Linker drug, and preparation method and use of antibody-drug conjugate thereof

Abstract

The invention relates to the field of drugs, in particular to a linker drug conjugate, an antibody drug conjugate, and a preparation method and application thereof. Specifically, the invention relates to a connexon drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt, the connexon drug conjugate has a structure as shown in formula I, M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne fragment, x is a connecting fragment composed of 1-5 amino acids or derivatives thereof, and D is cytotoxin. The linker drug conjugate disclosed by the invention can release Payload through an enzymolysis reaction under the condition that a hydrophobic structure PAB is removed, so that the hydrophobicity of Linker-Rug can be reduced, the content of a polymer generated during preparation of ADC can be reduced, and corresponding cell killing activity (M-X-D formula I) can also be shown.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610174223.2 (22) Application Date 2024.01.29 (66) Domestic Priority Data 202310093383.0 2023.01.31 CN 202310947390.2 2023.07.28 CN (62) Divisional Application Data 202480009220.2 2024.01.29 (71) Applicant Lepu Biotechnology Co., Ltd. Address Room C280, Building 1, No. 1628, Suzhao Road, Minhang District, Shanghai 201114 (72) Inventors Li Hongfeng, Liu Wei, Ma Tian, ​​Liu Wenchao, Wu Xiangyu, Wang Yanchun, Xu Zhenyi, Hu Chaohong, Qin Minmin (74) Patent Agency Beijing Kunrui Law Firm 11494 Patent Attorney Chen An (51) Int.Cl. A61K 47 / 65 (2017.01) A61K 31 / 4745 (2006.01) A61K 47 / 68 (2017.01) A61P 35 / 00 (2006.01) C07D 207 / 452 (2006.01) C07D 491 / 22 (2006.01) C07D 495 / 22 (2006.01) C07C 33 / 16 (2006.01) (54) Invention Title: A Class of Linker Drugs and Preparation Methods and Applications of Their Antibody-Drug Conjugates (57) Abstract: This invention relates to the pharmaceutical field, specifically to linker drug conjugates, antibody drug conjugates, their preparation methods and applications. Specifically, this invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the linker drug conjugate has the structure shown in Formula I, wherein M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne fragment, X is a linker fragment consisting of 1 to 5 amino acids or derivatives thereof, and D is a cytotoxic agent. The linker drug conjugate of this invention can release the payload through enzymatic hydrolysis after removing the hydrophobic PAB structure, thereby reducing the hydrophobicity of the linker-drug and decreasing the polymer content generated during ADC preparation, while also exhibiting corresponding cytotoxic activity, M-X-D Formula I. Claims (5 pages), Description (46 pages), Serial Number (electronic publication) CN 122031713 A 2026.05.15 CN 1 22 03 17 13 A 1. A linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I, M-X-D Formula I Wherein, M is a component containing maleimide (m)The chemical structure of the fragment or cyclooctyne fragment; X is a linker fragment consisting of 1 to 5 amino acids or their derivatives, preferably selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-(2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and their deuterated derivatives; D is a cytotoxin. 2. The linker drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the chemical structure comprising the maleimide (m) fragment is selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), M-(PEG)n, MC-(PEG)n, MCC-(PEG)n, as shown below: Wherein, n represents the degree of polymerization of PEG, preferably an integer between 2 and 12; the chemical structure comprising the cyclooctyne fragment is selected from BCN, DIBO, DIBAC, BARAC, etc., as shown below: Claims 1 / 5 page 2 CN 122031713 A. 3. The linker drug conjugate of any one of claims 1-2, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group; preferably, the hydroxyl (-OH), primary amine, or secondary amine group...Cytotoxins containing (-NH2) or secondary amine (-NHR) groups are selected from camptothecin-based topoisomerase I inhibitors, Daunorubicin-based topoisomerase II inhibitors, calendula derivatives, molecular colloidal compounds, and immunosuppressants; more preferably, the cytotoxins containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups are selected from Exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), belotecone, MMAE and its derivatives, MMAF and its derivatives, lenalidomide, rapamycin and its derivatives; most preferably, the cytotoxins containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups are selected from Exatecan, belotecone, and rapamycin. 4. The linker drug conjugate of any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the linker drug conjugate is MCC-AAQ-Exatecan and has the following structure: Claims 2 / 5, page 3, CN 122031713, A 5. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, said antibody-drug conjugate being formed by conjugating an antibody with a linker drug conjugate as described in any one of claims 1-4; preferably, said antibody-drug conjugate has the structure shown in Formula II, Ab-(L-D)p Formula II Wherein, Ab is an antibody; L is a linker with a structure of M'-X, wherein M' is linked to Ab and X is linked to D; M' is a chemical structure formed by linking M as defined in any one of claims 1-4 to Ab via a maleimide group; X is a linker fragment consisting of 1-5 amino acids or derivatives thereof as defined in any one of claims 1-4; D is a cytotoxin as defined in any one of claims 1-4; p is any value between 2 and 8. 6. The antibody-drug conjugate of claim 5, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody is selected from murine antibodies, rabbit antibodies, phage-display antibodies, yeast-display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies; preferably, the antibody is a monoclonal antibody selected from: HER2 antibodies and EGFR antibodies. 7. A pharmaceutical composition comprising the linker drug conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody-drug conjugate of claim 5 or 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt;Preferably, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapy drug, and an immunosuppressant for treating tumors; preferably, the pharmaceutical composition further comprises at least one pharmaceutical excipient. 8. Use of the linker drug conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody drug conjugate of claim 5 or 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors. 9. A method for preparing the linker drug conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps: (1) preparing a linker fragment X consisting of 1-5 amino acids or derivatives thereof; (2) linking X to a chemical structure M comprising a maleimide (m) fragment or a cyclooctyne fragment to obtain M-X; (3) linking cytotoxin D to M-X to obtain the linker drug conjugate M-X-D. 10. The method of claim 9, wherein the linker drug conjugate is MCC-AAQ-Exatecan, the method comprising the following steps: (1) reacting Fmoc-Gln with Exatecan to obtain intermediate one, wherein the structural formula of intermediate one is as follows: Claims 3 / 5 page 4 CN 122031713 A, (2) removing the Fmoc protecting group from intermediate one via DEA to obtain intermediate two, wherein the structural formula of intermediate two is as follows:, (3) reacting intermediate two with Fmoc-Ala-Ala to obtain intermediate three, wherein the structural formula of intermediate three is as follows:, (4) removing the Fmoc protecting group from intermediate three to obtain intermediate four, wherein the structural formula of intermediate four is as follows: Claims 4 / 5 page 5 CN 122031713 A, (5) condensing intermediate four with MCC to obtain the product, wherein the structural formula of the product is as follows:. Claims 5 / 5 Page 6 CN 122031713 A Method for preparing and applying a class of linker drugs and their antibody-drug conjugates

[0001] This application is a divisional application of Chinese Patent Application No. 202480009220.2, filed on January 29, 2024, entitled "Method for preparing and applying a class of linker drugs and their antibody-drug conjugates".

[0002] Cross-reference to related applications

[0003] This application is based on and claims priority to CN Application No. 202310093383.0, filed on January 31, 2023, and CN Application No. 202310947390.2, filed on July 28, 2023.The contents of this application are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates to the field of pharmaceuticals, specifically to linker drug conjugates, antibody drug conjugates, their preparation methods, and applications. Background Art

[0005] Research on antibody-drug conjugates (ADCs) can be traced back to the 1980s, but it wasn't until 2000 that the first ADC (trade name Mylotarg, developed by Pfizer) was approved by the FDA for the treatment of acute myeloid leukemia. However, due to limitations in conjugation technology, targeting, and efficacy, the complete ADC was unstable in the blood, leading to lethal toxicity, and was withdrawn from the market in 2010. This cast a further shadow over the already uncertain ADC drug research.

[0006] However, Seagen, through improvements to its existing technology, developed a novel antibody-drug conjugate (ADC) of brentuximab vedotin (SGN-35, trade name Adcetris), which was approved by the FDA in 2011 for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. In 2013, ADCs achieved another breakthrough when Ado-trastuzumab emtansine (T-DM1, trade name Kadcyla), jointly developed by Genentech and ImmunoGen, was approved by the FDA for HER2-positive breast cancer, becoming the first ADC targeting solid tumors. With the successful development of these two drugs, ADCs have once again entered the research field with great interest.

[0007] In recent years, ADCs have developed rapidly, with 15 ADC drugs currently approved for marketing and more than 200 ADCs entering clinical trials. ADCs are playing an increasingly important role in the field of targeted cancer therapy.

[0008] Antibody-drug conjugates (ADCs) consist of three parts: antibody (Ab), linker, and small molecule cytotoxic drug (payload / warhead). The antibody portion of an ADC should have tumor specificity, good endocytosis efficiency, good antigen affinity, and be non-immunogenic or low-immunogenic; IgG1 is the most commonly used antibody portion. The warhead portion is generally a cytotoxic drug that acts on microtubules, DNA, or RNA, such as camptothecin, maytansine, or olistatin, and needs to have sufficient toxicity, sufficient water solubility, and its target site is located inside the cell. The linker that connects the antibody and the small molecule cytotoxic drug is required to be stable in the bloodstream, rapidly disintegrate within the cell, and efficiently release the toxin. Summary of the Invention

[0009] The inventors of this application have creatively designed a new linker structure, thereby providing the present invention.

[0010] Linker Drug Conjugate Specification 1 / 46 pages 7 CN122031713 A

[0011] A first aspect of the present invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I,

[0012] M-X-D

[0013] Formula I

[0014] wherein,

[0015] M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragment (such as BCN, DIBO, DIBAC, BARAC, etc.);

[0016] X is a linker fragment consisting of 1 to 5 amino acids or their derivatives, preferably selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala‑Ala‑Ala‑(3‑cyano‑alanine), Ala‑Ala‑(3‑cyano‑alanine), Ala‑(3‑cyano‑ alanine), 3‑cyano‑alanine, Ala‑Ala‑Ala‑Pro, Ala‑Ala‑Pro, Ala‑Pro, Pro, Pro‑Asn, Asn‑ Pro, Lys, Lys‑Asn, Lys‑Pro, Ala‑Ala‑Ala‑Ala‑Gln, Ala‑Ala‑Ala‑Gln, Ala‑Ala‑Gln, Ala‑ Gln, Ala‑Ala‑Ala‑Ala‑(2‑amino‑4‑cyanobutanoic acid), Ala‑Ala‑Ala‑(2‑amino‑4‑ cyanobutanoic acid) acid), Ala‑Ala‑(2‑amino‑4‑cyanobutanoic acid), Ala‑(2‑amino‑4‑ cyanobutanoic acid) (0017) D is a cytotoxin.

[0018] In the linker drug conjugate of the present invention, the linker is composed of a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.) and a linker fragment consisting of 1 to 5 amino acids or their derivatives, and has the following characteristics: stable in blood circulation, can rapidly disintegrate in cells or tumor microenvironment, and efficiently release toxins.

[0019] Containing maleimide (m)The chemical structure of the fragment can be selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), M-(PEG)n, MC-(PEG)n, MCC-(PEG)n, etc., and the structures are shown below:

[0020] Specification 2 / 46 pages 8 CN 122031713 A

[0021] Wherein, n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

[0022] In some embodiments, the chemical structure of the maleimide (m) fragment is 6-maleiminohexanoyl (MC). In some embodiments, the chemical structure of the maleimide (m) fragment is MC-(PEG)n, wherein n is selected from 4, 8, 12. The inventors have found that a lower degree of polymerization of PEG is beneficial to reducing the polymer content (HMW%) in the ADC prepared by the linker drug conjugate to a certain extent; therefore, the degree of polymerization n of PEG is preferably 4.

[0023] The chemical structure containing the cyclooctyne fragment can be selected from BCN, DIBO, DIBAC, BARAC, etc., and the structures are shown below:

[0024] .

[0025] For most currently under-development ADCs, the PAB structure (p-aminobenzyl-carbamoyl) is an essential structure for the effective release of payload as a self-releasing fragment in the linker. However, the hydrophobicity of the PAB structure tends to generate more polymers during ADC preparation. Therefore, further research and development of new linker structures are needed to overcome the above problems.

[0026] To this end, the inventors of this application have discovered through experimental research that if D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group, then the amino acid fragment contained in the linker drug conjugate of this application is linked to a payload containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) structure. This allows the payload to be released via enzymatic hydrolysis after the hydrophobic PAB structure is removed. This reduces the hydrophobicity of the linker-drug, decreases the polymer content generated during ADC preparation, and also exhibits corresponding cell-killing activity.

[0027] Therefore, in some embodiments, D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group.

