,3-dienoyl)benzylamine)hexanoic acid
  • the compound 6-(3,5-di(3-butynylaminomethyl)benzylamine)hexanoic acid was dissolved in 1,4-dioxane (0.5 mL) and potassium carbonate solution (1 mL), and stirred at 40°C for 1 hour.
  • the mixture was extracted with ethyl acetate (10 mL ⁇ 2), the pH of the aqueous layer was adjusted to 4-5, and the mixture was extracted with ethyl acetate (10 mL ⁇ 2). After concentration, 10 mg of the target product 6-(3,5-di(butane-2,3-dienoyl)benzylamine)hexanoic acid was obtained as a white solid.
  • HATU (20.3 mg, 0.0534 mmol) and DIPEA (12 mg, 0.009 mmol) were added to a 1 mL DMF solution of NH2 -VC-MMAE (20 mg, 0.0178 mmol) and LK1 (10 mg, 0.035 mmol). After addition, the reaction solution was stirred at room temperature overnight. Purification by preparative HPLC gave compound LP1 (5 mg, yield 19.2%) as a white solid.
  • LP1-3 (20 mg, 0.0178 mmol) and LK2 (10 mg, 0.035 mmol) were dissolved in DMF (1 mL), and HATU (20.3 mg, 0.0534 mmol) and DIPEA (12 mg, 0.009 mmol) were added. After the addition was completed, the reaction solution was stirred at room temperature overnight. LP2 (3.8 mg, yield 14.9%) was obtained as a white solid by purification by preparative HPLC.
  • LK3 (14.2 mg, 0.036 mmol), HOBt (8.4 mg, 0.063 mmol) and EDCI (7.4 mg, 0.072 mmol) were dissolved in DMF (2 mL) solution and stirred at 0°C for 15 minutes.
  • LP1-3 (30 mg, 0.018 mmol) was added to the solution and stirred at 0°C for 5 hours. After the reaction was completed, the mixture was purified by preparative HPLC to obtain LP3 (4.98 mg, yield 12.4%) as a white solid.
  • LK4 (3.54 mg, 0.009 mmol), HOBt (2.1 mg, 0.016 mmol) and EDCI (1.85 mg, 0.002 mmol) were dissolved in DMF (0.5 mL) and stirred at 0°C for 15 minutes.
  • LP1-3 (5 mg, 0.0045 mmol) was added to the solution and reacted at 0°C for 5 hours. After the reaction was completed, the mixture was purified by preparative high performance liquid chromatography to obtain compound LP4 (0.71 mg, yield 10.6%) as a white solid.
  • 6-(3,5-bis(butyl-2,3-diaminomethyl)benzylamine)hexanoic acid 8 mg, 0.019 mmol
  • HOBt 6 mg, 0.044 mmol
  • EDCI 10 mg, 0.052 mmol
  • DMF 0.5 mL
  • NH 2 -VC-MMAE 15 mg, 0.013 mmol
  • LCMS showed the target product, and the target product LP5 (2.0 mg, 10.0%) was purified by preparative HPLC as a white solid.
  • Triethylamine (0.5 mL) was added to a solution of LP10-1 (88 mg, 0.0828 mmol) in DMF (1.5 mL). After the addition, the reaction solution was stirred at room temperature overnight. LCMS showed that the reaction was complete. The mixture was concentrated to give a residue, which was purified by C18 (0-60% ACN in aqueous solution) to give LP10-2 (45 mg, 65% yield) as a white solid.
  • the following are examples of methods for preparing and analyzing antibody drug conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments currently described. For example, the antibody is not limited to the antibodies currently displayed, but can also be any other antibody.
  • Adjust the concentration of the antibody solution to 5-10 mg/mL and the pH to about 7.5. Take a clean centrifuge tube as a reaction container, place the monoclonal antibody (1.0 eq.) in the tube, add TCEP (5 eq. to 8 eq.) and mix well, insert it into a constant temperature mixer, and incubate at 20-25°C for 2-16 hours to completely reduce the interchain disulfide bonds of the antibody. Add an equal volume of 500 mM Tris, pH 8.8 buffer to raise the pH to 8.0, or replace the antibody into 25 mM Na 2 B 4 O 7 , pH 8.0 buffer through a desalting column, and then add a toxin linker solution (5 eq.
  • High performance liquid chromatography e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.
  • Mobile phase Mobile phase A (MPA): 1.5M (NH4) 2 SO 4 +50mM potassium phosphate (pH 7.0); Mobile phase B (MPB): 50mM sodium phosphate (pH 7.0)/isopropanol (75:25V/V); Elution was performed according to the following elution program (5%-95%), wherein, from 0 to 2 min, the volume of mobile phase A was 100%-95%, and the volume of mobile phase B was 0%-5%; from 2 to 22 min, the volume of mobile phase A was 95%-5%, and the volume of mobile phase B was 5%-95%; from 22 to 24 min, the volume of mobile phase A was 5%-0%, and the volume of mobile phase B was 95%-100%; from 24 to 26 min, the volume of mobile phase A was 0%-100%, and the volume of mobile phase B was 100%-0%; from 26 to 30 min, the volume of mobile phase A was 100%-100%, and the volume of mobile phase B was 0%-0%.
  • MPA Mobile phase A
  • MPB 50
  • Detection conditions Set the mobile phase flow rate to 1 ml/min, the detection wavelength to 280 nm, and the column temperature to 30 °C.
  • Chromatographic column C18 3.5 ⁇ m 4.6 ⁇ 150mm (manufacturer: Waters).
  • Mobile phase Mobile phase A (MPA): 0.1% TFA-H 2 O; Mobile phase B (MPB): 0.1% TFA-ACN; Elution was performed according to the following elution procedure (5%-95%), wherein the volume of mobile phase A was 90%-20% from 0 to 30 min, and the volume of mobile phase B was 10%-80%; the volume of mobile phase A was 20%-90% from 30 to 31 min, and the volume of mobile phase B was 80%-10%; the volume of mobile phase A was 90%-90% from 31 to 35 min, and the volume of mobile phase B was 10%-10%
  • Detection conditions Set the mobile phase flow rate to 0.5 ml/min, the detection wavelength to 254 nm, and the column temperature to 30 °C.
  • Preparation example of reagent I Measure 30 mL of anhydrous methanol and 50 mL of acetonitrile, weigh 10 g of sodium chloride, mix in a container, stir at room temperature for more than 1 hour, and then let stand for 1 hour. Take the supernatant, filter with a 0.22 ⁇ m organic membrane, store at room temperature, and the validity period is 3 months.
  • Example of preparing reagent II measure 100 mL of reagent I, 15 mL of DMSO, and 85 mL of ADC sample storage buffer respectively, mix them evenly in a container, and store at room temperature. The shelf life is 2 months.
  • Sample test Take 85 ⁇ g of the coupled sample, mix the sample with 3 ⁇ l DMSO for 5 minutes, then take 60 ⁇ l of reagent I and add it to the sample system, mix for 5-10 minutes, centrifuge at 2000rpm for 2 minutes, take 20 ⁇ L of the supernatant, inject it into the HPLC, elute using the above elution procedure, and record the chromatogram.
  • Free Drug (mol/mol%) residual small molecule molar concentration/antibody molar concentration ⁇ 100.
  • Serial dilution of the standard dilute the standard to 20000, 16000, 10000, 4000, 1000, 200, 40, 20 ng/ml with 50% methanol in water.
  • Sample pre-treatment Take 20 ⁇ L sample, then add 20 ⁇ L blank buffer and 240 ⁇ L extractant. After vortex mixing, let stand until stratification, then take 160 ⁇ L supernatant and put it into a 96-well plate to dry, add 200 ⁇ L 50% acetonitrile water, mix well, and finally enter LC-MS/MS analysis.
  • Pre-treatment of standard products Take 2 ⁇ L of working solution and add it to 38 ⁇ L of blank buffer, then add 240 ⁇ L of extractant. Vortex and let stand until stratification, take 160 ⁇ L of supernatant and put it into a 96-well plate to dry, add 200 ⁇ L of 50% acetonitrile water and mix well.
  • a standard curve was made based on the concentration of the standard and the corresponding mass spectrometry response value.
  • the residual amount of free linker-payload was calculated through the linear fitting curve (formula) and the mass spectrometry response value of the detected sample.
  • High performance liquid chromatography e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.
  • Mobile phase 50mM PB + 200mM Arg (pH 6.80) + 10% IPA, eluted according to the following elution procedure, 0-30min mobile phase A volume is 100%-100%;
  • Detection conditions Set the mobile phase flow rate to 0.5 ml/min, the detection wavelength to 280 nm, and the column temperature to 26 °C.
  • Electrophoresis apparatus Tianneng EPS-600.
  • Imaging system analyzer Tianneng 4600SF
  • results analysis The presence of ⁇ 50kDa bands indicates that the thiol-bridging coupling is effective (H, L).
  • the 75kDa band represents the portion consisting of a pair of heavy chains and light chains connected by bridging thiol groups (LH).
  • the band formed by two heavy chains connected by one or two pairs of bridging thiol groups is 100kDa (HH).
  • the 125kDa band represents that only one heavy chain and light chain are not bridged (LHH).
  • the 150kDa band represents that all chains are bridged antibodies (LHHL).
  • Liquid chromatography column PLRP-S, 1000A column (Agilent 8 ⁇ m, 2.1 ⁇ 150mm)
  • the mobile phase B was 0.1% formic acid in acetonitrile.
  • the flow rate was 0.4 mL/min.
  • Mass spectrometry was used to analyze positive ions with the following parameters: scanning range 500-5000 m/z,
  • TCEP (10 mmol/L, 6 eq.) aqueous solution was added to trastuzumab (1.0 mg, 38.9 mg/mL, 1.0 eq.) in 20 mM PBS, 1 mM EDTA, pH 7.4 buffer, and reduced at 22°C for 2 hours; then TCEP was removed by desalting column and the buffer was replaced with 25 mM Na 2 B 4 O 7 , pH 8.0 buffer, and LP1 (10 mg/mL, 6 eq.) DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • HIC-DAR 4.05, SEC purity: 99.2%, Free Linker Payload (mol/mol%): not detected.
  • the single distributed component (DAR4) in the obtained ADC-1 accounts for 88.58%, and the aggregate is less than 1%.
  • Figures 3 and 4 show that the components of "half antibody” (DAR4) formed by the bridging coupling between the light chain and the heavy chain and the bridging coupling within the heavy chain are the main ones.
  • TCEP (2 mg/mL, 6 eq.) aqueous solution was added to trastuzumab (1.0 mg, 38.9 mg/mL, 1.0 eq.) in 25 mM Na 2 B 4 O 7 , 25 mM NaCl, 1 mM DTPA, pH 7.4 buffer, and reduced at 22°C for 2 hours; then an equal volume of 500 mM Tris, pH 8.8 buffer was added, and then LP2 (10 mg/mL, 7 eq.) in DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • HIC-DAR 3.86, SEC purity: 93.68%, Free Linker Payload (mol/mol%): not detected.
  • DAR 4: Rt 16.99min (ratio: 54.92%)
  • DAR 5: Rt 18.90min (ratio: 15.29%).
  • the proportion of the single distributed component (DAR4) in the obtained ADC-2 is 54.92%, which is reduced compared with ADC-1, and the aggregates are greater than 6%.
  • TCEP (10 mmol/L, 6 eq.) aqueous solution was added to trastuzumab (1.0 mg, 20 mg/mL, 1.0 eq.) in 20 mM PBS, 1 mM EDTA, pH 7.4 buffer, and reduced at 22°C for 2 hours; then TCEP was removed by desalting column and the buffer was replaced with 25 mM Na 2 B 4 O 7 , pH 8.0 buffer, and LP3 (10 mg/mL, 5 eq.) in DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • HIC-DAR 4.1, SEC purity: 98.3%, Free Linker Payload (mol/mol%): not detected.
  • the single distribution component (DAR4) in the obtained ADC-3 accounts for 68.5%, which is reduced compared with ADC-1, and the aggregate is 1.7%.
  • Figure 9 shows that the components of "half-antibody” (DAR4) formed by the bridge coupling between the light chain and the heavy chain and the bridge coupling within the heavy chain are the main ones.
  • TCEP (2 mg/mL, 6 eq.) aqueous solution was added to trastuzumab (1.0 mg, 38.9 mg/mL, 1.0 eq.) in 25 mM Na 2 B 4 O 7 , 25 mM NaCl, 1 mM DTPA, pH 7.4 buffer, and reduced at 22°C for 2 hours; then an equal volume of 500 mM Tris, pH 8.8 buffer was added, and then LP5 (10 mg/mL, 6 eq.) in DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • the proportion of the single distributed component (DAR4) in the obtained ADC-5 is 50.24%, which is reduced compared with ADC-1, and the aggregate is 1.1%.
  • the proportion of the "half antibody” (DAR4) component formed by the bridging coupling between the light chain and the heavy chain and the bridging coupling within the heavy chain is also reduced compared with ADC-1.
  • TCEP (2 mg/mL, 8 eq.) aqueous solution was added to trastuzumab (0.9 mg, 10 mg/mL, 1.0 eq.) in 50 mM PBS pH 7.4 buffer, and reduced at 22°C for 18 hours; then the antibody was replaced in sodium borate buffer at pH 8, LP10 (10 mg/mL, 7.0 eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 18 hours. After purification by activated carbon treatment three times, the buffer of the desired antibody drug conjugate was ultrafiltered five times to obtain ADC-10 (C ADC (mg/mL): 5.528, V (mL): 0.05, yield 30.7%).
  • ADC-10 C ADC (mg/mL): 5.528, V (mL): 0.05, yield 30.7%).
  • HIC-DAR 3.98, SEC purity: 100%, Free Linker Payload (mol/mol%): not detected.
  • TCEP (10 mmol/L, 6 eq.) aqueous solution was added to 20 mM PBS, 1 mM EDTA, pH 7.4 buffer containing C226S mutation trastuzumab (1.0 mg, 20 mg/mL, 1.0 eq.), and reduced at 22°C for 2 hours; then TCEP was removed by desalting column and the buffer was replaced with 25 mM Na 2 B 4 O 7 , pH 8.0 buffer, and LP3 (10 mg/mL, 3 eq.) DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • the C226S mutated trastuzumab described in the present invention refers to a trastuzumab heavy chain Eu numbering 226th cysteine mutation to serine.
  • HIC-DAR 3.00, SEC purity: 98.1%, Free Linker Payload (mol/mol%): not detected.
  • the single distribution component (DAR3) in the obtained ADC-25 accounts for 74.33%, and the aggregates account for 1.9%.
  • TCEP (10 mmol/L, 6 eq.) aqueous solution was added to 20 mM PBS, 1 mM EDTA, pH 7.4 buffer containing C226S mutation trastuzumab (1.0 mg, 20 mg/mL, 1.0 eq.), and reduced at 22°C for 2 hours; then TCEP was removed by desalting column and the buffer was replaced with 25 mM Na 2 B 4 O 7 , pH 8.0 buffer, and LP1 (10 mg/mL, 6 eq.) DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • HIC-DAR 2.94, SEC purity: 96.3%, Free Linker Payload (mol/mol%): not detected.
  • the single distribution component (DAR3) in the obtained ADC-26 accounts for 75.13%, and the aggregates account for 3.7%.
  • TCEP (10 mmol/L, 6 eq.) aqueous solution was added to 20 mM PBS, 1 mM EDTA, pH 7.4 buffer of Ifinatamab (1.0 mg, 15 mg/mL, 1.0 eq.), and reduced at 22°C for 2 hours; then TCEP was removed by desalting column and the buffer was replaced with 25 mM Na 2 B 4 O 7 , pH 8.0 buffer, and LP10 (10 mg/mL, 6 eq.) DMSO solution was added, and the coupling reaction was carried out at 25°C for 16 hours.
  • HIC-DAR 4, SEC purity: 98.8%, Free Linker Payload (mol/mol%): not detected.
  • the single distributed component (DAR4) in the obtained ADC-27 accounts for 100%, and the aggregate is 1.2%.
  • Figures 24 and 25 show that the components of the "half antibody” (DAR4) formed by the bridging coupling between the light chain and the heavy chain and the bridging coupling within the heavy chain are the main ones.
  • TCEP 10 mg/mL, 12 eq.
  • aqueous solution was added to 50 mM PBS pH 7.0 buffer of trastuzumab (5 mg, 21.07 mg/mL, 1.0 eq.), and reduced at 22°C for 2 hours; then, DMSO solution of Deruxtecan (10 mg/mL, 10 eq.) was added, and the coupling reaction was carried out at 22°C for 1 hour.
  • the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain Comparative Example-1 (C ADC (mg/mL): 6.74, V (mL): 0.6, yield 80.9%).
  • TCEP 10mM, 12eq. aqueous solution was added to 50mM PBS pH 7.0 buffer of Ifinatamab (20mg, 21.07mg/mL, 1.0eq.), and reduced at 22°C for 2 hours; then Deruxtecan (10mg/mL, 10eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain Comparative Example-2 (C ADC (mg/mL): 7.47, V (mL): 2.2, yield 82.2%).
  • Example 22 In vitro plasma stability experiment of ADC based on different linkers
  • This example evaluates the plasma stability of antibody-drug conjugates based on bi-ene linkers and maleimide linkers, and the monitoring indicator is the change of DAR value over time.
  • the tested antibody-drug conjugate was mixed with human plasma at a final concentration of 0.3 mg/ml and incubated in a 37°C incubator. 50 ⁇ L of samples were taken at each set time point (0, 1, 2, 4, 7, 14 days) and frozen at -80°C. After sampling, Elisa was used to determine the antibody-drug conjugate and total antibody concentration. The method is as follows: first, the antigen B7-H3 (Sino Biological, 11188-H08H) and anti-Dxd antibody (GenScript, A02217) were coated overnight in the Elisa plate wells to determine the concentration of total antibody and ADC, respectively.
  • ADC-27 based on the bivalent ene linker is very stable in plasma, and the DAR value remains basically unchanged, while the DAR value of the antibody-drug conjugate based on the maleimide linker (Comparative Example-2) continues to decrease during the incubation with plasma, and after 2 weeks, the DAR value is reduced by half. Therefore, the bivalent ene linker is more stable and has the potential for further improving safety and efficacy.
  • Example 23 Tumor cell proliferation inhibition experiment of antibody-drug conjugates based on bivalent ene bridging
  • NCI-N87 cells were seeded in a 96-well plate (excluding the edge wells filled with PBS), and the cells were cultured in 80 ⁇ L of appropriate culture medium and placed in a constant temperature cell culture incubator at 37°C and 5% CO 2. After the cells were cultured overnight in a constant temperature incubator (37°C, 5% CO 2 ), 20 ⁇ L of diluted bisene antibody-drug conjugate solution was added to each well to make the final concentration of the conjugate range from 0.2pM to 12.5nM or 3.0pM to 200nM. After the cells were incubated with the conjugate for 5 days, the 96-well plate was equilibrated at room temperature for 30 minutes.
  • ADCs showed certain anti-tumor activity.
  • the in vitro cell killing activity of ADC-1, ADC-2, ADC-3, ADC-5, ADC-10, ADC-25, ADC-26 and ADC-27 was comparable to that of Comparative Example-1.
  • the present invention provides a new double ene linker, which can be simply used for site-specific coupling of antibodies based on covalent thiol bridging, and can be applied to most antibodies. Compared with the cumbersome recombinant modification of antibodies, the method of site-specific coupling sites using the double ene linker in the present invention does not require recombinant modification of antibodies, so it can save a lot of time, cost and resources, and has a broader application prospect.
  • the novel bivalent olefin linker provided by the present invention can be covalently bridged and coupled with an antibody by a simple chemical method.
  • the DAR value distribution of the conjugate obtained by using the linker of the present invention is narrower, the generated product has higher uniformity, and the proportion of the single distributed component (DAR4) of the obtained conjugate can exceed 80%, and some can even exceed 95%, and the "half-antibody" (DAR4) component is the main component.
  • novel biolefin linker provided by the present invention can also be covalently bridged and coupled with most antibody mutants, for example, with mutants at position 226 (Eu numbering) or 229 (Eu numbering) of the heavy chain. Since the cysteine that forms the disulfide bond between the heavy chains is mutated to other amino acids, a uniform DAR3 antibody-drug conjugate can be produced.
  • the antibody-drug conjugates based on the novel biolefin connector provided by the present invention have comparable in vitro cell killing activity and better in vitro plasma stability compared with the antibody-drug conjugates based on the maleimide connector.

