Multi-drug link combined drug conjugate and application thereof

Abstract

The invention provides a multi-drug link combined drug conjugate and application thereof. Specifically disclosed is a drug conjugate represented by formula I or a pharmaceutically acceptable salt thereof. According to the drug conjugate provided by the invention, different effective loads can be connected to the same antibody, so that a synergistic effect is achieved or drug resistance can be overcome. # imgabs0 #

Description

[0001] This application claims the priority of Chinese Patent Application No. 2024102309333 with the filing date of February 29, 2024. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

[0002] The present invention relates to a drug conjugate with a multi-drug linkage combination and its application. Background Art

[0003] Cancer has become the second largest health threat globally. For decades, chemotherapy based on cytotoxic drugs has been the main method for treating various cancers. These cytotoxic drugs include DNA base analogs, microtubule protein inhibitors (paclitaxel and vincristine derivatives), etc. However, most of these chemotherapy drugs have a low therapeutic index and serious side effects, which are usually attributed to the non-specific exposure of drugs to off-target tissues.

[0004] Antibody-drug conjugates (ADCs) consist of antibodies, payloads, and linkers, combining the advantages of precise targeting of antibodies and high-efficiency killing of payloads (such as cytotoxic drugs), showing great clinical therapeutic value. So far, multiple ADCs have been approved for marketing or are in the clinical research stage. With the expansion of targets and indications, ADCs are leading a new era of targeted cancer therapy and are expected to become a substitute for traditional chemotherapy in the future.

[0005] Although multiple ADC drugs have been developed so far and their efficacy has been greatly improved, currently, the ADC payload generally uses a single type of small molecule compound directly linked to the antibody through a linker, and there are still problems such as drug resistance, adverse reactions, and insufficient blood stability. Its development still faces various challenges.

[0006] Novel ADCs can combine different types of payloads, connect two different payloads to the same antibody, and can precisely control the ratio of the payloads. Connecting two anti-tumor drugs with different mechanisms to the same antibody has a richer target type, more choices, and different mechanisms of action. To further develop anti-tumor drugs with excellent anti-tumor efficacy and safety, it is of great significance to study ADCs that connect different payloads. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a drug conjugate with a multi-drug linkage combination and its application in order to overcome the problems in the prior art that the structure of antibody-drug conjugates is single and there are problems such as drug resistance and adverse reactions. The drug conjugate with a multi-drug linkage combination can connect different payloads to the same antibody, thereby exerting a synergistic effect or being able to overcome drug resistance.

[0008] The present invention provides a drug conjugate of formula I or a pharmaceutically acceptable salt thereof,

[0009]

[0010] wherein Z is a chemical bond or the a-end is connected to Ab;

[0011] Y is one or more CH 2 independently substituted by Y 1 of -(CH 2 ) p -, wherein Y 1 are each independently -O-, -S-, -NH-, -C(=O)-, -C(=O)NH-, p is 1 - 24;

[0012] U is C 1-6 alkylene or the b-end is connected to CH; R 1a and R 1b are independently C 1-6 alkylene;

[0013] m is 1, 2 or 3;

[0014] When m is 1, X is a chemical bond;

[0015] When m is 2, X is the c-end is connected to a carbonyl group;

[0016] When m is 3, X is the c-end is connected to a carbonyl group;

[0017] W 1 and W 2 are independently a chemical bond or the e-end is connected to L 1 or L 3 ;

[0018] n1, n2, n3, n4, n5, n6, n7, n8, n9, n10 and n11 are independently any integer between 1 and 24;

[0019] L 1 and L 3 are independently a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0020] L 2 and L 4 are independently the f-end is connected to D 1 or D 2 ;

[0021] D1 and D2 are different and are each independently a bioactive molecule fragment, and the bioactive molecule is a cytotoxic drug molecule, a tubulin inhibitor, an ATR inhibitor, a PARP inhibitor or a ChK1 inhibitor;

[0022] q is any value between 1 and 16;

[0023] Ab is an antibody or an antigen-binding fragment thereof.

[0024] The present invention provides a drug conjugate of formula I or a pharmaceutically acceptable salt thereof,

[0025]

[0026] wherein Z is a chemical bond or the a-terminus is linked to Ab;

[0027] Y is one or more CH 2 independently substituted by Y 1 -(CH 2 ) p -, where Y 1 are each independently -O-, -NH-, -C(=O)-, -C(=O)NH-, p is 1 - 24;

[0028] U is C 1-6 alkylene or the b-terminus is linked to CH; R 1a and R 1b are each independently C 1-6 alkylene;

[0029] m is 1, 2 or 3;

[0030] When m is 1, X is a chemical bond;

[0031] When m is 2, X is the c-terminus is linked to a carbonyl group;

[0032] When m is 3, X is the c-terminus is linked to a carbonyl group;

[0033] W 1 and W 2 are each independently a chemical bond or the e-terminus is linked to L 1 or L 3 ;

[0034] n1, n2, n3, n4, n5, n6, n7, n8, n9, n10 and n11 are each independently any integer between 1 and 24;

[0035] L1 and L 3 are independently a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0036] L 2 and L 4 are independently the f-terminus is linked to D 1 or D 2 ;

[0037] D1 and D2 are different and are each independently a bioactive molecular fragment, and the bioactive molecule is a cytotoxic drug molecule, a tubulin inhibitor, an ATR inhibitor, a PARP inhibitor or a ChK1 inhibitor;

[0038] q is any value between 1 and 16;

[0039] Ab is an antibody or an antigen-binding fragment thereof.

[0040] In certain preferred embodiments of the present invention, R 1a and R 1b in which the C 1-6 alkylene group is preferably a C 1-4 alkylene group, such as a methylene group or -CH 2 CH 2 CH 2 CH 2 -.

[0041] In certain preferred embodiments of the present invention, n1, n2, n3, n4, n5, n6, n7, n8, n9, n10 and n11 are each independently any integer between 1 and 6 or between 8 and 16.

[0042] In certain preferred embodiments of the present invention, p is any integer between 4 and 16.

[0043] In certain preferred embodiments of the present invention, n1 is any integer between 1 and 6.