[0028] In some embodiments, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group may be selected from topoisomerase I inhibitors such as camptothecin compounds and their derivatives (e.g., eczema, 14-aminocamptothecin, 9-aminocamptothecin, belotecone, etc.), doxorubicin compounds and their derivatives (e.g., daunorubicin), etc.The cytotoxic agents include hemimerase II inhibitors, maytansine compounds and their derivatives, calichiomycin compounds and their derivatives, docalimicin compounds (page 3 / 46, CN 122031713 A), DNA alkylating agents such as pyrrolobenzodiazepine compounds and their derivatives (such as PBD), calendulatin compounds and their derivatives (such as MMAE and its derivatives or MMAF and its derivatives), microtubule inhibitors, Protac compounds and molecular gel compounds (such as lenalidomide), rapamycin and its derivatives, and other immunosuppressants, as well as various other small molecule compounds with cytotoxic activity. In some embodiments, the cytotoxic agent containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from eczemacon, belotecane, and rapamycin. In some embodiments, the cytotoxic agent containing a hydroxyl (-OH) group is rapamycin. In some embodiments, the cytotoxic agent containing a secondary amine (-NHR) group is belotecan. In some embodiments, the cytotoxic agent containing a primary amine (-NH2) group is exatecan.

[0029] In some embodiments, the linker fragment composed of the 1 to 5 amino acids or their derivatives in Formula I is selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala‑Ala‑Gln, Ala‑Gln, Ala‑Ala‑Ala‑Ala‑(2‑amino‑4‑cyanobutanoic acid), Ala‑Ala‑ Ala‑(2‑amino‑4‑cyanobutanoic acid), Ala‑Ala‑(2‑amino‑4‑cyanobutanoic acid),Ala- (2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and their deuterated derivatives.

[0030] The amino acid monomers or their derivatives in Formula I, or fragments composed of multiple amino acids or their derivatives, can be recognized by enzymes in the lysosomes of tumor cells and dissociate the carboxyl group in these compounds from the ester or amide bond (peptide bond) formed with compounds containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR), without requiring additional self-cleaving fragments.

[0031] In some embodiments, the linker fragment consisting of the 1 to 5 amino acids or their derivatives in Formula I is selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-L-alanine), Ala-Ala-Ala-(3-cyano-L-alanine), Ala-Ala-(3-cyano-L-alanine), Ala-(3-cyano-L-alanine), 3-cyano-L-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Ala-Gln, Ala- Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Ala-Ala-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), Ala-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), Ala-((S)-2-amino-4-cyanobutanoic acid), (S)-2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln.

[0032] In some embodiments, the linker fragment is selected from linker fragments containing asparagine (Asn), such as Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, Lys-Asn. In some embodiments, the linker fragment is Ala-Ala-Asn (AAN).

[0033] In some embodiments, the linker drug conjugate of the present invention is MCC-AAQ-Exatecan, which has the following structure: Specification 4 / 46 pages 10 CN 122031713 A

[0034] .

[0035] Antibody drug conjugate

[0036] A second aspect of the present invention relates to an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, said antibody drug conjugate being formed by conjugating the linker drug conjugate of the first aspect of the present invention with an antibody.

[0037] In some embodiments, the antibody-drug conjugate has the structure shown in Formula II,

[0038] Ab-(L-D)p

[0039] Formula II

[0040] Wherein,

[0041] Ab is an antibody;

[0042] L is a linker with the structure M'-X, wherein M' is linked to Ab and X is linked to D;

[0043] M' is a chemical structure formed by linking M to Ab via a maleimide group as defined above;

[0044] X is a linker fragment consisting of 1 to 5 amino acids or their derivatives as defined above;

[0045] D is a cytotoxin as defined above;

[0046] p is any value between 2 and 8 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, ... 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or such as 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).

[0047] In Formula II, L-D represents the linker and cytotoxin covalently linked to form an L-D molecule; Ab-(L-D)p represents p L-D molecules covalently coupled to Ab.

[0048] In this invention, the drug-antibody ratio (DAR) refers to the number of drug molecules coupled to the antibody (e.g., p in Formula II). The number of drug molecules contained in the antibody-drug conjugates described herein can be an integer or a decimal. Regardless of...Whether it is an integer or a decimal, it refers to the average number of drug molecules conjugated to each antibody. "p is any value between 2 and 8" means that p can be any integer selected from 2 to 8 (including the endpoints 2 and 8), or any decimal selected from 2 to 8, such as 2.3, 3.9, 4.0, and 4.2. At the same time, those skilled in the art will understand that even using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches are not necessarily exactly the same, for example, they can fluctuate within a range of no more than 0.5.

[0049] The drug-antibody ratio (DAR) can be verified by conventional analytical methods, such as mass spectrometry, ELISA assay, HIC, and HPLC. The quantitative distribution of ADC with respect to p can also be determined. In some cases, the separation, purification, and verification of homogeneous ADCs with a certain p value from ADCs with other drug loads can be achieved by means such as reversed-phase HPLC or electrophoresis.

[0050] In this invention, when the linker drug conjugate is covalently coupled to the antibody Ab, one method is to use the maleimide in the specification (page 5 / 46, CN 122031713 A) to couple with the thiol group in the antibody to form a -S-succinimide structure. For example, when MCC-AAQ-Exatecan is covalently coupled to the Ab, the structural formula of the resulting ADC is as follows:

[0051] .

[0052] In the maleimide linker ADC formed above, -S- can be an external thiol group introduced through engineering modification, or it can be a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0053] Another method is to use cyclooctyne to couple with the azide group (-N3) introduced in the modified antibody to form a triazole structure. For example, when BCN-AAQ-Exatecan is covalently coupled to Ab, the structure of the resulting ADC is as follows:

[0054]

[0055] There are no particular limitations on the antibodies that can be used in this invention, and they can be selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0056] In some embodiments, the antibody is a monoclonal antibody, and is not limited to: HER2 antibody, EGFR antibody.

[0057] In some embodiments, the antibody is a HER2 antibody. In some embodiments, the antibody consists of a light chain and a heavy chain, the light chain comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.As shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0058] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 7. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 8. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0059] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 10. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12. Specification 6 / 46 pages 12 CN 122031713 A

[0060] In some embodiments, the antibody is an EGFR antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0061] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 19. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 20. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 21.

[0062] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 22. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 23. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.

[0063] Information on some sequences involved in this invention is provided in Table 1.

[0064] Table 1: Sequence Description 7 / 46 pages 13 CN 122031713 A

[0065] Description 8 / 46 pages 14 CN 122031713 A

[0066]

[0067] Pharmaceutical Compositions and Pharmaceutical Use

[0068] A third aspect of the invention provides a pharmaceutical composition comprising the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable...Acceptable salt, solvate, or solvate of said salt.

[0069] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant for treating tumors.

[0070] In some embodiments, said chemotherapeutic agent is, for example, adriamycin, cyclophosphamide, taxanes (such as taxol, docetaxel), capecitabine, gemcitabine, vinorelbine, tamoxifen, aromatase inhibitors (renin, fenprox, fenprox), 5-FU plus leucovorin, irinotecan, oxaliplatin, cisplatin, carboplatin, estradiol, Novantrone, prednisone, vincristine, doxorubicin, prednisone, etc., or combinations thereof.

[0071] In some embodiments, the immunotherapeutic agent is, for example, a PD-1 antibody, a PD-L1 antibody, an EGFR monoclonal antibody, a CD20 monoclonal antibody, a HER2 monoclonal antibody (e.g., trastuzumab, Trastuzumab biosimilar, Trastuzumab-dkst), or a combination thereof. Specification 9 / 46 pages 15 CN 122031713 A

[0072] In some embodiments, the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tebufenozide; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prazosin, selenophora, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, imidazolidin, cyclophosphamide, fingolimod, etc.

[0073] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0074] A fourth aspect of the invention provides the use of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors.

[0075] A fifth aspect of the present invention provides a method for treating and / or preventing tumors, comprising: administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0076] The term "tumor" as used in this invention refers to a lesion formed by excessive proliferation of cells in a localized organ or tissue, including hematologic malignancies and solid tumors, including benign and malignant tumors. In some embodiments, the tumor is selected from tumors that are positive for or highly express the following markers: HER2, EGFR.

[0077] In some embodiments, the tumor is selected from: breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, spinal canal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, oral cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer (including squamous cell carcinoma and basal cell carcinoma), hemangioma, glioma, and brain glioma.

[0078] Preparation Method

[0079] A sixth aspect of the present invention provides a method for preparing the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, the method comprising the following steps:

[0080] 1. Preparing a linker fragment X consisting of 1 to 5 amino acids or derivatives thereof;

[0081] 2. Linking X with a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain M-X;

[0082] 3. Linking cytotoxin D with M-X to obtain the linker drug conjugate M-X-D.

[0083] The above preparation method will be further described below using MCC-AAQ-Exatecan as an example. An exemplary preparation process of MCC-AAQ-Exatecan is as follows:

[0084] 1. Reaction of Fmoc-Gln with Exatecan yields intermediate one, wherein the structural formula of intermediate one is as follows: Specification 10 / 46 page 16 CN 122031713 A

[0085]

[0086] 2. Removal of the Fmoc protecting group from intermediate one via DEA yields intermediate two, wherein the structural formula of intermediate two is as follows:

[0087]

[0088] 3. Reaction of intermediate two with Fmoc-Ala-Ala yields intermediate three, wherein the structural formula of intermediate three is as follows:

[0089]

[0090] 4. Removal of the Fmoc protecting group from intermediate three yields intermediate four, wherein the structural formula of intermediate four is as follows: Specification 11 / 46 page 17 CN 122031713 A

[0091]

[0092] 5. The intermediate four is condensed with MCC to obtain the product, wherein the product structure is as follows:

[0093]

[0094] The seventh aspect of the present invention provides a method for preparing the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps:

[0095] 1. Take a certain weight of monoclonal antibody, dilute it with PBS buffer and adjust the antibody pH to about 7.5; detect the protein concentration, weigh the net weight of the antibody solution, and calculate the total protein; add TCEP solution to the antibody, react at room temperature, and continuously mix;

[0096] 2. Add an excess of cytotoxic drug solution to the reduced antibody solution, mix well, and react at room temperature, continuously mixing; after the reaction is completed, add an excess of N-acetylcysteine ​​solution to the reaction solution, react at room temperature, and continuously mix;

[0097] 3. Purify the conjugate product using an ultrafiltration centrifuge tube, replace it in a storage solution, and then filter it with a sterile filter to obtain the antibody-drug conjugate.

[0098] In addition, the present invention also provides the following technical solutions:

[0099] Linker drug conjugate

[0100] The eighth aspect of the present invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I,

[0101] M-X-D

[0102] Formula I

[0103] Wherein, Specification 12 / 46 pages 18 CN 122031713 A

[0104] M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragment (such as BCN, DIBO, DIBAC, BARAC, etc.);

[0105] X is a linker fragment composed of 1 to 4 amino acids, selected from Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln;

[0106] D is a cytotoxin containing a primary amine (-NH2) group.

[0107] In the linker drug conjugate of the present invention, the linker is composed of a linker fragment consisting of a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.) and 1 to 4 amino acids, which has the following characteristics: stable in blood circulation, can rapidly disintegrate in cells or tumor microenvironment, and efficiently release toxins.

[0108] Contains maleimide (m)The chemical structure of the fragment can be selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (mcc), 6-maleiminohexanoyl (mc), m-(PEG)n, mc-(PEG)n, mcc-(PEG)n, etc., and the structures are shown below:

[0109]

[0110] Wherein, n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

[0111] In some embodiments, the chemical structure containing the maleimide (m) fragment is 6-maleiminohexanoyl (mc). In some embodiments, the chemical structure containing the maleimide (m) fragment is mc-(PEG)n, wherein n is selected from 4, 8, 12. The inventors have found that a lower degree of polymerization of PEG is beneficial to reducing the polymer content (HMW%) in the ADC prepared by the linker drug conjugate to a certain extent; therefore, the degree of polymerization n of PEG is preferably 4.

[0112] The chemical structure containing the cyclooctyne fragment can be selected from BCN, DIBO, DIBAC, BARAC, etc., and the structures are shown below: Specification 13 / 46 pages 19 CN 122031713 A

[0113] .

[0114] The linker fragment composed of 1 to 4 amino acids in Formula I is selected from Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln. The above-mentioned amino acid monomers or fragments composed of multiple amino acids can be recognized by specific enzymes in the lysosomes of tumor cells and dissociate the amide bond (peptide bond) formed between the carboxyl group and the compound containing a primary amine in this type of compound, without the need for additional self-immolative moiety.

[0115] In some embodiments, the linker fragment is selected from linker fragments containing asparagine (Asn), such as Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, Lys-Asn. Asparagine (Asn) can be recognized by ligumains, which are not only present in the lysosomes of tumor cells but also widely distributed outside the tumor cells, thus enabling the extracellular release of small molecules to exert a by-stander effect and have a good killing effect on low-endocytosis target cells. In some embodiments, the linker fragment is Ala-Ala-Asn (AAN).