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Abstract

一种双联烯连接子、连接子与药物缀合物、抗体偶联药物及其用途。双联烯连接子，其特征在于，其结构如通式Ⅰ所示。一种新的双联烯连接子，其可以简便的基于共价巯基桥接定点偶联抗体，其可以应用于大多数抗体，与对抗体进行繁琐的重组改造相比，使用的双联烯连接子定点偶联位点的方法不需要重组改造抗体，所以可以节约大量的时间、成本和资源，应用前景更广泛。

Description

双联烯连接子、连接子与药物缀合物、抗体偶联药物及其用途 技术领域
本发明涉及双联烯连接子、连接子与药物缀合物、抗体偶联药物及其用途。
背景技术
抗体药物偶联物(ADCs)，其设计思想源于德国科学家Paul Ehrlich在1913年首次提出的“Magic bullets“(魔法子弹)概念，该设想的核心理念是通过适当的载体将药物靶向输送到患病部位。由于受限于抗体及高活性细胞毒性药物技术的制约，所以直到2000年第一个用于治疗急性髓样白血病(AML)的抗体-药物偶联物(MylotargTM)才被FDA批准上市。然而，随着抗体人源化，可裂解连接子技术的不断发展，以及新型毒素分子的出现，到目前为止，美国食品药品监督管理局(FDA)已经批准了14种抗体药物偶联物(ADC)用于治疗血癌和实体瘤。尽管现有上市的抗体药物偶联物(ADCs)在临床治疗上取得了巨大成功，然而许多广泛使用的抗体偶联方法仍然有很大的局限性。成分均一的ADC的制备是一个仍需要克服的挑战。常见的偶联方法是利用赖氨酸残基或半胱氨酸部分(通过链间二硫键的还原)和亲电试剂之间的化学反应。由于上述化学反应目前无法做到有选择性，导致偶联数目和位点不确定，因此生成的偶联物不是均一的产物，存在多种组分。而每个不同DAR值的ADC都有不同的药代动力学、毒理学和疗效。这种多样化的ADC混合物不仅难以开发，而且还可能伴随着不理想的治疗窗口。
二硫化物桥接是形成均一ADC的一种策略。不同的试剂已经被用于二硫化物桥接，包括双硫酮，下一代马来酰亚胺(NGMs，是一类由两个卤化物或硫酚离去基团修饰的马来酰亚胺试剂)，哒嗪二酮，例如二溴甲基喹喔啉，亚芳基二丙腈，二乙烯基嘧啶(DVP)(Chem.Soc.Rev.,2021,50,1305-1353)，RC生物技术公司的二丙烯酰基六氢三嗪(CN107921030)，上海新理念生物医药科技有限公司的双马来酰亚胺(CN103933575)和台湾浩鼎生技股份有限公司的双砜基(CN110290800)等。二硫化物桥接尽管取得了以上的进展，但是仍然存在诸如成分不完全单一，比如DAR4的ADC是“半抗体”和“全抗体”的混合物，没有办法做到主要成分是“半抗体”或者“全抗体”，或DAR4的ADC含量偏低比等问题。
综上所述，本领域仍亟需高效、简单、实用的二硫化物桥接的化学偶联方法，在达到定点偶联目的的同时还可以保持或提高抗体-药物偶联物的药效、稳定性和安全性等性质。
发明内容
本发明的目的是提供一种能够与大多数抗体进行共价桥接的新型双联烯连接子。此外，本发明还提供了抗体/活性剂偶联方法及其应用，例如在抗体药物偶联物中的应用。
本发明的第一方面，提供一种双联烯连接子，其特征在于，包括如通式(Ⅰ)所示的化合物或同位素标记物：
其中：所述L1选自
M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
L2选自及中的一个或多个；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数。
根据本发明的一个实施方案：所述通式(Ⅰ)中，
所述L1选自
所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
所述L2选自及中的一个或多个；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数。
根据本发明的一个实施方案：所述通式(Ⅰ)中，
所述L1选自
所述M选自 所述n1与n2不相同，n3与n4相同或不相同，n5与n6相同或不相同；所述为单一手性的组分或者消旋体；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
L2选自及中的一个或多个；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数。
根据本发明的一个实施方案：所述通式(Ⅰ)中，
所述L1选自
M选自、
所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
L2选自n7＝1-10的整数，n8＝1-10的整数。
根据本发明的一个实施方案：所述通式(Ⅰ)中的所述双联烯连接子选自以下结构式化合物至少一个：


本发明另一方面是提供一种连接子与药物缀合物，其特征在于，前述的双联烯连接子中的羧基与中L3的氨基酸残基中的氨基通过酰胺键键合而形成的化合物；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，所述D通过D中的硫、氢或氧与L3共价结合。
根据本发明的一个实施例，所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂。
所述微管蛋白抑制剂包括但不限于一甲基澳瑞他汀(英文名称：monomethyl auristatin)、美登素(英文名称：maytansinoid)、美登醇(英文名称：maytansinol)或DM1美登素。所述一甲基澳瑞他汀可以是一甲基澳瑞他汀E或一甲基澳瑞他汀F等；所述RNA聚合酶抑制剂包括但不限于α-鹅膏蕈碱等；所述拓扑异构酶抑制剂包括但不限于依托泊苷、替尼泊苷、安吖啶、SN-38或依喜替康等；所述插入剂包括但不限于吡咯苯并二氮杂卓(英文名称：pyrrolobenzodiazepine)(PBD)等；所述DNA-反应剂包括但不限于卡奇霉素、天赐米星类(英文名称：tiancimycins)或其他烯二炔等；所述DNA烷化剂包括但不限于曲贝替定或卢比替定等海鞘素类衍生物。
本发明的另一方面是提供一种连接子与药物缀合物，其特征在于，包括如通式(Ⅱ)所示结构的化合物：
其中：所述L1选自
所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
所述L2选自及中的一个或多个；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，
其中D通过D中的硫、氮或氧与L3共价结合。
根据本发明的一个实施方案：所述通式(Ⅱ)中，
所述L1选自
所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
所述L2选自及中的一个或多个；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，
其中D通过D中的硫、氮或氧与L3共价结合。
根据本发明的一个实施方案：所述通式(Ⅱ)中， 所述L1选自
所述M选自 所述n1与n2不相同，n3与n4相同或不相同，n5与n6相同或不相同；所述为单一手性的组分或者消旋体；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
L2选自及中的一个或多个；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
D为细胞毒性类小分子药物或其他活性剂失去一个或多个原子或基团所形成的片段，
其中D通过D中的硫、氮或氧与L3共价结合。
根据本发明的一个实施方案：所述通式(Ⅱ)中，
所述L1选自
M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
L2选自n7＝1-10的整数，n8＝1-10的整数；
L3为
D为细胞毒性类小分子药物或其他活性剂失去一个或多个原子或基团所形成的片段，
其中D通过D中的硫、氮或氧与L3共价结合。
根据本发明的一个实施方案：所述通式(Ⅱ)中，
所述L1选自
M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
L2选自n7＝1-10的整数，n8＝1-10的整数；
L3为
所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂。
所述微管蛋白抑制剂包括但不限于一甲基澳瑞他汀(英文名称：monomethyl auristatin)、美登素(英文名称：maytansinoid)、美登醇(英文名称：maytansinol)或DM1美登素。所述一甲基澳瑞他汀包括但不限于一甲基澳瑞他汀E或一甲基澳瑞他汀F等；所述RNA聚合酶抑制剂包括但不限于α-鹅膏蕈碱等；所述拓扑异构酶抑制剂包括但不限于依托泊苷、替尼泊苷、安吖啶、SN-38或依喜替康等；所述插入剂包括但不限于吡咯苯并二氮杂卓(英文名称：pyrrolobenzodiazepine)(PBD)等；所述DNA-反应剂包括但不限于卡奇霉素、天赐米星类(英文名称：tiancimycins)或其他烯二炔等；所述DNA烷化剂包括但不限于曲贝替定或卢比替定等海鞘素类衍生物。
根据本发明的一个实施方案：所述的通式(Ⅱ)化合物选自以下表格中的化合物至少一个:



本发明的另一方面是提供一种抗体药物偶联物，其特征在于，由前述的连接子与药物缀合物中的双联烯基团和Ab还原之后的巯基基团进行偶联形成偶联物；
所述Ab是抗体、抗体片段或蛋白。
根据本发明的一个实施方案，所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体、其抗原结合片段、免疫活性部分或上述抗体的突变体。
本发明的另一方面是提供一种抗体药物偶联物，其特征在于，包括如通式(Ⅲ)所示的结构：
所述L1选自
所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
所述L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，
其中D通过D中的硫、氮或氧与L3共价结合。
所述n16选自1-20之间的任意数值；
所述Ab是抗体、抗体片段或蛋白。
其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体、其抗原结合片段、免疫活性部分或上述抗体的突变体。
根据本发明的一个实施方案：所述通式(Ⅲ)中，
所述L1选自
所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
所述L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝
1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
所述细胞毒性类小分子药物或活性剂为细胞毒性类小分子药物或其他活性剂失去一个或多个原子或基团所形成的片段；
其中D通过D中的硫、氮或氧与L3共价结合。
所述n16选自1-20之间的任意数值；
所述Ab是抗体、抗体片段或蛋白。
其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体、其抗原结合片段、免疫活性部分或上述抗体的突变体。
根据本发明的一个实施方案：所述通式(Ⅲ)中，
所述L1选自
所述M选自 所述n1与n2不相同，n3与n4相同或不相同，n5与n6相同或不相 同；所述为单一手性的组分或者消旋体；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
所述L3为 n14＝1-20的整数，n15＝1-20的整数；
D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段；
其中D通过D中的硫、氮或氧与L3共价结合；
所述n16选自1-20之间的任意数值；
所述Ab是抗体、抗体片段或蛋白。
其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体、其抗原结合片段、免疫活性部分或上述抗体的突变体。
根据本发明的一个实施方案：
所述L1选自
M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
L2选自n7＝1-10的整数，n8＝1-10的整数；
L3为
所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；
所述n16选自1-20之间的任意数值；
所述Ab是抗体、抗体片段或蛋白。
所述微管蛋白抑制剂包括但不限于一甲基澳瑞他汀(英文名称：monomethyl auristatin)、美登素(英文名称：maytansinoid)、美登醇(英文名称：maytansinol)或DM1美登素。所述一甲基澳瑞他汀可以是一甲基澳瑞他汀E或一甲基澳瑞他汀F等；所述RNA聚合酶抑制剂包括但不限于α-鹅膏蕈碱等；所述拓扑异构酶抑制剂包括但不限于依托泊苷、替尼泊苷、安吖啶、SN-38或依喜替康等；所述插入剂包括但不限于吡咯苯并二氮杂卓(英文名称：pyrrolobenzodiazepine)(PBD)等；所述DNA-反应剂包括但不限于卡奇霉素、天赐米星类(英文名称：tiancimycins)或其他烯二炔等；所述DNA烷化剂包括但不限于曲贝替定或卢比替定等海鞘素类衍生物。
其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体、其抗原结合片段、免疫活性部分或上述抗体的突变体。
根据本发明的一个实施方案：所述通式(Ⅲ)中，
所述L1选自
M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
L2选自n7＝1-10的整数，n8＝1-10的整数；
L3为
所述n16选自1-20之间的任意数值；
所述细胞毒性类小分子药物为炎症性疾病药物、神经系统疾病药物或心血管疾病药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；
所述Ab为阿昔单抗、阿仑单抗、阿奈妥单抗、阿特珠单抗、阿维鲁单抗、巴利昔单抗、贝伐单抗、博纳吐单抗、本妥昔单抗、卡妥索单抗、西妥昔单抗、西妥珠单抗、考妥昔单抗、达利珠单抗、达雷木单抗、地宁妥珠单抗、地诺单抗、德帕妥昔珠单抗、地努妥昔单抗、德瓦鲁单抗、埃罗妥珠单抗、因福土单抗、格巴妥木单抗、吉妥珠单抗、替伊莫单抗、英达妥昔单抗、英度妥单抗、奥英妥珠单抗、伊匹单抗、拉贝珠单抗、拉妥珠单抗、拉妥昔单抗、利法妥珠单抗、洛沃妥珠单抗、米拉组单抗、米妥昔单抗、那妥昔单抗、耐昔妥珠单抗、尼妥珠单抗、纳武单抗、奥滨尤妥珠单抗、奥法木单抗、奥拉单抗、奥马珠单抗、帕利珠单抗、帕尼单抗、帕曲妥单抗、派姆单抗、帕妥珠单抗、匹那妥珠单抗、泊洛妥珠单抗、雷莫芦单抗、洛伐妥珠单抗、沙西妥珠单抗、司妥昔单抗、司曲妥单抗、索非妥珠单抗、伐达妥昔单抗、沃瑟妥珠单抗、曲妥珠单抗、抗B7-H3单抗、抗CD4抗体、抗CD5抗体、抗CD13抗体、抗CD30抗体或上述抗体在重链226位或229位上的突变体、其抗原结合片段、免疫活性部分。
根据本发明的一个实施方案：所述通式(Ⅲ)中，
所述L1选自
M选自所述n1与n2相同，n3与n4相同,n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
L2选自n7＝1-10的整数，n8＝1-10的整数；
L3为
所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；
所述n16选自2-6之间的任意数值；
所述Ab是选自帕曲妥单抗、沙西妥珠单抗、米妥昔单抗、西妥昔单抗、曲妥珠单抗、抗B7-H3单抗、西妥珠单抗或上述抗体在重链Eu编号226位或Eu编号229位上的突变体。
所述突变体为使重链和重链之间形成二硫键的半胱氨酸突变为丝氨酸。
根据本发明的一个实施方案：所述通式(Ⅲ)中的所述抗体偶联药物选自下述结构式的化合物至少一个：