[0044] In certain preferred embodiments of the present invention, Z is a chemical bond or the a-terminus is linked to Ab.

[0045] In certain preferred embodiments of the present invention, Z is

[0046] In certain preferred embodiments of the present invention, Z is a chemical bond, the a-terminus is linked to Ab.

[0047] In certain preferred embodiments of the present invention, n2 is any integer between 8 and 16, such as 12.

[0048] In certain preferred embodiments of the present invention, Y is

[0049] The g end is connected to Z; y1 and y2 are independently any integer between 8 and 16, and y3 is independently any integer between 0 and 6.

[0050] In certain preferred embodiments of the present invention, Y is

[0051] The g end is connected to Z.

[0052] In certain preferred embodiments of the present invention, Y is The g end is connected to Z.

[0053] In certain preferred embodiments of the present invention, Y is

[0054] The g end is connected to Z.

[0055] In certain preferred embodiments of the present invention, U is -CH 2 CH 2 - or The b end is connected to CH.

[0056] In certain preferred embodiments of the present invention, n3, n4, n5 and n6 are independently any integer between 1 and 6.

[0057] In certain preferred embodiments of the present invention, when m is 2, X is The c end is connected to the carbonyl group.

[0058] In certain preferred embodiments of the present invention, n7, n8, n9 and n10 are independently any integer between 1 and 6.

[0059] In certain preferred embodiments of the present invention, when m is 3, X is The c end is connected to the carbonyl group.

[0060] In certain preferred embodiments of the present invention, W 1 and W 2 are independently a chemical bond, The e end is connected to L 1 or L 3 connected.

[0061] In certain preferred embodiments of the present invention, the dipeptide residue is

[0062] The h-terminus is connected to L 2 or L 4 connected.

[0063] In certain preferred embodiments of the present invention, the tripeptide residue is The h-terminus is connected to L 2 or L 4 connected.

[0064] In certain preferred embodiments of the present invention, the tetrapeptide residue is The h-terminus is connected to L 2 or L 4 connected.

[0065] In certain preferred embodiments of the present invention, L 1 and L 3 are independently The h-terminus is connected to L 2 or L 4 connected.

[0066] In certain preferred embodiments of the present invention, D 1 is a topoisomerase I inhibitor fragment, an ATR inhibitor fragment or a PARP inhibitor fragment.

[0067] In certain preferred embodiments of the present invention, D 2 is a topoisomerase I inhibitor fragment, a tubulin inhibitor fragment or an ATR inhibitor fragment.

[0068] In certain preferred embodiments of the present invention, D 1 is

[0069] In certain preferred embodiments of the present invention, D 2 is

[0070] In certain preferred embodiments of the present invention, q is any value between 1 and 8.

[0071] In certain preferred embodiments of the present invention, q is 3.87, 6.70 or 6.74.

[0072] In certain preferred embodiments of the present invention, q is 0.8, 1.3, 1.4, 1.6, 1.7, 1.8 or 2.0.

[0073] In certain preferred embodiments of the present invention, q is 0.8, 1.3, 1.4, 1.6, 1.7, 1.8, 2.0, 3.87, 6.70 or 6.74.

[0074] In certain preferred embodiments of the present invention, Ab is a HER2 antibody, an antigen-binding fragment of a HER2 antibody, or a HER2 antibody with a transpeptidase tag at the C-terminus of the heavy chain.

[0075] In certain preferred embodiments of the present invention, Ab is trastuzumab, trastuzumab with N297A mutation, or trastuzumab with a transpeptidase tag at the C-terminus of the heavy chain.

[0076] In certain preferred embodiments of the present invention, Ab is trastuzumab, trastuzumab with N297A mutation, or trastuzumab with an LPETG tag at the C-terminus of the heavy chain.

[0077] In certain preferred embodiments of the present invention, Ab is trastuzumab with N297Q mutation.

[0078] In certain preferred embodiments of the present invention, Ab is trastuzumab, trastuzumab with N297A mutation, trastuzumab with N297Q mutation, or trastuzumab with an LPETG tag at the C-terminus of the heavy chain.

[0079] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is the drug conjugate represented by Formula I-1,

[0080]

[0081] wherein, Z is a chemical bond or the a-terminus is connected to Ab;

[0082] Y is the g-terminus is connected to Z;

[0083] W 1 and W 2 are independently a chemical bond or the e-terminus is connected to L 1 or L 3 ;

[0084] L 1 and L 3 are independently the h-terminus is connected to L 2 or L 4 ;

[0085] L 2 and L 4 are independently the f-terminus is connected to D1 or D 2 Connect;

[0086] n1 is any integer between 1 and 6;

[0087] n11 is any integer between 8 and 16;

[0088] D1 and D2 are different and are each independently

[0089] q is any value between 1 and 8;

[0090] Ab is a HER2 antibody, an antigen-binding fragment of a HER2 antibody, or a HER2 antibody with a transpeptidase tag at the C-terminus of the heavy chain. In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is the drug conjugate represented by Formula I-1,

[0091]

[0092] wherein, Z is a chemical bond or the a-terminus is connected to Ab;

[0093] Y is the g-terminus is connected to Z;

[0094] W 1 and W 2 are independently a chemical bond or the e-terminus is connected to L 1 or L 3 Connect;

[0095] L 1 and L 3 are independently the h-terminus is connected to L 2 or L 4 Connect;

[0096] L 2 and L 4 are independently the f-terminus is connected to D 1 or D 2 Connect;

[0097] n1 is any integer between 1 and 6;

[0098] n11 is any integer between 8 and 16;

[0099] D1 and D2 are different and are each independently

[0100] q is any value between 1 and 8;

[0101] Ab is a HER2 antibody, an antigen-binding fragment of a HER2 antibody, or a HER2 antibody with a transpeptidase tag at the C-terminus of the heavy chain.

[0102] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is the drug conjugate represented by Formula I-2 or I-3,

[0103]

[0104] wherein Z, Y, W1, W2, L1, L3, L2, L4, D1, D2, q, Ab, n3, n4, n5, n6, n7, n8, n9, and n10 are as described above.