[0116] The primary amine-containingCytotoxins containing the (-NH2) group can be selected from various small molecule compounds with cytotoxic activity, such as topoisomerase I inhibitors (e.g., eczemacin, 14-aminocamptothecin, 9-aminocamptothecin, etc.), topoisomerase II inhibitors (e.g., doxorubicin, maytansine, calichiomycin, docarmicin, and pyrrolobenzodiazepines and their derivatives (e.g., PBD), microtubule inhibitors (e.g., demethyl MMAE or demethyl MMAF, etc.), Protac compounds, and molecular gel compounds (e.g., lenalidomide, etc.). In some embodiments, the cytotoxin containing the primary amine (-NH2) group is eczemacin.

[0117] In some embodiments, the cytotoxic substance containing a primary amine (-NH2) group is selected from Exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), demethyl MMAE, demethyl MMAF, Daunorubicin, and lenalidomide.

[0118] In some embodiments, the linker drug conjugate of the present invention is mc-AAN-Exatecan, which has the following structure:

[0119] .

[0120] Antibody drug conjugate

[0121] The ninth aspect of the present invention relates to an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, said antibody drug conjugate being formed by conjugating the linker drug conjugate of the eighth aspect of the present invention with an antibody.

[0122] In some embodiments, the antibody-drug conjugate has the structure shown in Formula II,

[0123] Ab-(L-D)p

[0124] Formula II

[0125] Wherein,

[0126] Ab is an antibody;

[0127] L is a linker with the structure M'-X, wherein M' is linked to Ab and X is linked to D;

[0128] M' is the chemical structure formed by linking M to Ab via a maleimide group as defined above;

[0129] X is a linker consisting of 1 to 4 amino acids as defined above;

[0130] D is a cytotoxin containing a primary amine (-NH2) group as defined above;

[0131] p is any value between 2 and 8 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, ...4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or such as 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).

[0132] In Formula II, L-D represents the linker and cytotoxin covalently linked to form an L-D molecule; Ab-(L-D)p represents p L-D molecules covalently coupled to Ab.

[0133] In this invention, the drug-antibody ratio (DAR) refers to the number of drug molecules coupled to the antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugates described herein can be an integer or a decimal. Whether integer or decimal, it refers to the average number of drug molecules coupled to each antibody. "p is any value between 2 and 8" means that p can be any integer selected from 2 to 8 (including the endpoints 2 and 8), or any decimal selected from 2 to 8, such as 2.3, 3.9, 4.0, 4.2. At the same time, those skilled in the art will understand that even using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches are not necessarily completely the same, for example, they can fluctuate within a range of no more than 0.5. Instructions for Use, Pages 15 / 46, 21 CN 122031713 A

[0134] The drug-antibody ratio (DAR) can be verified by conventional analytical methods, such as mass spectrometry, ELISA assay, HIC, and HPLC. The quantitative distribution of the ADC in terms of p can also be determined. In some cases, the separation, purification, and verification of homogeneous ADCs with a certain p value from ADCs with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.

[0135] In this invention, when the linker drug conjugate is covalently coupled to the antibody Ab, one method is to form a -S-succinimide structure by coupling maleimide to the thiol group in the antibody. For example, when mc-AAN-Exatecan is covalently coupled to the Ab, the resulting ADC has the following structural formula:

[0136]

[0137] In the maleimide-linked ADC formed above, -S- can be an external thiol group introduced through engineering modification, or it can be a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0138] Another approach is to link the antibody with the chemical structure of cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.), wherein the cyclooctyne is coupled with the azide group (-N3) introduced into the modified antibody to form a triazole structure. For example, when BCN-AAN-Exatecan is covalently coupled to the Ab, the structure of the resulting ADC is shown below:

[0139] .

[0140] There are no particular limitations on the antibodies that can be used in this invention, and they can be selected from mouse antibodies, rabbit antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0141] In some embodiments, the antibody is a monoclonal antibody, and is not limited to: HER2 antibody, EGFR antibody.

[0142] In some embodiments, the antibody is a HER2 antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, the light chain comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0143] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 7. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 8. In some embodiments described on pages 16 / 46 of CN 122031713 A, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0144] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 10. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.

[0145] In some embodiments, the antibody is an EGFR antibody. In some embodiments, the antibody consists of a light chain and a heavy chain, the light chain comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0146] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 19. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 20. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 21.

[0147] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 22. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 23. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.

[0148] Information on some sequences involved in this invention is provided in Table 1 above.

[0149] Pharmaceutical Compositions and Pharmaceutical Uses

[0150] A tenth aspect of the invention provides a pharmaceutical composition comprising the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0151] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant for treating tumors.

[0152] In some embodiments, the chemotherapeutic agent is, for example, adriamycin, cyclophosphamide, taxanes (such as taxol, docetaxel), capecitabine, gemcitabine, vinorelbine, tamoxifen, aromatase inhibitors (renin, fenofibrate, arnoxin), 5-FU plus leucovorin, irinotecan, oxaliplatin, cisplatin, carboplatin, estradiol, Novantrone, prednisone, vincristine, doxorubicin, prednisone, etc., or combinations thereof.

[0153] In some embodiments, the immunotherapeutic agent is, for example, a PD-1 antibody, a PD-L1 antibody, an EGFR monoclonal antibody, a CD20 monoclonal antibody, a HER2 monoclonal antibody (e.g., trastuzumab, Trastuzumab biosimilar, Trastuzumab-dkst), or a combination thereof.

[0154] In some embodiments, the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tebufenozide; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide.Amide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prazosin, selenophora, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, imidazolidin, cyclophosphamide, fingolimod, etc.

[0155] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0156] The eleventh aspect of the present invention provides the use of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors. Specification 17 / 46 pages 23 CN 122031713 A

[0157] The twelfth aspect of the present invention provides a method for treating and / or preventing tumors, comprising: administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0158] The term "tumor" as used in the present invention refers to a lesion formed by excessive proliferation of local cells in an organ or tissue, including hematologic malignancies and solid tumors, including benign tumors and malignant tumors. In some embodiments, the tumor is selected from tumors that are positive for or highly express the following markers: HER2, EGFR.

[0159] In some embodiments, the tumor is selected from: breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, spinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, oral cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer (including squamous cell carcinoma and basal cell carcinoma), hemangioma, glioma, and brain glioma.

[0160] Preparation Method

[0161] The thirteenth aspect of the present invention provides a method for preparing the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps:

[0162] 1. Preparing a linker fragment X consisting of 1 to 4 amino acids;

[0163] 2. Linking X with a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain M-X;

[0164] 3. Adding a primary amine (-NH2) to the linker fragment...The cytotoxic D of the group is linked to M-X to obtain the linker drug conjugate M-X-D.

[0165] The above preparation method will be further explained below using mc-AAN-Exatecan as an example. An exemplary preparation process of mc-AAN-Exatecan is as follows:

[0166] 1. Fmoc-Ala-OH is activated with HOSu and then reacted with L-Ala to obtain intermediate one, wherein the structural formula of intermediate one is as follows:

[0167]

[0168] 2. Intermediate one is activated with HOSu and then reacted with L-Asn to obtain intermediate two, wherein the structural formula of intermediate two is as follows:

[0169] Specification 18 / 46 pages 24 CN 122031713 A

[0170] 3. Intermediate two is subjected to a de-Fmoc reaction to obtain intermediate three, wherein the structural formula of intermediate three is as follows:

[0171]

[0172] 4. Intermediate three is reacted with 6-(maleimide)hexanoic acid succinimide ester to obtain intermediate four, wherein the structural formula of intermediate four is as follows:

[0173]

[0174] 5. The intermediate tetrahydropalmatine (THM) is condensed with Exatecan to obtain the product, wherein the product has the following structural formula:

[0175] .

[0176] The fourteenth aspect of the present invention provides a method for preparing the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, the method comprising the following steps:

[0177] 1. Take a certain weight of monoclonal antibody, dilute it with PBS buffer and adjust the antibody pH to about 7.5; detect the protein concentration, weigh the net weight of the antibody solution, and calculate the total protein; add TCEP solution to the antibody, react at room temperature, and continuously shake well;

[0178] 2. Add an excess of cytotoxic drug solution to the reduced antibody solution, mix well and react at room temperature, continuously mix well; after the reaction is completed, add an excess of N-acetylcysteine ​​solution to the reaction solution, react at room temperature, and continuously mix well;

[0179] 3. Purify the conjugate product using an ultrafiltration centrifuge tube, replace it in a storage solution, and then filter it with a sterile filter to obtain the antibody-drug conjugate.

[0180] Beneficial Effects

[0181] In some embodiments, the linker provided by the present invention can release the payload through enzymatic hydrolysis after removing the hydrophobic PAB structure. This can reduce the hydrophobicity of the linker-drug, reduce the polymer content generated during ADC preparation, and also exhibit corresponding cell-killing activity. Detailed Embodiments

[0182] Terminology Definitions Specification 19 / 46 pages 25 CN 122031713 A

[0183] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand.Please understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the embodiments, they are performed under conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products.

[0184] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used herein are all widely used terms and conventional procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of related terms are provided below.

[0185] In this invention, unless otherwise stated, any numerical range should be understood to include any value within the range or any subrange.

[0186] In this invention, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of identical polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). The light chains of antibodies can be divided into two classes: κ and λ. Heavy chains can be classified into five types: μ, δ, γ, α, and ε. Based on the heavy chain type, antibodies can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids. The heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the C1q component of the complement system. The VH and VL regions can be further subdivided into highly denatured regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form antibody-binding sites. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917;The definition in Chothia et al. (1989) Nature 342:878-883.

[0187] In some embodiments, the drug module is coupled to the antibody in a coupling reaction less than the theoretical maximum. Generally, antibodies do not contain many free and reactive cysteine ​​thiols that can link the drug module; in fact, most cysteine ​​thiols in antibodies are present in the form of disulfide bridges. In some embodiments, antibodies can be reduced under partially or completely reducing conditions with reducing agents such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to produce reactive cysteine ​​thiols.

[0188] In this invention, the term "pharmaceutically acceptable salt" means (i) the salt formed by the acidic functional group present in the couplings provided by this invention and a suitable inorganic or organic cation (base), including but not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenylethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. And (ii) the salts formed by the basic functional groups present in the couplings provided by the present invention and suitable inorganic or organic anions (acids), including but not limited to, hydrohalides, such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, such as benzenesulfonates, p-benzenesulfonic acid salts, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0189] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic substance with a suitable acid providing a pharmaceutically acceptable anion, or by reacting an adequate amount of an acidic substance with a suitable base providing a pharmaceutically acceptable cation.

[0190] In this invention, solvates represent these forms of the antibody-drug conjugates of the invention: complexes in solid or liquid form formed by coordination with solvent molecules. Hydrates are a type of solvate.Specifically, it has coordinated water molecules. In this invention, hydrates are preferred solvates.

[0191] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be obvious to those skilled in the art according to the disclosure of this invention. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed to them at the doses and concentrations used.

[0192] In this invention, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions, and refer to substances that, apart from the active ingredient, have undergone reasonable safety assessments and are included in pharmaceutical preparations. In addition to excipients, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release, and are important components that may affect the quality, safety, and efficacy of pharmaceuticals. According to their source, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their function and use, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. According to their route of administration, they can be divided into oral, injection, mucosal, transdermal or local administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0193] In this invention, the pharmaceutical composition can be formulated into various suitable dosage forms according to the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0194] The term "treatment" as used herein generally refers to obtaining the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or based on the partial or complete stabilization or cure of the disease.And / or side effects resulting from the disease may be therapeutic. As used herein, “treatment” encompasses any treatment of a patient’s disease, including: (a) prevention of disease or symptoms occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its development; or (c) relief of the symptoms of the disease, i.e., causing the disease or symptoms to regress.

[0195] In this invention, “subject” refers to a vertebrate. In some embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, a mammal refers to a human.

[0196] In this invention, “effective amount” refers to the amount that effectively achieves the desired therapeutic or preventative effect at the necessary dose and time. The “therapeutic effective amount” of the substance / molecule of this invention may vary depending on factors such as an individual’s disease state, age, sex, and weight, and the ability of the substance / molecule to elicit the desired response in the individual. Therapeutic effective doses also encompass the amount by which the therapeutic benefit of the substance / molecule outweighs any toxic or harmful consequences. "Prophylactic effective doses" refer to the amount at which the desired preventative effect is effectively achieved at the necessary dose and time. Typically, but not necessarily, the preventative effective dose will be lower than the therapeutic effective dose because the preventative dose is administered to the subject before or early in the course of the disease. In the case of cancer, a therapeutically effective dose of a drug may reduce the number of cancer cells; shrink the tumor volume; inhibit (i.e., slow down, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent.

