Diallene linker, linker-drug conjugate, antibody-drug conjugate, and use thereof

Abstract

A diallene linker, a linker-drug conjugate, an antibody-drug conjugate, and a use thereof. The diallene linker is characterized in that the structure of the diallene linker is as shown in general formula I. The novel diallene linker can conveniently implement bridged site-specific conjugation with an antibody on the basis of a covalent thiol, and can be applied to most antibodies. Compared with tedious recombinant engineering of antibodies, a used method for site-specific conjugation at a site by means of the diallene linker does not require recombinant engineering of antibodies, and thus can save significant time, costs and resources, and have wider prospects for application.

Description

Bis-ene linker, linker-drug conjugate, antibody-drug conjugate and uses thereof Technical Field

[0001] The present invention relates to a biolefin linker, a linker-drug conjugate, an antibody-drug conjugate and uses thereof. Background Art

[0002] The design concept of antibody-drug conjugates (ADCs) originated from the concept of "magic bullets" first proposed by German scientist Paul Ehrlich in 1913. The core idea of ​​this concept is to deliver drugs to the diseased site through appropriate carriers. Due to the limitations of antibody and highly active cytotoxic drug technology, it was not until 2000 that the first antibody-drug conjugate (Mylotarg) for the treatment of acute myeloid leukemia (AML) was developed. TM ) were approved for marketing by the FDA. However, with the continuous development of antibody humanization, cleavable linker technology, and the emergence of novel toxin molecules, the US Food and Drug Administration (FDA) has approved 14 antibody-drug conjugates (ADCs) to date for the treatment of blood cancers and solid tumors. Despite the tremendous success of currently marketed antibody-drug conjugates (ADCs) in clinical therapy, many widely used antibody conjugation methods still have significant limitations. The preparation of ADCs with uniform composition remains a challenge that needs to be overcome. Common conjugation methods utilize chemical reactions between lysine residues or cysteine ​​moieties (via reduction of interchain disulfide bonds) and electrophilic reagents. Because these chemical reactions are currently not selective, the number and sites of conjugation are uncertain, resulting in non-uniform conjugates with 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 also may be accompanied by a suboptimal therapeutic window.

[0003] Disulfide bridging is a strategy for forming homogeneous ADCs. Various reagents have been used for disulfide bridging, including disulfide ketones, next-generation maleimides (NGMs, a class of maleimide reagents modified with two halide or thiol leaving groups), pyridazinediones such as dibromomethylquinoxaline, arylidene dipropionitrile, divinylpyrimidine (DVP) (Chem. Soc. Rev., 2021, 50, 1305-1353), diacryl hexahydrotriazine (CN107921030) from RC Biotechnology, bismaleimide (CN103933575) from Shanghai New Concept Biopharmaceutical Technology Co., Ltd., and bissulfone (CN110290800) from Taiwan Hao Ding Biotechnology Co., Ltd. Despite the above progress in disulfide bridging, there are still problems such as incomplete composition. For example, DAR4's ADC is a mixture of "half antibody" and "full antibody", and there is no way to make the main component "half antibody" or "full antibody", or the ADC content of DAR4 is relatively low.

[0004] In summary, there is an urgent need in this field for efficient, simple, and practical disulfide-bridging chemical coupling methods that can achieve the purpose of site-specific coupling while maintaining or improving the efficacy, stability, and safety of antibody-drug conjugates.

[0005] Summary of the Invention

[0006] The present invention aims to provide a novel bivalent olefin linker capable of covalently bridging with most antibodies. Furthermore, the present invention provides methods for antibody / active agent coupling and applications thereof, such as in antibody-drug conjugates.

[0007] In a first aspect of the present invention, there is provided a biolefin linker, characterized in that it comprises a compound or an isotope-labeled substance as represented by the general formula (I):

[0008] Wherein: said L1 is selected from

[0009] M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0010] L2 is selected from and One or more; n7 = an integer of 1-20, n8 = an integer of 1-20, n9 = an integer of 1-20, n 10= integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20.

[0011] According to one embodiment of the present invention: in the general formula (I),

[0012] The L1 is selected from

[0013] The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0014] The L2 is selected from and One or more; n7 = an integer of 1-20, n8 = an integer of 1-20, n9 = an integer of 1-20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20.

[0015] According to one embodiment of the present invention: in the general formula (I),

[0016] The L1 is selected from

[0017] The M is selected from Said n1 is different from n2, n3 is the same as or different from n4, n5 is the same as or different from n6; said A single chiral component or racemate; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0018] L2 is selected from and One or more; n7 = an integer of 1-20, n8 = an integer of 1-20, n9 = an integer of 1-20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12= integer from 1 to 20, n 13 = integer from 1 to 20.

[0019] According to one embodiment of the present invention: in the general formula (I),

[0020] The L1 is selected from

[0021] M is selected from

[0022] Said n1 is the same as n2, n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, n4=an integer from 0 to 8;

[0023] L2 is selected from n7=an integer from 1 to 10, and n8=an integer from 1 to 10.

[0024] According to one embodiment of the present invention: the biolefin linker in the general formula (I) is selected from at least one of the following compounds:

[0025] Another aspect of the present invention is to provide a linker-drug conjugate, characterized in that the carboxyl group in the aforementioned bis-ene linker is A compound formed by bonding the amino group of the amino acid residue in L3 via an amide bond;

[0026] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0027] D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent, and D is covalently bonded to L3 via sulfur, hydrogen or oxygen in D.

[0028] According to one embodiment of the present invention, 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.

[0029] 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; the RNA polymerase inhibitors include but are not limited to α-amanitin; the topoisomerase inhibitors include but are not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan; the intercalating agents include but are not limited to pyrrolobenzodiazepine (PBD); the DNA-reactive agents include but are not limited to calicheamicin, tiancimycins or other enediynes; the DNA alkylating agents include but are not limited to ecteinascidin derivatives such as trabectedin or rubectin.

[0030] 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):

[0031] Wherein: said L1 is selected from

[0032] The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0033] The L2 is selected from and One or more; n7 = an integer of 1-20, n8 = an integer of 1-20, n9 = an integer of 1-20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20;

[0034] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0035] D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent.

[0036] Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

[0037] According to one embodiment of the present invention: in the general formula (II),

[0038] The L1 is selected from

[0039] The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0040] The L2 is selected from and One or more; n7 = an integer of 1-20, n8 = an integer of 1-20, n9 = an integer of 1-20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20;

[0041] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0042] D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent.

[0043] Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

[0044] According to one embodiment of the present invention: in the general formula (II), L1 is selected from

[0045] The M is selected from Said n1 is different from n2, n3 is the same as or different from n4, n5 is the same as or different from n6; said A single chiral component or racemate; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0046] L2 is selected from and One or more; n7 = an integer of 1-20, n8 = an integer of 1-20, n9 = an integer of 1-20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20;

[0047] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0048] 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.

[0049] Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

[0050] According to one embodiment of the present invention: in the general formula (II),

[0051] The L1 is selected from

[0052] M is selected from Said n1 is the same as n2, n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, n4=an integer from 0 to 8;

[0053] L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10;

[0054] L3 is

[0055] 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.

[0056] Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

[0057] According to one embodiment of the present invention: in the general formula (II),

[0058] The L1 is selected from

[0059] M is selected from Said n1 is the same as n2, n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, n4=an integer from 0 to 8;

[0060] L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10;

[0061] L3 is

[0062] 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.

[0063] The tubulin inhibitors include but are not limited to monomethyl auristatin, maytansinoid, maytansinol or DM1 maytansine. The monomethyl auristatins include but are not limited to monomethyl auristatin E or monomethyl auristatin F; the RNA polymerase inhibitors include but are not limited to α-amanitin; the topoisomerase inhibitors include but are not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan; the intercalating agents include but are not limited to pyrrolobenzodiazepine (PBD); the DNA-reactive agents include but are not limited to calicheamicin, tiancimycins or other enediynes; the DNA alkylating agents include but are not limited to ecteinascidin derivatives such as trabectedin or rubectin.

[0064] According to one embodiment of the present invention: the compound of general formula (II) is selected from at least one of the compounds in the following table:

[0065] 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 bis-ene group in the drug conjugate and the sulfhydryl group after reduction of the Ab;

[0066] The Ab is an antibody, antibody fragment or protein.

[0067] According to one embodiment of the present invention, the antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody, an antigen-binding fragment thereof, an immunologically active portion or a mutant of the above antibodies.

[0068] 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):

[0069] The L1 is selected from

[0070] The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0071] The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20;

[0072] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0073] D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent.

[0074] Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

[0075] The n 16 Select any value between 1-20;

[0076] The Ab is an antibody, antibody fragment or protein.

[0077] The antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody, an antigen-binding fragment thereof, an immunologically active portion or a mutant of the above antibodies.

[0078] According to one embodiment of the present invention: in the general formula (III),

[0079] The L1 is selected from

[0080] The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0081] The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 =

[0082] Integer from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20;

[0083] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0084] The cytotoxic small molecule drug or active agent is a fragment formed by losing one or more atoms or groups of the cytotoxic small molecule drug or other active agent;

[0085] Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

[0086] The n 16 Select any value between 1-20;

[0087] The Ab is an antibody, antibody fragment or protein.

[0088] The antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody, an antigen-binding fragment thereof, an immunologically active portion or a mutant of the above antibodies.

[0089] According to one embodiment of the present invention: in the general formula (III),

[0090] The L1 is selected from

[0091] The M is selected from Said n1 is different from n2, n3 is the same as or different from n4, n5 is the same as or different from n6; said A single chiral component or racemate; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10;

[0092] L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20;

[0093] The L3 is n 14 = integer from 1 to 20, n 15 = an integer from 1 to 20;

[0094] D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent;

[0095] Wherein D is covalently bonded to L3 through the sulfur, nitrogen or oxygen in D;

[0096] The n 16 Select any value between 1-20;

[0097] The Ab is an antibody, antibody fragment or protein.

[0098] The antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody, an antigen-binding fragment thereof, an immunologically active portion or a mutant of the above antibodies.

[0099] According to one embodiment of the present invention:

[0100] The L1 is selected from

[0101] M is selected from Said n1 is the same as n2, n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, n4=an integer from 0 to 8;

[0102] L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10;

[0103] L3 is

[0104] 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;

[0105] The n 16 Select any value between 1-20;

[0106] The Ab is an antibody, antibody fragment or protein.