[0105] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is the drug conjugate represented by Formula I-2 or I-3,

[0106]

[0107] wherein Z is a chemical bond or the a-terminus is connected to Ab;

[0108] Y is the g-terminus is connected to Z; y1 and y2 are each independently any integer between 8 and 16, and y3 is independently any integer between 0 and 6;

[0109] W 1 and W 2 are each independently a chemical bond or the e-terminus is connected to L 1 or L 3 ;

[0110] L 1 and L 3 are each independently the h-terminus is connected to L 2 or L 4 ;

[0111] L 2 and L 4 are each independently the f-terminus is connected to D 1 or D 2 ;

[0112] D1 and D2 are different and are each independently

[0113] q is any value between 1 and 16;

[0114] Ab is a HER2 antibody, an antigen-binding fragment of a HER2 antibody, or a HER2 antibody with a transpeptidase tag at the C-terminus of the heavy chain.

[0115] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is any of the following structures:

[0116]

[0117]

[0118]

[0119] Wherein, Her2(N297A) is trastuzumab with N297A mutation, and Her2(HC)-LPET is trastuzumab with a transpeptidase tag at the C-terminus of the heavy chain.

[0120] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is any of the following structures:

[0121]

[0122]

[0123] Wherein, Her2(N297A) is trastuzumab with N297A mutation, and Her2(N297Q) is trastuzumab with N297Q mutation.

[0124] Those skilled in the art can understand that Z is linked to the carboxylic acid or LPETG contained in the antibody itself. For example, in the above drug conjugate, the carbonyl group is formed after the carboxylic acid contained in the antibody itself is linked to Z.

[0125] Those skilled in the art can understand that Z is linked to the carboxylic acid, LPETG or sulfhydryl group contained in the antibody itself. For example, in the above drug conjugate, the carbonyl group is formed after the carboxylic acid contained in the antibody itself is linked to Z.

[0126] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is any of the following structures:

[0127]

[0128]

[0129] Wherein, Her2(HC)-LPET is trastuzumab with a transpeptidase tag at the C-terminus of the heavy chain.

[0130] In certain preferred embodiments of the present invention, the drug conjugate represented by Formula I is any of the following structures:

[0131]

[0132]

[0133]

[0134] Wherein, Her2(N297A) is trastuzumab with N297A mutation, and Her2(N297Q) is trastuzumab with N297Q mutation.

[0135] The present invention provides a pharmaceutical composition comprising the drug conjugate represented by Formula I as described above or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

[0136] The present invention provides the use of the drug conjugate represented by Formula I as described above or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described above, in the preparation of a drug for treating and / or preventing diseases related to abnormal cell activities. The diseases related to abnormal cell activities are preferably lung cancer, urethral cancer, colorectal cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

[0137] The present invention provides a drug linker represented by Formula II or a pharmaceutically acceptable salt thereof,

[0138]

[0139] Wherein, the definitions of U, X, W 1 、W 2 、L 1 、L 2 、L 3 、L 4 、D 1 、D 2 and m are as defined above; Y 1 is

[0140] y1, y2, y4 and y5 are independently any integer between 8 and 16, and y3 is independently any integer between 0 and 6.

[0141] The present invention provides a drug linker represented by Formula II or a pharmaceutically acceptable salt thereof,

[0142]

[0143] Among them, U, X, W 1 , W 2 , L 1 , L 2 , L 3 , L 4 , D 1 , D 2 and the definitions of m are as described above; Y 1 is

[0144] y1 and y2 are independently any integer between 8 and 16, and y3 is independently any integer between 0 and 6.

[0145] In certain preferred embodiments of the present invention, the drug conjugate shown in Formula II is the drug conjugate shown in Formula II-1, II-2 or II-3,

[0146]

[0147]

[0148] Among them, Y 1 , W 1 , W 2 , L 1 , L 2 , L 3 , L 4 , D 1 , D 2 have the definitions as described above.

[0149] In certain preferred embodiments of the present invention, the drug conjugate shown in Formula II is any of the following structures:

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] Definition of terms

[0161] The "-" at the end of a group means that the group is connected to other fragments in the molecule through this site.

[0162] In the structure fragment It means that the structural fragment is connected to other fragments in the molecule through this site.

[0163] The compounds of the present invention may exist in certain stereoisomeric forms. and dotted wedge key Indicates the absolute configuration of a stereocenter. Use a straight solid bond and straight dashed key Indicates the cis-trans configuration of a stereocenter.

[0164] The term "plurality" refers to 2, 3, 4 or 5.

[0165] When any variable (such as the group Y 1 ) appears multiple times in the definition of a compound, their definitions are independent of each other and do not affect each other. For example, 3 CH 2 Independently by Y 1 The substituted -(CH 2 ) p - means -(CH 2 ) p -CH in 2 Will be 3 Y 1 Replace, 3 Y 1 The definitions are independent of each other and do not affect each other.

[0166] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, sulfates, mesylate salts, and the like.

[0167] The term "therapeutically effective amount" refers to the amount of a compound administered to a patient that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the compound, the type of disease, the severity of the disease, the age of the patient, etc., but can be adjusted by those skilled in the art according to the circumstances.

[0168] The term "pharmaceutical excipient" refers to excipients and additives used in the production of drugs and the formulation of prescriptions, and is all substances contained in a pharmaceutical preparation except the active ingredient.

[0169] The term "treatment" refers to any of the following situations: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that triggers the disease; (3) slowing down the development of one or more biological manifestations of a disease.

[0170] The term "prevention" refers to reducing the risk of developing a disease.

[0171] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0172] The reagents and raw materials used in the present invention are all commercially available.

[0173] The positive and progressive effects of the present invention are as follows: The present invention provides a drug conjugate of a multi-drug linkage combination and its application. The drug conjugate of the multi-drug linkage combination can link different payloads to the same antibody, has better synergistic effects or can overcome drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0174] Figure 1 It is the SDS gel electrophoresis diagram of ADC5 - ADC8.

[0175] Figure 2 It is the SKBR3 curve of ADC6 - ADC8.