[0197] In this invention, the 20 common amino acids and their abbreviations follow their usual usage. See Immunology-A Synthesis (2nd edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0198] In this invention, if X is Ala-Ala-Gln, it means that X is a linker fragment composed of Ala, Ala, and Gln sequentially linked to form peptide bonds. Those skilled in the art will understand that it does not limit the specific stereoconfiguration of Ala, Ala, and Gln, such as L-type or D-type. Similarly, if X is Ala-Ala-(3-cyano-alanine), it means that X is a linker fragment composed of Ala, Ala, and (3-cyano-alanine) sequentially linked to form peptide bonds. Those skilled in the art will also understand that it does not limit the specific stereoconfiguration of Ala, Ala, and Gln, such as L-type or D-type.The specific stereochemical configuration of Ala, Ala, and (3-cyano-alanine) is defined, such as L-type or D-type, or S-type or R-type. Other similar limitations of X in this invention are understood with reference to the foregoing.

[0199] In this invention, if X is selected from Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine) and their deuterated derivatives, it means that X can be Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine), a deuterated derivative of Ala-Ala-Gln, or a deuterated derivative of Ala-Ala-(3-cyano-alanine).

[0200] The term "deuterated compound" refers to a compound in which one or more (e.g., 2, 3, 4 or more) hydrogen atoms (the position of the hydrogen atoms is not specifically limited, and can be hydrogen atoms on alkyl groups, amino groups, etc.) are replaced by deuterium atoms.

[0201] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0202] "Humanized" antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody), wherein residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0203] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations may be introduced, for example, by PCR-mediated mutation, the effects of which on antibody binding or other functional properties can be assessed using the in vitro or in vivo tests described herein. Typically, conserved mutations are introduced. Such mutations may be amino acid substitutions, additions, or deletions. Additionally, the mutations within the CDRs typically do not exceed one or two.

[0204] The invention will be further explained below with reference to specific embodiments, but these embodiments are not intended to limit the scope of the invention.

[0205] Example 1: Preparation of the linker drug MCC-AAQ-Exatecan

[0206] 1. Synthesis of intermediate one Fmoc-Gln-Exatecan Specification 22 / 46 pages 28 CN 122031713 A

[0207] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, followed by DMF (1 ml), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.). The reaction was carried out at room temperature for 2 hours, and HPLC monitoring showed no residual starting material. After the reaction, the product was purified by medium-pressure column chromatography (DCM / MeoH). The product was collected, concentrated, and intermediate one was obtained.

[0208] The reaction formula is as follows:

[0209]

[0210] 2. Synthesis of intermediate two Gln-Exatecan

[0211] Intermediate one Fmoc-Gln-Exatecan (130 mg 1.0 eq.) was added to a reaction flask, followed by DCM (2 ml). The mixture was stirred at room temperature. DEA (0.5 ml) was added, and the reaction was carried out at room temperature. After 4 hours, HPLC monitoring showed no residual starting material. Stop the reaction, slowly add MTBE (10 ml), a large amount of solid precipitates, stir for 30 min. Filter, wash with MTBE, get a grayish-white solid, dry, and get 95 mg of intermediate two.

[0212] The reaction formula is as follows:

[0213]

[0214] 3. Synthesis of intermediate three Fmoc-Ala-Ala-Gln-Exatecan

[0215] Add intermediate two Gln-Exatecan (100 mg 1.0 eq.) to the reaction flask, DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg 1.0 eq.), TBTU (68.4 mg 1.2 eq.), DIEA (34.4 mg 1.5 eq.), react at room temperature for 3 hours, monitor with HPLC, and the reaction ends. Purify by column chromatography (DCM / MeOH) under medium pressure, collect the product to get intermediate three.

[0216] The reaction formula is as follows:

[0217]

[0218] 4. Synthesis of intermediate four Ala-Ala-Gln-Exatecan

[0219] Intermediate three Fmoc-Ala-Ala-Gln-Exatecan (140 mg 1.0 eq.) was added to a reaction flask, and DCM (2 ml) was added. The mixture was stirred at room temperature. DEA (0.5 ml) was added, and the reaction was carried out at room temperature. After 4 hours, the reaction was monitored by HPLC and the reaction was completed. MTBE (10 ml) was slowly added, and a large amount of solid precipitated. The mixture was stirred for 30 min. The mixture was filtered, washed with MTBE, and a grayish-white solid was obtained. After drying, intermediate four was obtained.

[0220] The reaction formula is as follows:

[0221]

[0222] 5. Synthesis of product MCC-Ala-Ala-Gln-Exatecan

[0223] Intermediate tetra(Ala-Ala-Gln-Exatecan) (50 mg 1.0 eq.) was added to a reaction flask, followed by DMF (1 mL), MCC (18 mg 1.1 eq), HATU (80.8 mg, 3.0 eq), and DIEA (36.6 mg, 4.0 eq.). The reaction was carried out at room temperature for 2 hours, monitored by HPLC, until no intermediate tetra(Ala-Gln-Exatecan) remained, at which point the reaction was stopped. The product was purified by Prep-HPLC, collected, concentrated to dryness, and the final product was obtained (NMR data are shown below).

[0224] Reaction formula

[0225]

[0226] 1H NMR (400 MHz, DMSO-d6) δ 8.17-8.26 (d, J = 2.0 Hz, 1H), 7.83-7.91 (d, J = 2.0 Hz, 2H), 7.65-7.74 (d, J = 2.0 Hz, 2H), 7.21-7.28 (d, J = 8.0 Hz, 2H), 6.98-7.04 (m, 2H), 5.43-5.50 (m, 1H), 5.36-5.43 (m, 2H), 5.15-5.24 (m, 1H), 5.01-5.11 (m, 1H), 4.14-4.24(m,1H), 3.97-4.08(m,1H), 3.47-3.57(m,1H), 3.20-3.27(m,1H), 3.05-3.16 (m,2H), 2.30-2.36(m,2H), 2.00-2.15(m,4H), 1.77-1.92(m,3H), 1.65-2.00(m,5H), 1.45- 1.64(m,4H), 1.09-1.24(m,5H), 0.95-1.07(m,4H), 0.79-0.94(m,5H).

[0227] Example 2: Preparation of linker drug MCC-Ala-Ala-Asn-Exatecan

[0228] 1. Synthesis of intermediate one

[0229] Fmoc-Ala-Ala-OH (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine) was activated with HOSu (N-hydroxysuccinimide) and then reacted with L-Asn (L-asparagine) to obtain intermediate one.

[0230] The specific steps are as follows: Fmoc-Ala-Ala-OH (1.0 eq), HOSu (1.3 eq), and THF (10 v / w) were added to the reaction flask at room temperature. The temperature was controlled at room temperature, and DCC (1.3 eq) was slowly added under stirring. The reaction was carried out at room temperature and detected by HPLC. When the residual Fmoc-Ala-Ala-OH was small (area normalization method), the reaction solution was filtered, and the filter cake was washed with THF (2.5 v / w). Purified water (11 g / L) was added to the filtrate.0.5 v / w), add L-Asn (1.1 eq), sodium bicarbonate solid (1.0 eq), stir the reaction at room temperature, and detect the Fmoc-Ala-Ala-OSu residue by HPLC. Then add citric acid monohydrate (1.0 eq) and stir. Concentrate the reaction solution to remove most of the solvent. Add DMF (4 v / w) to dissolve the product. After filtration, perform reverse phase preparation through preparative liquid phase. Concentrate the preparation solution until no obvious droplets flow out. Extract with ethyl acetate 4 times. Concentrate the ethyl acetate phase to dryness to obtain intermediate one. The reaction formula is as follows:

[0231]

[0232] 2. Synthesis of intermediate two

[0233] Add DEA (diethylamine) to intermediate one to remove Fmoc to obtain intermediate two.

[0234] The specific steps are as follows: Add intermediate one (1.0 eq) to the reaction flask at room temperature, add DMF (15 v / w), control the temperature at room temperature, add DEA (3 v / w) dropwise, react at room temperature, detect with HPLC until intermediate one is free of residue, concentrate to remove DMF, add DCM (40 v / w), purified water (40 v / w), stir and separate the liquid, concentrate the aqueous phase to dryness to obtain intermediate two, the reaction formula is as follows:

[0235]

[0236] 3. Synthesis of intermediate three

[0237] Intermediate two is reacted with 4-(N-maleimide methyl)cyclohexanecarboxylic acid-N-succinimide ester to obtain intermediate three.

[0238] The specific steps are as follows: At room temperature, add intermediate II (1.0 eq), 4-(N-maleimide methyl)cyclohexanecarboxylic acid-N-succinimide ester (1.1 eq), DMF (22 v / w), water (9.5 v / w), and DIPEA (1.0 eq) to the reaction flask, and then react at room temperature until there is little residue of intermediate II (QDA detection). After the reaction is completed, concentrate to remove most of the solvent, add water (50 v / w), EA (50 v / w), add 1.0 eq DIEA, stir to dissolve, and let stand to separate the liquid. Extract the aqueous phase twice with EA (50 v / w*2), concentrate the aqueous phase to dryness, and obtain intermediate III. The reaction formula is as follows:

[0239]

[0240] 4. Synthesis of the product

[0241] Intermediate III and eczema mesylate (CAS No.: 169869-90-3) are condensed by HATU to obtain intermediate IV.

[0242] The specific steps are as follows: At room temperature, intermediate trioxide (1.2 eq) is added to the reaction flask, followed by DMF (15 v / w), and then eczema mesylate (1.0 eq), TBTU (1.4 eq), and DIPEA (4.5 eq). The reaction is then carried out at room temperature and monitored by HPLC. After the reaction is completed, the solution is purified by prep-HPLC, concentrated, extracted with DCM, and concentrated to obtain the product (NMR is shown below). The reaction formula is as follows:Specification, pages 25 / 46, 31 CN 122031713 A

[0243]

[0244] 1H NMR (400 MHz, DMSO-d6) δ 8.15-8.25 (d, J = 2.0 Hz, 1H), 7.85-8.0 (d, J = 7.5 Hz, 2H), 7.7-7.85 (d, J = 2.0 Hz, 1H), 7.33-7.4 (d, J = 8.0 Hz, 1H), 7.28-7.32 (m, 1H), 6.98-7.05 (d, J = 8.0 Hz, 2H), 6.85-6.95 (m, 1H), 6.45-6.57 (m, 1H), 5.45-5.55 (m,1H), 5.40-5.45(m,1H), 5.20-5.27(m,1H), 4.40-4.50(m,1H), 3.95-4.05(m,1H), 3.20- 3.27(m,2H), 3.10-3.18(m,2H), 2.86-2.97(m,2H), 2.36-2.43(m,3H), 2.15-2.25(m,1H), 1.95-2.10(m,3H), 1.80-1.93(m,3H), 1.73-1.78(m,1H), 1.55-1.70(m,3H), 1.45-1.55(m, 3H), 1.0-1.3 (m, 10H), 0.80-0.95 (m, 5H).

[0245] Example 3: Preparation of linker drug MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan

[0246] 1. Synthesis of intermediate one (3-cyano-L-alanine)-Exatecan

[0247] Fmoc-Asn-OH (40 mg 1.2 eq) was added to the reaction flask, followed by DMF (1.5 ml), TBTU (45 mg 1.5 eq), and DIEA (60 mg 2.5 eq). The mixture was stirred until completely dissolved. After stirring for 30 min, icetane mesylate (50 mg 1.0 eq) was added and stirred for 4 hrs. HPLC detection showed no reaction. TBTU (45 mg, 1.5 eq) and DIEA (60 mg, 2.5 eq) were added to continue the reaction at room temperature. HPLC monitoring showed that no Exatecan remained, and Fmoc-(3-cyano-L-alanine)-Exatecan accounted for approximately 70%. The reaction was stopped, and purification was carried out under medium pressure. The product was collected. It was concentrated to dryness under reduced pressure at 30°C to obtain the product.

[0248] The above product was added to a reaction flask, and DCM (4 ml) was added and stirred. DEA (1 ml) was added, and the mixture was stirred at room temperature to dissolve.Complete. HPLC monitoring showed no raw material residue. 10 ml of MTBE was added and stirred at room temperature, resulting in the precipitation of a large amount of solid. Filtration yielded a grayish-white solid. The filter cake was washed with MTBE and dried to obtain 35 mg of solid, which is intermediate one.

[0249] The reaction formula is as follows:

[0250]

[0251] 2. Synthesis of intermediate two MCC-Ala-Ala-OH

[0252] SMCC (460 mg, 1.1 eq) was added to the reaction flask, followed by NH2-Ala-Ala-OH (200 mg, 1.0 eq), DMF (4 ml, 20 V), and stirring. Then DIPEA (161.4 mg, 1.0 eq) was added. The mixture was heated to 60 °C. As the reaction proceeded, the solution gradually dissolved. The reaction was allowed to proceed for at least 8 hours. LC-MS analysis was performed, and the reaction was stopped. The mixture was then cooled to room temperature. DMF was removed by concentration under reduced pressure at 35°C, followed by the addition of purified water (4 ml, 20 V) and DIPEA (161.4 mg, 1.0 eq). EA (4 ml, 20 V) was added, and the mixture was stirred and washed. The organic phase was discarded. EA (2 ml, 10 V) was added again for extraction twice. The organic phase was discarded. The aqueous phase was retained, and the pH was adjusted to 1-2 with 2M hydrochloric acid, resulting in the precipitation of a large amount of white solid. The mixture was stirred for one hour. The solid was filtered to obtain a white solid. The solid was washed with purified water and dried. 320 mg of white solid, intermediate two, was obtained.