n16选自2-6之间的任意数值。
本发明所述的通式(Ⅲ)结构式及各ADC-1到ADC-29结构式中的S是抗体Ab中含有的硫原子。
本发明所述的微管蛋白抑制剂包括但不限于一甲基澳瑞他汀(英文名称：monomethyl auristatin)、美登素(英文名称：maytansinoid)、美登醇(英文名称：maytansinol)或DM1美登素。所述一甲基澳瑞他汀可以 是一甲基澳瑞他汀E或一甲基澳瑞他汀F等；所述RNA聚合酶抑制剂包括但不限于α-鹅膏蕈碱等；所述拓扑异构酶抑制剂包括但不限于依托泊苷、替尼泊苷、安吖啶、SN-38或依喜替康等；所述插入剂包括但不限于吡咯苯并二氮杂卓(英文名称：pyrrolobenzodiazepine)(PBD)等；所述DNA-反应剂包括但不限于卡奇霉素、天赐米星类(英文名称：tiancimycins)或其他烯二炔等；所述DNA烷化剂包括但不限于曲贝替定或卢比替定等海鞘素类衍生物。
本发明所述的阿昔单抗英文名称为Abciximab；阿仑单抗英文名称为Alemtuzumab；阿奈妥单抗英文名称为Anetumab；阿特珠单抗英文名称为Atezolizumab；阿维鲁单抗英文名称为Avelumab；巴利昔单抗英文名称为Basiliximab；贝伐单抗英文名称为Bevacizumab；博纳吐单抗英文名称为Blinatomumab；本妥昔单抗英文名称为Brentuximab；卡妥索单抗英文名称为Catumaxomab；西妥昔单抗英文名称为Cetuximab；西妥珠单抗英文名称为Cirmtuzumab；考妥昔单抗英文名称为Coltuximab；达利珠单抗英文名称为Daclizumab；达雷木单抗英文名称为Daratumumab；地宁妥珠单抗英文名称为Denintuzumab；地诺单抗英文名称为Denosumab；德帕妥昔珠单抗英文名称为Depatuxizumab；地努妥昔单抗英文名称为Dinutuximab；德瓦鲁单抗英文名称为Durvalumab；埃罗妥珠单抗英文名称为Elotuzumab；因福土单抗英文名称为Enfortumab；格巴妥木单抗英文名称为Glembatumumab；吉妥珠单抗英文名称为Gemtuzumab；替伊莫单抗英文名称为Ibritumomab；英达妥昔单抗英文名称为Indatuximab；英度妥单抗英文名称为Indusatumab；奥英妥珠单抗英文名称为Inotuzumab；伊匹单抗英文名称为Ipilimumab；拉贝珠单抗英文名称为Labetuzumab；拉妥珠单抗英文名称为Ladiratuzumab；拉妥昔单抗英文名称为Laprituximab；利法妥珠单抗英文名称为Lifastuzumab；洛沃妥珠单抗英文名称为Lorvotuzumab；米拉组单抗英文名称为Milatuzumab；米妥昔单抗(Mirvetuximab)、那妥昔单抗(Naratuximab)、耐昔妥珠单抗(Necitumumab)、尼妥珠单抗英文名称为Nimotuzumab；纳武单抗英文名称为Nivolumab；奥滨尤妥珠单抗英文名称为Obinutuzumab；奥法木单抗英文名称为Ofatumumab；奥拉单抗英文名称为Olaratumab；奥马珠单抗英文名称为Omalizumab；帕利珠单抗英文名称为Palivizumab；帕尼单抗英文名称为Panitumumab；帕曲妥单抗英文名称为Patritumab；派姆单抗英文名称为Pembrolizumab；帕妥珠单抗英文名称为Pertuzumab；匹那妥珠单抗英文名称为Pinatuzumab；泊洛妥珠单抗英文名称为Polatuzumab；雷莫芦单抗英文名称为Ramucirumab；洛伐妥珠单抗英文名称为Rovalpituzumab；沙西妥珠单抗英文名称为Sacituzumab；司妥昔单抗英文名称为Siltuximab；司曲妥单抗英文名称为Sirtratumab；索非妥珠单抗英文名称为Sofituzumab；伐达妥昔单抗英文名称为Vadastuximab；沃瑟妥珠单抗英文名称为Vorsetuzumab；曲妥珠单抗英文名称为Trastuzumab；抗B7-H3单抗选自Ifinatamab，中文译名伊非那他单抗。
前述的抗体药物偶联物在用于治疗癌症中的用途。
所述癌症包括淋巴瘤、母细胞瘤、肉瘤、白血病或淋巴恶性瘤、鳞状细胞癌(如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌和肺的鳞状细胞癌)、腹膜癌、肝癌、胃癌、胃部癌(包括胃肠癌)、胰腺癌、脑胶质瘤、宫颈瘤、卵巢癌、口腔癌、膀胱癌、泌尿道癌症、乳腺癌、HER2阳性乳腺癌、结肠癌、直肠癌、结直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、多发性骨髓瘤、B-细胞淋巴瘤、脑癌、头颈部癌和相关转移性肿瘤。
本发明的主要优点在于：
1、本发明提供了一种新的双联烯连接子，其可以简便的基于共价巯基桥接定点偶联抗体，其可以应用于大多数抗体，与对抗体进行繁琐的重组改造相比，使用本发明中的双联烯连接子定点偶联位点的方法不需要重组改造抗体，所以可以节约大量的时间、成本和资源，应用前景更广泛。
2、本发明提供的新型双联烯连接子，可通过简单的化学方法与抗体共价桥接偶联，所生成的抗体-药物偶联物与传统的随机偶联方法生成的抗体-药物偶联物相比，使用本发明的连接子得到的偶联物DAR值分布更窄，生成的产品均一性更高，获得的偶联物单一分布的组份(DAR4)占比可以超过80％，有些甚至可以超过95％，而且“半抗体”(DAR4)的组分是占主要的。
3、本发明提供的新型双联烯连接子，也可以和大多数抗体的突变体，例如和在重链226位(Eu编号)或229位(Eu编号)上的突变体共价桥接偶联，由于重链和重链之间形成二硫键的半胱氨酸突变为其他氨基酸，从而可以产生均一的DAR3的抗体-药物偶联物。
4、在抗体相同的情况下，基于本发明提供的新型双联烯连接头的抗体-药物偶联物和马来酰亚胺连接 头的抗体-药物偶联物相比，具有相当的体外细胞杀伤活性，和更好的体外血浆稳定性。
附图说明
图1为本发明实施例中制备的ADC-1HIC图。从图1可以看出，获得的偶联物单一分布的组份(DAR4)占比可以超过80％。
图2为本发明实施例中制备的ADC-1SEC图。
图3为本发明实施例中制备的ADC-1MS图。
图4为本发明实施例中制备的ADC-1SDS-PAGE图。
图5为本发明实施例中制备的ADC-2HIC图。
图6为本发明实施例中制备的ADC-2SEC图。
图7为本发明实施例制备的ADC-3HIC图。
图8为本发明实施例制备的ADC-3SEC图。
图9为本发明实施例制备的ADC-3SDS-PAGE图。
图10为本发明实施例制备的ADC-5HIC图。
图11为本发明实施例制备的ADC-5SEC图。
图12为本发明实施例制备的ADC-5SDS-PAGE图。
图13为本发明实施例制备的ADC-10HIC图。从图13可以看出，获得的偶联物单一分布的组份(DAR4)占比可以超过95％。
图14为本发明实施例制备的ADC-10SEC图。
图15为本发明实施例制备的ADC-10SDS-PAGE图。
图16为本发明实施例制备的ADC-25HIC图。
图17为本发明实施例制备的ADC-25SEC图。
图18为本发明实施例制备的ADC-25SDS-PAGE图。
图19为本发明实施例制备的ADC-26HIC图。
图20为本发明实施例制备的ADC-26SEC图。
图21为本发明实施例制备的ADC-26SDS-PAGE图。
图22为本发明实施例中制备的ADC-27HIC图。从图22可以看出，获得的偶联物单一分布的组份(DAR4)占比可以超过95％。
图23为本发明实施例中制备的ADC-27SEC图。
图24为本发明实施例中制备的ADC-27MS图。
图25为本发明实施例中制备的ADC-27SDS-PAGE图。
图26为基于不同连接头的ADC体外血浆稳定性实验图。
具体实施方式
以下是本专利所述化合物(包括连接子和连接子-药物缀合物)和抗体药物偶联物的合成和分析的示例。必须注意，这些实施例并不限制本发明的保护范围。
除非另外说明，所有的无水试剂都是直接从供应商购买的，并在氮气下保存。
本专利所述“半抗体”和“全抗体”的定义和示意图见下：
“全抗体”如右图所示，为轻链-重链之间桥接和重链-重链之间桥接偶联形成：
“半抗体”如右图所示，为轻链-重链之间桥接和重链之内桥接偶联形成：
连接子的合成与表征
实施例1：化合物LK1的合成
步骤1：6-(((苄氧基)羰基)氨基)己酸-2-(三甲基甲硅烷基)乙酯的合成。
向6-(((苄氧基)羰基)氨基)己酸(5.0g，0.0188mmol)和EDCI(3.98g，0.0207mol)以及HOBt(2.80g，0.0207mol)的30mL二氯甲烷溶液中加入2-(三甲基甲硅烷基)乙烷-1-醇(2.23mg，0.0188mmol)与DIPEA(4.87g，0.0376mol)。加完后，混合物在室温下搅拌2小时。将混合物浓缩并重新溶解在MTBE中，用盐水、饱和碳酸氢钠洗涤，浓缩，残余物通过硅胶快速柱层析纯化(用乙酸乙酯/庚烷＝1:3洗脱)，得到白色固体状的产物(5.5g，产率79.8％)。
LC-MS(ESI)m/z:338.0[M-2CH3+3H]+。
1H NMR(300MHz,DMSO-d6)δ7.33(q,J＝5.6,4.1Hz,5H),7.21(d,J＝7.5Hz,1H),4.99(s,2H),4.19–4.01(m,2H),2.96(q,J＝6.5Hz,2H),2.23(t,J＝7.3Hz,2H),1.57–1.44(m,2H),1.38(t,J＝7.4Hz,2H),1.24(d,J＝7.0Hz,3H),1.02–0.85(m,2H),0.01(d,J＝2.4Hz,9H)。
步骤2：6-氨基己酸-2-(三甲基硅烷基)乙酯的合成
用30mL MeOH和浓HCl(0.74g，7.4mmol)稀释2-(三甲基硅烷基)6-(((苄氧基)羰基)氨基)己酸乙酯(2.5g，6.8mmol)，然后在N2下加入0.3g 5％Pd/C。将悬浮液抽真空并用H2置换数次。将混合物在1MPa H2下搅拌1小时。悬浮液通过硅藻土垫过滤，用MeOH洗涤。将合并的滤液浓缩至干，得到油状液体，直接用于下一步，未进行进一步纯化。
1H NMR(400MHz,DMSO-d6)δ7.87(s,3H),4.10(t,J＝8.4Hz,2H),2.74(s,2H),2.27(t,J＝7.4Hz,3H),1.53(h,J＝7.2Hz,5H),1.31(q,J＝7.9Hz,2H),0.94(t,J＝8.4Hz,2H),0.03(d,J＝1.8Hz,8H)。
步骤3：合成3-((叔丁基羰基)氨基)-2-((叔丁基羰基)氨基)甲基丙酸甲酯
在3-氨基-2-(氨基甲基)丙酸甲酯(900mg,4.383mmol)的1,4-二氧六环/H2O(30mL，1:1)溶液中加入Na2CO3(2.33g，21.9mmol)，搅拌0.5小时，冷却至0℃。在反应混合物中加入Boc2O(3.9g，17.53mmol)，室温搅拌过夜。反应完成后，用乙酸乙酯(50mL×3)萃取。有机层用无水硫酸钠干燥，浓缩。柱层析(庚烷/乙酸乙酯＝10:1-5:1)纯化，得到无色油状目标产物(1.23g，83.9％)。
1H NMR(400MHz,Chloroform-d)δ5.23(s,1H),3.70(s,3H),3.56(q,J＝15.7,12.1Hz,2H),3.21(dd,J＝13.4,6.8Hz,2H),2.74(p,J＝5.6Hz,1H),1.43(s,19H)。
步骤4：合成3-((叔丁基羰基)氨基)-2-((叔丁基羰基)氨基)甲基丙酸
在MeOH/H2O(2:1,12mL)的3-((叔丁基羰基)氨基)-2-((叔丁基羰基)氨基)甲基丙酸甲酯(1.23g,3.7mmol)溶液中，控温在0℃，加入LiOH˙H2O(776.3mg，18.5mmol)。在室温下搅拌0.5小时，用1N HCl调节pH为3-4。用乙酸乙酯(50mL×3)萃取，用饱和食盐水洗涤有机层。分离有机层，用无水硫酸钠干燥。浓缩后用于下一步，无需进一步提纯。
1H NMR(400MHz,Chloroform-d)δ5.38(s,1H),3.65–3.15(m,4H),2.76(q,J＝5.7Hz,1H),1.43(s,19H).
步骤5：合成2-(三甲基硅基)乙基-6-(3-(叔丁基羰基)氨基)-2-((叔丁基羰基)氨基)甲基丙胺)己酸酯
在3-(叔丁基羰基)氨基)-2-(叔丁基羰基)氨基)甲基丙酸(1.0g，3.14mmol)的二氯甲烷(20mL)溶液中加入EDCI(658mg，3.45mmol)和HOBt(463.3mg，3.45mmol)，2-(三甲基硅基)乙基6-氨基己酸酯(1.46g，31.19mmol)。将混合物在室温下搅拌过夜。用5％柠檬酸溶液(50mL×3)清洗，然后用水(50mL×3)清洗。有机层用无水硫酸钠干燥，浓缩，柱层析(乙酸乙酯/正庚烷＝1:1)纯化，得到白色固体的目标产物(870mg，52.1％)。
1H NMR(400MHz,Chloroform-d)δ6.54(s,1H),5.51(s,2H),4.23–4.14(m,2H),3.54–3.42(m,2H),3.31–3.20(m,4H),2.57(p,J＝5.4Hz,1H),2.32(t,J＝7.4Hz,2H),1.71–1.62(m,2H),1.60–1.50(m,3H),1.47(s,18H),1.43–1.34(m,3H),1.05–0.96(m,2H),0.07(s,9H)。
步骤6：合成2-(三甲基硅基)乙基-6-(3-氨基-2-(氨基甲基)丙胺)己酸二盐酸盐
在2-(三甲基硅基)乙基6-(3-(叔丁基羰基)氨基)-2-(叔丁基羰基)氨基)甲基丙胺)己酸酯(500mg，0.94mmol)的乙酸乙酯(7mL)溶液中加入2N HCl/EA(2.4ml)。将混合物室温搅拌2天。浓缩除去溶剂，加入乙腈进行打浆处理，将固体过滤干燥，得到目标产物(300mg，79.1％)白色固体盐。
1H NMR(400MHz,Chloroform-d)δ8.16(s,4H),4.14(t,J＝8.4Hz,2H),3.58(s,4H),3.21(s,2H),2.64(s,2H),2.29(s,2H),1.48(d,J＝93.5Hz,6H),1.04–0.92(m,2H),0.04(s,10H)。
步骤7：合成2-(三甲基硅基)乙基-6-(3-(丁-3-氨基基)-2-(丁-3-氨基基)丙胺)己酸酯
在丁-3-乙酸(623.6mg，7.4mmol)的二氯甲烷(10mL)混悬液加入草酰氯(939.1mg，7.4mmol)。室温搅拌15min后，将反应液冷却至0℃，加入0.15mL DMF。将反应混合物在0℃下搅拌，然后在室温下再搅拌90min。将上述溶液滴入2-(三甲基硅基)乙基6-(3-氨基-2-(氨基甲基)丙胺)己酸二盐酸盐溶液(300mg，0.74mmol)的饱和碳酸氢钠溶液(10mL)中，反应混合物在室温条件下继续搅拌2h。反应混合物用二氯甲烷(50mL×3)萃取，浓缩。柱层析(乙酸乙酯/正庚烷＝1:1)纯化，得到目标产物(292mg，85％)为白色固体。
LC-MS(ESI)m/z:464[M+H]+。
1H NMR(400MHz,Chloroform-d)δ7.52(s,2H),6.67(s,1H),4.19–4.12(m,2H),3.71(ddd,J＝14.1,7.8,4.7Hz,2H),3.31–3.24(m,3H),3.23(d,J＝2.7Hz,5H),2.63(p,J＝5.5Hz,1H),2.41(t,J＝2.7Hz,2H),2.28(t,J＝7.4Hz,2H),1.67(s,8H),1.62(q,J＝7.6Hz,2H),1.53(p,J＝7.3Hz,2H),1.39–1.32(m,3H),1.04–0.93(m,2H),0.04(s,9H)。
步骤8：合成2-(三甲基硅基)乙基-6-(3-(丁-2,3-二氨基)-2-(丁-2,3-二氨基甲基)丙胺)己酸酯
将2-(三甲基硅基)乙基6-(3-(丁-3-氨基氨基)-2-(丁-3-氨基甲基)丙胺)己酸盐(292mg，0.64mmol)溶解在18％的K2CO3水溶液/二氧六环(8mL/8mL)的溶液中。将反应混合物加热至40℃，保持2小时，加入柠檬酸(60mL,10％水)和乙酸乙酯(500mL×3)萃取，有机层用无水硫酸钠干燥，浓缩得到(210mg，71.9％)为油状化合物。
1H NMR(400MHz,Chloroform-d)δ7.08(t,J＝6.6Hz,2H),6.96(s,1H),5.63(t,J＝6.6Hz,2H),5.29(dd,J＝6.6,1.6Hz,4H),4.19–4.10(m,2H),3.80–3.71(m,2H),3.21(dt,J＝14.1,5.6Hz,4H),2.63(q,J＝5.2,4.3Hz,1H),2.34–2.23(m,3H),1.63(p,J＝7.5Hz,2H),1.52(p,J＝7.3Hz,2H),1.42–1.30(m,3H),1.01–0.94(m,2H),0.04(s,9H)。
步骤9：合成LK1
将2-(三甲基硅基)乙基-6-(3-(丁-2,3-二氨基)-2-(丁-2,3-二氨基甲基)丙胺)己酸酯(60mg，0.129mmol)溶于TFA/DCM(1:1,1mL)中，室温搅拌1小时。浓缩去溶剂，用C18柱(ACN/H2O)纯化，得到20mg白色固体。
LC-MS(ESI)m/z:364[M+H]+。
1H NMR(400MHz,DMSO-d6)δ11.95(s,1H),7.84(q,J＝5.6Hz,3H),5.75(t,J＝6.6Hz,2H),5.28(d,J＝6.6Hz,4H),3.18(dh,J＝26.9,6.3Hz,5H),3.02(q,J＝6.6Hz,2H),2.57(t,J＝6.9Hz,1H),2.18(t,J＝7.4Hz,2H),1.47(p,J＝7.4Hz,2H),1.38(p,J＝7.2Hz,2H),1.24(q,J＝8.8,8.2Hz,2H)。
实施例2：化合物LK2的合成
步骤1：2,3-双((叔丁氧羰基)氨基)丙酸甲酯的合成
向2,3-二氨基丙酸甲酯二盐酸盐(2.5g,13.1mmol)的1,4-二氧六环/H2O(30mL,1:1)的溶液中加入Na2CO3(6.9g,65.7mmol)。反应液在0℃条件下搅拌0.5小时。向反应液中加入Boc2O(11.4g,52.4mmol)，室温条件下搅拌过夜。反应完成后，用乙酸乙酯(50mL×3)萃取。合并有机层，用无水硫酸钠干燥有机层并浓缩。残留物通过硅胶快速柱层析(庚烷/乙酸乙酯＝10:1到5:1)纯化，获得目标产物(2.0g,产率47.7％)为无色油状物。
1H NMR(400MHz,DMSO-d6)δ7.06(d,J＝7.8Hz,1H),6.84(t,J＝6.3Hz,1H),4.03(qd,J＝7.1,6.6,3.8Hz,1H),3.59(s,3H),3.25(ddt,J＝19.4,13.7,6.6Hz,2H),1.37(d,J＝5.2Hz,18H)。