[0107] 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; the RNA polymerase inhibitors include but are not limited to α-amanitin; the topoisomerase inhibitors include but are not limited to etoposide, teniposide, amsacrine, SN-38 or exitecan; the intercalating agents include but are not limited to pyrrolobenzodiazepine (PBD); the DNA-reactive agents include but are not limited to calicheamicin, tiancimycins or other enediynes; the DNA alkylating agents include but are not limited to ecteinascidin derivatives such as trabectedin or rubectin.

[0108] The antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody and a multispecific antibody, an antigen-binding fragment thereof, an immunologically active portion or a mutant of the above antibodies.

[0109] According to one embodiment of the present invention: in the general formula (III),

[0110] The L1 is selected from

[0111] M is selected from Said n1 is the same as n2, n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, n4=an integer from 0 to 8;

[0112] L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10;

[0113] L3 is

[0114] The n 16 Select any value between 1-20;

[0115] 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;

[0116] The Ab is abciximab, alemtuzumab, anetuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatumomab, brentuximab, catumaxomab, cetuximab, cetuximab, coxutuximab, daclizumab, daratumumab, deninutuzumab, denosumab, depatuxizumab, dinutuximab, durvalumab, elotuzumab, infortumumab, gebatuzumab, gemtuzumab, ibritumomab tiuxetan, indalatuzumab, indoximab, inutuzumab, ipilimumab, labetuzumab, latuzumab, latuximab, rifatuzumab, lovatuzumab, miratumumab, metuximab, Natuximab, nesituzumab, nimotuzumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pertrastuzumab, pembrolizumab, pertuzumab, pinatuzumab, polotuzumab, ramucirumab, lovatuzumab, tasitumab, setuximab, setuximab, sofetozumab, vadatuximab, worsotuzumab, trastuzumab, anti-B7-H3 monoclonal antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody, anti-CD30 antibody, or mutants of the above antibodies at position 226 or 229 of the heavy chain, antigen-binding fragments, or immunologically active portions thereof.

[0117] According to one embodiment of the present invention: in the general formula (III),

[0118] The L1 is selected from

[0119] M is selected from Said 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;

[0120] L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10;

[0121] L3 is

[0122] 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;

[0123] The n 16 Select any value between 2-6;

[0124] The Ab is selected from pertratuzumab, 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.

[0125] The mutant is a mutant in which the cysteine ​​that forms a disulfide bond between heavy chains is mutated to serine.

[0126] According to one embodiment of the present invention: the antibody-drug conjugate in the general formula (III) is selected from at least one compound of the following structural formula:

[0127] n 16 Select any value between 2-6.

[0128] In the general structural formula (III) and the structural formulas of ADC-1 to ADC-29 of the present invention, S is a sulfur atom contained in the antibody Ab.

[0129] The tubulin inhibitors described in the present invention 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; the RNA polymerase inhibitors include but are not limited to α-amanitin; the topoisomerase inhibitors include but are not limited to etoposide, teniposide, amsacrine, SN-38, or exitecan; the intercalating agents include but are not limited to pyrrolobenzodiazepine (PBD); the DNA-reactive agents include but are not limited to calicheamicin, tiancimycins, or other enediynes; and the DNA alkylating agents include but are not limited to ecteinascidin derivatives such as trabectedin or rubectin.

[0130] 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 cetuximab is Cirmtuzumab; Coltuximab is called Coltuximab; Daclizumab is called Daclizumab; Daratumumab is called Daratumumab; Denintuzumab is called Denintuzumab; Denosumab is called Depatuxizumab; Dinutuximab is called Dinutuximab; Durvalumab is called Durvalumab; Elotuzumab is called Elotuzumab; Enfortumab is called Enfortumab; G lembatumumab; Gemtuzumab is the English name of Gemtuzumab; Ibritumomab is the English name of Titumomab; Indatuximab is the English name of Indusatumab; Inotuzumab is the English name of Inotuzumab; Ipilimumab is the English name of Ipilimumab; Labetuzumab is the English name of Ladiratuzumab; Laprituximab is the English name of Lifastuzumab; Lovotouzumab is the English name of It is called Lorvotuzumab; Milatuzumab is called Milatuzumab; Mirvetuximab, Naratuximab, Necitumumab and Nimotuzumab are called Nivolumab; Obinutuzumab is called Obinutuzumab; Ofatumumab is called Ofatumumab; Olaratumab is called Olaratumab; Omalizumab is called Omalizumab; Palivizumab is called Palivizumab;The English name of panitumumab is Panitumumab; the English name of patritumab is Patritumab; the English name of pembrolizumab is Pembrolizumab; the English name of pertuzumab is Pertuzumab; the English name of pinatuzumab is Pinatuzumab; the English name of polotuzumab is Polatuzumab; the English name of ramucirumab is Ramucirumab; the English name of lovatuzumab is Rovalpituzumab; the English name of sacituzumab is Sacituzumab; the English name of siltuximab is Siltuximab; the English name of sirtratumab is Sirtratumab; the English name of sofituzumab is Sofituzumab; the English name of vadastuximab is Vadastuximab; the English name of vorsetuzumab is Vorsetuzumab; the English name of trastuzumab is Trastuzumab; the anti-B7-H3 monoclonal antibody is Ifinatamab, which is translated into Chinese as Ifinatamab. ;

[0131] Use of the aforementioned antibody-drug conjugate in treating cancer.

[0132] The cancers include lymphoma, blastoma, sarcoma, leukemia or lymphoid malignancy, 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.

[0133] The main advantages of the present invention are:

[0134] 1. The present invention provides a new double-ene linker that can be used to conveniently site-couple antibodies based on covalent thiol bridging. It can be applied to most antibodies. Compared with the tedious recombinant modification of antibodies, the method of using the double-ene linker site-coupling site 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.

[0135] 2. The novel bivalent olefin linker provided by the present invention can be covalently bridged and coupled with an antibody by a simple chemical method. Compared with the antibody-drug conjugates generated by traditional random coupling methods, the conjugates obtained using the linker of the present invention have a narrower distribution of DAR values ​​and higher product uniformity. 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.

[0136] 3. The novel biolefin linker provided by the present invention can also be covalently bridged and coupled with most antibody mutants, for example, 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.

[0137] 4. When the antibodies are the same, the antibody-drug conjugates based on the novel bis-ene linker provided by the present invention have comparable in vitro cell killing activity and better in vitro plasma stability than the antibody-drug conjugates based on the maleimide linker. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] 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 the single distributed component (DAR4) of the obtained conjugate can exceed 80%.

[0139] FIG2 is a SEC diagram of ADC-1 prepared in an embodiment of the present invention.

[0140] FIG3 is a MS diagram of ADC-1 prepared in an embodiment of the present invention.

[0141] FIG4 is an SDS-PAGE image of ADC-1 prepared in an embodiment of the present invention.

[0142] FIG5 is a diagram of ADC-2HIC prepared in an embodiment of the present invention.

[0143] FIG6 is a graph of ADC-2SEC prepared in an embodiment of the present invention.

[0144] FIG7 is a diagram of ADC-3HIC prepared in an embodiment of the present invention.

[0145] FIG8 is a graph of ADC-3SEC prepared in an embodiment of the present invention.

[0146] FIG9 is an SDS-PAGE image of ADC-3 prepared in an embodiment of the present invention.

[0147] FIG10 is a diagram of ADC-5HIC prepared in an embodiment of the present invention.

[0148] FIG11 is a graph of ADC-5SEC prepared in an embodiment of the present invention.

[0149] FIG12 is an SDS-PAGE image of ADC-5 prepared in an embodiment of the present invention.

[0150] Figure 13 is a graph of ADC-10 HIC 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%.

[0151] FIG14 is a graph of ADC-10SEC prepared in an embodiment of the present invention.

[0152] FIG15 is an SDS-PAGE image of ADC-10 prepared in an embodiment of the present invention.

[0153] FIG16 is a diagram of ADC-25HIC prepared in an embodiment of the present invention.

[0154] FIG17 is a graph of ADC-25SEC prepared in an embodiment of the present invention.

[0155] FIG18 is an SDS-PAGE image of ADC-25 prepared in an embodiment of the present invention.

[0156] FIG19 is a diagram of ADC-26HIC prepared in an embodiment of the present invention.

[0157] FIG20 is a graph of ADC-26SEC prepared in an embodiment of the present invention.

[0158] FIG21 is an SDS-PAGE image of ADC-26 prepared in an embodiment of the present invention.

[0159] 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 distributed component (DAR4) of the obtained conjugate can exceed 95%.

[0160] FIG23 is a graph of ADC-27SEC prepared in an embodiment of the present invention.

[0161] FIG24 is a MS diagram of ADC-27 prepared in an embodiment of the present invention.

[0162] FIG25 is an SDS-PAGE image of ADC-27 prepared in an example of the present invention.

[0163] FIG26 is a graph showing the in vitro plasma stability experiment of ADCs based on different connectors. DETAILED DESCRIPTION

[0164] The following are examples of the synthesis and analysis of the compounds (including linkers and linker-drug conjugates) and antibody drug conjugates described in this patent. It should be noted that these examples do not limit the scope of protection of the present invention.

[0165] Unless otherwise stated, all anhydrous reagents were purchased directly from the suppliers and stored under nitrogen.

[0166] The definitions and schematic diagrams of "half antibodies" and "full antibodies" described in this patent are as follows:

[0167] The "full 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:

[0168] "Half-antibody" is shown in the figure on the right, which is formed by the bridge between the light chain and the heavy chain and the bridge within the heavy chain:

[0169] Synthesis and characterization of linkers

[0170] Example 1: Synthesis of compound LK1

[0171] Step 1: Synthesis of 2-(trimethylsilyl)ethyl 6-(((benzyloxy)carbonyl)amino)hexanoate.

[0172] To a solution of 6-(((benzyloxy)carbonyl)amino)hexanoic acid (5.0 g, 0.0188 mmol), EDCI (3.98 g, 0.0207 mol), and HOBt (2.80 g, 0.0207 mol) in 30 mL of dichloromethane were added 2-(trimethylsilyl)ethan-1-ol (2.23 mg, 0.0188 mmol) and DIPEA (4.87 g, 0.0376 mol). After the addition was complete, the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and redissolved in MTBE, washed with brine and saturated sodium bicarbonate, and concentrated. The residue was purified by flash column chromatography on silica gel (eluted with ethyl acetate / heptane = 1:3) to give the product as a white solid (5.5 g, 79.8% yield).