[0253] The reaction formula is as follows:

[0254]

[0255] 3. Product Synthesis MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan

[0256] Intermediate 1 (3-cyano-L-alanine)-Exatecan (35 mg 1.0 eq) was added to a reaction flask, DMF (1 mL), and then intermediate 2 MCC-Ala-Ala-OH (28 mg 1.1 eq), TBTU (25.3 mg 1.2 eq), and DIPEA (12.8 mg 1.5 eq) were added. After reacting at room temperature for 2 hours, HPLC monitoring showed that intermediate 1 was not remaining, so the reaction was stopped. Prep-HPLC purification was performed, and the product was collected. The product was concentrated to dryness. A yellow glassy solid was obtained, which is the product (NMR is shown below).

[0257] The reaction formula is as follows:

[0258]

[0259] 1H NMR (400 MHz, DMSO-d6) δ 8.45-8.55 (d, J = 2.0 Hz, 1H), 8.23-8.29 (d, J = 2Hz, 2H), 7.95-8.03 (d, J = 2.0 Hz, 1H), 7.84-7.92 (d, J = 8.0 Hz, 1H), 7.76-7.84(m,1H), 7.27-7.33(d, J =8.0 Hz,1H), 6.98-7.02(m,1H), 5.47-5.56(m,1H), 5.40-5.45(m,1H), 5.26-5.36(m,1H), 5.1-5.2(m ,1H), 4.44-4.55(m,1H), 4.07-4.19(m,1H), 3.88-4.0(m,2H), 3.19-3.26(m,2H), 3.13-3.18(m,1H), 2.90-3.0(m ,2H),2.76-2.88(m, 2H), 2.36-2.43 (m, 3H), 2.00-2.12 (m, 2H), 1.80-1.93 (m, 3H), 1.80-1.92 (m, 2H), 1.55-1.70 (m, 3H), 1.13-1.26 (m, 5H), 1.05-1.12 (m, 3H), 0.80-0.95 (m, 5H).

[0260] Example 4: Synthetic route of linker drug MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0261] 1. Synthesis of intermediate one ((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0262] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-cyanobutyric acid (79.8 mg 1.1 eq) and icetane mesylate (110 mg 1.0 eq) were added to a reaction flask, followed by DMF (1.5 ml), TBTU (79.73 mg, 1.5 eq), and DIEA (80 mg 2.5 eq). The reaction was carried out at room temperature for 3 hours under HPLC monitoring. After the reaction was completed, 9 ml of ACN was added and stirred at room temperature. Then, 1.0 ml of DEA was added (see page 27 / 46 of the instruction manual, CN 122031713 A), and the reaction was continued at room temperature for 1 hour. Samples were taken for monitoring, and no raw materials remained. 10 ml of MTBE was added. Stirring was performed, and no obvious solid precipitation was observed. Stirring was performed for 1 hour, and no significant change was observed.

[0263] The solvent was removed by concentration under reduced pressure at 30°C. A large amount of solid precipitated during the concentration process, and only a small amount of solvent, about 3 ml, was removed. Stop concentration and continue stirring at room temperature for 1 hour. Filter and wash the solid with MTBE. Dry to obtain a grayish-white solid, i.e., intermediate one.

[0264] The reaction formula is as follows:

[0265]

[0266] 2. Synthesis of intermediate two: MCC-Ala-Ala-OH

[0267] Add SMCC (460 mg, 1.1 eq) to the reaction flask, then add NH2-Ala-Ala-OH (200 mg, 1.0 eq) and DMF (4 ml, 20V). Stir, then add DIPEA (161.4 mg, 1.0 eq). Heat to 60°C and react for 8 hours. Sample and analyze by LC-MS. After the reaction is complete, cool to room temperature. Concentrate under reduced pressure at 35°C to remove DMF, then add purified water (4 ml, 20V) and DIPEA (161.4 mg, 1.0 eq). Add EA (4 ml, 20V) and wash with stirring. Discard the organic phase. Add EA (2 ml, 10V) and wash twice. Discard the organic phase. Retain the aqueous phase and adjust the acidity to pH 1-2 with 2M hydrochloric acid. A large amount of white solid precipitates. Stir for one hour. Filter to obtain a white solid. Wash the solid with purified water. Dry. Obtain 320 mg of white solid, which is intermediate two.

[0268] The reaction formula is as follows:

[0269]

[0270] 3. Product Synthesis MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0271] Intermediate 1 ((S)-2-amino-4-cyanobutanoic acid)-Exatecan (25 mg 1.0 eq) was added to a reaction flask, DMF (1 mL), and then intermediate 2 MCC-Ala-Ala-OH (19 mg , 1.1 eq), TBTU (17.7 mg , 1.2 eq), and DIPEA (8.88 mg , 1.5 eq) were added. After reacting at room temperature for 2 hours, the reaction was monitored by HPLC and the reaction ended. The product was purified by Prep-HPLC and collected. Most of the acetonitrile was removed by concentration. The product was lyophilized to obtain a yellow solid (NMR is shown below). Specification page 28 / 46 34 CN 122031713 A

[0272] The reaction formula is as follows:

[0273]

[0274] 1H NMR (400 MHz, DMSO-d6) δ 8.30-8.37 (d, J =2.0 Hz, 1H), 7.87-7.96 (d, J =2.0 Hz, 2H), 7.75-7.86 (d, J =2.0 Hz, 2H), 7.27-7.33 (d, J =8.0 Hz, 1H), 6.98- 7.02(m,2H), 5.45-5.54(m,1H), 5.40-5.45(m,2H), 5.23-5.32(m,1H), 5.04-5.13(m,1H),4.25–4.37 (m, 1H), 3.97–4.09 (m, 1H), 3.56–3.66 (m, 4H), 3.10–3.26 (m, 4H), 2.35–2.46 (m, 4H), 2.00–2.13 (m, 3H), 1.77–1.92 (m, 3H), 1.66–1.75 (m, 2H), 1.56–1.64 (m, 3H), 1.45–1.53 (m, 1H), 1.12–1.26 (m, 4H), 1.01–1.13 (m, 4H), 0.80–0.94 (m, 4H).

[0275] Example 5: Linker drug MCC-Ala-Ala-Ala-Ala-Gln-Exatecan Synthetic route

[0276] 1. Synthesis of intermediate one MCC-Ala-Ala-Ala-Ala-Gln-OH

[0277] H-Gln(Trt)-OH 2-CTC resin (1.0 g, 0.5 mmol, 0.5 mmol / g) was added to a solid-phase reactor, and DCM (20 mL) was added. The resin was shaken in a shaker for 20 min to fully swell. The resin was filtered and washed three times with DMF. 20 mL of DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol) and HOBT (3.0 eq, 202 mg, 1.5 mmol) was added. After shaking well, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added, and the mixture was reacted in a shaker for 1 hr. The Kaiser test for resin color was negative. The resin was filtered and washed six times with DMF. 20 mL of 20% piperidine / DMF solution was added, and the mixture was reacted in a shaker for 15 min. The mixture was filtered again, and 20 mL of 20% piperidine / DMF solution was added once more. The mixture was reacted in a shaker for 15 min. The Kaiser test for resin color was positive. The resin was washed six times with DMF to obtain H-Ala-Gln(Trt)-2-CTC-resin.

[0278] Add 20 mL of DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol) to a reaction tube containing H-Ala-Gln (Trt)-2-CTC-resin. After shaking well, add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) and react in a shaker for 1 hr.The Kaiser test for resin color was negative. The resin was filtered and washed six times with DMF. 20 mL of 20% piperidine / DMF solution was added, and the mixture was reacted in a shaker for 15 min. The mixture was filtered, and another 20 mL of 20% piperidine / DMF solution was added, and the mixture was reacted in a shaker for 15 min. The Kaiser test for resin color was positive. The resin was washed six times with DMF to obtain H-Ala-Ala-Gln(Trt)-2-CTC- resin.

[0279] To a reaction tube containing H-Ala-Ala-Gln(Trt)-2-CTC-resin, add 20 mL of DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol), shake well, and then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). React in a shaker for 1 hour. The resin color was negative according to Kaiser assay. Filter and wash the resin 6 times with DMF. Add 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min, filter, and add another 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min. The resin color was positive according to Kaiser test. Wash the resin 6 times with DMF to obtain H-Ala-Ala-Ala-Gln (Trt)-2- CTC-resin. Instructions for Use, pages 29 / 46, 35 CN 122031713 A

[0280] To a reaction tube containing H-Ala-Ala-Ala-Gln (Trt)-2-CTC-resin, add 20 mL of DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol), shake well, and then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). React in a shaker for 1 hour. The resin color was negative according to Kaiser assay. Filter and wash the resin 6 times with DMF. Add 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min, filter, and add another 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min. The resin color was positive according to Kaiser assay. Wash the resin 6 times with DMF to obtain H-Ala-Ala-Ala-Ala-Gln (Trt)-2-CTC-resin.

[0281] Add H-Ala-Ala-Ala-Ala-Gln...In a reaction tube containing (Trt)-2-CTC-resin, 20 mL of DMF solution containing SMCC (3.0 eq, 501 mg, 1.5 mmol) was added, and after shaking, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added. The mixture was reacted in a shaker for 1 hour. The resin color was negative according to Kaiser assay. The resin was filtered and washed 6 times with DMF, 3 times with MeOH, and 3 times with MTBE. The resin was dried in a vacuum drying oven at 30°C for 1 hour to obtain MCC-Ala-Ala-Ala-Ala-Gln (Trt)-2-CTC-resin (1.42 g, 0.5 mmol).

[0282] The resin obtained above was transferred to a 50 mL centrifuge tube, and 15 mL of cold 95% TFA / Tis solution was added. The mixture was reacted in a shaker for 1 hour. Filter and slowly add the filtrate to a cold MTBE solution (150 mL), let stand for 1 hour, a white precipitate will precipitate, centrifuge and wash the precipitate 3 times with cold MTBE solution. Collect the washed precipitate and dry it in a vacuum drying oven at 30°C for 4 hours to obtain a white solid, namely intermediate MCC-Ala-Ala-Ala-Ala-Gln-OH.

[0283] The reaction formula is as follows:

[0284]

[0285] 2. Product synthesis MCC-Ala-Ala-Ala-Ala-Gln-Exatecan

[0286] Add intermediate MCC-Ala-Ala-Ala-Ala-Gln-OH (244 mg, 2.0 eq) to a reaction flask, then add DMF (2 mL), iccinotecan sulfonate (100 mg, 1.0 eq), TBTU (132 mg, 2.2 eq), and DIPEA (73 mg, 3.0 eq). The reaction was stirred at room temperature for 1 hour. HPLC analysis showed that 10% of Exatecan remained.

[0287] Prep-HPLC purification was performed, and the product was collected. The solution was concentrated and dried to obtain a yellow solid (NMR data are shown below).

[0288] The reaction formula is as follows:

[0289]

[0290] 1H NMR (400 MHz, DMSO-d6) δ 8.25-8.33 (d, J = 2.0 Hz, 1H), 7.87-7.95 (d, J = 2.0 Hz, 2H), 7.76-7.86 (d, J = 2.0 Hz, 2H), 7.28-7.34 (d, J = 8.0 Hz, 1H), 7.20-7.26 (m, 1H), 6.98-7.02 (m, 1H), 6.73-6.76 (m, 1H), 5.47-5.55 (m, 1H), 5.40-5.41 (m, 1H), (See specification 30 / 46 pages 36 CN)122031713 A 5.26‑5.36(m,2H), 5.05‑5.15 (m,2H), 4.12‑4.20(m,2H), 4.03‑4.09(m,1H), 3.87‑3.94(m, 1H), 3.20-3.27(m,2H), 3.12-3.18(m,1H), 2.65-2.68(m,1H), 2.36-2.43(m,2H), 2.31-2.35 (m,1H), 2.02-2.13(m,2H), 1.78-1.93(m,3H), 1.6-1.76(m,2H), 1.56-1.64(m,2H), 1.21- 1.27 (m, 1H), 1.06–1.20 (m, 6H), 0.85–0.94 (m, 3H).