[0176] Figure 3 It is the MDA - MB - 231 curve of ADC6 - ADC8. DETAILED DESCRIPTION OF THE INVENTION

[0177] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0178] The abbreviations of the reagents used in the examples are shown in the following table:

[0179] Abbreviation Full name DMF N,N-Dimethylformamide HOBt 1-Hydroxybenzotriazole DIPEA N,N-Diisopropylethylamine Fmoc 9-Fluorenylmethyloxycarbonyl vcPAB Val-Cit-PAB (Valinyl-Citrullinyl-para-aminobenzyl ester) PNP p-Nitrophenolcarbonyl HATU 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate COT 2-(Cyclooct-2-yn-1-yloxy)acetyl OSu Succinimidyl active ester group LCMS Liquid chromatography-mass spectrometry

[0180] Example 1 Synthesis of the single-drug amino linking unit LPA

[0181] Step 1: Preparation of vcPAB-Irinotecan

[0182]

[0183] To a solution of Fmoc-vcPAB-PNP (0.10 g, 0.13 mmol) in DMF (2 mL) were successively added irinotecan (57 mg, 0.13 mmol), HOBt (22 mg, 0.16 mmol) and DIPEA (21 mg, 0.16 mmol). The reaction mixture was stirred at room temperature for 3 hours. After LCMS showed the disappearance of the starting materials, diethylamine (1 mL) was added and the mixture was stirred at room temperature for another 1 hour. The reaction mixture was neutralized to pH 6 with dilute hydrochloric acid (1 N), and then purified by reversed-phase flash chromatography (elution with 0 - 50% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)) to give yellow solid vcPAB-irinotecan (87 mg, yield 80%). ESI m / z: 841.2 (M + H) + 。

[0184] Step 2: Preparation of Drug Linker LPA1

[0185]

[0186] To a DMF solution (1 mL) of N-Fmoc-PEG 4 -COOH (CAS: 557756-85-1, 54 mg, 0.11 mmol) were successively added HATU (46 mg, 0.12 mmol), DIPEA (16 mg, 0.12 mmol) and vcPAB-irinotecan (87 mg, 0.10 mmol). The reaction mixture was stirred at room temperature for 1 hour, then diethylamine (0.5 mL) was added and the mixture was stirred at room temperature for another 1 hour. The reaction mixture was neutralized to pH 6 with dilute hydrochloric acid (1 N), and then purified by reversed-phase flash chromatography (elution with 0 - 50% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)) to give yellow solid LPA1 (66 mg, yield 61%). ESI m / z: 1088.3 (M + H) + 。

[0187] The preparation processes of the single-drug amino linking units LPA2, LPA3, LPA4 and LPA5 in Table 1 are similar to that of LPA1, except that different payloads and corresponding amino linkers were used.

[0188] Table 1

[0189]

[0190] Example 2 Synthesis of Monodrug Cyclooctynyl (COT) Linker Unit LPB

[0191]

[0192] The starting material COT-PEG4-vcPAB-PNP was synthesized in large quantities by the method reported in WO2020146541 and reserved. The preparation process of the known compound LPB3 is as described in WO2022015656.

[0193] Preparation of Drug Linker LPB1

[0194]

[0195] To a DMF solution (2 mL) of COT-PEG4-vcPAB-PNP (0.10 g, 0.11 mmol) were successively added the PARP inhibitor ABT888 (26 mg, 0.11 mmol), HOBt (33 mg, 0.21 mmol), and DIPEA (20 mg, 0.16 mmol). After the reaction mixture was stirred at room temperature for 2 hours, it was directly separated by reversed-phase flash liquid chromatography (C18 column, eluted with 0 - 60% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain white solid LPB1 (88 mg, yield 80%). ESI m / z: 1062.4 (M+H) + 。

[0196] The preparation process of LPB2 is similar to that of LPB1, except that the ATR inhibitor M6620 was used to react with COT-PEG4-vcPAB-PNP.

[0197] Table 2

[0198]

[0199] The 1H NMR data of LPB1 and LPB2 are shown in Table 3 below.

[0200] Table 3

[0201]

[0202]

[0203] Example 3: Synthesis of Bisdrug (1:1) Amino Linker Unit

[0204] Step 1: Synthesis of Compound 2a

[0205]

[0206] To a DMF solution (2 mL) of N-Fmoc-Gly3-OH (Compound 1a, CAS: 170941-79-4, 0.10 g, 0.24 mmol) were successively added HATU (0.11 g, 0.29 mmol), DIPEA (37 mg, 0.29 mmol), and azido lysine (46 mg, 0.27 mmol). After the reaction mixture was stirred at room temperature for 1 hour, it was directly separated by reversed-phase flash liquid chromatography (C8 column, eluted with 0-60% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)) to obtain white solid Compound 2a (80 mg, yield 58%). ESI m / z: 556.3 (M+H) + 。

[0207] Step 2: Synthesis of Compound 3a

[0208]

[0209] To a DMF solution (1 mL) of Compound 2a (19 mg, 33 μmol) were successively added HATU (14 mg, 36 μmol), DIPEA (5 mg, 36 μmol), and LPA1 (33 mg, 30 μmol). The reaction mixture was stirred at room temperature for 1 hour, neutralized to pH 6 with dilute hydrochloric acid (1 N), and then separated by reversed-phase flash liquid chromatography (C18 column, eluted with 0-60% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)) to obtain light yellow solid Compound 3a (38 mg, yield 77%). ESI m / z: 818.3 (M / 2+H) + 。

[0210] Step 3: Synthesis of Compound LP211

[0211]

[0212] To a DMF solution (1 mL) of Compound 3a (10 mg, 6.1 μmol) was added LPB1 (13 mg, 12 μmol). The reaction mixture was stirred overnight at room temperature and then directly separated by reversed-phase flash liquid chromatography (C8 column, eluted with 0-65% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)) to obtain yellow solid Fmoc-LP211. ESI m / z: 899.7 (M / 3+H) + 。

[0213] The obtained compound Fmoc-LP211 was dissolved in DMF (1 mL), and diethylamine (0.2 mL) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour until LCMS showed complete removal of Fmoc. After the resulting solution was neutralized to pH 6 with dilute hydrochloric acid (1 N), it was separated by reversed-phase flash chromatography (eluted with a C8 column, 0 - 60% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound LP211 (3 mg, yield 20%). ESI m / z: 1237.9 (M / 2 + H) + 。

[0214] The preparation processes of the remaining dual-drug (1:1) amino-linking units LP212, LP213, LP214, and LP215 in Table 4 were similar to that of LP211, with the only difference being the use of different LPAs or LPBs and the corresponding compound 1b. The analytical data of these dual-drug (1:1) amino-linking units are shown in Table 5 below.