[0173] LC-MS(ESI)m / z:338.0[M-2CH3+3H] + .

[0174] 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).

[0175] Step 2: Synthesis of 2-(trimethylsilyl)ethyl 6-aminohexanoate

[0176] Ethyl 2-(trimethylsilyl)-6-(((benzyloxy)carbonyl)amino)hexanoate (2.5 g, 6.8 mmol) was diluted with 30 mL of MeOH and concentrated HCl (0.74 g, 7.4 mmol), and then 0.3 g of 5% Pd / C was added under N2. The suspension was evacuated and replaced with H2 several times. The mixture was stirred under 1 MPa of H2 for 1 hour. The suspension was filtered through a pad of celite and washed with MeOH. The combined filtrates were concentrated to dryness to give an oily liquid, which was used directly in the next step without further purification.

[0177] 1 H 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).

[0178] Step 3: Synthesis of methyl 3-((tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropanoate

[0179] To a solution of methyl 3-amino-2-(aminomethyl)propanoate (900 mg, 4.383 mmol) in 1,4-dioxane / H₂O (30 mL, 1:1) was added Na₂CO₃ (2.33 g, 21.9 mmol), stirred for 0.5 hours, and cooled to 0°C. Boc₂O (3.9 g, 17.53 mmol) was added to the reaction mixture, and stirred at room temperature overnight. After completion of the reaction, the mixture was extracted with ethyl acetate (50 mL x 3). The organic layer was dried over anhydrous sodium sulfate and concentrated. Purification by column chromatography (heptane / ethyl acetate = 10:1 to 5:1) afforded the desired product (1.23 g, 83.9%) as a colorless oil.

[0180] 1 H 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).

[0181] Step 4: Synthesis of 3-((tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropanoic acid

[0182] To a solution of methyl 3-((tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropanoate (1.23 g, 3.7 mmol) in MeOH / H2O (2:1, 12 mL), the temperature was controlled at 0°C, and LiOH was added. ˙ H2O (776.3 mg, 18.5 mmol). Stir at room temperature for 0.5 h and adjust the pH to 3-4 with 1N HCl. Extract with ethyl acetate (50 mL x 3) and wash the organic layer with saturated brine. Separate the organic layer and dry it over anhydrous sodium sulfate. Concentrate and use in the next step without further purification.

[0183] 1 H 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).

[0184] Step 5: Synthesis of 2-(trimethylsilyl)ethyl-6-(3-(tert-butylcarbonyl)amino)-2-((tert-butylcarbonyl)amino)methylpropylamino)hexanoate

[0185] EDCI (658 mg, 3.45 mmol) and HOBt (463.3 mg, 3.45 mmol) and 2-(trimethylsilyl)ethyl 6-aminohexanoate (1.46 g, 31.19 mmol) were added to a solution of 3-(tert-butylcarbonyl)amino)-2-(tert-butylcarbonyl)amino)methylpropanoic acid (1.0 g, 3.14 mmol) in dichloromethane (20 mL). The mixture was stirred at room temperature overnight. The mixture was washed with 5% citric acid solution (50 mL × 3) and then with water (50 mL × 3). The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (ethyl acetate / n-heptane = 1:1) to obtain the target product (870 mg, 52.1%) as a white solid.

[0186] 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).

[0187] Step 6: Synthesis of 2-(trimethylsilyl)ethyl-6-(3-amino-2-(aminomethyl)propylamino)hexanoic acid dihydrochloride

[0188] To a solution of 2-(trimethylsilyl)ethyl 6-(3-(tert-butylcarbonyl)amino)-2-(tert-butylcarbonyl)amino)methylpropylamino)hexanoate (500 mg, 0.94 mmol) in ethyl acetate (7 mL) was added 2N HCl / EA (2.4 ml). The mixture was stirred at room temperature for 2 days. The solvent was removed by concentration, and the mixture was slurried with acetonitrile. The solid was filtered and dried to obtain the desired product (300 mg, 79.1%) as a white solid salt.

[0189] 1 H 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).

[0190] Step 7: Synthesis of 2-(trimethylsilyl)ethyl-6-(3-(butyl-3-amino)-2-(butyl-3-amino)propylamino)hexanoate

[0191] To a suspension of butane-3-acetic acid (623.6 mg, 7.4 mmol) in dichloromethane (10 mL) was added oxalyl chloride (939.1 mg, 7.4 mmol). After stirring at room temperature for 15 minutes, the reaction solution was cooled to 0°C and 0.15 mL of DMF was added. The reaction mixture was stirred at 0°C and then at room temperature for an additional 90 minutes. The above solution was added dropwise to a solution of 2-(trimethylsilyl)ethyl 6-(3-amino-2-(aminomethyl)propylamino)hexanoic acid dihydrochloride (300 mg, 0.74 mmol) in saturated sodium bicarbonate solution (10 mL). The reaction mixture was stirred at room temperature for another 2 hours. The reaction mixture was extracted with dichloromethane (50 mL x 3) and concentrated. Purification by column chromatography (ethyl acetate / n-heptane = 1:1) afforded the desired product (292 mg, 85%) as a white solid.

[0192] LC-MS (ESI) m / z: 464 [M+H] + .

[0193] 1 H NMR(400MHz,Chloroform-d)δ7.52(s,2H),6.67(s,1H),4.19–4.12(m,2H),3.71(d dd,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).

[0194] Step 8: Synthesis of 2-(trimethylsilyl)ethyl-6-(3-(butyl-2,3-diamino)-2-(butyl-2,3-diaminomethyl)propylamino)hexanoate

[0195] 2-(Trimethylsilyl)ethyl 6-(3-(butyl-3-aminoamino)-2-(butyl-3-aminomethyl)propylamine)hexanoate (292 mg, 0.64 mmol) was dissolved in an 18% aqueous K2CO3 solution / dioxane (8 mL / 8 mL). The reaction mixture was heated to 40°C for 2 hours, then extracted with citric acid (60 mL, 10% water) and ethyl acetate (500 mL x 3). The organic layer was dried over anhydrous sodium sulfate and concentrated to yield 210 mg, 71.9% of the compound as an oil.

[0196] 1H NMR(400MHz,Chloroform-d)δ7.08(t,J=6.6Hz,2H),6.96(s,1H),5.63(t,J=6.6H z,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=7.5 Hz, 2H), 1.52 (p, J = 7.3Hz, 2H), 1.42–1.30 (m, 3H), 1.01–0.94 (m, 2H), 0.04 (s, 9H).

[0197] Step 9: Synthesize LK1

[0198] 2-(Trimethylsilyl)ethyl-6-(3-(butyl-2,3-diamino)-2-(butyl-2,3-diaminomethyl)propylamino)hexanoate (60 mg, 0.129 mmol) was dissolved in TFA / DCM (1:1, 1 mL) and stirred at room temperature for 1 hour. The solvent was concentrated and the mixture was washed with C 18 Column purification (ACN / H2O) gave 20 mg of a white solid.

[0199] LC-MS (ESI) m / z: 364 [M+H] + .

[0200] 1 H 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).

[0201] Example 2: Synthesis of compound LK2

[0202] Step 1: Synthesis of methyl 2,3-bis((tert-butyloxycarbonyl)amino)propionate

[0203] To a solution of methyl 2,3-diaminopropionate dihydrochloride (2.5 g, 13.1 mmol) in 1,4-dioxane / H₂O (30 mL, 1:1) was added Na₂CO₃ (6.9 g, 65.7 mmol). The reaction was stirred at 0°C for 0.5 hours. Boc₂O (11.4 g, 52.4 mmol) was added to the reaction mixture and stirred at room temperature overnight. After completion of the reaction, the mixture was extracted with ethyl acetate (50 mL x 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel flash column chromatography (heptane / ethyl acetate = 10:1 to 5:1) to obtain the desired product (2.0 g, 47.7% yield) as a colorless oil.

[0204] 1 H 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).

[0205] Step 2: Synthesis of 2,3-bis((tert-butyloxycarbonyl)amino)propionic acid

[0206] To a solution of methyl 2,3-bis((tert-butoxycarbonyl)amino)propanoate (1.23 g, 3.7 mmol) in MeOH / H₂O (2:1, 12 mL) at 0°C was added LiOH﹒H₂O (776.3 mg, 18.5 mmol). Stir at room temperature for 0.5 hours, and adjust the pH to 3-4 with 1N HCl. Extract with ethyl acetate (50 mL x 3), and wash the organic phase with saturated brine. Separate the organic phase, dry it over anhydrous sodium sulfate, concentrate it, and use it in the next step without further purification.

[0207] 1 H 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).

[0208] Step 3: Synthesis of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-bis((tert-butoxycarbonyl)amino)propionamido)hexanoate

[0209] 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) were added EDCI (658 mg, 3.45 mmol) and HOBt (463.3 mg, 3.45 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was washed with aq. 5% citric acid (50 mL × 3) and then with water (50 mL × 3). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel flash column chromatography (ethyl acetate / n-heptane = 1:1) to obtain the desired product (1.0 g, 58.8% yield) as a white solid.

[0210] 1 H 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).

[0211] Step 4: Synthesis of 2-(trimethylsilyl)ethyl-9,10-diamino-8-oxodecanoic acid dihydrochloride

[0212] To a solution of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-bis((tert-butoxycarbonyl)amino)propionamido)hexanoate (500 mg, 0.94 mmol) in ethyl acetate (7 mL) was added 2N HCl / ethyl acetate (2.4 mL, 5 eq). The mixture was stirred at room temperature for 2 days. The solvent was removed by concentration and a solid was precipitated with acetonitrile. The solid was filtered and dried to afford (230 mg, 75.2% yield) as a white solid.

[0213] 1 H 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).