[0291] Example 6: Synthetic route of linker drug MCC-Ala-Ala-Gln-Belotecan

[0292] Synthetic route of MCC-Ala-Ala-Gln-Belotecan

[0293]

[0294] 1. Synthesis of intermediate 3 Fmoc-Gln-Belotecan

[0295]

[0296] Intermediate 1 (212 mg, 0.575 mmol, 5.0 eq), Belotecan (50 mg, 0.115 mmol, 1.0 eq), HATU (219 mg, 0.575 mmol, 5 eq) were added to a reaction flask, DMF (3 mL) was added and shaken to dissolve, and DIEA (190) was added at 10°C. μL, 1.15 mmol, 10 eq), stirred at 10°C for 1 hr. HPLC detection showed that the Belotecan reaction was complete. The product was prepared, purified, and collected. It was concentrated and lyophilized to obtain a yellow solid product 3 Fmoc-Gln-Belotecan (51 mg, purity: 98.71%, yield: 55.84%). MS (ESI): m / z 784.3 [M+H]+.

[0297] 2. Synthesis of intermediate 4 Gln-Belotecan

[0298]

[0299] Intermediate 3 Fmoc-Gln-Belotecan (51 mg, 0.065 mmol, 1.0 eq) was added to a reaction flask, DMF (4 mL) and diethylamine (1 mL) were added, and the mixture was stirred at 20°C for 2 hr. HPLC detection showed that no raw material remained. The reaction solution was concentrated under vacuum, and DMF (2 mL) was added. After preparative purification, the solution was concentrated and lyophilized to obtain a yellow solid, 4 Gln-Belotecan (24 mg, purity: 91.96%, yield: 60.40%). MS (ESI): m / z 562.34 [M+H]+.

[0300] 3 Intermediate 5Synthesis of MCC-Ala-Ala-OH

[0301] Specification 31 / 46 pages 37 CN 122031713 A

[0302] The raw materials 5-1 MCC-OSu (9.5 g, 59.31 mmol, 1.0 eq) and 5-2 Ala-Ala-OH (21.8 g, 65.24 mmol, 1.1 eq) were added to a reaction flask. DMF (190 mL) was added at 25°C, followed by DIEA (9.8 mL, 59.31 mmol, 1.0 eq). The mixture was stirred at 60°C for 8 hours. The reaction was confirmed to be complete by HPLC. The reaction solution was cooled to 25°C and concentrated under vacuum. Then, H2O (190 mL), DIEA (9.8 mL), and EA (190 mL) were added sequentially. The mixture was stirred at 25°C for 1 hour and then transferred to a separatory funnel for separation. The aqueous phase was separated and extracted twice with EA (95 mL). The EA layer was removed, and 2M hydrochloric acid solution (71.3 mL) was slowly added dropwise to the aqueous phase. After stirring at 25°C for 1 hr, the mixture was filtered. The resulting solid was dried under vacuum to give a white solid 5 MCC-Ala-Ala-OH (16.55 g, purity: 95%, yield: 73.54%). MS (ESI): m / z 380.2 [M+H]+.

[0303] 4. Synthesis of MCC-Ala-Ala-Gln-Belotecan

[0304]

[0305] Intermediate 4 (15 mg, 0.027 mmol, 1.0 eq), 5 MCC-Ala-Ala-OH (15.2 mg, 0.04 mmol, 1.5 eq), and HOAT (7.3 mg, 0.054 mmol, 2.0 eq) were added to a reaction flask. DMF (2 mL) was added and shaken to dissolve the intermediate. DIC (8.0 μL, 0.054 mmol, 2.0 eq) was added at 10°C, and the mixture was stirred at 20°C for 3 hours. HPLC analysis showed that the reaction of intermediate 4 was complete. The product was purified and collected. It was concentrated and lyophilized to obtain a yellow solid product, MCC-Ala-Ala-Gln-Belotecan (11 mg, purity: 96.42%, yield: 43.03%). MS(ESI): m / z 462.5[M+2H] / 2+. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J= 10.4 Hz, 1H), 8.19 (d, J= 8.0 Hz, 1H), 8.15 (d, J= 8.0 Hz, 1H), 7.92-7.87 (m, 4H), 7.78 (t, J= 8.0 Hz, 1H) , 7.35 (s , 1H), 7.21 (brs, 1H), 6.99 (s, 2H), 6.77 (brs, 1H), 5.46‑5.43 (m, 4H), 4.79‑4.76 (m, 1H), 4.35‑4.28 (m, 1H), 4.26‑4.21 (m, 2H) , 3.42‑3.38 (m, 2H) , 3.23 (d, J= 6.8 Hz, 2H) , 3.22‑3.18 (m, 1H) , 2.22‑2.07 (m, 3H) , 1.93‑1.84 (m , 3H) , 1.75‑1.66 (m , 3H), 1.62-1.59 (m, 2H), 1.27-1.26 (m, 4H), 1.23-1.21 (m, 5H), 1.18-1.16 (m, 6H), 0.90-0.86 (m, 6H).

[0307] In addition, those skilled in the art can also obtain other linker drug conjugates of the present invention by referring to the preparation methods of Examples 1 to 6 above, for example, MCC-AAQ-Rapamycin,

[0308] Specification 32 / 46 pages 38 CN 122031713 A

[0309] MCC-AAQ-Rapamycin.

[0310] Example 7: Preparation of EGFR Antibody-Drug Conjugate

[0311] I. Preparation of Antibody-Drug Conjugate EGFR Antibody-MCC-AAQ-Exatecan

[0312] 1. Take EGFR antibody and adjust the antibody pH to about 7.5 with Tris-EDTA solution; use Nanodrop to detect the protein concentration and weigh the net weight of the antibody solution to calculate the total protein. Add TCEP solution to the antibody, place it on a 3D shaker, and react at room temperature for more than 120 min, continuously mixing to completely reduce the disulfide bonds between the antibody chains.

[0313] 2. Add excess MCC-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix well, place it on a 3D shaker, and react at room temperature for more than 30 min, continuously mixing. After the reaction is complete, add excess N-acetylcysteine ​​solution to the reaction solution, place it on a 3D shaker, and react at room temperature for more than 30 min, continuously mixing.

[0314] 3. The conjugation product was purified using a 30KD ultrafiltration centrifuge tube and displaced into a storage solution (10mM Histidine, pH 5.5), with a displacement factor greater than 1000 times. Then, it was filtered through a 0.22µm sterile filter to obtain the antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan, which was stored at 4°C.

[0315] 4. The protein concentration of the antibody-drug conjugate was detected by UV / BCA, DAR was detected by HIC, and purity was detected by SEC.

[0316] II. Preparation of other EGFR antibody-drug conjugates

[0317] The method for preparing antibody-drug conjugates with other linker drug conjugates of the present invention (such as the linker drug conjugate shown in Formula I) and EGFR antibody can refer to the preparation method of the antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan described above.

[0318] Example 8: Preparation of HER2 antibody-drug conjugate

[0319] The method for preparing antibody-drug conjugates (such as the antibody-drug conjugate HER2 antibody-MCC-AAQ-Exatecan) with the linker drug conjugate of the present invention (such as the linker drug conjugate shown in Formula I) and HER2 antibody can refer to the preparation method of the antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan described in Example 7 above.

[0320] Example 9: In vitro activity test of different antibody-drug conjugates

[0321] In vitro activity tests were performed on different antibody-drug conjugates of the present invention to detect their cell-killing activity.

[0322] The test method is as follows:

[0323] Target cells were plated in a certain number into a 96-well black-bottomed cell culture plate. After incubation for 24 hours, samples with gradient dilutions were added. After incubation for another 72 hours or 144 hours, a colorimetric reagent was used for color development for 60 minutes, and the plate was read using a microplate reader. The more viable cells, the higher the signal value. Based on the different signal values ​​obtained at different concentration gradient points, a four-parameter fitting curve was formed on the microplate reader to obtain the C value, i.e., the IC50 value.

[0324] Some data are as follows.

[0325] Table 1 Activity data of MCC-AAQ-Exatecan ADC in MDA-MB-468 cell line Specification 33 / 46 pages 39 CN 122031713 A

[0326]

[0327] In addition, the present invention also provides the following embodiments.

[0328] Example 10: Preparation of linker drug conjugate mc-AAN-Exatecan

[0329] 1. Synthesis of intermediate one

[0330] Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-L-alanine, CAS No.: 35661-39-3) was activated with HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6), and then reacted with L-Ala (L-alanine, CAS No.: 56-41-7) to obtain intermediate one; specifically, Fmoc-Ala-OH (3g, 1.0eq) and HOSu (1.45g, 1.3eq) were added to the reaction flask, and 21mL of [unclear text] was added.THF was used to control the temperature at room temperature. DCC (2.59 g, 1.3 eq) was slowly added under stirring. The reaction was then carried out at room temperature and monitored by HPLC. After the reaction was completed, the reaction solution was filtered and the filter cake was washed with THF (6 mL). Purified water (15 mL) was added to the filtrate, followed by L-Ala (1.12 g, 1.3 eq) and sodium bicarbonate solid (0.81 g, 1.0 eq). The reaction was stirred at room temperature and monitored by HPLC. After the reaction was completed, citric acid (2.02 g, 1.0 eq) was added and stirred. The reaction solution was then extracted with ethyl acetate, the organic phase was concentrated, DMF (12 mL) was added to dissolve the product, and the product was filtered and passed through pre-HPLC. The preparation solution was concentrated until no obvious droplets flowed out, and then extracted three times with ethyl acetate. The ethyl acetate phase was concentrated to dryness to obtain intermediate one 1.75 g. The yield of this step was 57%, and the reaction formula is as follows:

[0331]

[0332] 2. Synthesis of intermediate two

[0333] Intermediate one (N-[fluorenemethoxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) was synthesized by HOSu After activation, N-hydroxysuccinimide (CAS No.: 6066-82-6) was reacted with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain intermediate two. Specifically, intermediate one (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) were added to the reaction flask. The temperature was controlled at room temperature, and DCC (0.61 g, 1.3 eq) was slowly added under stirring. The reaction was carried out at room temperature and monitored by HPLC. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with THF (2 mL). Add purified water (10 mL) to the filtrate, add L-Asn (0.34 g, 1.1 eq), sodium bicarbonate solid (0.19 g, 1.0 eq), stir the reaction at room temperature, monitor with HPLC, after the reaction is completed, add citric acid monohydrate (0.48 g, 1.0 eq) and stir, concentrate the reaction solution to remove most of the solvent, prepare the purified residue, concentrate the prepared solution until no obvious droplets flow out, extract with ethyl acetate, concentrate the organic phase to dryness to obtain intermediate II 826 mg, the yield of this step is 73%, the reaction formula is as follows: Instruction manual 34 / 46 pages 40 CN 122031713 A

[0334]

[0335] 3. Synthesis of intermediate III

[0336] After adding DEA (diethylamine, CAS No.: 109-89-7) to intermediate II to remove Fmoc, intermediate III is obtained; specifically, add intermediate II (100 mg), DMF (1 0.5 mL), control the temperature at room temperature, add DEA (300 μL), and react at room temperature under HPLC monitoring until intermediate II is completely removed. Concentrate to remove DMF, add DCM (4 mL) and purified water (4 mL), stir and separate the layers.The aqueous phase was concentrated to dryness to obtain 92 mg of intermediate three. The yield of this step was 166% (including partial solvent encapsulation). The reaction formula is as follows:

[0337]

[0338] 4. Synthesis of intermediate four

[0339] Intermediate three was reacted with succinimide 6-(maleimide)hexanoate (CAS No.: 55750-63-5) to obtain intermediate four; specifically, intermediate three (92 mg, 1.0 eq), succinimide 6-(maleimide)hexanoate (135 mg, 1.3 eq), DMF (1.5 mL), and DIPEA (0.059 mL, 1.0 eq) were added to the reaction flask. The reaction was monitored by HPLC. After the reaction was completed, the solution was purified and concentrated to obtain 56 mg of intermediate four. The yield of this step was 35.7%. The reaction formula is as follows:

[0340]

[0341] 5. Synthesis of the product

[0342] Intermediate 4 was obtained by condensation reaction with eczemacon mesylate (CAS No.: 169869-90-3). Specifically, at room temperature, intermediate 4 (26 mg, 1.0 eq) was added to a reaction flask, followed by DMF (1.5 mL), then eczemacon mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg, 1.5 eq), HATU (31.8 mg, 1.5 eq), DMAP (0.7 mg, 0.1 eq), and DIPEA (29.2 μL, 3.0 eq), and the reaction was carried out at room temperature. HPLC monitoring was performed. After the reaction was completed, the product was purified and concentrated to obtain 16.5 mg of product (NMR results are shown below). The yield of this step was 33.5%, and the reaction formula is as follows:

[0343]

[0344] 1H NMR (400 MHz, DMSO-d6) δ 8.22-8.29 (d, J =2.0 Hz, 1H), 7.94-8.05 (d, J =2.0 Hz, 3H), 7.83-7.88 (d, J =2.0 Hz, 1H), 7.75-7.82 (d, J =2.0 Hz, 1H), 7.34-7.40 (m, 1H), 7.28-7.33 (m, 1H), 6.96-7.02 (d, J =8.0 Hz, 1H). Hz, 2H), 6.87-6.94 (m, 1H), 5.42-5.55 (m, 1H), 5.41-5.46 (m, 2H), 5.21-5.26 (m, 1H), 4.41-4.49 (m, 1H), 4.00-4.12 (m, 2H), 3.32-3.40 (m, 2H), 3.12-3.17 (m, 2H), 2.86-2.96 (m, 2H), 2.71-2.76 (m, 2H), 2.36-2.43 (m, 2H), (See instruction manual, pages 35 / 46, 41 CN)122031713 A 2H), 2.14-2.24 (m, 1H), 1.97-2.07 (m, 3H), 1.81-1.93 (m, 2H), 1.36-1.52 (m, 4H), 1.05-1.25 (m, 8H), 0.83-0.93 (m, 3H).