[0215]

[0216] Table 4

[0217]

[0218]

[0219]

[0220] Table 5 Analytical Data of Dual-Drug (1:1) Amino-Linking Units

[0221]

[0222]

[0223] Example 4: Synthesis of Dual-Drug (1:1) COT-Linking Unit

[0224] Synthesis of Compound LP223

[0225]

[0226] To a DMF solution (1 mL) of compound LP213 (10 mg, 3 μmol) were successively added COT-OSu (1.3 mg, 4.5 μmol) and DIPEA (0.5 mL). After the reaction mixture was stirred at room temperature for 2 hours, it was directly separated by reversed-phase flash chromatography (eluted with a C8 column, 0 - 60% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound LP223 (3 mg, yield 31%). ESI m / z: 1073.0 (M / 3 + H)+.

[0227] The preparation processes of the remaining dual-drug (1:1) COT linking units LP224 and LP225 in Table 6 are similar to that of LP223, with the only difference being the use of different dual-drug (1:1) amino linking units. The mass spectrometry data of LP223, LP224, and LP225 are shown in Table 6. Synthesis of dual-drug (1:1) BCN linking unit

[0228] LP226:

[0229]

[0230] To a DMF solution (1 mL) of compound LP214 (10 mg, 3 μmol) were successively added BCN-PNP (CAS: 1263166-91-1, 1 mg, 3 μmol) and DIPEA (0.8 μL). After the reaction mixture was stirred at room temperature for 2 hours, it was directly separated by reversed-phase flash liquid chromatography (eluted with a C8 column, 0 - 50% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound LP226 (7 mg, yield 66%). ESI m / z: 1130.3 (M / 3 + H) + 。

[0231] Synthesis of dual-drug (1:1) Mc linking unit

[0232] LP227:

[0233]

[0234] To a DMF solution (1 mL) of compound LP214 (10 mg, 3 μmol) were successively added Mc-OSu (CAS: 55750-61-3, 1 mg, 4 μmol) and DIPEA (0.8 μL). After the reaction mixture was stirred at room temperature for 2 hours, it was directly separated by reversed-phase flash liquid chromatography (eluted with a C8 column, 0 - 53% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound LP227 (5.7 mg, yield 53%). ESI m / z: 1117.4 (M / 3 + H) + 。

[0235] Table 6. Dual-drug (1:1) COT linking unit, BCN linking unit, and Mc linking unit

[0236]

[0237]

[0238] Example 5: Synthesis of dual-drug (2:1) amino linking unit

[0239] Step 1: Synthesis of compound 5a

[0240]

[0241] In a DMF solution (30 mL) of compound N-Fmoc-Glu-OH (4a, CAS: 121343-82-6, 7.5 mg, 20 mmol), amino-PEG3-azide (8.8 mg, 41 mmol), HATU (19 g, 49 mmol) and DIPEA (16 g, 0.12 mol) were added successively. The reaction solution was stirred at room temperature for 2 hours, and LCMS showed that the reaction was completed. The obtained reaction solution was poured into saturated brine (200 mL) and extracted with ethyl acetate (200 mL x 3). After the organic phases were combined, they were washed with saturated brine (200 mL x 2) and dried over anhydrous sodium sulfate, and then the clear solution was concentrated under reduced pressure at low temperature. The concentrated solution was separated by reverse-phase flash liquid chromatography (eluted with a C18 column, 0-60% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)), and the white solid compound Fmoc-5a (13 g, ESI m / z: 770.2 (M+H) + ) was obtained. The obtained solid was dissolved in DMF (40 mL), diethylamine (3.4 g, 33 mmol) was added to this solution, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the Fmoc protecting group had been completely removed. The reaction solution was poured into saturated brine (200 mL) and extracted with ethyl acetate (200 mL x 3). After the organic phases were combined, they were washed with saturated brine (200 mL x 2) and dried over anhydrous sodium sulfate, and then the clear solution was concentrated under reduced pressure at low temperature. The concentrate was separated by reverse-phase flash liquid chromatography (eluted with a C18 column, 0-40% acetonitrile / trifluoroacetic acid aqueous solution (0.1%)), and the white solid compound 5a (13 g, yield 68% (calculated from 4a)) was obtained. ESI m / z: 548.2 (M+H) + .

[0242] Step 2: Synthesis of compound 6a

[0243]

[0244] In a DMF solution (30 mL) of compound 5a (4.5 g, 8.3 mmol), N-Fmoc-PEG 3-COOH (CAS: 867062-95-1, 4.0 g, 9 mmol), HATU (7.6 g, 20 mmol) and DIPEA (6.5 g, 50 mmol). The reaction mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was completed. The resulting reaction mixture was poured into saturated brine (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 2) and dried over anhydrous sodium sulfate. The clear solution was concentrated under reduced pressure at low temperature. The concentrate was separated by reversed-phase flash chromatography (C18 column, eluted with 0 - 60% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound Fmoc-6a (5 g, ESI m / z: 973.1 (M+H) + ). The resulting solid was dissolved in DMF (20 mL). Diethylamine (1.1 g, 10 mmol) was added to this solution and stirred at room temperature for 2 hours. LCMS showed that the Fmoc protecting group had been completely removed. The reaction mixture was poured into saturated brine (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, and the clear solution was concentrated under reduced pressure at low temperature. The concentrate was separated by reversed-phase flash chromatography (C18 column, eluted with 0 - 40% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound 6a (2.9 g, yield 50% (calculated from 5a)). ESI m / z: 751.5 (M+H) + .