[0214] Step 5: Synthesis of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-di(but-3-ynamido)propionamido)hexanoate

[0215] To a suspension of but-3-ynoic acid (623.6 mg, 7.4 mmol) in dichloromethane (10 mL) was added oxalyl chloride (939.1 mg, 7.4 mmol). After stirring at room temperature for 15 minutes, the reaction solution was cooled to 0°C and DMF (0.15 mL) was added. The mixture was then stirred at room temperature for 90 minutes. This solution was added dropwise to 2-(trimethylsilyl)ethyl-9,10-diamino-8-oxodecanoic acid dihydrochloride (300 mg, 0.74 mmol) in 10 mL of saturated sodium bicarbonate solution and stirred at room temperature for 2 hours. The mixture was extracted with dichloromethane (50 mL x 3), and the organic phases were combined, dried, and concentrated. Purification by silica gel flash column chromatography (ethyl acetate / n-heptane = 1:1) afforded the desired product (292 mg, 85% yield) as a white solid.

[0216] 1 H 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).

[0217] Step 6: Synthesis of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-di(buta-2,3-dienamido)propionamido)hexanoate

[0218] A solution of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-di(but-3-ynamido)propionamido)hexanoate (292 mg, 0.64 mmol) and KCO (18% aq., 8 mL) in 1,4-dioxane (8 mL) was heated to 40°C and stirred for 2 hours. Citric acid (60 mL, 10% aqueous solution) and ethyl acetate (500 mL x 3) were then added. The organic layers were combined, dried, and concentrated to afford the desired product (210 mg, 72% yield) as an oil.

[0219] 1 H 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).

[0220] Step 7: 6-(2,3-Di(buta-2,3-dienamido)propionamido)hexanoic acid

[0221] A solution of ethyl 2-(trimethylsilyl)ethyl-6-(2,3-di(buta-2,3-dienylamido)propionamido)hexanoate (70 mg, 0.129 mmol) in TFA / DCM (1:1, 1 mL) was stirred at room temperature for 1 hour. The solvent was concentrated and the product was purified on a C18 column (ACN / H2O) to afford the desired product (30 mg, 55%) as a white solid.

[0222] Example 3: Synthesis of compound LK3

[0223] Step 1: Synthesis of 2-(trimethylsilyl)ethyl 6-(3,5-dinitrobenzamido)hexanoate

[0224] To a solution of LK1-3 (455 mg, 1.97 mmol) and triethylamine (300 mg, 2.96 mmol) in 20 mL of dichloromethane was slowly added 3,5-dinitrobenzoyl chloride (500 mg, 2.16 mmol) at 0°C. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction was monitored for completion by LCMS. The reaction solution was washed sequentially with 1N HCl, saturated NaHCO3, and brine. The organic phases were combined, dried over sodium sulfate, and filtered. The residue was concentrated and purified by silica gel flash column chromatography to obtain the title compound (429 mg, 51% yield) as a yellow solid.

[0225] 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).

[0226] Step 2: Synthesis of 2-(trimethylsilyl)ethyl 6-(3,5-diaminobenzamido)hexanoate

[0227] To a solution of 2-(trimethylsilyl)ethyl 6-(3,5-dinitrobenzamido)hexanoate (420 mg, 1.15 mmol) in methanol (10 mL) was added 10% wet Pd / C (42 mg, 10% w / w). The mixture was stirred at room temperature overnight under H2 protection, filtered, and concentrated to obtain the desired product (377 mg, crude) as a yellow solid.

[0228] LC-MS (ESI) m / z: 366 [M+H] + .

[0229] 1 H 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).

[0230] Step 3: Synthesis of 2-(trimethylsilyl)ethyl-6-(3,5-di(but-3-ynylmethylamino)benzylamino)hexanoate

[0231] To a suspension of but-3-ynoic acid (850.8 mg, 10.12 mmol) in dichloromethane (50 mL) was added oxalyl chloride (1.28 g, 10.12 mmol). After stirring at room temperature for 15 minutes, the reaction solution was cooled to 0°C and a catalytic amount of DMF (0.15 mL) was added. The temperature was slowly raised to room temperature and stirred for an additional 90 minutes. This solution was added dropwise to a solution of 2-(trimethylsilyl)ethyl 6-(3,5-diaminobenzamido)hexanoate (370 mg, 1.012 mmol) in saturated sodium bicarbonate (10 mL). The mixture was stirred at room temperature for 2 hours. The mixture was extracted with dichloromethane (50 mL x 3), and the organic layers were combined, dried, filtered, and concentrated to obtain a yellow oily compound.

[0232] 1 H 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).

[0233] Step 4: Synthesis of 2-(trimethylsilyl)ethyl-6-(3,5-di(buta-2,3-dienamido)benzamido)hexanoate

[0234] A solution of 2-(trimethylsilyl)ethyl-6-(3,5-di(but-3-ynylmethylamino)benzylamino)hexanoate (1.15 g, 2.32 mol) in KCO (18% aqueous solution, 10 mL) was mixed with 1,4-dioxane (10 mL). The reaction mixture was heated to 40°C and stirred for 2 hours. Citric acid (60 mL, 10% aqueous solution) and ethyl acetate (500 mL x 3) were then added. The organic phases were combined, dried, filtered, and concentrated to yield 1.15 g of crude oil.

[0235] 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,2 H),1.58-1.69(m,4H),1.43-1.35(m,2H),1.01–0.92(m,2H),0.02(s,9H).

[0236] Step 5: Synthesis of 6-(3,5-di(buta-2,3-dienylamino)benzamido)hexanoic acid

[0237] 2-(Trimethylsilyl)ethyl-6-(3,5-di(buta-2,3-dienamido)benzamido)hexanoate (1.15 g, 2.32 mmol) was dissolved in TFA / DCM (1:1, 1 mL) and stirred at room temperature for 1 hour. The solvent was concentrated and the product was purified on a C18 column (ACN / H2O) to obtain the desired product as a white solid (330 mg, 35.9% yield over 3 steps).

[0238] 1 H 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),5.8 9(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).

[0239] Example 4: Synthesis of compound LK4

[0240] Step 1: Synthesis of 2-(trimethylsilyl)ethyl-6-(3,4-dinitrobenzamido)hexanoate

[0241] To a solution of LK1-3 (884 mg, 3.82 mmol) and triethylamine (527 mg, 5.2 mmol) in dichloromethane (15 mL) was slowly added 3,4-dinitrobenzoyl chloride (800 mg, 3.47 mmol) dropwise at 0°C. The reaction was stirred at room temperature for 2 hours, and the reaction was monitored for completion by LCMS. The reaction solution was washed sequentially with 1N HCl, saturated NaHCO₃, and brine. The organic phases were combined, dried over sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the desired product (1.38 g, 93% yield) as a yellow solid. LC-MS (ESI) m / z: 448 [M+Na] + .

[0242] 1 H 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).

[0243] Step 2: Synthesis of 2-(trimethylsilyl)ethyl 6-(3,4-diaminobenzamido)hexanoate

[0244] A solution of 2-(trimethylsilyl)ethyl-6-(3,4-dinitrobenzamido)hexanoate (1.23 g, 2.9 mmol), iron powder (812 mg, 14.5 mmol), and NH₄Cl (1.55 g, 29 mmol) in 12 mL of EtOH / H₂O (5 / 1, v / v) was added to a sealed tube and heated to 110°C for 1 hour. After the reaction was complete, the mixture was filtered and washed with ethyl acetate and water. The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel flash column chromatography to obtain the title compound (485 mg, 45.9% yield) as a yellow solid.

[0245] LC-MS (ESI) m / z: 366 [M+H] + .

[0246] Step 3: Synthesis of 2-(trimethylsilyl)ethyl-6-(3,4-di(but-3-ynamido)benzamido)hexanoate

[0247] To a suspension of but-3-ynoic acid (644 mg, 7.66 mmol) in dichloromethane (30 mL) was added oxalyl chloride (972 mg, 7.66 mmol). 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 stirred for 90 minutes. This solution was added dropwise to a solution of 2-(trimethylsilyl)ethyl 6-(3,4-diaminobenzamido)hexanoate (280 mg, 0.766 mmol) in saturated sodium bicarbonate (10 mL) and stirred at room temperature for 2 hours. The mixture was extracted with dichloromethane (50 mL x 3), dried, filtered, and concentrated. The title compound (270 mg, 71% yield) was purified by silica gel flash column chromatography to obtain a yellow solid.

[0248] 1 H 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),4.2 0–4.12(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).

[0249] Step 4 and Step 5: Synthesis of 6-(3,4-di(buta-2,3-dienylamido)benzamido)hexanoic acid

[0250] 2-(Trimethylsilyl)ethyl-6-(3,4-di(but-3-ynamido)benzamido)hexanoate (50 mg, 0.1 mmol) was dissolved in TFA / DCM (1:1, 2 mL) and stirred at room temperature for 1 hour. The solvent was concentrated, and the residue was dissolved in 1,4-dioxane (10 mL). K2CO3 (18% aqueous solution, 10 mL) was added. The reaction mixture was heated to 40°C and stirred for 2 hours. Citric acid (60 mL, 10% aqueous solution) and ethyl acetate (500 mL x 3) were then added. The organic phases were combined, dried, filtered, concentrated, and purified on a C18 column to afford the desired product (38 mg, 95.2% yield over two steps) as a yellow solid.

[0251] LC-MS (ESI) m / z: 398 [M+H] + .

[0252] 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),5.6 9(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).

[0253] Example 5: Synthesis of compound LK5

[0254] Step 1: Synthesis of 3,5-bis((tert-butylcarbonyl)amino)methyl)benzoic acid

[0255] Dissolve 3,5-bis(aminomethyl)benzoic acid dihydrochloride (450 mg, 1.78 mmol) and sodium hydroxide (216 mg, 5.4 mmol) in water (1 mL). Add (Boc)2O (1164 mg, 5.3 mmol) dissolved in 1,4-dioxane (16 mL) to the reaction mixture and stir overnight. Then, extract with ethyl acetate (20 mL x 2). HCl is added to the aqueous layer to adjust the pH to 4-5. Extract with ethyl acetate (30 mL x 2), dry over anhydrous sodium sulfate, and concentrate to yield 3,5-bis((tert-butylcarbonyl)amino)methyl)benzoic acid (410 mg, 60.5% yield) as a white solid.

[0256] Characterization data:

[0257] LC-MS (ESI) m / z: 403.2 [M+Na] + .

[0258] 1 H 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).