[0345] Example 11: Preparation of linker drug conjugate mc-Ala-Ala-Gln-Exatecan

[0346] Synthesis of intermediate one Fmoc-Gln-Exatecan

[0347] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.), Exatecan (100 mg) 1.0 eq.) was added to a reaction flask, followed by DMF (1 ml), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.). The reaction was carried out at room temperature. HPLC monitoring showed no residual starting material. After the reaction was completed, silica gel was added for concentration and the mixture was purified by column chromatography. The product was collected, concentrated to dryness, and 140 mg of product was obtained with a purity of 95% (approximately 4% of the byproduct Hobt of TBTU was not completely removed), yielding 94%.

[0348] The reaction formula is as follows:

[0349]

[0350] 1. Synthesis of intermediate two Gln-Exatecan

[0351] Intermediate one Fmoc-Gln-Exatecan (130 mg 1.0 eq.) was added to a reaction flask, followed by DCM (2 ml). The mixture was stirred at room temperature and could not be completely dissolved. DEA (0.5 ml) was added and the reaction was carried out at room temperature. HPLC monitoring showed no residual starting material. Stop the reaction, slowly add MTBE (10 ml), a large amount of solid precipitates, stir for 30 min. Filter to obtain a grayish-white solid, dry to obtain 95 mg, purity 96%, yield 94%.

[0352] The reaction formula is as follows:

[0353]

[0354] 2. Synthesis of intermediate three Fmoc-Ala-Ala-Gln-Exatecan

[0355] Add intermediate two Gln-Exatecan (100 mg 1.0 eq.) to the reaction flask, DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg 1.0 eq.), TBTU (68.4 mg 1.2 eq.), DIEA (34.4 mg 1.5 eq.), and react at room temperature. HPLC monitoring showed no intermediate two remaining, so stop the reaction. Add silica gel and mix, purify by column chromatography, collect the product to obtain 140 mg, purity 95%, yield 85%.

[0356] The reaction formula is as follows: Specification 36 / 46 pages 42 CN 122031713 A

[0357]

[0358] 3. Synthesis of intermediate four Ala-Ala-Gln-Exatecan

[0359] The intermediate trif-Fmoc-Ala-Ala-Gln-Exatecan (140 mg 1.0 eq.) was added to a reaction flask, along with DCM (2 ml) and DEA (0.5 ml). The reaction was carried out at room temperature. HPLC monitoring showed no residual starting material. The reaction was stopped, and MTBE (10 ml) was slowly added, resulting in the precipitation of a large amount of solid. The mixture was stirred for 30 min. Filtration yielded a grayish-white solid, which was dried to obtain 70 mg of the product. Purity: 96%; Yield: 66%.

[0360] The reaction formula is as follows:

[0361]

[0362] 4. Product Synthesis mc-Ala-Ala-Gln-Exatecan

[0363] Intermediate tetra(Ala-Ala-Gln-Exatecan) (50 mg 1.0 eq.) was added to a reaction flask, DMF (1 mL), and then 6-(maleimide)hexanoic acid succinimide ester (21.8 mg 1.1 eq.) and DIEA (12.1 mg 1.5 eq.) were added. The reaction was carried out at room temperature. HPLC monitoring was performed until intermediate tetra(Ala-Ala-Gln-Exatecan) was completely removed, and the reaction was stopped. The product was purified, collected, concentrated to dryness, and 35 mg of product was obtained (NMR results are shown below). Purity: 98%; Yield: 55%.

[0364] The reaction formula is as follows:

[0365]

[0366] 1H NMR (400 MHz, DMSO-d6) δ 8.28-8.36 (d, J = 2.0 Hz, 1H), 7.90-7.96 (d, J = 2.0 Hz, 2H), 7.75-7.87 (d, J = 2.0 Hz, 2H), 7.27-7.33 (d, J = 2.0 Hz, 1H), 7.20-7.25 (m, 1H), 6.97-7.03 (d, J = 8.0 Hz, 2H), 6.69-6.75 (m, 1H), 6.49-6.53 (m, 1H), 5.46- 5.55(m,1H), 5.40-5.45(m,2H), 5.24-5.34(m,1H), 5.04-5.14(m,1H), 4.31-4.38(m,1H), 4.02-4.23(m,2H), 3.86-3.96(m,1H), 3.40-3.49(m,1H), 3.33-3.40(m,2H), 3.12-3.20(m, 1H), 2.36-2.42(m,2H), 2.15-2.25(m,1H), 2.00-2.12(m,4H), 1.71-1.93(m,3H), 1.36-1.52 (m, 4H), 1.01–1.20 (m, 10H), 0.83–0.92 (m, 3H).

[0367] Example XII: Preparation of linker drug conjugation mc-Pro-Exatecan

[0368] 1. Synthesis of Intermediate 1 Fmoc-Pro-Exatecan Specification 37 / 46 pages 43 CN 122031713 A

[0369] Fmoc-Pro-OH (N-fluorenylmethoxycarbonyl-L-proline) (41 mg 1.3 eq.) and Exatecan (50 mg 1.0 eq.) were added to a reaction flask, and DMF (1 ml) and DIEA (49 mg 4 eq.) were added to dissolve them. HATU (46 mg 1.3 eq.) and HOBt (12.7 mg 1.0 eq.) were added. The reaction was carried out at room temperature and monitored by HPLC. No raw material remained. After the reaction was completed, silica gel was added to concentrate and mix the sample. The product was purified by column chromatography. The product was collected, concentrated to dryness, and 50 mg of product was obtained with a purity of 95% and a yield of 73%.

[0370] The reaction formula is as follows:

[0371]

[0372] 2. Synthesis of intermediate two Pro-Exatecan

[0373] Intermediate one Fmoc-Pro-Exatecan (50 mg 1.0 eq.) was added to a reaction flask, along with DCM (1 ml) and DEA (0.2 ml). The reaction was carried out at room temperature and monitored by HPLC. No raw material remained. The reaction was stopped, and MTBE (10 ml) was slowly added. A large amount of solid precipitated, and the mixture was stirred for 30 min. The mixture was filtered to obtain a grayish-white solid, which was dried to obtain 30 mg of a product with a purity of 96% and a yield of 86%.

[0374] The reaction formula is as follows:

[0375]

[0376] 3. Synthesis of the product mc-Pro-Exatecan

[0377] Intermediate II Pro-Exatecan (30 mg 1.0 eq.) was added to a reaction flask, DMF (1 mL), then 6-(maleimide)hexanoic acid succinimide ester (21.2 mg 1.1 eq.) and DIEA (12.1 mg 1.5 eq.) were added. The reaction was carried out at room temperature and monitored by HPLC until no intermediate II remained. The reaction was then stopped. Silica gel was added and the sample was stirred. The product was purified by column chromatography and 25 mg of product was collected (NMR results are shown below). Purity: 96% Yield: 56%.

[0378] The reaction formula is as follows:

[0379]

[0380] 1H NMR (400 MHz, DMSO-d6) δ 8.17-8.26 (d, J = 2.0 Hz, 1H), 7.80-7.90 (d, J = 2.0 Hz, 2H), 7.21-7.28 (d, J = 8.0 Hz, 1H), 6.98-7.04 (m, 1H), 5.01-5.11 (m,1H), 4.14-4.24(m,1H), 3.97-4.08(m,1H), 3.47-3.57(m,1H), 3.20-3.27(m,1H), 3.05-3.16 (m,2H), 2.30-2.36(m,2H), 2.00-2.15(m,4H), 1.77-1.92(m,3H), 1.65-2.00(m,6H), 1.45- 1.64(m,4H), 1.09-1.24(m,4H), 0.95-1.07(m,2H), 0.79-0.94(m,3H).

[0381] Example 13: Preparation of antibody-conjugate HER2 antibody-mc-AAN-Exatecan (DAR8)

[0382] 1. Take 10 mg of HER2 antibody, dilute it with PBS buffer to a concentration of 5 mg / mL, and adjust the pH of the antibody to about 7.5 with Tris-EDTA solution; detect the protein concentration with Nanodrop, weigh the net weight of the antibody solution, and calculate the total protein. Add 8 molar amounts of TCEP solution to the antibody, place it on a 3D shaker, and react at room temperature for more than 120 min, continuously mixing.

[0383] 2. Add excess mc-AAN-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix well, place it on a 3D shaker, and react at room temperature for more than 30 min, continuously mixing. After the reaction is complete, add excess N-acetylcysteine ​​solution to the reaction solution, place it on a 3D shaker, and react at room temperature for more than 30 min, continuously mixing.

[0384] 3. The conjugate product was purified using a 15 mL 30 KD ultrafiltration centrifuge tube and displaced into a storage solution (10 mM Histidine, pH 5.5), with a displacement factor greater than 1000 times. Then, it was filtered through a 0.22 μm sterile filter to obtain the antibody-drug conjugate HER2 antibody-mc-AAN-Exatecan, which was stored at 4 °C.

[0385] 4. The protein concentration of the antibody-drug conjugate was detected by UV / BCA, DAR was detected by HIC, and purity was detected by SEC.

[0386] Example 14: In vitro activity test of different antibody-drug conjugates

[0387] Referring to the process of Example 13, the linker drug conjugate mc-AAN-Exatecan was conjugated with HER2 antibody, EGFR antibody and non-specific binding antibody (Anti-HEL) to prepare corresponding ADCs, and the linker drug conjugate mc-Pro-Exa was conjugated with HER2 antibody to prepare ADCs, and the cell killing activity was detected.

[0388] The test method is as follows:

[0389] Target cells were plated in a certain quantity into a 96-well black transparent cell culture plate. After 24 hours of incubation, serially diluted samples were added, and after another 72 hours of incubation, a chromogenic reagent was used for 60 minutes of development. The plate was then read using a microplate reader. The higher the number of viable cells, the higher the signal value. Based on the different signal values ​​obtained at different concentration gradient points, a four-parameter fitting curve was generated on the microplate reader to obtain the C value, i.e., the IC50 value.

[0390] Detailed data are as follows.

[0391] Table 2 Activity data of mc-AAN-Exatecan ADC in different cell lines

[0392]

[0393] Table 3 Comparison of activity data of Exatecan ADC and Payload Specification 39 / 46 pages 45 CN 122031713 A

[0394]

[0395] Table 4 Activity data of mc-Pro-Exa ADC in breast cancer cell lines

[0396]

[0397] Example 15: Preparation and detection analysis of ADC using linkers or cytotoxins with different structures

[0398] Referring to the preparation process of Linker-Payload in Example 10 and ADC stock solution in Example 13, the inventors prepared the following ADC stock solution and detected and analyzed the basic quality characteristics of the obtained ADC stock solution.

[0399] After conjugation with the same HER2 antibody, the DAR values ​​were all close to 8. Based on the analysis of the detection data of these ADCs, the following conclusions can be drawn:

[0400] 1. Compared with the ADCs containing PAB fragments, the polymer content (HMW%) of the ADCs prepared by the Linker-Payload without PAB fragments is significantly lower than that of the ADCs prepared by the Linker-Payload containing PAB fragments, indicating that the PAB fragments are not conducive to the preparation of ADCs by this type of Linker-Payload. The specific data are shown in Table 5;

[0401] 2. The Payload containing the primary amine (-NH2) structure can be released through the Linker without the PAB fragment, while the Payload containing the non-primary amine group (-NH-(i-Pr) structure) cannot be released through the Linker without the PAB fragment. The specific data are shown in Table 6.

[0402] The inventors studied the polymer content in ADCs obtained by coupling mc-Pro-PAB-Exatecan with antibody TF (i.e., TF-mAb-H39 in CN201610705557.4) and mc-Pro-Exatecan with HER2 antibody. They found that the PAB structure had a very significant impact. After removing PAB from the linker, the polymer content could be reduced from about 70-80% to 1%.