步骤2：2,3-双((叔丁氧羰基)氨基)丙酸的合成
在0℃条件下，向2,3-双((叔丁氧羰基)氨基)丙酸甲酯(1.23g,3.7mmol)的MeOH/H2O(2:1,12mL)溶液中加入LiOH﹒H2O(776.3mg,18.5mmol)。在室温条件搅拌0.5小时，用1N HCl将pH调整为3-4。用乙酸乙酯(50mL×3)萃取，饱和食盐水洗涤有机相。分离有机相并用无水硫酸钠干燥。浓缩并用于下一步，无需进一步纯化。
1H NMR(400MHz,DMSO-d6)δ6.80(dd,J＝16.6,7.0Hz,2H),3.96(q,J＝6.6Hz,1H),3.27–3.15(m,2H),1.37(d,J＝3.9Hz,20H)。
步骤3：2-(三甲基甲硅烷基)乙基-6-(2,3-双((叔丁氧基羰基)氨基)丙酰胺基)己酸乙酯的合成
向2,3-双(叔丁氧羰基)氨基)丙酸(1.0g,3.14mmol)，LK1-3(1.46g,31.19mmol)的二氯甲烷(20mL)溶液中加入EDCI(658mg,3.45mmol)和HOBt(463.3mg,3.45mmol)。在室温条件下搅拌过夜。用aq.5％柠檬酸(50mL×3)洗涤反应液，然后用水(50mL×3)洗涤，有机相用无水硫酸钠干燥，浓缩有机相，并通过硅胶快速柱层析(乙酸乙酯/正庚烷＝1:1)纯化，获得目标产物(1.0g，产率58.8％)为白色固体。
1H NMR(400MHz,Chloroform-d)δ6.66(s,1H),5.77(s,1H),5.14(s,1H),4.19–4.10(m,4H),3.56–3.40(m,3H),3.25(dp,J＝13.7,6.5Hz,2H),2.27(t,J＝7.5Hz,2H),1.63(p,J＝7.5Hz,3H),1.52(p,J＝7.3Hz,2H),1.02–0.94(m,2H),0.04(s,11H)。
步骤4：2-(三甲基甲硅烷基)乙基-9,10-二氨基-8-氧癸酸二盐酸盐的合成
向2-(三甲基甲硅烷基)乙基-6-(2,3-双((叔丁氧基羰基)氨基)丙酰胺基)己酸乙酯(500mg,0.94mmol)的乙酸乙酯(7mL)溶液中加入2N HCl/乙酸乙酯(2.4mL,5eq)。室温条件下搅拌2天。浓缩以去除溶剂和并用乙腈沉淀固体。将固体过滤并干燥，获得(230mg，产率75.2％)为白色固体。
1H NMR(400MHz,Deuterium Oxide)δ4.29(dd,J＝6.2,5.4Hz,1H),4.25–4.18(m,2H),3.58–3.46(m,2H),3.37–3.26(m,1H),3.22–3.12(m,1H),2.35(t,J＝7.3Hz,2H),1.57(dp,J＝22.6,7.3Hz,4H),1.33(qd,J＝7.3,6.3,4.1Hz,2H),1.04–0.96(m,2H),0.01(s,9H)。
步骤5：2-(三甲基甲硅烷基)乙基-6-(2,3-二(丁-3-炔酰胺基)丙酰胺基)己酸乙酯的合成
向丁-3-炔酸(623.6mg,7.4mmol)的二氯甲烷(10mL)悬浮液中加入草酰氯(939.1mg,7.4mmol)。在室温条件下搅拌15分钟后，将反应液冷却到0℃并加入DMF(0.15mL)。且随后在室温条件下搅拌90 分钟。将该溶液逐滴添加至2-(三甲基甲硅烷基)乙基-9,10-二氨基-8-氧癸酸二盐酸盐(300mg,0.74mmol)于10mL饱和碳酸氢钠溶液中，在室温条件下搅拌2小时。用二氯甲烷(50mL×3)萃取，合并有机相，干燥，浓缩。利用硅胶快速柱层析(乙酸乙酯/正庚烷＝1:1)纯化，获得目标产物(292mg，产率85％)为白色固体。
1H NMR(400MHz,Chloroform-d)δ7.75(d,J＝6.2Hz,1H),7.30(s,1H),6.96(s,1H),4.45–4.36(m,1H),4.22–4.09(m,2H),3.81(ddd,J＝14.3,7.1,3.7Hz,1H),3.52(dt,J＝14.3,5.5Hz,1H),3.28(d,J＝7.0Hz,1H),3.26(d,J＝2.7Hz,3H),3.24(d,J＝2.6Hz,2H),2.40(dt,J＝6.3,2.7Hz,2H),2.28(t,J＝7.4Hz,2H),1.63(p,J＝7.5Hz,7H),1.41–1.29(m,4H),1.02–0.94(m,2H),0.04(s,9H)。
步骤6：2-(三甲基甲硅烷基)乙基-6-(2,3-二(丁-2,3-二烯酰胺基)丙酰胺基)己酸乙酯的合成
将2-(三甲基甲硅烷基)乙基-6-(2,3-二(丁-3-炔酰胺基)丙酰胺基)己酸乙酯(292mg,0.64mmol)和K2CO3(18％aq，8mL)的1,4-二氧六环(8mL)溶液加热到40℃并搅拌2小时，再加入柠檬酸(60mL,10％水溶液)和乙酸乙酯(500mL×3)。合并有机层，干燥，浓缩得到目标产物(210mg，产率72％)为油状物。
1H NMR(400MHz,Chloroform-d)δ7.45(d,J＝5.7Hz,1H),7.12(s,1H),6.72(s,1H),5.64(q,J＝6.4Hz,2H),5.35–5.23(m,4H),4.42–4.34(m,1H),4.20–4.10(m,2H),3.80(ddd,J＝14.4,7.2,3.2Hz,1H),3.53(dt,J＝14.3,5.5Hz,1H),3.24(dt,J＝7.9,6.3Hz,2H),2.27(t,J＝7.5Hz,2H),1.52(q,J＝7.4Hz,2H),1.36(s,3H),1.01–0.94(m,2H),0.04(s,9H)。
步骤7：6-(2,3-二(丁-2,3-二烯酰胺基)丙酰胺基)己酸
将2-(三甲基甲硅烷基)乙基-6-(2,3-二(丁-2,3-二烯酰胺基)丙酰胺基)己酸乙酯(70mg,0.129mmol)的TFA/DCM(1:1,1mL)溶液在室温条件下搅拌1小时。浓缩去除溶剂，通过C18柱(ACN/H2O)纯化，得到目标产物(30mg，55％)为白色固体。
实施例3：化合物LK3的合成
步骤1：6-(3,5-二硝基苯甲酰胺基)己酸-2-(三甲基甲硅烷基)乙酯的合成
在0℃条件下，向LK1-3(455mg,1.97mmol)和三乙胺(300mg,2.96mmol)的(20mL)二氯甲烷溶液中缓慢滴入3,5-二硝基苯甲酰氯(500mg,2.16mmol)。滴加完毕后，在室温条件下搅拌2小时，通过LCMS监测反应完成。用1N HCl、饱和NaHCO3和盐水依次洗涤反应液。合并有机相，用硫酸钠干燥有机层并过滤。浓缩并通过硅胶快速柱层析纯化，获得目标化合物(429mg，产率51％)为黄色固体。
1H NMR(400MHz,Chloroform-d)δ9.15(t,J＝2.1Hz,1H),9.04(s,1H),9.05(s,1H),7.05(m,1H),4.22–4.16(m,2H),3.59(q,J＝6.5Hz,2H),2.36(t,J＝6.8Hz,2H),1.66-1.74(m,4H),1.46-1.52(m,2H),1.03–0.94(m,2H),0.04(s,9H)。
步骤2：6-(3,5-二氨基苯甲酰胺基)己酸-2-(三甲基甲硅烷基)乙酯的合成
向6-(3,5-二硝基苯甲酰胺基)己酸2-(三甲基甲硅烷基)乙酯(420mg,1.15mmol)的甲醇(10mL)溶液中加入10％湿Pd/C(42mg,10％w/w)。并在H2保护条件下室温搅拌过夜，过滤，浓缩，获得目标产物(377mg，粗品)为黄色固体。
LC-MS(ESI)m/z:366[M+H]+。
1H NMR(400MHz,Methanol-d4)δ6.51(s,1H),6.50(s,1H),6.29(t,J＝2.0Hz,1H),4.59 (brs,4H),4.25–4.15(m,2H),3.36-3.38(m,2H),2.35(t,J＝7.4Hz,2H),1.60-1.73(m,4H),1.41-1.47(m,2H),1.04–0.98(m,2H),0.08(s,9H)。
步骤3：2-(三甲基甲硅烷基)乙基-6-(3,5-二(丁-3-炔甲氨基)苯甲氨基)己酸酯的合成
向丁-3-炔酸(850.8mg,10.12mmol)的(50mL)二氯甲烷悬浮液中加入草酰氯(1.28g,10.12mmol)。在室温条件下搅拌15分钟后，将反应液冷却到0℃并加入催化量的DMF(0.15mL)。将温度缓慢升到室温并再搅拌90分钟。将该溶液逐滴加入到6-(3,5-二氨基苯甲酰胺基)己酸2-(三甲基甲硅烷基)乙酯(370mg,1.012mmol)的饱和碳酸氢钠(10mL)溶液中。在室温条件下搅拌2小时。用二氯甲烷(50mL×3)萃取，合并有机层，干燥，过滤，浓缩，获得黄色油状化合物。
1H NMR(400MHz,Chloroform-d)δ8.45(s,2H),8.06(t,J＝2.1Hz,1H),7.78(s,1H),7.77(s,1H),6.53-6.56(m,1H),4.18–4.10(m,2H),3.40-3.46(m,2H),3.38(d,J＝2.8Hz,4H),2.49(t,J＝2.7Hz,2H),2.29(t,J＝7.4Hz,2H),1.59-1.69(m,4H),1.36-1.44(m,2H),1.00–0.93(m,2H),0.02(s,9H)。
步骤4：2-(三甲基甲硅烷基)乙基-6-(3,5-二(丁-2,3-二烯酰胺基)苯甲酰胺基)己酸酯的合成
将2-(三甲基甲硅烷基)乙基-6-(3,5-二(丁-3-炔甲氨基)苯甲氨基)己酸酯(1.15g,2.32mol)的K2CO3(18％水溶液，10mL)溶液与1,4-二氧六环(10mL)混合。将反应液加热到40℃并搅拌2小时，加入柠檬酸(60mL,10％水溶液)和乙酸乙酯(500mL×3)。合并有机相，干燥，过滤，浓缩得到(1.15g，粗品)油状物。
1H NMR(400MHz,Chloroform-d)δ8.06(t,J＝2.0Hz,1H),8.00(brs,2H),7.77(s,1H),7.76(s,1H),6.64(t,J＝5.8Hz,1H),5.76(t,J＝6.6Hz,2H),5.37(d,J＝6.6Hz,4H),4.18–4.13(m,2H),3.39-3.44(m,2H),2.28(t,J＝7.4Hz,2H),1.58-1.69(m,4H),1.43-1.35(m,2H),1.01–0.92(m,2H),0.02(s,9H)。
步骤5：6-(3,5-二(丁-2,3-二烯酰胺基)苯甲酰胺基)己酸的合成
将2-(三甲基甲硅烷基)乙基-6-(3,5-二(丁-2,3-二烯酰胺基)苯甲酰胺基)己酸酯(1.15g,2.32mmol)溶解于TFA/DCM(1:1,1mL)的溶液中并在室温条件下搅拌1小时。浓缩去除溶剂，通过C18柱(ACN/H2O)纯化，获得目标产物为白色固体(330mg，3步产率为35.9％)。
1H NMR(400MHz,DMSO-d6)δ9.70(s,1H),8.12(t,J＝1.9Hz,1H),7.72(d,J＝2.0Hz,2H),7.69(s,1H),7.66(t,J＝5.8Hz,1H),5.89(t,J＝6.5Hz,2H),5.19(d,J＝6.5Hz,4H),3.23-3.28(m,2H),2.19(d,J＝7.6Hz,2H),1.59-1.48(m,4H),1.36-1.29(m,2H)。
实施例4：化合物LK4的合成
步骤1：2-(三甲基甲硅烷基)乙基-6-(3,4-二硝基苯甲酰胺基)己酸酯的合成
在0℃条件下，向LK1-3(884mg,3.82mmol)和三乙胺(527mg,5.2mmol)的二氯甲烷(15mL)溶液中缓慢滴入3,4-二硝基苯甲酰氯(800mg,3.47mmol)，反应液在室温条件下搅拌2小时，通过LCMS监测反应完成。用1N HCl、饱和NaHCO3和盐水依次洗涤反应液。合并有机相，硫酸钠干燥，过滤，浓缩并通过硅胶快速柱层析纯化，获得目标产物(1.38g，产率93％)为黄色固体。LC-MS(ESI)m/z:448[M+Na]+。
1H NMR(400MHz,Chloroform-d)δ8.46(d,J＝1.8Hz,1H),8.26(dd,J＝8.3,1.8Hz,1H),7.96(d,J＝8.3Hz,1H),7.30(s,1H),4.20–4.12(m,2H),3.50(q,J＝6.6Hz,2H),2.32(t,J＝7.0Hz,2H),1.70-1.62(m,4H),1.47-1.40(m,2H),1.01–0.93(m,2H),0.03(s,9H)。
步骤2:6-(3,4-二氨基苯甲酰胺基)己酸-2-(三甲基甲硅烷基)乙酯的合成
将2-(三甲基甲硅烷基)乙基-6-(3,4-二硝基苯甲酰胺基)己酸酯(1.23g,2.9mmol)、铁粉(812mg,14.5mmol)和NH4Cl(1.55g,29mmol)的12mL EtOH/H2O(5/1,v/v)溶液加入密封管中，并加热至110℃反应1小时。在反应完成后，过滤，乙酸乙酯和水洗涤。合并有机层，用无水硫酸钠干燥，过滤，浓缩，通过硅胶快速柱层析纯化获得目标化合物(485mg，产率为45.9％)为黄色固体。
LC-MS(ESI)m/z:366[M+H]+。
步骤3:2-(三甲基甲硅烷基)乙基-6-(3，4-二(丁-3-炔酰胺基)苯甲酰胺基)己酸酯的合成
向丁-3-炔酸(644mg,7.66mmol)的二氯甲烷(30mL)悬浮液中加入草酰氯(972mg,7.66mmol)，在室温条件下搅拌15分钟后，将反应液冷却到0℃并加入DMF(0.15mL)。反应液逐渐升温至室温继续搅拌90分钟。将该溶液逐滴加入至6-(3,4-二氨基苯甲酰胺基)己酸2-(三甲基甲硅烷基)乙酯(280mg,0.766mmol)的饱和碳酸氢钠(10mL)的溶液中，在室温条件下搅拌2小时。用二氯甲烷(50mL×3)萃取，干燥，过滤，浓缩。通过硅胶快速柱层析纯化获得目标化合物(270mg，产率71％)为黄色固体。
1H NMR(400MHz,Chloroform-d)δ8.82(s,1H),8.74(s,1H),7.82(d,J＝2.0Hz,1H),7.68–7.57(m,2H),6.52-6.49(m,1H),4.20–4.12(m,2H),3.47–3.38(m,4H),2.49(m,2H),2.30(t,J＝7.3Hz,2H),1.47–1.27(m,6H),1.01–0.94(m,2H),0.04(s,9H)。
步骤4和步骤5：6-(3,4-二(丁-2,3-二烯酰胺基)苯甲酰胺基)己酸的合成
将2-(三甲基甲硅烷基)乙基-6-(3，4-二(丁-3-炔酰胺基)苯甲酰胺基)己酸酯(50mg,0.1mmol)溶解于TFA/DCM(1:1,2mL)中并在室温条件下搅拌1小时。浓缩去除溶剂，残留物溶解于1,4-二氧六环(10mL)中，并加入K2CO3(18％水溶液，10mL)，反应液加热到40℃并搅拌2小时，再加入柠檬酸(60mL,10％水溶液)和乙酸乙酯(500mL×3)。合并有机相，干燥，过滤，浓缩，通过C18柱纯化，得到目标产物(38mg，两步产率为95.2％)为黄色固体。
LC-MS(ESI)m/z:398[M+H]+。
1H NMR(400MHz,Chloroform-d)δ9.35–9.31(m,1H),9.20–9.16(m,1H),7.78(m,1H),7.64(m,1H),7.57(m,1H),5.69(s,2H),5.20(d,J＝6.6Hz,4H),3.99(d,J＝7.4Hz,2H),3.28(m,2H),2.19-3.32-3.25(m,2H),1.92-1.88(m,4H)。
实施例5：化合物LK5的合成
步骤1:合成3,5-二((叔丁基羰基)氨基)甲基)苯甲酸
将3,5-二(氨基甲基)苯甲酸二盐酸盐(450mg，1.78mmol)和氢氧化钠(216mg,5.4mmol)溶于水(1mL)。将溶解在1,4-二氧六环(16mL)中的(Boc)2O(1164mg，5.3mmol)加入反应液中搅拌过夜，然后加入乙酸乙酯(20mL×2)萃取。然后在水层中加入HCl调节pH为4-5，用乙酸乙酯(30mL×2)萃取，无水硫酸钠干燥，浓缩得到3,5-二((叔丁基羰基)氨基)甲基)苯甲酸(410mg，产率：60.5％)为白色固体。
表征数据：
LC-MS(ESI)m/z:403.2[M+Na]+。
1H NMR(400MHz,DMSO-d6)δ12.85(s,1H),7.70(s,2H),7.45-7.42(m,2H),7.33(s,1H),4.16(s,4H),1.39(s,18H)。
步骤2:合成2-(三甲基硅基)乙基-6-(3,5-二((叔丁基羰基)氨基)甲基)苄胺)己酸酯
将3,5-二((叔丁基羰基)氨基)甲基苯甲酸(200mg，0.53mmol)、2-(三甲基硅基)乙基-6-氨基己酸酯(120mg，0.52mmol)和HATU(300mg，0.79mmol)的混合物在室温下搅拌10min，加入DIEA(280μL，1.62 mmol)。将所得混合物搅拌4小时。乙酸乙酯萃取两次(30mL×2)，用水洗乙酸乙酯层4次。无水硫酸钠干燥后，浓缩，得到粗产物2-(三甲基硅基)乙基-6-(3,5-二((叔丁基羰基)氨基)甲基)苄胺)己酸酯290mg，为白色固体。
表征数据：
LC-MS(ESI)m/z:394.2[M+H-200]+。
1H NMR(400MHz,DMSO-d6)δ8.38(m,1H),7.53(s,2H),7.41-7.38(m,2H),7.22(s,1H),4.113-4.05(m,6H),3.24-3.19(m,2H),2.25(t,J＝8,2H),1.53-1.48(m,4H),1.37(s,18H),1.28-1.22(m,2H),0.93-0.89(m,2H),0.0(s,9H)。
步骤3:2-(三甲基硅基)乙基-6-(3,5-二(氨基甲基)苄胺)己酸酯
向粗品2-(三甲基硅基)乙基-6-(3,5-二((叔丁基羰基)氨基)甲基)苄基)己酸酯(290mg)中加入氯化氢的乙酸乙酯溶液(3mL，2M)，在室温下反应30min。反应液直接用制备液相分离纯得化2-(三甲基硅基)乙基6-(3,5-二(氨基甲基)苄胺)己酸酯80mg，为油状物。
表征数据：
LC-MS(ESI)m/z:394.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.38(s,1H),7.53(s,2H),7.41-7.38(m,2H),7.22(s,1H),4.13-4.05(m,6H),2.25(t,J＝8Hz,2H),1.53-1.48(m,4H),1.37(s,18H),1.28-1.22(m,2H),0.93-0.89(m,2H),0.0(s,9H)。
步骤4:2-(三甲基硅基)乙基-6-(3,5-二(3-丁炔酰胺基甲基)苄胺)己酸酯
将3-丁炔酸(42mg，0.51mmol)溶于二氯甲烷(2mL)中，在0℃条件下加入草酰氯(85μL，1.02mmol)。反应液在室温条件下搅拌1小时后。将反应液加入到2-(三甲基硅基)乙基-6-(3,5-二(氨基甲基)苄胺)己酸酯(20mg,0.051mmol)的饱和碳酸氢钠水溶液(5mL)中搅拌2小时。在溶液中加入二氯甲烷(20mL×2)萃取，二氯甲烷层用无水硫酸钠干燥，浓缩得到粗产品2-(三甲基硅基)乙基-6-(3,5-二(3-丁炔酰胺基甲基)苄胺)己酸酯28mg,为灰色固体。