[0245] Step 3: Synthesis of compound 7a

[0246]

[0247] To a DMF solution (20 mL) of compound 6a (2.9 g, 3.9 mmol) were successively added Fmoc-L-glutamic acid methyl ester (1.5 g, 3.9 mmol), HATU (3.6 g, 9.4 mmol) and DIPEA (3.1 g, 23 mmol). The reaction mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was completed. The resulting reaction mixture was poured into saturated brine (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 2) and dried over anhydrous sodium sulfate. The clear solution was concentrated under reduced pressure at low temperature to obtain a white concentrate (2 g, ESI m / z: 1116.4 (M+H) +)。The obtained concentrate was dissolved in DMF (10 mL). Diethylamine (0.19 g, 3.6 mmol) was added to this solution, and the mixture was stirred at room temperature for 2 hours. LCMS showed that the Fmoc protecting group had been completely removed. The reaction solution was poured into saturated brine (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, and the clear solution was concentrated under reduced pressure at low temperature. The obtained concentrate was dissolved in dichloromethane (20 mL). Di-tert-butyl dicarbonate (Boc-anhydride) (0.45 g, 2 mmol) and triethylamine (0.5 g, 5.4 mmol) were successively added to this solution. After the reaction solution was stirred at room temperature for 6 hours, water (50 mL) was added, and the mixture was extracted with dichloromethane (50 mL x 3). The combined organic phases were washed with water (50 mL x 2), dried over anhydrous sodium sulfate, and the clear solution was concentrated under reduced pressure at low temperature. The concentrate was separated by reverse-phase flash liquid chromatography (eluted with a C18 column, 0 - 50% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain a white solid compound (1.25 g, ESI m / z: 994.6 (M+H) + )。This white solid was dissolved in tetrahydrofuran (20 mL), and an aqueous lithium hydroxide solution (0.3 g, 13 mmol / 5 mL water) was slowly added to this solution. After the reaction solution was stirred at room temperature for 2 hours, it was diluted with saturated brine (50 mL) and extracted with ethyl acetate (50 mL x 3). The combined extracts were washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, and the clear solution was concentrated under reduced pressure at low temperature. The concentrate was separated by reverse-phase flash liquid chromatography (eluted with a C18 column, 0 - 50% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain a white solid compound (0.95 g). This white solid was dissolved in dichloromethane, and trifluoroacetic acid (2 mL) was added dropwise to this solution. After the reaction solution was stirred at room temperature for 1 hour, it was concentrated under reduced pressure at low temperature and dried in vacuo to obtain an oily compound 7a (0.80 g, trifluoroacetate, total yield 21%). ESI m / z: 880.6 (M+H) + .

[0248] Step 4: Synthesis of Compound 8a

[0249]

[0250] To a solution of N-Fmoc-Gly3-OH (Compound 1a) (49 mg, 0.12 mmol) in DMF (1 mL) were successively added HATU (49 mg, 0.13 mmol), DIPEA (17 mg, 0.13 mmol), and Compound 7a (0.10 g, 0.11 mmol). After the reaction mixture was stirred at room temperature for 1 h, it was directly separated by reversed-phase flash liquid chromatography (eluted with 0 - 50% acetonitrile / aqueous trifluoroacetic acid solution (0.1%) on a C18 column) to afford Compound 8a as a colorless liquid (0.10 g, yield 70%). ESI m / z: 637.2 (M / 2 + H) + 。

[0251] Step Five: Synthesis of Compound 9a

[0252]

[0253] To a solution of Compound 8a (29 mg, 23 μmol) in DMF (1 mL) were successively added HATU (11 mg, 28 μmol), DIPEA (4 mg, 28 μmol), and the single-drug amino linker unit LPA1 (25 mg, 23 μmol). After the reaction mixture was stirred at room temperature for 1 h, dilute hydrochloric acid (1 N) was added dropwise until the pH reached 6, and then it was directly separated by reversed-phase flash liquid chromatography (eluted with 0 - 60% acetonitrile / aqueous trifluoroacetic acid solution (0.1%) on a C8 column) to afford Compound 9a as a light yellow solid (37 mg, yield 69%). ESI m / z: 1172.3 (M / 2 + H) + 。

[0254] Step Six: Synthesis of Compound 311

[0255]

[0256] Compound 9a (14 mg, 6 μmol) was dissolved in a mixed solution of acetonitrile and water (1 mL / 1 mL), and LPB1 (22 mg, 21 μmol) was added to this solution. After the reaction mixture was stirred at room temperature for three days, it was directly separated by reversed-phase flash liquid chromatography (eluted with 0 - 60% acetonitrile / aqueous trifluoroacetic acid solution (0.1%) on a C8 column) to afford Fmoc-LP311 as a white solid (10 mg, ESI m / z: 1117 (M / 4 + H) +)。The resulting compound Fmoc-LP311 was dissolved in DMF (1 mL), and diethylamine (0.2 mL) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour until LCMS showed complete removal of Fmoc. After the resulting solution was neutralized to pH 6 with dilute hydrochloric acid (1 N), it was separated by reversed-phase flash liquid chromatography (elution with a C8 column, 0 - 55% acetonitrile / aqueous trifluoroacetic acid solution (0.1%)) to obtain the white solid compound LP311 (4.7 mg, yield 19%). ESI m / z: 1061.7 (M / 4 + H) + 。

[0257] The preparation processes of the remaining dual-drug (2:1) amino-linking units LP312, LP313, LP314, and LP315 in Table 7 were similar to that of LP311, with the only difference being the use of different dual-drug (1:1) amino-linking units or different compound 1b. The analytical data of the dual-drug (2:1) amino-linking units are shown in Table 8.

[0258] Table 7. Dual-drug (2:1) amino-linking units

[0259]

[0260]

[0261]

[0262] Table 8. Analytical data of dual-drug (2:1) amino-linking units

[0263]

[0264]

[0265] Example 6: Synthesis of dual-drug (2:1) COT-linking units

[0266] The preparation of the dual-drug (2:1) COT-linking units LP324 and LP325 was similar to that of the dual-drug (1:1) COT-linking unit LP223, with the only difference being the use of LP314 or LP315 instead of LP213.