[0259] Step 2: Synthesis of 2-(trimethylsilyl)ethyl-6-(3,5-di((tert-butylcarbonyl)amino)methyl)benzylamine)hexanoate

[0260] A mixture of 3,5-bis((tert-butylcarbonyl)amino)methylbenzoic acid (200 mg, 0.53 mmol), 2-(trimethylsilyl)ethyl-6-aminohexanoate (120 mg, 0.52 mmol), and HATU (300 mg, 0.79 mmol) was stirred at room temperature for 10 minutes. DIEA (280 μL, 1.62 mmol) was added. The resulting mixture was stirred for 4 hours. Extraction was performed twice with ethyl acetate (30 mL x 2), and the ethyl acetate layer was washed four times with water. After drying over anhydrous sodium sulfate, the mixture was concentrated to yield 290 mg of crude 2-(trimethylsilyl)ethyl-6-(3,5-bis((tert-butylcarbonyl)amino)methyl)benzylamino)hexanoate as a white solid.

[0261] Characterization data:

[0262] LC-MS (ESI) m / z: 394.2 [M+H-200] + .

[0263] 1 H 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).

[0264] Step 3: 2-(Trimethylsilyl)ethyl-6-(3,5-bis(aminomethyl)benzylamine)hexanoate

[0265] To the crude product 2-(trimethylsilyl)ethyl-6-(3,5-bis((tert-butylcarbonyl)amino)methyl)benzyl)hexanoate (290 mg) was added a solution of hydrogen chloride in ethyl acetate (3 mL, 2 M) and allowed to react at room temperature for 30 minutes. The reaction solution was directly purified by preparative liquid phase separation to yield 80 mg of 2-(trimethylsilyl)ethyl-6-(3,5-bis(aminomethyl)benzylamino)hexanoate as an oil.

[0266] Characterization data:

[0267] LC-MS (ESI) m / z: 394.2 [M+H] + .

[0268] 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).

[0269] Step 4: 2-(Trimethylsilyl)ethyl-6-(3,5-bis(3-butynamidomethyl)benzylamine)hexanoate

[0270] 3-Butynoic acid (42 mg, 0.51 mmol) was dissolved in dichloromethane (2 mL), and oxalyl chloride (85 μL, 1.02 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then added to a saturated sodium bicarbonate solution (5 mL) of 2-(trimethylsilyl)ethyl-6-(3,5-bis(aminomethyl)benzylamino)hexanoate (20 mg, 0.051 mmol) and stirred for 2 hours. Dichloromethane (20 mL x 2) was added to the solution for extraction. The dichloromethane layer was dried over anhydrous sodium sulfate and concentrated to yield 28 mg of crude 2-(trimethylsilyl)ethyl-6-(3,5-bis(3-butynamidomethyl)benzylamino)hexanoate as a gray solid.

[0271] Characterization data:

[0272] LC-MS(ESI)m / z:498.2[M+3H-2CH3] + .

[0273] 1 H 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).

[0274] Step 5: 6-(3,5-bis(3-butynylamidomethyl)benzylamine)hexanoic acid

[0275] Crude 2-(trimethylsilyl)ethyl-6-(3,5-bis(3-butynamidomethyl)benzylamino)hexanoate (28 mg) was dissolved in DCM / TFA (1 mL / 1 mL) and reacted for 1 hour. The reaction solution was concentrated to obtain 20 mg of the target compound, 6-(3,5-bis(3-butynamidomethyl)benzylamino)hexanoic acid, as an oil.

[0276] Characterization data:

[0277] LC-MS (ESI) m / z: 426.2 [M+H] + .

[0278] 1 H NMR(400MHz,DMSO-d6)δ8.51-8.43(m,3H),7.60(s,2H),7.28(s,1H),4.30(d,J=4Hz,2H),3.2 4-3.18(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).

[0279] Step 6: 6-(3,5-di(butane-2,3-dienoyl)benzylamine)hexanoic acid

[0280] The compound 6-(3,5-bis(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 x 2). The pH of the aqueous layer was adjusted to 4-5, and the mixture was extracted with ethyl acetate (10 mL x 2). After concentration, 10 mg of the target product, 6-(3,5-bis(butane-2,3-dienoyl)benzylamine)hexanoic acid, was obtained as a white solid.

[0281] Characterization data:

[0282] LC-MS (ESI) m / z: 426.2 [M+H] + .

[0283] 1 H 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).

[0284] Synthesis and characterization of linker-drug conjugates

[0285] Example 6: Synthesis of Compound LP1

[0286] Step 1: Synthesis of LP1-2:

[0287] To a solution of (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)1-oxobutan-2-yl)carbamate (587 mg, 0.770 mmol) in DMF (10 mL) at 0°C was added (S)-N-((3R,4S,4S,5S)-1-(S)-2-((1R,2R)-3-nitrophenoxy)carbonyl -(1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (500 mg, 0.690 mmol), HOBt (211 mg, 1.53 mmol) and DIEA (198 mg, 1.53 mmol). After addition, the mixture was stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give a residue, which was purified by C 18 Purification by HPLC (0-60% ACN in aqueous solution) afforded LP1-2 (600 mg, 64% yield) as a white solid.

[0288] Characterization data:

[0289] LC-MS (ESI) m / z: 1346.8 [M+H] + .

[0290] Step 2: Synthesis of LP1-3:

[0291] At 0°C, 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)-5-ureapentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropyl-2-amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl) To a solution of 3-(methyl)-3-methoxy-5-methyl-1-oxo-4-yl(methyl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate (600 mg, 0.45 mmol) in DMF (5 mL) was added diethylamine (66.0 mg, 0.90 mmol). After the addition was complete, the mixture was stirred at 0°C for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give a residue, which was purified by C 18 Purification (0-45% acetonitrile in H2O) afforded LP1-3 (300 mg, 59% yield) as a white solid.

[0292] Characterization data:

[0293] LC-MS (ESI) m / z: 1123.8 [M+H] + .

[0294] Step 3: Synthesis of LP1:

[0295] To a solution of NH2-VC-MMAE (20 mg, 0.0178 mmol) and LK1 (10 mg, 0.035 mmol) in 1 mL of DMF were added HATU (20.3 mg, 0.0534 mmol) and DIPEA (12 mg, 0.009 mmol). After the addition was complete, the reaction mixture was stirred at room temperature overnight. Purification by preparative HPLC afforded compound LP1 (5 mg, 19.2% yield) as a white solid.

[0296] LC-MS (ESI) m / z: 734 [M / 2+H] + .

[0297] 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,4 H),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).

[0298] Example 7: Synthesis of Compound LP2

[0299] Synthetic LP2:

[0300] LP1-3 (20 mg, 0.0178 mmol) and LK2 (10 mg, 0.035 mmol) were dissolved in DMF (1 mL), followed by the addition of HATU (20.3 mg, 0.0534 mmol) and DIPEA (12 mg, 0.009 mmol). After the addition, the reaction mixture was stirred at room temperature overnight. Purification by preparative HPLC afforded LP2 (3.8 mg, 14.9% yield) as a white solid.

[0301] LC-MS (ESI) m / z: 727 [M / 2+H] +

[0302] Example 8: Synthesis of Compound LP3

[0303] Synthetic LP3:

[0304] 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) and stirred at 0°C for 15 minutes. LP1-3 (30 mg, 0.018 mmol) was added to the solution, and the mixture was stirred at 0°C for 5 hours. After the reaction was complete, the mixture was purified by preparative HPLC to obtain LP3 (4.98 mg, 12.4% yield) as a white solid. LC-MS (ESI) m / z: 752 [M / 2+H] + .

[0305] 1 H 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,1 H), 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).

[0306] Example 9: Synthesis of Compound LP4

[0307] Synthetic LP4:

[0308] 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 the mixture was allowed to react at 0°C for 5 hours. After completion of the reaction, the mixture was purified by preparative HPLC to yield compound LP4 (0.71 mg, 10.6% yield) as a white solid.

[0309] LC-MS (ESI) m / z: 752 [M / 2+H] +

[0310] Example 10: Synthesis of Compound LP5

[0311] Synthetic LP5:

[0312] 6-(3,5-Bis(butyl-2,3-diaminomethyl)benzylamine)hexanoic acid (8 mg, 0.019 mmol), HOBt (6 mg, 0.044 mmol), and EDCI (10 mg, 0.052 mmol) were dissolved in DMF (0.5 mL), and the reaction mixture was stirred at room temperature for 10 minutes. NH2-VC-MMAE (15 mg, 0.013 mmol) was added to the reaction mixture, and stirring was continued for 4 hours. LCMS indicated the desired product, which was purified by preparative HPLC to yield the desired product, LP5 (2.0 mg, 10.0%), as a white solid.

[0313] Characterization data:

[0314] LC-MS (ESI) m / z: 1530.98 [M+H] + .

[0315] 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),2.06-2.0 0(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).

[0316] Example 11: Synthesis of compound LP10

[0317] Step 1: Synthesis of LP10-2:

[0318] To a solution of LP10-1 (88 mg, 0.0828 mmol) in DMF (1.5 mL) was added triethylamine (0.5 mL). After the addition was complete, 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 C 18 Purification by HPLC (0-60% ACN in aqueous solution) afforded LP10-2 (45 mg, 65% yield) as a white solid.

[0319] Characterization data:

[0320] LC-MS (APCI) m / z: 841.20 [M+H] + .

[0321] 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.

[0322] Step 2: Synthesis of LP10:

[0323] To a 2 mL DMF solution of LP10-2 (45 mg, 53.6 mmol) and LK1 (9.9 mg, 27.2 mmol) was added HOBt (6.4 mg, 47.7 mmol) and EDCI (10.4 mg, 54.5 mmol). After the addition was complete, the reaction mixture 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 C 18 Purification by HPLC (0-60% ACN in aqueous solution) afforded LP10 (20 mg, 32% yield) as a white solid.

[0324] Characterization data:

[0325] LC-MS (ESI) m / z: 1186.17 [M+H] + .

[0326] 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).

[0327] Preparation and characterization of antibody-drug conjugates

[0328] The following are examples of methods for preparing and analyzing antibody-drug conjugates. It should be noted that the following examples are merely partial examples and are not limited to the examples described herein. For example, the antibodies described are not limited to the antibodies currently presented but may be any other antibodies.