表征数据：
LC-MS(ESI)m/z:498.2[M+3H-2CH3]+。
1H NMR(400MHz,DMSO-d6)δ8.47-8.41(m,3H),7.58(s,2H),7.26(s,1H),4.28(d,J＝4Hz,4H),4.10-4.05(m,2H),3.40-3.29(m,6H),3.00(d,J＝4Hz,2H),2.25(t,J＝8Hz,18H),1.53-1.36(m,4H),1.32-1.28(m,4H),0.98-0.84(m,2H)。
步骤5:6-(3,5-二(3-丁炔酰胺基甲基)苄胺)己酸
将2-(三甲基硅基)乙基-6-(3,5-二(3-丁炔酰胺基甲基)苄胺)己酸酯(28mg)化合物粗品溶解在DCM/TFA(1mL/1mL)中反应1小时。将反应液浓缩，得到目标化合物6-(3,5-二(3-丁炔酰胺基甲基)苄胺)己酸20mg，为油状化合物。
表征数据：
LC-MS(ESI)m/z:426.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.51-8.43(m,3H),7.60(s,2H),7.28(s,1H),4.30(d,J＝4Hz,2H),3.24-3.18(m,6H),3.02(d,J＝4Hz,2H),2.23(t,J＝8Hz,1H),1.54-1.49(m,4H),1.32-1.26(m,6H)。
步骤6:6-(3,5-二(丁-2,3-二烯酰)卞胺)己酸
将化合物6-(3,5-二(3-丁炔酰胺基甲基)苄胺)己酸溶于1,4-二氧六烷(0.5mL)和碳酸钾溶液(1mL)中，在40℃下搅拌1小时。用乙酸乙酯(10mL×2)萃取，水层pH调节至4-5，混合后用乙酸乙酯(10mL×2)萃取，浓缩后得到目标产物6-(3,5-二(丁-2,3-二烯酰)卞胺)己酸10mg，为白色固体。
表征数据：
LC-MS(ESI)m/z:426.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.46(m,3H),7.61(s,2H),7.27(s,1H),5.81-5,78(m,2H),5.33(d,J＝4Hz,4H),4.33(d,J＝4Hz,2H),2.22-2.19(m,2H),1.52-1.50(m,4H),1.30-1.24(m,6H)。
连接子-药物缀合物的合成与表征
实施例6：化合物LP1的合成
步骤1：合成LP1-2:
在0℃下，向(9H-芴-9-基)甲基((S)-3-甲基-1-(((S)-1-((4-硝基苯氧基)羰基)氧基)甲基)苯基)氨基)-1-氧代-5-脲基戊烷-2-基)氨基)1-氧代丁-2-基)氨基甲酸酯(587mg，0.770mmol)在DMF(10mL)中的溶液中加入(S)-N-((3R，4S，4S，5S)-1-(S)-2-((1R，2R)-3-(1S，2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)-N，3-二甲基-2-((S)-3-甲基-2-(甲氨基)丁酰胺)丁酰胺(500mg，0.690mmol)、HOBt(211mg，1.53mmol)和DIEA(198mg，1.53mmol)。添加后，将混合物在25℃下搅拌12小时。LCMS显示反应完成。将混合物浓缩，得到残留物，经C18(水溶液中0～60％ ACN)纯化，得到LP1-2(600mg，产率64％)，为白色固体。
表征数据：
LC-MS(ESI)m/z:1346.8[M+H]+。
步骤2：合成LP1-3:
在0℃下，向4-((S)-2-((S)-2-((((9H-芴-9-基)甲氧基)羰基)氨基)-3-甲基丁酰胺)-5-脲戊酰胺基)苄基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙基-2-氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代-4-基(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)氨基甲酸酯(600mg，0.45mmol)的DMF(5mL)溶液中加入二乙胺(66.0mg，0.90mmol)。加完后，将混合物在0℃下搅拌2小时。LCMS显示反应完全。将混合物浓缩得到残余物，用C18(0-45％乙腈在H2O中)纯化得到LP1-3(300mg，产率59％)为白色固体。
表征数据：
LC-MS(ESI)m/z:1123.8[M+H]+。
步骤3：合成LP1:
向NH2-VC-MMAE(20mg，0.0178mmol)和LK1(10mg，0.035mmol)的1mL DMF溶液中加入HATU(20.3mg，0.0534mmol)和DIPEA(12mg，0.009mmol)。加完后，反应液在室温下搅拌过夜。用制备高效液相色谱法纯化，得到化合物LP1(5mg，产率19.2％)为白色固体。
LC-MS(ESI)m/z:734[M/2+H]+。
1H NMR(400MHz,DMSO-d6)δ9.99(s,1H),8.11(d,J＝7.3Hz,1H),7.94–7.78(m,4H),7.57(d,J＝8.1Hz,2H),7.28(dd,J＝15.7,6.7Hz,6H),7.17(d,J＝7.1Hz,1H),6.02(s,1H),5.74(t,J＝6.6Hz,2H),5.42(s,2H),5.27(d,J＝6.5Hz,4H),5.01(d,J＝22.4Hz,2H),4.48(s,1H),4.45–4.32(m,1H),4.29–4.14(m,1H),3.99(s,2H),3.76(d,J＝8.4Hz,1H),3.20(dd,J＝18.4,8.8Hz,8H),3.10(s,1H),3.06–2.92(m,4H),2.85(d,J＝15.0Hz,1H),2.14(dd,J＝17.8,7.8Hz,2H),1.55–1.32(m,6H),1.00(ddd,J＝11.8,9.8,6.6Hz,5H),0.91–0.70(m,23H)。
实施例7：化合物LP2的合成
合成LP2:
将LP1-3(20mg,0.0178mmol)和LK2(10mg,0.035mmol)溶解于DMF(1mL)中,再加入HATU(20.3mg,0.0534mmol)和DIPEA(12mg,0.009mmol)。添加完毕后，反应液在室温下搅拌过夜。通过制备高效液相色谱法纯化，得到LP2(3.8mg,产率14.9％)为白色固体。
LC-MS(ESI)m/z:727[M/2+H]+
实施例8：化合物LP3的合成
合成LP3:
将LK3(14.2mg,0.036mmol)、HOBt(8.4mg,0.063mmol)和EDCI(7.4mg,0.072mmol)溶解于DMF(2mL)溶液中，在0℃搅拌15分钟。向溶液中加入LP1-3(30mg,0.018mmol)，在0℃条件下搅拌5小时。反应完成后，混合物通过制备高效液相色谱法纯化，得到LP3(4.98mg,产率12.4％)为白色固体。LC-MS(ESI)m/z:752[M/2+H]+。
1H NMR(400MHz,DMSO-d6)δ10.13(s,2H),9.96(s,1H),8.36(s,1H),8.10(d,J＝2.0Hz,1H),8.07(d,J＝7.5Hz,1H),7.79(d,J＝8.6Hz,1H),7.69(d,J＝2.0Hz,2H),7.58(d,J＝8.7Hz,2H),7.36–7.22(m,7H),7.17(t,J＝7.4Hz,1H),5.98(t,J＝6.5Hz,3H),5.43(d,J＝6.5Hz,4H),5.39(s,3H),5.33(d,J＝4.9Hz,1H),5.04(t,J＝18.0Hz,2H),4.49(s,1H),4.40(dd,J＝18.0,6.7Hz,2H),4.27(d,J＝11.0Hz,1H),4.20(dd,J＝14.7,7.0Hz,1H),4.06–3.94(m,2H),3.21(dd,J＝19.2,7.8Hz,9H),3.11(s,1H),2.98(td,J＝15.1,14.3,7.7Hz,5H),2.86(d,J＝14.2Hz,3H),2.28(t,J＝7.5Hz,1H),2.16(ddd,J＝21.8,14.1,7.3Hz,2H),2.02–1.91(m,1H),1.51(q,J＝7.0Hz,5H),1.32(d,J＝16.2Hz,2H),1.07–0.97(m,6H),0.91–0.72(m,27H)。
实施例9：化合物LP4的合成
合成LP4:
将LK4(3.54mg,0.009mmol)、HOBt(2.1mg,0.016mmol)和EDCI(1.85mg,0.002mmol)溶解于DMF(0.5mL)中,在0℃条件下搅拌15分钟。向溶液中加入LP1-3(5mg,0.0045mmol)，在0℃下反应5小时。反应完成后，混合物通过制备高效液相色谱法纯化，得到化合物LP4(0.71mg,产率10.6％)为白色固体。
LC-MS(ESI)m/z:752[M/2+H]+
实施例10：化合物LP5的合成
合成LP5:
将6-(3,5-双(丁-2,3-二胺甲基)苄胺)己酸(8mg，0.019mmol)、HOBt(6mg，0.044mmol)和EDCI(10mg，0.052mmol)溶解在DMF(0.5mL)中，反应液在室温下搅拌10min。NH2-VC-MMAE(15mg,0.013mmol)加入反应液中，继续搅拌4小时，LCMS显示目标产物，经制备高效液相色谱纯化得到目标产物LP5(2.0mg，10.0％)为白色固体。
表征数据：
LC-MS(ESI)m/z:1530.98[M+H]+。
1H NMR(400MHz,DMSO-d6)δ9.90(s,1H),8.45-8.42(m,3H),7.62-7.58(m,5H),7.32-7.22(m,10H),7.10(s,1H),6.97(s,1H),5.99-5.95(m,1H),5.83-5.79(m,2H),5.41(s,2H),5.33-5.32(m,4H),4.34(d,J＝8Hz,5H),3.38-2.85(m,42H),2.34(s,4H),2.08(s,2H),2.06-2.00(m,4H),1.55-1.52(m,7H),1.30-1.24(m,12H),1.06-0.98(m,13H),0.85-0.75(m,32H)。
实施例11：化合物LP10的合成
步骤1:合成LP10-2:
向LP10-1(88mg,0.0828mmol)的DMF(1.5mL)溶液中加入三乙胺(0.5mL)。加完后，反应液室温搅拌过夜。LCMS显示反应完成。将混合物浓缩，得到残留物，经C18(水溶液中0～60％ ACN)纯化，得到LP10-2(45mg，65％产率)，为白色固体。
表征数据：
LC-MS(APCI)m/z:841.20[M+H]+。
1H NMR(400MHz,MeOD-d4)δ7.99(s,3H),7.56-7.46(m,2H),7.33-7.09(m,4H),5.73(dd,J＝8.4,4.6Hz,1H),5.55(d,J＝16.3Hz,1H),5.36-5.19(m,1H),4.65(d,J＝10.6Hz,1H),4.44(dd,J＝8.5,6.2Hz,1H),4.30-4.11(m,1H),4.08-3.99(m,1H),3.96-3.81(m,3H),3.67(d,J＝1.0Hz,1H),3.54(s,1H),3.28-3.16(m,9H),2.37(d,J＝1.8Hz,2H),0.97(t,J＝7.4Hz,2H)ppm。
步骤2:合成LP10:
向LP10-2(45mg,53.6mmol)和LK1(9.9mg,27.2mmol)的2mL DMF溶液中加入HOBt(6.4mg,47.7mmol)和EDCI(10.4mg,54.5mmol)。加完后，反应液室温搅拌过夜。LCMS显示反应完成。将混合物浓缩，得到残留物，经C18(水溶液中0～60％ ACN)纯化，得到LP10(20mg，32％产率)，为白色固体。
表征数据：
LC-MS(ESI)m/z:1186.17[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.63(t,J＝6.6Hz,1H),8.51(d,J＝8.8Hz,1H),8.37-8.26(m,1H),8.12(d,J＝8.0Hz,1H),8.06-8.02(m,1H),8.00(t,J＝5.7Hz,1H),7.87-7.79(m,2H),7.29-7.13(m,5H),5.75(t,J＝6.6Hz,2H),5.33-5.14(m,4H),4.64(d,J＝6.6Hz,4H),4.54 -4.39(m,2H),3.83-3.57(m,6H),3.28-3.09(m,5H),3.07-2.94(m,4H),2.39(d,J＝1.9Hz,3H),2.22-2.14(m,2H),2.14-2.05(m,2H),1.92-1.79(m,2H),1.53-1.43(m,2H),1.41-1.31(m,2H),1.28-1.19(m,2H),0.87(t,J＝7.3Hz,3H)。
抗体药物偶联物的制备与表征
以下是抗体药物偶联物的制备和分析表征方法的示例。应当注意，以下示例仅是部分实施例，不限于当前所描述的实施例。例如，所述抗体不限于当前所展示的抗体，也可以是其他的任何抗体。
制备双联烯共价桥接的抗体药物偶联物的通用偶联过程
将抗体溶液浓度调节至5-10mg/mL，pH调整到7.5左右，取干净离心管作为反应容器，将单克隆抗体(1.0eq.)置于管中，加入TCEP(5eq.至8eq.)混匀后，插入恒温混匀仪中，在20-25℃下孵育2-16小时使抗体链间二硫键被完全还原。加入等体积500mM Tris,pH 8.8的缓冲液将pH提高到8.0，或者通过脱盐柱将抗体置换到25mM Na2B4O7,pH 8.0缓冲液中，再加入浓度为10mg/mL DMSO溶解的毒素连接子溶液(5eq.至8eq.)，按照最终反应液总体积的10-20％补充DMSO，震荡混匀后置于加热制冷型恒温混匀仪上反应，25℃偶联16-24小时。
使用300mg/mL的葡聚糖包被活性炭(厂家：Sigma)去除反应液内未与抗体偶联的残留毒素连接子，在反应液内加入体积10％的活性炭溶液，混匀后在室温下孵育1小时，离心取上清，检测其中残留的毒素连接子含量，如游离毒素连接子含量大于1％，应重复上述活性炭处理过程多次，直至检测结果达到标准(通常处理3次即可满足要求)。处理完成后，将所需抗体药物偶联物的缓冲液通过3-6次超滤替换为合适的存储缓冲液，并保存于-80℃。
抗体药物偶联物的通用表征方法
(a)HIC-HPLC分析ADC的DAR值
高效液相色谱仪：e2695高效液相色谱系统或1260安捷伦液相色谱仪。
色谱柱：MabPacTMHIC-Butyl 5μm 4.6×100mm(厂家：Thermo)。
流动相：流动相A(MPA)：1.5M(NH4)2SO4+50mM磷酸钾盐(pH7.0)；流动相B(MPB)：50mM磷酸钠盐(pH 7.0)/异丙醇(75:25V/V)；按照以下洗脱程序(5％-95％)进行洗脱，其中，0-2min流动相A体积为100％-95％，流动相B体积为0％-5％；2-22min流动相A体积为95％-5％，流动相B体积为5％-95％；22-24min流动相A体积为5％-0％，流动相B体积为95％-100％；24-26min流动相A体积为0％-100％，流动相B体积为100％-0％；26-30min流动相A体积为100％-100％，流动相B体积为0％-0％。
检测条件：设置流动相流速为1ml/min，检测波长为280nm，柱温30℃。
实验步骤：取偶联后样品50μg(体积依据样品浓度而定)，注入高效液相色谱仪，利用上述洗脱程序洗脱，并记录色谱图。
DAR值计算公式：DAR＝Σ(relative peak area×number of loaded drugs)/100
(b)C18-HPLC分析Free Linker Payload(mol/mol％)值
高效液相色谱仪：e2695高效液相色谱系统。
色谱柱：C18 3.5μm 4.6×150mm(厂家：Waters)。
流动相：流动相A(MPA)：0.1％ TFA-H2O；流动相B(MPB)：0.1％ TFA-ACN；按照以下洗脱程序(5％-95％)进行洗脱，其中，0-30min流动相A体积为90％-20％，流动相B体积为10％-80％；30-31min流动相A体积为20％-90％，流动相B体积为80％-10％；31-35min流动相A体积为90％-90％，流动相B体积为10％-10％
检测条件：设置流动相流速为0.5ml/min，检测波长为254nm，柱温30℃。
试剂I配制示例：量取30mL无水甲醇和50mL乙腈，称取10g氯化钠，混合于容器中，室温下搅拌1小时以上，然后静置1小时。取上清液，用0.22μm有机膜过滤，常温保存，有效期3个月。
试剂II配制示例：分别量取100mL试剂I、15mL DMSO、85mL ADC样品保存Buffer，混合均匀于一容器中，常温保存，有效期2个月。
实验步骤：
样品溶液制备：
1)游离毒素参照品：取供试品对应的毒素作为参照品，用上述试剂Ⅱ配制成终浓度为1.0mg/ml。
2)取上述浓度为1.0mg/ml毒素参照品10μl，加入到90μl试剂Ⅱ中，配置成浓度为100μg/ml；然后按照一下表格用试剂Ⅱ依次稀释样品，配制所需浓度的样品。
注：将上述配制所需浓度的样品浓度从小到大的顺序进样HPLC。
样品测试：取偶联后样品85μg，用3μl DMSO混合样品5min，然后取试剂Ⅰ 60μl加入到样品体系中，混匀5-10min，2000rpm离心2min，取上清液20μL，注入高效液相色谱仪，利用上述洗脱程序洗脱，并记录色谱图。
根据计算所得标准二元一次回归曲线方程，计算出ADC中游离毒素的含量，
Free Drug(mol/mol％)＝残留小分子摩尔浓度/抗体摩尔浓度×100。
(c)LC-MS/MS方法测定游离linker-payload含量
内参化合物：50ng/ml甲苯磺丁脲、拉贝洛尔、依托非沙胺和丁螺环酮
萃取剂：乙酸乙酯
标准品梯度稀释：用50％的甲醇水溶液稀释标准品到20000，16000，10000，4000，1000，200，40，20ng/ml。
样品上机前处理：取20μL样品，然后加入20μL空白缓冲液和240μL萃取剂。涡旋混合后，静置至分层，然后取160μL上清液放入96孔板中吹干，加入200μL 50％乙腈水，混合均匀，最后进入LC-MS/MS分析。
标准品上机前处理：取2μL工作液加入38μL空白缓冲液中，再加入240μL萃取剂。涡旋混匀后静置至分层，取160μL上清液放入96孔板中吹干，加入200μL 50％乙腈水，混匀。
质谱条件：