[0267]

[0268] LP324 ESI m / z: 1297.3 (M / 4 + H) +

[0269]

[0270] LP325 ESI m / z: 1407.0 (M / 4 + H) +

[0271] Example 7: Synthesis of Dual-Drug (3:1) Linking Unit

[0272] Step 1: Synthesis of Compound 11a

[0273]

[0274] The operation was carried out according to the similar preparation steps of Compound 7a, with the only difference being that Compound 10a (CAS: 220431-61-8) was used instead of Compound 4a and 10 equivalents of amino-PEG3-azide were used, and a light yellow oily Compound 11a (1.5 g, total yield 10%) was obtained. ESI m / z: 747.0 (M / 2 + H) + 。

[0275] Step 2: Synthesis of Compound 12a

[0276]

[0277] The operation was carried out according to the similar preparation steps of Compound 8a, with the only difference being that Compound 11a was used instead of Compound 7a, and a light yellow oily Compound 12a (1.0 g, yield 47%) was obtained. ESI m / z: 1046.5 (M / 2 + H) + 。

[0278] Step 3: Synthesis of Compound LP424

[0279]

[0280]

[0281] The operation was carried out according to the similar preparation steps of the dual-drug (2:1) amino linking unit LP314, with the only difference being that Compound 12a was used instead of Compound 9a and the equivalent number of LPB1 was increased to 6, and a white solid Compound LP414 was obtained. Then, the operation was carried out according to the similar preparation steps of the dual-drug (2:1) COT linking unit LP324, using LP414 instead of LP314, and a white solid LP424 (2 mg, two-step yield 8%) was obtained. ESI m / z: 1109 (M / 6 + H) + 。

[0282] The preparation processes of the remaining dual-drug (3:1) linking units LP425, LP426, and LP427 in Table 9 were similar to that of LP424, with the only difference being that different single-drug amino linking units LPA and the corresponding single-drug COT linking units LPB were used. The analysis data of the dual-drug (3:1) linking units are shown in Table 9.

[0283] Table 9: Dual-drug (3:1) Linker Unit

[0284]

[0285]

[0286]

[0287]

[0288]

[0289] Example 8: Synthesis of Antibody-Drug Conjugate by One-step Coupling Method with Transglutaminase (MTG)

[0290] General Operating Procedure for One-step Coupling Method with Transglutaminase (MTG)

[0291] Dialyze the Herceptin antibody with N297A mutation in sodium borate buffer (25 mM, pH 7.4) for 24 hours, changing the buffer twice during this period. Quantify the collected antibody using ultraviolet spectrophotometry. If the concentration is too low, concentrate it to a concentration > 1.0 mg / ml by ultrafiltration. Add the sodium borate buffer solution of MTG enzyme (Jiangsu Yiming Biology, purified by gel filtration, 4495.5 U / g) (0.98 mg / mL, final concentration 0.017 U / mg Ab), and the DMSO solution (50 mM) of the linker unit with a primary amino group. React the mixture at 37 °C for 96 hours. Follow the reaction by SDS gel electrophoresis. The reaction product is passed through a desalting column chromatography to remove uncoupled small molecules and DMSO, and the drug-antibody ratio (DAR) is analyzed by LC-MS, and the aggregation degree is analyzed by SEC-HPLC.

[0292]

[0293] Example 9: Synthesis of Antibody-Drug Conjugate by Two-step Coupling Method with Transglutaminase (MTG)

[0294] General Operating Procedure for Two-step Coupling Method with Transglutaminase (MTG)

[0295] The first-step reaction is as described in Example 8. First, under the action of MTG, the Herceptin antibody with N297A or N297Q mutation is coupled with a linker containing a primary amino group and an azide group (such as NH 2 -PEG 3 -N 3, (CAS: 134179 - 38 - 7) was coupled to obtain an antibody with an azide group. In the second step, the antibody with an azide group was mixed with a linker unit with an alkyne group (such as COT) in sodium borate buffer (25 mM, pH 7.4) at a molar ratio of 1:10 to 1:30. The mixture was reacted at 25 or 37 °C for 24 - 96 hours, and the reaction was monitored by SDS gel electrophoresis, and the conjugate was purified by desalting column chromatography. The SDS gel electrophoresis pattern of ADC5 - 8 is as shown in Figure 1 shown.

[0296]

[0297]

[0298]

[0299] Example 10: Synthesis of Antibody - Drug Conjugate by Sortase A Coupling Method

[0300] The pre - synthesized Herceptin antibody with a sortase tag (STtag, LPETG) at the C - terminus of the heavy chain (modifying the antibody according to the general method in the reference Nature Protocols, 10(3), 508 - 516.) was mixed with sortase A and a dual - drug linker unit with a continuous glycine (Gly - gly - gly) sequence at a molar ratio of 1:0.5:50 in sodium borate buffer at 25 mM, pH 7.4, and the concentration of the antibody in the mixture was 1 - 5 mg / mL. The mixture was reacted at 35 °C for 48 hours. After the reaction, the sortase with a histidine tag (His - tag) was removed by nickel column chromatography and the excess small molecules were removed by desalting column chromatography. The conjugation rate of the conjugate product was detected by LC - MS method.

[0301]

[0302] Example 11: Synthesis of Antibody - Drug Conjugate by Maleimide - Thiol Method

[0303] The commonly used method for coupling maleimide with Herceptin antibody is the maleimide - thiol method, and the general steps are as follows:

[0304] Antibody Pretreatment

[0305] Select a suitable buffer: Use histidine buffer, phosphate buffer, etc., such as histidine - hydrochloride buffer, with a concentration generally ranging from 1 mM to 30 mM, preferably 20 mM.

[0306] Add a protecting agent: 1% to 10% (w / v) sucrose can be added to the buffer to protect the antibody activity.

[0307] Reducing antibody: Add a reducing agent to break the interchain disulfide bonds of the Herceptin antibody, generating free sulfhydryl groups. Commonly used reducing agents such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), and the molar ratio of the reducing agent to the antibody is controlled at 1:1 to 4:1, preferably 2:1 to 3:1. The reaction temperature is greater than 10 °C, such as 10.5 °C to 20 °C, the pH is 5 to 9, preferably 6 to 8; the reaction time is 1 to 10 hours, preferably 2 to 4 hours.