[0329] General conjugation process for the preparation of bivalently bridged antibody-drug conjugates

[0330] Adjust the antibody solution to a concentration of 5-10 mg / mL and a pH of approximately 7.5. Place the monoclonal antibody (1.0 eq.) in a clean centrifuge tube as the reaction vessel. Add TCEP (5 to 8 eq.) and mix thoroughly. Incubate the tube at 20-25°C on a thermomixer for 2-16 hours to completely reduce the interchain disulfide bonds. Raise the pH to 8.0 by adding an equal volume of 500 mM Tris, pH 8.8 buffer, or replace the antibody with 25 mM Na2B4O7, pH 8.0 buffer via a desalting column. Add the toxin linker solution (5 to 8 eq.) dissolved in DMSO at a concentration of 10 mg / mL. Supplement the reaction with DMSO to 10-20% of the final volume. Vortex the reaction mixture and incubate on a thermomixer with heating and cooling. Allow the reaction to proceed at 25°C for 16-24 hours.

[0331] Use 300 mg / mL dextran-coated activated carbon (manufacturer: Sigma) to remove residual toxin linkers in the reaction solution that are not coupled to the antibody. Add 10% by volume of activated carbon solution to the reaction solution, mix well, incubate at room temperature for 1 hour, centrifuge and take the supernatant to test the residual toxin linker content. If the free toxin linker content is greater than 1%, the above activated carbon treatment process should be repeated multiple times until the test results meet the standard (usually 3 treatments are sufficient). After treatment, the buffer of the desired antibody drug conjugate is replaced with an appropriate storage buffer by 3-6 ultrafiltrations and stored at -80°C.

[0332] General Characterization Methods for Antibody Drug Conjugates

[0333] (a) DAR value of ADC analyzed by HIC-HPLC

[0334] High performance liquid chromatography: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0335] Column: MabPac TM HIC-Butyl 5 μm 4.6×100 mm (manufacturer: Thermo).

[0336] Mobile phase: Mobile phase A (MPA): 1.5M (NH4)2SO4 + 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%.

[0337] 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.

[0338] Experimental steps: Take 50 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0339] DAR value calculation formula: DAR = Σ (relative peak area × number of loaded drugs) / 100

[0340] (b) C18-HPLC analysis of Free Linker Payload (mol / mol%)

[0341] High performance liquid chromatography: e2695 high performance liquid chromatography system.

[0342] Chromatographic column: C18 3.5 μm 4.6×150 mm (manufacturer: Waters).

[0343] Mobile phase: Mobile phase A (MPA): 0.1% TFA-H2O; Mobile phase B (MPB): 0.1% TFA-ACN; Elution was performed according to the following elution program (5%-95%): 0-30 min: 90%-20% mobile phase A, 10%-80% mobile phase B; 30-31 min: 20%-90% mobile phase A, 80%-10% mobile phase B; 31-35 min: 90%-90% mobile phase A, 10%-10% mobile phase B

[0344] 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.

[0345] Reagent I preparation example: 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 at least 1 hour, then let it stand for 1 hour. Remove the supernatant, filter it through a 0.22 μm organic membrane, and store at room temperature. It is valid for 3 months.

[0346] Reagent II preparation example: Measure 100 mL of Reagent I, 15 mL of DMSO, and 85 mL of ADC Sample Storage Buffer, mix well in a container, and store at room temperature. The shelf life is 2 months.

[0347] Experimental steps:

[0348] Sample solution preparation:

[0349] 1) Free toxin reference: Take the toxin corresponding to the test sample as the reference and prepare it with the above reagent II to a final concentration of 1.0 mg / ml.

[0350] 2) Take 10 μl of the 1.0 mg / ml toxin reference sample and add it to 90 μl of Reagent II to prepare a concentration of 100 μg / ml. Then, dilute the sample with Reagent II according to the table below to prepare the sample of the desired concentration.

[0351] Note: Inject the samples prepared above to the required concentration into HPLC in ascending order.

[0352] Sample test: Take 85 μg of the coupled sample and mix it with 3 μl DMSO for 5 minutes. Then take 60 μl of reagent I and add it to the sample system. Mix for 5-10 minutes. Centrifuge at 2000 rpm for 2 minutes. Take 20 μL of the supernatant and inject it into the high performance liquid chromatograph. Elute using the above elution procedure and record the chromatogram.

[0353] According to the calculated standard binary linear regression curve equation, the content of free toxin in ADC was calculated.

[0354] Free Drug (mol / mol%) = residual small molecule molar concentration / antibody molar concentration × 100.

[0355] (c) Determination of free linker-payload content by LC-MS / MS

[0356] Internal reference compounds: 50 ng / ml tolbutamide, labetalol, etofesamide, and buspirone

[0357] Extraction solvent: ethyl acetate

[0358] Serial dilution of the standard: dilute the standard to 20,000, 16,000, 10,000, 4,000, 1,000, 200, 40, and 20 ng / ml with 50% methanol in water.

[0359] Sample pre-treatment: Take 20 μL of sample, then add 20 μL of blank buffer and 240 μL of extraction agent. After vortex mixing, let stand until layers separate. Then, take 160 μL of supernatant, place it in a 96-well plate, air dry, add 200 μL of 50% acetonitrile water, mix thoroughly, and finally perform LC-MS / MS analysis.

[0360] Standard pre-treatment: Add 2 μL of working solution to 38 μL of blank buffer, then add 240 μL of extraction agent. Vortex to mix thoroughly, let stand until layers separate. Transfer 160 μL of supernatant to a 96-well plate, air dry, and add 200 μL of 50% acetonitrile water. Mix thoroughly.

[0361] Mass spectrometry conditions:

[0362] Data processing: 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 by linear fitting the curve (formula) and the mass spectrometry response value of the test sample.

[0363] (d) SEC-HPLC analysis of ADC aggregates

[0364] High performance liquid chromatography: e2695 high performance liquid chromatography system or 1260 Agilent liquid chromatograph.

[0365] Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm)

[0366] Mobile phase: 50 mM PB + 200 mM Arg (pH 6.80) + 10% IPA, elution was performed according to the following elution program, 0-30 min mobile phase A volume was 100%-100%;

[0367] 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.

[0368] Experimental steps: Take 20 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

[0369] Calculation formula: Monomer purity (%) = A monomer / A total × 100%; polymer purity (%) = A polymer / A total × 100%

[0370] (e) Reducing SDS-PAGE analysis of the coupling mode of the ADC linker and the composition of the ADC

[0371] Electrophoresis apparatus: Tianneng EPS-600.

[0372] Acrylamide gel: GenScript M00656

[0373] Imaging system analyzer: Tianneng 4600SF

[0374] Experimental steps: Mix 10 μg of sample with the corresponding loading buffer and heat in a boiling water bath for 5 minutes; add the sample and standard protein (10 μl / well) to 4-20 gradient gel comb wells in sequence, and electrophorese for 60 minutes at a constant voltage of 120 volts; remove the gel, rinse it with deionized water, then add an appropriate amount of Coomassie Brilliant Blue G250 staining solution to cover the gel, and stain it on a shaker for 0.5 hours; rinse the stained gel three times with deionized water, and shake it on a horizontal shaker for 30 minutes each time; remove the destained gel and transfer it to an imager to record the gel image.

[0375] Results analysis: The presence of ≥50 kDa bands indicates that the thiol-bridging coupling is effective (H, L). The 75 kDa band represents the portion consisting of a heavy chain and a light chain connected by a pair of bridging thiol groups (LH). The band formed by two heavy chains connected by one or two pairs of bridging thiol groups is 100 kDa (HH). The 125 kDa band represents that only one heavy chain and light chain are not bridged (LHH). Finally, the 150 kDa band represents that all chains are bridged (LHHL).

[0376] (f) Analysis of the bridged product using LC / TOF (Agilent, 6230LC / TOF) liquid chromatography-mass spectrometry system

[0377] Liquid chromatography column: PLRP-S, 1000A column (Agilent 8μm, 2.1×150mm)

[0378] Mobile phase A was 0.1% formic acid

[0379] Mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 0.4 mL / min.

[0380] Mass spectrometry analysis of positive ions, setting parameters: scanning range 500-5000m / z,

[0381] Gas temperature: 320℃

[0382] VCap: 4500V

[0383] Fragmentor: 200V

[0384] Skimmer: 65V.

[0385] Example 12: Preparation of Antibody Drug Conjugate ADC-1

[0386] n 16 =4.05

[0387] According to the general coupling process for preparing bivalent olefin-bridged antibody-drug conjugates, 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; TCEP was then removed by desalting column and the buffer was replaced with 25 mM Na2B4O7, pH 8.0 buffer, followed by the addition of LP1 (10 mg / mL, 6 eq.) in DMSO, and the coupling reaction was carried out at 25°C for 16 hours. After purification by dextran-coated activated carbon twice, the buffer of the desired antibody-drug conjugate was ultrafiltered three times to obtain ADC-1 (CADC (mg / mL):6.06, V (mL):0.08, yield 48.5%).

[0388] HIC-DAR: 4.05, SEC purity: 99.2%, Free Linker Payload (mol / mol%): not detected.

[0389] DAR 3: Rt = 14.85 min (ratio: 3.28%), DAR 4: Rt = 16.65 min (ratio: 88.58%), DAR 5: Rt = 18.20 min (ratio: 8.15%).

[0390] As can be seen from Figures 1 and 2 , the single-distributed component (DAR4) in the resulting ADC-1 accounts for 88.58%, and aggregates are less than 1%. In addition, Figures 3 and 4 show that the "half-antibody" (DAR4) components formed by light chain-heavy chain bridges and intra-heavy chain bridges are the main components.

[0391] Example 13: Preparation of Antibody Drug Conjugate ADC-2

[0392] n 16 =3.86

[0393] According to the general coupling process for preparing bivalent olefin-bridged antibody drug conjugates, 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 Na2B4O7, 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, followed by a DMSO solution of LP2 (10 mg / mL, 7 eq.), and the coupling reaction was carried out at 25°C for 16 hours. After purification by dextran-coated activated carbon twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-2 (C ADC (mg / mL): 5.2, V (mL): 0.8, yield 41.6%).

[0394] HIC-DAR: 3.86, SEC purity: 93.68%, Free Linker Payload (mol / mol%): not detected.

[0395] DAR 3: Rt = 15.84 min (ratio: 29.79%), DAR 4: Rt = 16.99 min (ratio: 54.92%), DAR 5: Rt = 18.90 min (ratio: 15.29%).

[0396] As can be seen from Figures 5 and 6, 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%.