数据处理：根据标准品的浓度和相应的质谱响应值做出标准曲线，通过线性拟合曲线(公式)以及检测样品的质谱相应值，计算得到游离linker-payload的残留量。
(d)SEC-HPLC分析ADC的聚集体
高效液相色谱仪：e2695高效液相色谱系统或1260安捷伦液相色谱仪。
色谱柱：Waters Xbridge BEH200 SEC(7.8×300mm,3.5μm)
流动相：50mM PB+200mM Arg(pH 6.80)+10％ IPA，按照以下洗脱程序进行洗脱，0-30min流动相A体积为100％-100％；
检测条件：设置流动相流速为0.5ml/min，检测波长为280nm，柱温26℃。
实验步骤：取偶联后样品20μg(体积依据样品浓度而定)，注入高效液相色谱仪，利用上述洗脱程序洗脱，并记录色谱图。
计算公式：单体纯度(％)＝A单体/A总×100％；聚体纯度(％)＝A聚体/A总×100％
(e)还原性SDS-PAGE分析ADC的连接头的偶联方式及ADC的成分
电泳仪：天能EPS-600。
丙烯酰胺凝胶：金斯瑞M00656
成像系统分析仪：天能4600SF
实验步骤：将10微克的样品与相应的上样缓冲液混合，沸水浴上加热5分钟；按顺序将样品和标准蛋白(10微升/孔)加入4～20梯度胶梳孔中，在恒压120伏条件下电泳60分钟；将凝胶取出，用去离子水润洗一遍，然后加入适量考马斯亮蓝G250染色液没过凝胶，并在摇床上染色0.5小时；染色后的胶用去离子水冲洗三次,每次在水平摇床上震荡30分钟；脱色后的胶取出后转移到成像仪上记录凝胶图像。
结果分析：≥50kDa条带的存在说明巯基桥接偶联是有效的(H，L)。其中75kDa的条带代表由一对桥接巯基连接的重链和轻链(LH)组成的部分。由一对或两对桥接巯基连接的两条重链，形成的条带是100kDa(HH)。125kDa的条带代表只有一条重链和轻链之间没有形成桥接(LHH)。最后，150kDa的条带代表所有链都桥接抗体(LHHL)。
(f)采用LC/TOF(安捷伦，6230LC/TOF)液质联用系统分析桥接产物
液相色谱柱为：PLRP-S,1000A色谱柱(Agilent 8μm、2.1×150mm)
流动相A为0.1％甲酸
流动相B为0.1％甲酸乙腈。流速为0.4mL/min。
质谱分析正离子，设置参数为:扫描范围500-5000m/z，
气体温度：320℃
VCap：4500V
碎片发生器(fragmentor)：200V
取样器(Skimmer)：65V。
实施例12：抗体药物偶联物ADC-1的制备
n16＝4.05
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，往曲妥珠单抗(1.0mg,38.9mg/mL,1.0eq.)的20mM PBS,1mM EDTA,pH 7.4的缓冲液中加入TCEP(10mmol/L,6eq.)水溶液，在22℃下还原2小时；然后通过脱盐柱去除TCEP并置换缓冲液到25mM Na2B4O7,pH 8.0的缓冲液，再加入LP1(10mg/mL,6eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-1(CADC(mg/mL):6.06,V(mL):0.08，收率48.5％)。
HIC-DAR:4.05,SEC纯度:99.2％，Free Linker Payload(mol/mol％):not detected。
DAR 3:Rt＝14.85min(比例：3.28％),DAR 4:Rt＝16.65min(比例：88.58％),DAR 5:Rt＝18.20min(比例：8.15％)。
从图1和图2可以看出来，得到的ADC-1中单一分布的组份(DAR4)占比为88.58％，聚集体小于1％，此外图3和图4显示轻链-重链之间桥接和重链之内桥接偶联形成的“半抗体”(DAR4)的组分是占主要的。
实施例13：抗体药物偶联物ADC-2的制备
n16＝3.86
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，向曲妥珠单抗(1.0mg,38.9mg/mL,1.0eq.)的25mM Na2B4O7,25mM NaCl,1mM DTPA,pH 7.4的缓冲液中加入TCEP(2mg/mL,6eq.)水溶液，在22℃下还原2小时；然后加入等体积500mM Tris,pH 8.8的缓冲液，再加入LP2(10mg/mL,7eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-2(CADC(mg/mL):5.2,V(mL):0.8，收率41.6％)。
HIC-DAR:3.86,SEC纯度:93.68％，Free Linker Payload(mol/mol％):not detected。
DAR 3:Rt＝15.84min(比例：29.79％),DAR 4:Rt＝16.99min(比例：54.92％),DAR 5:Rt＝18.90min(比例：15.29％)。
从图5和图6可以看出来，得到的ADC-2中单一分布的组份(DAR4)占比为54.92％，相较于ADC-1，有所减少，聚集体大于6％。
实施例14：抗体药物偶联物ADC-3的制备
n16＝4.1
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，往曲妥珠单抗(1.0mg,20mg/mL,1.0eq.)的20mM PBS,1mM EDTA,pH 7.4的缓冲液中加入TCEP(10mmol/L,6eq.)水溶液，在22℃下还原2小时；然后通过脱盐柱去除TCEP并置换缓冲液到25mM Na2B4O7,pH 8.0的缓冲液，再加入LP3(10mg/mL,5eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-3(CADC(mg/mL):9.32,V(mL):0.07，收率65.24％)。
HIC-DAR:4.1,SEC纯度:98.3％，Free Linker Payload(mol/mol％):not detected。
DAR 2:Rt＝14.02min(比例：3.52％),DAR 3:Rt＝14.58min(比例：9.03％),DAR 4:Rt＝16.33min(比例：68.5％),DAR 5:Rt＝18.13min(比例：18.93％)。
从图7和图8可以看出来，得到的ADC-3中单一分布的组份(DAR4)占比为68.5％，相较于ADC-1，有所减少，聚集体1.7％。此外图9显示轻链-重链之间桥接和重链之内桥接偶联形成的“半抗体”(DAR4)的组分是占主要的。
实施例15：抗体药物偶联物ADC-5的制备
n16＝3.89
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，往曲妥珠单抗(1.0mg,38.9mg/mL,1.0eq.)的25mM Na2B4O7,25mM NaCl,1mM DTPA,pH 7.4的缓冲液中加入TCEP(2mg/mL,6eq.)水溶液，在22℃下还原2小时；然后加入等体积500mM Tris,pH 8.8的缓冲液，再加入LP5(10mg/mL,6eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-5(CADC(mg/mL):8.2,V(mL):0.5，收率41.0％)。
HIC-DAR:3.89
DAR 1：Rt＝11.05min(比例0.55％)；DAR 2:Rt＝12.80min(比例2.53％)；DAR 3：Rt＝15.36min(比例11.87％)；；DAR 4：Rt＝16.978mins(比例50.24％)；DAR 5:Rt＝16.978min(比例29.28％)。
从图10到图12可以看出来，得到的ADC-5中单一分布的组份(DAR4)占比为50.24％，相较于ADC-1，有所减少，聚集体为1.1％，另外轻链-重链之间桥接和重链之内桥接偶联形成的“半抗体”(DAR4)组分占比相较于ADC-1也有所降低。
实施例16：抗体药物偶联物ADC-10的制备
n16＝3.98
根据制备抗体药物偶联物的一般偶联过程，往曲妥珠单抗(0.9mg,10mg/mL,1.0eq.)的50mM PBS pH 7.4的缓冲液中加入TCEP(2mg/mL,8eq.)水溶液，在22℃下还原18小时；然后将抗体置换到pH 8的硼酸钠缓冲液中，加入LP10(10mg/mL,7.0eq.)的DMSO溶液，在22℃下偶联反应18小时。经活性炭处理3次纯化后，将所需抗体药物偶联物的缓冲液通过5次超滤得到ADC-10(CADC(mg/mL):5.528,V(mL):0.05，收率30.7％)。
HIC-DAR:3.98,SEC纯度:100％，Free Linker Payload(mol/mol％):not detected。
DAR 3:Rt＝12.01min(比例：1.64％),DAR 4:Rt＝13.48min(比例：98.36％),DAR 5:无。
从图13和图14可以看出来，得到的ADC-10中单一分布的组份(DAR4)占比为98.36％，相较于ADC-1，有所增加，未发现聚集体，此外图15显示轻链-重链之间桥接和重链之内桥接偶联形成的“半抗体”(DAR4)的组分是占主要的。
实施例17：抗体药物偶联物ADC-25的制备
n16＝3.00
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，往含C226S突变的曲妥珠单抗(1.0mg,20mg/mL,1.0eq.)的20mM PBS,1mM EDTA,pH 7.4的缓冲液中加入TCEP(10mmol/L,6eq.)水溶液，在22℃下还原2小时；然后通过脱盐柱去除TCEP并置换缓冲液到25mM Na2B4O7,pH 8.0的缓冲液，再加入LP3(10mg/mL,3eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-25(CADC(mg/mL):8.5,V(mL):0.07，收率59.5％)。本发明所述的C226S突变的曲妥珠单抗是指将曲妥珠单抗的重链Eu编号第226位的半胱氨酸突变为丝氨酸。
HIC-DAR:3.00,SEC纯度:98.1％，Free Linker Payload(mol/mol％):not detected。
DAR 2:Rt＝12.59min(比例：12.29％),DAR 3:Rt＝14.25min(比例：74.33％),DAR 4:Rt＝16.45min(比例：13.38％)。
从图16和图17可以看出来，得到的ADC-25中单一分布的组份(DAR3)占比为74.33％，聚集体1.9％。
实施例18：抗体药物偶联物ADC-26的制备
n16＝2.94
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，往含C226S突变的曲妥珠单抗(1.0mg,20mg/mL,1.0eq.)的20mM PBS,1mM EDTA,pH 7.4的缓冲液中加入TCEP(10mmol/L,6eq.)水溶液，在22℃下还原2小时；然后通过脱盐柱去除TCEP并置换缓冲液到25mM Na2B4O7,pH 8.0的缓冲液，再加入LP1(10mg/mL,6eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-26(CADC(mg/mL):8.50,V(mL):0.07，收率59.5％)。
HIC-DAR:2.94,SEC纯度:96.3％，Free Linker Payload(mol/mol％):not detected。
DAR 1:Rt＝10.88min(比例：1.95％),DAR 2:Rt＝12.53min(比例：12.32％),DAR 3:Rt＝14.18min(比例：75.13％)，DAR 4:Rt＝16.58min(比例：10.61％)。
从图19和图20可以看出来，得到的ADC-26中单一分布的组份(DAR3)占比为75.13％，聚集体3.7％。
实施例19：抗体药物偶联物ADC-27的制备
n16＝4.00
根据制备双联烯共价桥接的抗体药物偶联物的一般偶联过程，往伊非那他单抗(Ifinatamab)(1.0mg,15mg/mL,1.0eq.)的20mM PBS,1mM EDTA,pH 7.4的缓冲液中加入TCEP(10mmol/L,6eq.)水溶液，在22℃下还原2小时；然后通过脱盐柱去除TCEP并置换缓冲液到25mM Na2B4O7,pH 8.0的缓冲液，再加入LP10(10mg/mL,6eq.)的DMSO溶液，在25℃下偶联反应16小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到ADC-27(CADC(mg/mL):10.4,V(mL):0.07，收率72.8％)。
HIC-DAR:4,SEC纯度:98.8％，Free Linker Payload(mol/mol％):not detected。
DAR 4:Rt＝12.90min(比例：100％)。
从图22和图23可以看出来，得到的ADC-27中单一分布的组份(DAR4)占比为100％，聚集体1.2％，此外图24和图25显示轻链-重链之间桥接和重链之内桥接偶联形成的“半抗体”(DAR4)的组分是占主要的。
实施例20：抗体药物偶联物对比例-1的制备
n16＝7.96
根据制备抗体药物偶联物的一般偶联过程，往曲妥珠单抗(5mg,21.07mg/mL,1.0eq.)的50mM PBS pH 7.0的缓冲液中加入TCEP(10mg/mL,12eq.)水溶液，在22℃下还原2小时；然后加入Deruxtecan(10mg/mL,10eq.)的DMSO溶液，在22℃下偶联反应1小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到对比例-1(CADC(mg/mL):6.74,V(mL):0.6，收率80.9％)。
通过抗体药物偶联物的通用表征方法，得到了以下表征结果：
HIC-DAR:7.96,SEC纯度:97.7％，Free Linker Payload(mol/mol％):0.04221。
实施例21：抗体药物偶联物对比例-2的制备
n16＝4.5
根据制备抗体药物偶联物的一般偶联过程，往伊非那他单抗(Ifinatamab)(20mg,21.07mg/mL,1.0eq.)的50mM PBS pH 7.0的缓冲液中加入TCEP(10mM,12eq.)水溶液，在22℃下还原2小时；然后加入Deruxtecan(10mg/mL,10eq.)的DMSO溶液，在22℃下偶联反应1小时。经葡聚糖包被活性炭处理2次纯化后，将所需抗体药物偶联物的缓冲液通过3次超滤得到对比例-2(CADC(mg/mL):7.47,V(mL):2.2，收率82.2％)。
通过抗体药物偶联物的通用表征方法，得到了以下表征结果：
HIC-DAR:4.5,SEC纯度:99.92％，Free Linker Payload(mol/mol％):0.21077。
实施例22：基于不同连接头的ADC体外血浆稳定性实验
本实施例评估了基于双联烯连接头和基于马来酰亚胺连接头的抗体-药物偶联物的血浆稳定性，监测指标为DAR值随时间的变化。
将受试的抗体-药物偶联物按最终浓度为0.3mg/ml与人血浆混合，置于37℃恒温箱中孵育，在设定的各时间点(0,1,2,4,7,14天)分别取样50μL，取样后冷冻于-80℃，待取样结束，统一采用Elisa测定抗体-药物偶联物和总抗浓度。方法如下，首先在Elisa板孔内过夜包被抗原B7-H3(义翘神州，11188-H08H)和anti-Dxd抗体(金斯瑞，A02217)，分别用于测定总抗和ADC的浓度。第二天，用250μL PBS/T洗涤孔板3次之后，加入150μL 4％脱脂奶粉，在37℃下孵育2小时。之后将待测样品稀释液或者标准品加入Elisa孔内继续孵育2小时。再用250μL PBS/T洗涤3次，之后加入100μL Anti-human IgG二抗(金斯瑞，A01855)稀释液(1:3000),在37℃下孵育1小时。再次用250μL PBS/T洗涤3次，每孔加入50μL TMB显色液，在37℃下孵育5分钟显色，用100μL终止液终止显色反应。采集OD450nm光吸收信号，做出标准曲线，根据标准曲线计算出样品中抗体-药物偶联物和总抗浓度，进而计算得到DAR值变化曲线如图26所示。
由图26可知，基于双联烯连接头的ADC-27在血浆中非常稳定，DAR值基本保持不变，而基于马来酰亚胺连接头的抗体-药物偶联物(对比例-2)在与血浆孵育过程中DAR值持续下降，2周之后，DAR值降低了一半。因此双联烯连接头更稳定，具有进一步提高安全性和药效的应用潜力。
生物学评价
实施例23：基于双联烯共价桥接的抗体-药物偶联物的肿瘤细胞增殖抑制实验
将1500个NCI-N87细胞接种在96孔板中(排除PBS填充的边缘孔)，细胞培养在80μL合适的培养基中，并置于37℃、5％CO2的恒温细胞培养箱中。细胞在恒温培养箱(37℃,5％ CO2)中过夜培养后，每孔加入20μL稀释好的双联烯的抗体-药物偶联物溶液，使偶联物终浓度范围为0.2pM-12.5nM或3.0pM-200nM。细胞与偶联物共孵育5天后，将96孔板置于室温下平衡30min。每孔加入40μL CellTiter-(Promega,G7572)试剂，避光反应10min后，使用SpectraMax i3X酶标仪测定荧光值。并使用Graphpad Prism软件拟合发光值对偶联物浓度(nM)的曲线。
抗体-药物偶联物的体外细胞毒性(++++：<1nM；+++：1-10nM；++：10-200nM；+：>200nM)
大部分的ADC都表现出一定的抗肿瘤活性，例如在NCI-N87细胞中，ADC-1，ADC-2，ADC-3，ADC-5，ADC-10，ADC-25，ADC-26和ADC-27的体外细胞杀伤活性与对比例-1的体外细胞杀伤活性相当。
本发明的主要优点在于：
1、本发明提供了一种新的双联烯连接子，其可以简便的基于共价巯基桥接定点偶联抗体，其可以应用于大多数抗体，与对抗体进行繁琐的重组改造相比，使用本发明中的双联烯连接子定点偶联位点的方法不需要重组改造抗体，所以可以节约大量的时间、成本和资源，应用前景更广泛。
2、本发明提供的新型双联烯连接子，可通过简单的化学方法与抗体共价桥接偶联，所生成的抗体-药物偶联物与传统的随机偶联方法生成的抗体-药物偶联物相比，使用本发明的连接子得到的偶联物DAR值分布更窄，生成的产品均一性更高，获得的偶联物单一分布的组份(DAR4)占比可以超过80％，有些甚至可以超过95％，而且“半抗体”(DAR4)的组分是占主要的。
3、本发明提供的新型双联烯连接子，也可以和大多数抗体的突变体，例如和在重链226位(Eu编号)或229位(Eu编号)上的突变体共价桥接偶联，由于重链和重链之间形成二硫键的半胱氨酸突变为其他氨基酸，从而可以产生均一的DAR3的抗体-药物偶联物。
4、在抗体相同的情况下，基于本发明提供的新型双联烯连接头的抗体-药物偶联物和马来酰亚胺连接头的抗体-药物偶联物相比，具有相当的体外细胞杀伤活性，和更好的体外血浆稳定性。
以上仅为本发明较佳的实施例，并不用于局限本发明的保护范围，任何在本发明精神内的修改、等同替换或改进等，都涵盖在本发明的权利要求范围内。