[0403] Table 5 Analysis data on the effect of PAB fragment on HMW% residual amount Specification 40 / 46 pages 46 CN 122031713 A

[0404]

[0405] In studying the in vitro cytotoxic activity of ADCs conjugated with HER2 antibodies, mc-Pro-Belotecan and mc-Pro-Exatecan, respectively, it was found that the ADC conjugated with mc-Pro-Belotecan had no tumor cell killing activity, indicating that its structure is stable and cannot release the payload through enzymatic digestion. In contrast, the ADC conjugated with mc-Pro-Exatecan exhibited better cytotoxic activity, indicating that this structure can release the payload. Meanwhile, the ADC generated by conjugating mc-Pro-Exatecan with a non-binding antibody (without targeted endocytosis) had no specific cytotoxic activity, indicating that this structure is stable extracellularly and needs to be internalized into the cell before the payload can be released. In addition, by comparing the chemical structures of mc-Pro-Belotecan and mc-Pro-Exatecan, it can be found that the key difference between the two is that Belotecan is linked to Proline with a -NH-(i-Pr) structure, while Exatecan is linked to Proline with a -NH2 structure; therefore, it is inferred that if the amino acid fragment in the Linker is linked to the Payload containing the primary amine (-NH2) structure, the Payload can be released through enzymatic hydrolysis after the hydrophobic structure PAB is removed (for most ADCs currently under development, the PAB structure is an essential structure for effective release of the Payload as a self-releasing fragment); in this way, the hydrophobicity of the Linker-Drug can be reduced, the polymer content generated during ADC preparation can be reduced, and the corresponding cell-killing activity can also be exhibited.

[0406] Table 6 Data on the conjugation analysis of different Linker-Payloads with HER2 antibody

[0407]

[0408] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings already disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

[0409] This invention relates to the following embodiments.

[0410] 1. A linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I, Specification 41 / 46 pages 47 CN 122031713 A

[0411] M-X-D

[0412] Formula I

[0413] Wherein,

[0414] M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne fragment;

[0415] X is a linker fragment consisting of 1 to 5 amino acids or their derivatives, preferably selected from Ala-Ala-Ala-Ala-Asn,Ala‑Ala‑Ala‑Asn, Ala‑Ala‑Asn, Ala‑Asn, Asn, Ala‑Ala‑Ala‑Ala‑(3‑cyano‑alanine), Ala‑Ala‑Ala‑(3‑cyano‑alanine), Ala‑Ala‑(3‑cyano‑alanine), Ala‑(3‑cyano‑ alanine), 3‑cyano‑alanine, Ala‑Ala‑Ala‑Pro, Ala‑Ala‑Pro, Ala‑Pro, Pro, Pro‑Asn, Asn‑ Pro, Lys, Lys‑Asn, Lys‑Pro, Ala‑Ala‑Ala‑Ala‑Gln, Ala‑Ala‑Ala‑Gln, Ala‑Ala‑Gln, Ala‑ Gln, Ala‑Ala‑Ala‑Ala‑(2‑amino‑4‑cyanobutanoic Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-(2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and their deuterated derivatives;

[0416] D is a cytotoxin.

[0417] 2. The linker drug conjugate of Embodiment 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the chemical structure containing the maleimide (m) fragment is selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), M-(PEG)n, MC-(PEG)n, MCC-(PEG)n, and the structures are shown below:

[0418]

[0419] Wherein, n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

[0420] 3. The linker drug conjugate of Embodiment 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the chemical structure containing the cyclooctyne fragment is selected from BCN, DIBO, DIBAC, BARAC, etc., and the structures are shown below: Specification 42 / 46 pages 48 CN 122031713 A

[0421] .

[0422] 4. The linker drug conjugate of any one of embodiments 1-3, or a pharmaceutically acceptable salt, solvate, or thereof.The solvate of the salt, wherein the linker fragment consisting of 1 to 5 amino acids or their derivatives is selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala-Ala-Ala- Gln, Ala‑Gln, Ala‑Ala‑Ala‑Ala‑(2‑amino‑4‑cyanobutanoic acid), Ala‑Ala‑Ala‑(2‑ amino‑4‑cyanobutanoic acid), Ala‑Ala‑(2‑amino‑4‑cyanobutanoic acid), Ala‑(2‑ amino‑4‑cyanobutanoic acid), 2‑amino‑4‑cyanobutanoic acid acid, Gly‑Gly‑Gly‑Gln, Gly‑Gly‑Gln, Gly‑Gln, Gln and their deuterated compounds.

[0423] 5. The linker drug conjugate of any one of embodiments 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group;

[0424] Preferably, the cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from camptothecin-type topoisomerase I inhibitors, Daunorubicin-type topoisomerase II inhibitors, calendula derivatives, molecular colloidal compounds, and immunosuppressants;

[0425] More preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), belotecan, MMAE and its derivatives, MMAF and its derivatives, lenalidomide, rapamycin and its derivatives;

[0426] Most preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from Exatecan, beloteccan, and rapamycin.

[0427] 6. The linker drug conjugate of any one of embodiments 1-5, or a pharmaceutically acceptable salt, solvate, or solvate of the salt described in page 43 / 46 of the specification, CN 122031713 A, wherein the linker drug conjugate is MCC-AAQ-Exatecan, having the following structure:

[0428]

[0429] 7. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the antibody-drug conjugate is formed by conjugating an antibody with a linker drug conjugate as described in any one of embodiments 1-6;

[0430] Preferably, the antibody-drug conjugate has the structure shown in Formula II,

[0431] Ab-(L-D)p

[0432] Formula II

[0433] Wherein,

[0434] Ab is an antibody;

[0435] L is a linker with the structure M'-X, wherein M' is linked to Ab and X is linked to D;

[0436] M' is a chemical structure formed by linking M as defined in any one of embodiments 1-6 to Ab via a maleimide group;

[0437] X is a linker fragment consisting of 1 to 5 amino acids or their derivatives as defined in any one of embodiments 1-6;

[0438] D is a cytotoxic substance as defined in any one of embodiments 1-6;

[0439] p is any value between 2 and 8.

[0440] 8. The antibody-drug conjugate of embodiment 7, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody is selected from murine antibodies, rabbit antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies;

[0441] Preferably, the antibody is a monoclonal antibody, selected from: HER2 antibody and EGFR antibody.

[0442] 9. A pharmaceutical composition comprising the linker drug conjugate of any one of embodiments 1-6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody-drug conjugate of embodiment 6 or 7, or a pharmaceutically acceptable salt, solvate, or solvate of said salt;

[0443] Preferably, the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors;

[0444] Preferably, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0445] 10. The linker drug conjugate described in any one of embodiments 1-6, or a pharmaceutically acceptable salt or solvent thereof.The use of the linker drug conjugate of embodiment 7 or 8, or a pharmaceutically acceptable salt, solvate, or solvate of the salt, in the preparation of a drug for the prevention and / or treatment of tumors.

[0446] 11. A method for preparing the linker drug conjugate of any one of embodiments 1-6, or a pharmaceutically acceptable salt, solvate, or solvate of the salt, the method comprising the following steps:

[0447] 1. Preparing a linker fragment X consisting of 1 to 5 amino acids or derivatives thereof; Specification 44 / 46 pages 50 CN 122031713 A

[0448] 2. Linking X with a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain M-X;

[0449] 3. Linking cytotoxin D with M-X to obtain the linker drug conjugate M-X-D.

[0450] 12. The method of implementation scheme 11, wherein the linker drug conjugate is MCC-AAQ-Exatecan, the method includes the following steps:

[0451] 1. Reacting Fmoc-Gln with Exatecan to obtain intermediate one, wherein the structural formula of intermediate one is as follows:

[0452]

[0453] 2. Deprotecting intermediate one with Fmoc via DEA to obtain intermediate two, wherein the structural formula of intermediate two is as follows:

[0454]

[0455] 3. Reacting intermediate two with Fmoc-Ala-Ala to obtain intermediate three, wherein the structural formula of intermediate three is as follows:

[0456]

[0457] 4. Deprotecting intermediate three with Fmoc to obtain intermediate four, wherein the structural formula of intermediate four is as follows: Specification 45 / 46 pages 51 CN 122031713 A

[0458]

[0459] 5. The intermediate four is condensed with MCC to obtain the product, wherein the product structure is as follows:

[0460] . Specification 46 / 46 pages 52 CN 122031713 A Abstract The present invention relates to the field of pharmaceuticals, and in particular, relates to linker-drug conjugates, antibody-drug conjugates, as well as their preparation methods and applications. Specifically, the present invention relates to a linker-drug conjugate, or apharmaceutically acceptable salt, solvate, or solvate of the salt thereof. The linker-drug conjugate has a structure represented by Formula I, wherein M is a chemical structure containing a maleimide (m) moiety or a cyclooctyne moiety; X is a linking fragment composed of 1 to 5 amino acids or derivatives thereof; and D represents a cytotoxin. The linker-drug conjugate of the present invention can release the payload via enzymatic cleavage without incorporating the hydrophobic p-aminobenzyl (PAB) structure. It can reduce the hydrophobicity of Linker-Drug conjugates, lower the content of polymers generated during the preparation of antibody-drug conjugates (ADCs), and meanwhile retain potent cell-killing activity, M-X-D Formula I.

Claims

1. A linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I, MXD Formula I in, M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne fragment; X is a linker fragment consisting of 1 to 5 amino acids or their derivatives, preferably selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), Ala-(3-cyano-alanine). ine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro ,Ala-Ala-Ala-Ala-Gln,Ala-Ala-Ala-Gln,Ala-Ala-Gln,Ala-Gln,Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid),Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid),Ala-Ala-(2-amino-4-cyanobutanoic acid),Ala-(2-amino-4-cyanobutanoic acid),2-amino-4-cyanobutanoic acid acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and their deuterated compounds; D stands for cytotoxin.

2. The linker drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, The chemical structure containing the maleimide (m) fragment is selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), and M-(PEG). n MC-(PEG) n MCC-(PEG) n The structures are shown below: Wherein, n represents the degree of polymerization of PEG, preferably an integer between 2 and 12; the chemical structure containing the cyclooctylene fragment is selected from BCN, DIBO, DIBAC, BARAC, etc., and the structures are shown below: 。 3. The linker drug conjugate of any one of claims 1-2, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group; Preferably, the cytotoxin containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups is selected from camptothecin-type topoisomerase I inhibitors, Daunorubicin-type topoisomerase II inhibitors, calendula derivatives, molecular colloidal compounds, and immunosuppressants. More preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from Exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), belotecan, MMAE and its derivatives, MMAF and its derivatives, lenalidomide, rapamycin and its derivatives; Most preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from Exatecan, beloteccan, and rapamycin.

4. The linker drug conjugate of any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, The linker drug conjugate is MCC-AAQ-Exatecan, which has the following structure: 。 5. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody-drug conjugate is formed by conjugating an antibody with a linker drug conjugate according to any one of claims 1-4; Preferably, the antibody-drug conjugate has the structure shown in Formula II. Ab-(LD) p Formula II in, Ab represents antibodies; L is a connector with a structure of M'-X, where M' is connected to Ab and X is connected to D; M' is the chemical structure formed by M as defined in any one of claims 1-4 being linked to Ab via a maleimide group; X is a linker segment consisting of 1 to 5 amino acids or their derivatives as defined in any one of claims 1-4; D is a cytotoxic substance as defined in any one of claims 1-4; p is any value between 2 and 8.

6. The antibody-drug conjugate of claim 5, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody is selected from murine antibodies, rabbit antibodies, phage-display-derived antibodies, yeast-display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies; Preferably, the antibody is a monoclonal antibody, selected from: HER2 antibody and EGFR antibody.

7. A pharmaceutical composition comprising the linker drug conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody drug conjugate of claim 5 or 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt. Preferably, the pharmaceutical composition further comprises at least one of a chemotherapy drug, an immunotherapy drug, and an immunosuppressant for treating tumors; Preferably, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

8. Use of the linker drug conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody drug conjugate of claim 5 or 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors.

9. A method for preparing the linker drug conjugate according to any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps: (1). Prepare linker fragment X consisting of 1-5 amino acids or their derivatives; (2). X is connected to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain MX; (3). Cytotoxin D is linked to MX to obtain the linker drug conjugate MXD.

10. The method of claim 9, wherein the linker drug conjugate is MCC-AAQ-Exatecan, the method comprising the following steps: (1). Fmoc-Gln was reacted with Exatecan to obtain intermediate one, the structural formula of which is as follows: ， (2). Intermediate 1 is deprotected from the Fmoc protecting group via DEA to obtain intermediate 2, wherein the structural formula of intermediate 2 is as follows: ， (3). Intermediate 2 is reacted with Fmoc-Ala-Ala to obtain intermediate 3, the structural formula of which is as follows: ， (4). The intermediate ter was deprotected from the Fmoc protecting group to obtain intermediate 4, wherein the structural formula of intermediate 4 is as follows: ， (5). The intermediate four is condensed with MCC to obtain the product, the product structure of which is as follows: 。