[0397] Example 14: Preparation of Antibody Drug Conjugate ADC-3

[0398] n 16 =4.1

[0399] According to the general coupling process for preparing bivalent olefin-bridged antibody-drug conjugates, 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; TCEP was then removed by desalting column and the buffer was replaced with 25 mM Na2B4O7, pH 8.0 buffer, followed by the addition of LP3 (10 mg / mL, 5 eq.) in DMSO, and the coupling reaction was carried out at 25°C for 16 hours. After purification by dextran-coated activated carbon twice, the buffer of the desired antibody-drug conjugate was ultrafiltered three times to obtain ADC-3 (C ADC (mg / mL):9.32, V (mL):0.07, yield 65.24%).

[0400] HIC-DAR: 4.1, SEC purity: 98.3%, Free Linker Payload (mol / mol%): not detected.

[0401] DAR 2: Rt = 14.02 min (ratio: 3.52%), DAR 3: Rt = 14.58 min (ratio: 9.03%), DAR 4: Rt = 16.33 min (ratio: 68.5%), DAR 5: Rt = 18.13 min (ratio: 18.93%).

[0402] As can be seen in Figures 7 and 8, the single-distributed component (DAR4) in the resulting ADC-3 accounts for 68.5%, a decrease compared to ADC-1, with aggregates accounting for 1.7%. Furthermore, Figure 9 shows that the "half-antibody" (DAR4) components formed by light chain-heavy chain bridges and intra-heavy chain bridges are the predominant components.

[0403] Example 15: Preparation of Antibody Drug Conjugate ADC-5

[0404] n 16 =3.89

[0405] According to the general coupling process for preparing bivalent olefin-bridged antibody-drug conjugates, 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 Na2B4O7, 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, followed by LP5 (10 mg / mL, 6 eq.) in DMSO, and the coupling reaction was carried out at 25°C for 16 hours. After purification by dextran-coated activated carbon twice, the buffer of the desired antibody-drug conjugate was ultrafiltered three times to obtain ADC-5 (C ADC (mg / mL):8.2, V (mL):0.5, yield 41.0%).

[0406] HIC-DAR:3.89

[0407] DAR 1: Rt = 11.05 min (ratio 0.55%); DAR 2: Rt = 12.80 min (ratio 2.53%); DAR 3: Rt = 15.36 min (ratio 11.87%); DAR 4: Rt = 16.978 mins (ratio 50.24%); DAR 5: Rt = 16.978 min (ratio 29.28%).

[0408] As can be seen from Figures 10 to 12, the proportion of the single distributed component (DAR4) in the obtained ADC-5 is 50.24%, which is lower than that of ADC-1. The aggregate is 1.1%. In addition, the proportion of the "half-antibody" (DAR4) component formed by the bridge coupling between the light chain and the heavy chain and the bridge coupling within the heavy chain is also lower than that of ADC-1.

[0409] Example 16: Preparation of Antibody Drug Conjugate ADC-10

[0410] n 16 =3.98

[0411] According to the general coupling process for preparing antibody drug conjugates, 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.) in DMSO 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%).

[0412] HIC-DAR: 3.98, SEC purity: 100%, Free Linker Payload (mol / mol%): not detected.

[0413] DAR 3: Rt = 12.01 min (ratio: 1.64%), DAR 4: Rt = 13.48 min (ratio: 98.36%), DAR 5: none.

[0414] As can be seen from Figures 13 and 14 , the single-distributed component (DAR4) in the resulting ADC-10 accounts for 98.36%, an increase compared to ADC-1, and no aggregates are observed. Furthermore, Figure 15 shows that the "half-antibody" (DAR4) formed by light chain-heavy chain bridges and intra-heavy chain bridges is the predominant component.

[0415] Example 17: Preparation of Antibody Drug Conjugate ADC-25

[0416] n 16 =3.00

[0417] According to the general coupling process for preparing bivalently bridged antibody drug conjugates, TCEP (10 mmol / L, 6 eq.) aqueous solution was added to 20 mM PBS, 1 mM EDTA, pH 7.4 buffer containing C226S mutation (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 Na2B4O7, 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. After purification by dextran-coated activated carbon twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-25 (C ADC(mg / mL): 8.5, V (mL): 0.07, yield 59.5%). The C226S mutated trastuzumab of the present invention refers to a mutation of the cysteine ​​at position 226 of the Eu numbering of the heavy chain of trastuzumab to serine.

[0418] HIC-DAR: 3.00, SEC purity: 98.1%, Free Linker Payload (mol / mol%): not detected.

[0419] DAR 2: Rt = 12.59 min (ratio: 12.29%), DAR 3: Rt = 14.25 min (ratio: 74.33%), DAR 4: Rt = 16.45 min (ratio: 13.38%).

[0420] As can be seen from Figures 16 and 17, the single distribution component (DAR3) in the obtained ADC-25 accounts for 74.33%, and the aggregates account for 1.9%.

[0421] Example 18: Preparation of Antibody Drug Conjugate ADC-26

[0422] n 16 =2.94

[0423] According to the general coupling process for preparing bivalently bridged antibody drug conjugates, TCEP (10 mmol / L, 6 eq.) aqueous solution was added to 20 mM PBS, 1 mM EDTA, pH 7.4 buffer containing C226S mutation (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 Na2B4O7, 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. After purification by dextran-coated activated carbon twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-26 (C ADC (mg / mL):8.50, V (mL):0.07, yield 59.5%).

[0424] HIC-DAR: 2.94, SEC purity: 96.3%, Free Linker Payload (mol / mol%): not detected.

[0425] DAR 1: Rt = 10.88 min (ratio: 1.95%), DAR 2: Rt = 12.53 min (ratio: 12.32%), DAR 3: Rt = 14.18 min (ratio: 75.13%), DAR 4: Rt = 16.58 min (ratio: 10.61%).

[0426] As can be seen from Figures 19 and 20, the single distribution component (DAR3) in the obtained ADC-26 accounts for 75.13%, and the aggregates account for 3.7%.

[0427] Example 19: Preparation of Antibody Drug Conjugate ADC-27

[0428] n 16 =4.00

[0429] According to the general coupling process for preparing bivalent olefin-bridged antibody-drug conjugates, TCEP (10 mmol / L, 6 eq.) aqueous solution was added to a 20 mM PBS, 1 mM EDTA, pH 7.4 buffer solution of ifinatamab (1.0 mg, 15 mg / mL, 1.0 eq.) and reduced at 22°C for 2 hours; TCEP was then removed by desalting column and the buffer was replaced with a 25 mM Na2B4O7, pH 8.0 buffer solution, followed by the addition of LP10 (10 mg / mL, 6 eq.) in DMSO, and the coupling reaction was carried out at 25°C for 16 hours. After purification by dextran-coated activated carbon twice, the buffer solution of the desired antibody-drug conjugate was ultrafiltered three times to obtain ADC-27 (C ADC (mg / mL): 10.4, V (mL): 0.07, yield 72.8%).

[0430] HIC-DAR: 4, SEC purity: 98.8%, Free Linker Payload (mol / mol%): not detected.

[0431] DAR 4: Rt = 12.90 min (ratio: 100%).

[0432] As can be seen from Figures 22 and 23 , the single distributed component (DAR4) in the resulting ADC-27 accounts for 100%, and aggregates account for 1.2%. In addition, Figures 24 and 25 show that the "half-antibody" (DAR4) components formed by light chain-heavy chain bridging and intra-heavy chain bridging coupling are the main components.

[0433] Example 20: Preparation of Antibody Drug Conjugate Comparative Example-1

[0434] n 16 =7.96

[0435] According to the general coupling process for preparing antibody drug conjugates, 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 the mixture was reduced at 22°C for 2 hours; then, Deruxtecan (10 mg / mL, 10 eq.) 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-1 (C ADC (mg / mL): 6.74, V (mL): 0.6, yield 80.9%).

[0436] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0437] HIC-DAR: 7.96, SEC purity: 97.7%, Free Linker Payload (mol / mol%): 0.04221.

[0438] Example 21: Preparation of Antibody Drug Conjugate Comparative Example-2

[0439] n 16 =4.5

[0440] According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mM, 12 eq.) aqueous solution was added to a 50 mM PBS pH 7.0 buffer solution of Ifinatamab (20 mg, 21.07 mg / mL, 1.0 eq.), and the mixture was reduced at 22°C for 2 hours; then, a DMSO solution of Deruxtecan (10 mg / mL, 10 eq.) 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 solution 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%).

[0441] The following characterization results were obtained using the general characterization method for antibody drug conjugates:

[0442] HIC-DAR: 4.5, SEC purity: 99.92%, Free Linker Payload (mol / mol%): 0.21077.

[0443] Example 22: In vitro plasma stability experiments of ADCs based on different linkers

[0444] This example evaluates the plasma stability of antibody-drug conjugates based on a bis-ene linker and a maleimide linker, and the monitoring indicator is the change in DAR value over time.

[0445] The test antibody-drug conjugate was mixed with human plasma at a final concentration of 0.3 mg / ml and incubated at 37°C. At designated time points (0, 1, 2, 4, 7, and 14 days), 50 μL aliquots were collected and frozen at -80°C. After sampling, the antibody-drug conjugate and total antibody concentrations were determined using ELISA. The method was as follows: First, the ELISA plate wells were coated overnight with the antigen B7-H3 (Sino Biological, 11188-H08H) and anti-Dxd antibody (GenScript, A02217) for determination of total antibody and ADC concentrations, respectively. The next day, the plate was washed three times with 250 μL of PBS / T, followed by the addition of 150 μL of 4% nonfat dry milk and incubation at 37°C for 2 hours. The test sample dilution or standard was then added to the ELISA wells and incubated for another 2 hours. The cells were then washed three times with 250 μL of PBS / T, followed by the addition of 100 μL of anti-human IgG secondary antibody (GenScript, A01855) diluted 1:3000 and incubation at 37°C for 1 hour. The cells were washed three more times with 250 μL of PBS / T, and 50 μL of TMB colorimetric solution was added to each well. The cells were incubated at 37°C for 5 minutes to develop color, and the color development reaction was terminated with 100 μL of stop solution. The absorbance signal at 450 nm was collected and a standard curve was generated. The concentrations of the antibody-drug conjugate and total antibody in the samples were calculated based on the standard curve. The DAR curve is shown in Figure 26.