Claims (23)

  1. 双联烯连接子，其特征在于，包括通式(Ⅰ)结构所示的化合物或同位素标记物：
    其中：所述L1选自
    M选自 n1与n2相同或不相同，n3与n4相同或不相同,n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数。
  2. 根据权利要求1所述的双联烯连接子，其特征在于，所述通式(Ⅰ)中，
    所述L1选自
    所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    所述L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数。
  3. 根据权利要求1所述的双联烯连接子，其特征在于，所述通式(Ⅰ)中，
    所述L1选自
    所述M选自 所述n1与n2不相同，n3与n4相同或不相同，n5与n6相同或不相同；所述为单一手性的组分或者消旋体；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数。
  4. 根据权利要求1所述的双联烯连接子，其特征在于，所述通式(Ⅰ)中，
    所述L1选自
    M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
    L2选自n7＝1-10的整数，n8＝1-10的整数。
  5. 根据权利要求1所述的双联烯连接子，其特征在于，所述通式(Ⅰ)中的所述双联烯连接子选自以下结构式化合物至少一个：

  6. 一种连接子与药物缀合物，其特征在于，权利要求1至5任一权利要求所述的双联烯连接子中的羧基与中L3的氨基酸残基中的氨基通过酰胺键键合而形成的化合物；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段；其中D通过D中的硫、氮或氧与L3共价结合。
  7. 根据权利要求6所述的连接子与药物缀合物，其特征在于，所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；所述细胞毒性类小分子药物为治疗肿瘤疾病的药物。
  8. 一种连接子与药物缀合物，其特征在于，包括通式(Ⅱ)所示结构的化合物：
    其中：所述L1选自
    所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    所述L2选自及 中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段；
    其中D通过D中的硫、氮或氧与L3共价结合。
  9. 根据权利要求8所述的连接子与药物缀合物，其特征在于，所述通式(Ⅱ)中，
    所述L1选自
    所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    所述L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段；
    其中D通过D中的硫、氮或氧与L3共价结合。
  10. 根据权利要求8所述的连接子与药物缀合物，其特征在于，所述通式(Ⅱ)中，
    所述L1选自
    所述M选自 所述n1与n2不相同，n3与n4相同或不相同，n5与n6相同或不相同；所述为单一手性的组分或者消旋体；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    D为细胞毒性类小分子药物或其他活性剂失去一个或多个原子或基团所形成的片段；
    其中D通过D中的硫、氮或氧与L3共价结合。
  11. 根据权利要求8所述的连接子与药物缀合物，其特征在于，所述通式(Ⅱ)中，
    所述L1选自
    M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
    L2选自n7＝1-10的整数，n8＝1-10的整数；
    L3为
    D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段；
    其中D通过D中的硫、氮或氧与L3共价结合。
  12. 根据权利要求8所述的连接子与药物缀合物，其特征在于，所述通式(Ⅱ)中，
    所述L1选自
    M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
    L2选自n7＝1-10的整数，n8＝1-10的整数；
    L3为
    所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；
    所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂。
  13. 根据权利要求8所述的连接子与药物缀合物，其特征在于，所述的通式(Ⅱ)化合物选自以下表格中的化合物至少一个:



  14. 一种抗体药物偶联物，其特征在于，由权利要求8至13任一权利要求所述的连接子与药物缀合物中的双联烯基团和Ab还原之后的巯基基团进行偶联形成偶联物；
    所述Ab是抗体、抗体片段或蛋白。
  15. 一种抗体药物偶联物，其特征在于，包括如通式(Ⅲ)所示的结构：
    所述通式Ⅲ中，所述通式Ⅱ中双联烯基团的碳碳双键与还原抗体Ab中的巯基之间通过迈克尔加成反应形成C-S键相连接；
    所述L1选自
    所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    所述L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，其中D通过D中的硫、氮或氧与L3共价结合；
    所述n16选自1-20之间的任意数值；
    所述Ab是抗体、抗体片段或蛋白。
  16. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述通式(Ⅲ)中，
    所述L1选自
    所述M选自 n1与n2相同或不相同，n3与n4相同或不相同，n5与n6相同或不相同；n1＝0-10的整数，n2＝0-10的整数，n3＝0-10的整数，n4＝0-10的整数，n5＝1-10的整数，n6＝1-10的整数；
    所述L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    所述D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，其中D通过D中的硫、氮或氧与L3共价结合；
    所述n16选自1-20之间的任意数值；
    所述Ab是抗体、抗体片段或蛋白。
  17. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述通式(Ⅲ)中，
    所述L1选自
    所述M选自 所述n1与n2不相同，n3与n4相同或不相同，n5与n6相同或不相同；所述为单一手性的组分或者消旋体；n1＝0-10的整数,n2＝0-10的整数,n3＝0-10的整数,n4＝0-10的整数,n5＝1-10的整数,n6＝1-10的整数；
    L2选自及中的一个或多个组合；n7＝1-20的整数，n8＝1-20的整数，n9＝1-20的整数，n10＝1-20的整数，n11＝1-20的整数，n12＝1-20的整数，n13＝1-20的整数；
    所述L3为 n14＝1-20的整数，n15＝1-20的整数；
    D为细胞毒性类小分子药物或活性剂失去一个或多个原子或基团所形成的片段，其中D通过D中的硫、氮或氧与L3共价结合；
    所述n16选自1-20之间的任意数值；
    所述Ab是抗体、抗体片段或蛋白。
  18. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述通式(Ⅲ)中，
    所述L1选自
    M选自所述n1与n2相同，n3与n4相同，n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
    L2选自n7＝1-10的整数，n8＝1-10的整数；
    L3为
    所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；
    所述n16选自1-20之间的任意数值；
    所述Ab是抗体、抗体片段或蛋白。
  19. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述通式(Ⅲ)中，
    所述L1选自
    M选自所述n1与n2相同，n3与n4相同；n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
    L2选自n7＝1-10的整数，n8＝1-10的整数；
    L3为
    所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；
    所述n16选自1-20之间的任意数值；
    所述Ab为阿昔单抗、阿仑单抗、阿奈妥单抗、阿特珠单抗、阿维鲁单抗、巴利昔单抗、贝伐单抗、博纳吐单抗、本妥昔单抗、卡妥索单抗、西妥昔单抗、西妥珠单抗、考妥昔单抗、达利珠单抗、达雷木单抗、地宁妥珠单抗、地诺单抗、德帕妥昔珠单抗、地努妥昔单抗、德瓦鲁单抗、埃罗妥珠单抗、因福土单抗、格巴妥木单抗、吉妥珠单抗、替伊莫单抗、英达妥昔单抗、英度妥单抗、奥英妥珠单抗、伊匹单抗、拉贝珠单抗、拉妥珠单抗、拉妥昔单抗、利法妥珠单抗、洛沃妥珠单抗、米拉组单抗、米妥昔单抗、那妥昔单抗、耐昔妥珠单抗、尼妥珠单抗、纳武单抗、奥滨尤妥珠单抗、奥法木单抗、奥拉单抗、奥马珠单抗、帕利珠单抗、帕尼单抗、帕曲妥单抗、派姆单抗、帕妥珠单抗、匹那妥珠单抗、泊洛妥珠单抗、雷莫芦单抗、洛伐妥珠单抗、沙西妥珠单抗、司妥昔单抗、司曲妥单抗、索非妥珠单抗、伐达妥昔单抗、沃瑟妥珠单抗、曲妥珠单抗、抗B7-H3单抗、抗CD4抗体、抗CD5抗体、抗CD13抗体、抗CD30抗体或上述抗体在重链226位或229位上的突变体、其抗原结合片段、免疫活性部分。
  20. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述通式(Ⅲ)中，
    所述L1选自
    M选自所述n1与n2相同，n3与n4相同，n1＝0-8的整数，n2＝0-8的整数，n3＝0-8的整数，n4＝0-8的整数；
    L2选自n7＝1-10的整数，n8＝1-10的整数；
    L3为
    所述细胞毒性类小分子药物为治疗肿瘤疾病的药物；所述活性剂为微管蛋白抑制剂、RNA聚合酶抑制剂、拓扑异构酶抑制剂、插入剂、DNA-反应剂、DNA烷化剂或免疫抑制剂；
    所述n16选自2-6之间的任意数值；
    所述Ab是帕曲妥单抗、沙西妥珠单抗、米妥昔单抗、西妥昔单抗、曲妥珠单抗、抗B7-H3单抗、西妥珠单抗或上述抗体在重链226位或229位上的突变体。
  21. 根据权利要求15所述的抗体药物偶联物，其特征在于，所述通式(Ⅲ)中的所述抗体偶联药物选自下述结构式的化合物至少一个：






    n16选自2-6之间的任意数值。
  22. 权利要求15至21任一权利要求所述的抗体药物偶联物在用于治疗癌症中的用途。
  23. 根据权利要求22所述的抗体药物偶联物在用于治疗癌症中的用途，所述癌症包括淋巴瘤、母细胞瘤、肉瘤、白血病或淋巴恶性瘤、鳞状细胞癌、肺癌、腹膜癌、肝癌、胃癌、胃部癌、胰腺癌、脑胶质瘤、宫颈瘤、卵巢癌、口腔癌、膀胱癌、泌尿道癌症、乳腺癌、HER2阳性乳腺癌、结肠癌、直肠癌、结直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、多发性骨髓瘤、B-细胞淋巴瘤、脑癌、头颈部癌和相关转移性肿瘤。
PCT/CN2024/109676 2023-08-04 2024-08-03 双联烯连接子、连接子与药物缀合物、抗体偶联药物及其用途 Pending WO2025031305A1 (zh)

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CN105814213A (zh) * 2013-10-14 2016-07-27 西纳福克斯股份有限公司 经修饰的糖蛋白、蛋白质-缀合物及其制备方法
CN107921030A (zh) * 2015-08-14 2018-04-17 Rc生物技术公司 抗体‑药物偶联物的共价连接子及其制备方法与应用
CN110621673A (zh) * 2017-04-06 2019-12-27 杭州多禧生物科技有限公司 双链连接的细胞毒性药物偶联物
WO2023086028A2 (en) * 2021-11-10 2023-05-19 Nanyang Technological University Allenamide linkers for antibody-drug conjugate

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CN105814213A (zh) * 2013-10-14 2016-07-27 西纳福克斯股份有限公司 经修饰的糖蛋白、蛋白质-缀合物及其制备方法
CN107921030A (zh) * 2015-08-14 2018-04-17 Rc生物技术公司 抗体‑药物偶联物的共价连接子及其制备方法与应用
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