[0446] As shown in Figure 26, ADC-27, based on a bis-ene linker, is very stable in plasma, with its DAR value remaining essentially unchanged. However, the DAR value of the antibody-drug conjugate based on a maleimide linker (Comparative Example 2) continues to decrease during incubation with plasma, and after two weeks, the DAR value has been reduced by half. Therefore, the bis-ene linker is more stable and has the potential to further improve safety and efficacy.

[0447] Biological evaluation

[0448] Example 23: Tumor cell proliferation inhibition experiment of antibody-drug conjugates based on bivalent olefin covalent bridging

[0449] 1500 NCI-N87 cells were seeded in a 96-well plate (excluding the edge wells filled with PBS), cultured in 80 μL of appropriate culture medium, and placed in a constant temperature cell culture incubator at 37°C and 5% CO2. After the cells were cultured overnight in a constant temperature incubator (37°C, 5% CO2), 20 μL of diluted bis-ene 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 incubating the cells with the conjugate for 5 days, the 96-well plate was placed at room temperature for equilibration for 30 minutes. 40 μL of CellTiter- (Promega, G7572) reagent was reacted in the dark for 10 minutes, and fluorescence was measured using a SpectraMax i3X microplate reader. Graphpad Prism software was used to fit the curve of luminescence value versus conjugate concentration (nM).

[0450] In vitro cytotoxicity of antibody-drug conjugates (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM)

[0451] Most ADCs exhibited certain anti-tumor activity. For example, in NCI-N87 cells, 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.

[0452] The main advantages of the present invention are:

[0453] 1. The present invention provides a new double-ene linker that can be used to conveniently site-couple antibodies based on covalent thiol bridging. It can be applied to most antibodies. Compared with the tedious recombinant modification of antibodies, the method of using the double-ene linker site-coupling site 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.

[0454] 2. The novel bivalent olefin linker provided by the present invention can be covalently bridged and coupled with an antibody by a simple chemical method. Compared with the antibody-drug conjugates generated by traditional random coupling methods, the conjugates obtained using the linker of the present invention have a narrower distribution of DAR values ​​and higher product uniformity. 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.

[0455] 3. The novel biolefin linker provided by the present invention can also be covalently bridged and coupled with most antibody mutants, for example, 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.

[0456] 4. When the antibodies are the same, the antibody-drug conjugates based on the novel bis-ene linker provided by the present invention have comparable in vitro cell killing activity and better in vitro plasma stability than the antibody-drug conjugates based on the maleimide linker.

[0457] The above are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements or improvements within the spirit of the present invention are included in the scope of the claims of the present invention.

Claims

1. A biolefin linker, characterized in that: Including compounds or isotope-labeled substances represented by the general formula (I): Wherein: said L1 is selected from M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1=an integer of 0-10, n2=an integer of 0-10, n3=an integer of 0-10, n4=an integer of 0-10, n5=an integer of 1-10, n6=an integer of 1-10; L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20.

2. The bis-olefin linker according to claim 1, characterized in that: In the general formula (I), The L1 is selected from The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10; The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20.

3. The bis-ene linker according to claim 1, characterized in that: In the general formula (I), 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, n5 is the same as or different from n6; It is a single chiral component or a racemate; n1=an integer from 0 to 10, n2=an integer from 0 to 10, n3=an integer from 0 to 10, n4=an integer from 0 to 10, n5=an integer from 1 to 10, n6=an integer from 1 to 10; L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = an integer from 1 to 20.

4. The bis-olefin linker according to claim 1, characterized in that: In the general formula (I), The L1 is selected from M is selected from The n1 is the same as n2, and n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, and n4=an integer from 0 to 8; L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10.

5. The bis-olefin linker according to claim 1, characterized in that: The biolefin linker in the general formula (I) is selected from at least one of the following compounds:

6. A linker-drug conjugate, characterized in that: The carboxyl group in the biolefin linker according to any one of claims 1 to 5 is A compound formed by bonding the amino group of the amino acid residue of L3 via an amide bond; The L3 is n 14 = integer from 1 to 20, n 15 = 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; wherein D is covalently bonded to L3 via sulfur, nitrogen or oxygen in D.

7. The linker-drug conjugate according to claim 6, characterized in that: 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; and the cytotoxic small molecule drug is a drug for treating tumor diseases.

8. A linker-drug conjugate, characterized in that: The compound comprising the structure shown in the general formula (II): Wherein: said L1 is selected from The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10; The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20; The L3 is n 14 = integer from 1 to 20, n 15 = 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; Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

9. The linker-drug conjugate according to claim 8, characterized in that: In the general formula (II), The L1 is selected from The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10; The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20; The L3 is n 14 = integer from 1 to 20, n 15 = 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; Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

10. The linker-drug conjugate according to claim 8, characterized in that: In the general formula (II), 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, n5 is the same as or different from n6; It is a single chiral component or a racemate; n1=an integer from 0 to 10, n2=an integer from 0 to 10, n3=an integer from 0 to 10, n4=an integer from 0 to 10, n5=an integer from 1 to 10, n6=an integer from 1 to 10; L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20; The L3 is n 14 = integer from 1 to 20, n 15 = 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; Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

11. The linker-drug conjugate according to claim 8, characterized in that: In the general formula (II), The L1 is selected from M is selected from The n1 is the same as n2, and n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, and n4=an integer from 0 to 8; L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10; L3 D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent; Wherein D is covalently bonded to L3 via the sulfur, nitrogen or oxygen in D.

12. The linker-drug conjugate according to claim 8, characterized in that: In the general formula (II), The L1 is selected from M is selected from The n1 is the same as n2, and n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, and n4=an integer from 0 to 8; L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10; L3 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.

13. The linker-drug conjugate according to claim 8, characterized in that: The compound of general formula (II) is selected from at least one of the compounds in the following table:

14. An antibody-drug conjugate, characterized in that: The linker according to any one of claims 8 to 13 is coupled with the bifenal group in the drug conjugate and the thiol group after reduction of Ab to form a conjugate; The Ab is an antibody, an antibody fragment or a protein.

15. An antibody-drug conjugate, characterized in that: Including the structure shown in general formula (III): In the general formula III, the carbon-carbon double bond of the bi-alkenyl group in the general formula II is connected to the thiol group in the reduced antibody Ab by Michael addition reaction to form a CS bond; The L1 is selected from The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10; The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20; The L3 is n 14 = integer from 1 to 20, n 15 = 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, wherein D is covalently bonded to L3 via sulfur, nitrogen or oxygen in D; The 16 Select any value between 1-20; The Ab is an antibody, an antibody fragment or a protein.

16. The antibody-drug conjugate according to claim 15, characterized in that: In the general formula (III), The L1 is selected from The M is selected from n1 and n2 are the same or different, n3 and n4 are the same or different, n5 and n6 are the same or different; n1 = an integer from 0 to 10, n2 = an integer from 0 to 10, n3 = an integer from 0 to 10, n4 = an integer from 0 to 10, n5 = an integer from 1 to 10, n6 = an integer from 1 to 10; The L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20; The L3 is n 14 = integer from 1 to 20, n 15 = integer from 1 to 20; The D is a fragment formed by the loss of one or more atoms or groups of a cytotoxic small molecule drug or active agent, wherein D is covalently bonded to L3 via sulfur, nitrogen or oxygen in D; The 16 Select any value between 1-20; The Ab is an antibody, an antibody fragment or a protein.

17. The antibody-drug conjugate according to claim 15, characterized in that: In the general formula (III), 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, n5 is the same as or different from n6; It is a single chiral component or a racemate; n1 = an integer of 0-10, n2 = an integer of 0-10, n3 = an integer of 0-10, n4 = an integer of 0-10, n5 = an integer of 1-10, n6 = an integer of 1-10; L2 is selected from and One or more combinations of; n7 = integers from 1 to 20, n8 = integers from 1 to 20, n9 = integers from 1 to 20, n 10 = integer from 1 to 20, n 11 = integer from 1 to 20, n 12 = integer from 1 to 20, n 13 = integer from 1 to 20; The L3 is n 14 = integer from 1 to 20, n 15 = 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, wherein D is covalently bonded to L3 via sulfur, nitrogen or oxygen in D; The 16 Select any value between 1-20; The Ab is an antibody, an antibody fragment or a protein.

18. The antibody-drug conjugate according to claim 15, characterized in that: In the general formula (III), The L1 is selected from M is selected from Said 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; L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10; L3 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; The 16 Select any value between 1-20; The Ab is an antibody, an antibody fragment or a protein.

19. The antibody-drug conjugate according to claim 15, characterized in that: In the general formula (III), The L1 is selected from M is selected from The n1 is the same as n2, and n3 is the same as n4; n1=an integer from 0 to 8, n2=an integer from 0 to 8, n3=an integer from 0 to 8, and n4=an integer from 0 to 8; L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10; L3 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; The 16 Select any value between 1-20; 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, lovutuzumab, miratumumab, metuximab, Natuximab, necituzumab, nimotuzumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, pertrastuzumab, pembrolizumab, pertuzumab, pinatuzumab, polotuzumab, ramucirumab, lovatuzumab, tasitumab, setuximab, setuximab, sofetozumab, vadatuximab, worsotuzumab, trastuzumab, anti-B7-H3 monoclonal antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody, anti-CD30 antibody, or mutants of the above antibodies at position 226 or 229 of the heavy chain, antigen-binding fragments thereof, or immunologically active portions thereof.

20. The antibody-drug conjugate according to claim 15, characterized in that: In the general formula (III), The L1 is selected from M is selected from Said 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; L2 is selected from n7=an integer from 1 to 10, n8=an integer from 1 to 10; L3 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; The 16 Select any value between 2-6; The Ab is pertrastuzumab, saccituzumab, metuximab, cetuximab, trastuzumab, anti-B7-H3 monoclonal antibody, cetuximab or a mutant of the above antibodies at position 226 or 229 of the heavy chain.

21. The antibody-drug conjugate according to claim 15, characterized in that: 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 Select any value between 2-6.

22. Use of the antibody drug conjugate according to any one of claims 15 to 21 in treating cancer.

23. Use of the antibody drug conjugate according to claim 22 for treating cancer, wherein the cancer includes lymphoma, blastoma, sarcoma, leukemia or lymphatic malignancy, squamous cell carcinoma, lung cancer, peritoneal cancer, liver cancer, gastric cancer, gastric 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.

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