[0308] Preparation of linker-drug (or other functional molecule)

[0309] Selecting a linker: The linker should contain a maleimide group, such as SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester), Mal-PEG3-Val-Cit-PAB-PNP, etc.

[0310] Dissolving the linker: Dissolve it with a suitable organic solvent, such as acetone, dimethyl sulfoxide (DMSO), etc., or prepare an aqueous solution of an organic solvent, such as 50% aqueous acetone solution, 80% DMSO aqueous solution, etc.

[0311] Coupling reaction

[0312] Mixing reaction: Mix the antibody containing sulfhydryl groups with the dissolved linker-drug molecule. The molar ratio of the linker-drug to the antibody is generally 2:1 to 10:1, such as 3:1 to 7:1. React under certain temperature and pH conditions, the temperature is 10.5 °C to 20 °C, the pH is adjusted according to specific circumstances, and the reaction lasts for several hours to overnight.

[0313] Terminating the reaction: After the reaction is completed, an appropriate amount of terminator can be added or the reaction can be terminated by changing the reaction conditions (such as adjusting the pH, lowering the temperature, etc.).

[0314] Purification and analysis

[0315] Purification: Use methods such as gel filtration chromatography and affinity chromatography to remove unreacted linker-drug, reducing agent and other impurities to obtain the conjugate product.

[0316] Analysis and detection: Use means such as ultraviolet spectroscopy and mass spectrometry to analyze the drug-antibody conjugation ratio (DAR), purity, structural integrity, etc. of the conjugate to evaluate the conjugation effect.

[0317]

[0318] The drug-antibody ratios (DAR) of the antibody-drug conjugates prepared in Examples 8-11 above are as follows:

[0319]

[0320]

[0321] Example 12: Inhibitory Effect of Antibody-Drug Conjugates on Cells

[0322] The cell lines used for the proliferation inhibition assay were SK-BR-3 and MDA-MB-231; the cells were cultured in McCoy's 5a Medium + 10% FBS. In a 96-well plate, cells (80 μL, 1500 cells) were added to each well and incubated in a 37 °C carbon dioxide incubator for 24 hours. Then, different concentrations of the test compound (20 μL) were placed in an appropriate cell culture medium (total volume 0.1 mL) for treatment. The control wells contained cells and medium but no test compound. Incubate in a 37 °C carbon dioxide incubator for 144 hours. Take out the 96-well plate, observe the cell status under a microscope, stick a white bottom film, add CTG reagent (100 μL) to the wells, shake the plate at room temperature for 10 minutes, let it stand for 10 minutes, and record the luminescence with a Microplate Reader. The calculation formula for the inhibition rate is: Inhibition% = (1 - (T sample - T blank ) / (T DMSO - T blank )) × 100%. The test results are shown in Figure 2 and Figure 3 .

[0323] The IC 50 values of the tested ADCs are as follows.

[0324]

[0325] The results showed that all the tested ADCs had a strong inhibition rate in the Her2-positive cell SKBR3, while there was no inhibition rate in the Her2-negative cells.

Claims

1. A drug conjugate as shown in formula I or a pharmaceutically acceptable salt thereof, in, Z is a chemical bond or The a end is connected to Ab; Y is -(CH2) in which one or more CH2 groups are independently substituted by Y1 p -, wherein Y1 is independently -O-, -S-, -NH-, -C(=O)-, -C(=O)NH-, p is 1-24; U is C 1-6 Alkylene or The b end is connected to CH; R 1a and R 1b Independently for C 1-6 Alkylene; m is 1, 2 or 3; When m is 1, X is a chemical bond; When m is 2, X is The c-terminus is connected to the carbonyl group; When m is 3, X is The c-terminus is connected to the carbonyl group; W1 and W2 are independently a chemical bond or The e-terminal is connected to L1 or L3; n1, n2, n3, n4, n5, n6, n7, n8, n9, n10 and n11 are independently any integer between 1 and 24; L1 and L3 are independently a dipeptide residue, a tripeptide residue or a tetrapeptide residue; L2 and L4 are independently Terminal f is connected to D1 or D2; D1 and D2 are different and are independently bioactive molecule fragments, wherein the bioactive molecule is a cytotoxic drug molecule, a tubulin inhibitor, an ATR inhibitor, a PARP inhibitor or a ChK1 inhibitor; q is any value between 1 and 16; Ab is an antibody or an antigen-binding fragment thereof.

2. The drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions: (1) p is any integer between 4 and 16; (2) n1 is any integer between 1 and 6; (3) n2 is any integer between 8 and 16; (4) n3, n4, n5 and n6 are independently any integer between 1 and 6; (5) n7, n8, n9 and n10 are independently any integer between 1 and 6; (6) The dipeptide residue is The h end is connected to L2 or L4; (7) The tripeptide residue is The h end is connected to L2 or L4; (8) The tetrapeptide residue is The h end is connected to L2 or L4; (9) D1 is a topoisomerase I inhibitor fragment, an ATR inhibitor fragment or a PARP inhibitor fragment; (10) D2 is a topoisomerase I inhibitor fragment, a tubulin inhibitor fragment or an ATR inhibitor fragment; (11) q is any value between 1 and 8; (12) Ab is a HER2 antibody, an antigen-binding fragment of a HER2 antibody, or a HER2 antibody with a transpeptidase tag at the C-terminus of the heavy chain.

3. The drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions: (1) Z is a chemical bond, The a end is connected to Ab; (2)Y is g terminal is connected to Z; y1 and y2 are independently any integer between 8 and 16, and y3 is independently any integer between 0 and 6; preferably The g end is connected to Z; (3) U is -CH2CH2- or The b end is connected to CH; (4) When m is 2, X is The c-terminus is connected to the carbonyl group; (5) When m is 3, X is The c-terminus is connected to the carbonyl group; (6) W1 and W2 are independently chemical bonds, The e-terminal is connected to L1 or L3; (7) L1 and L3 are independently The h end is connected to L2 or L4; (8) D1 is (9) D2 is (10) q is 0.8, 1.3, 1.4, 1.6, 1.7, 1.8, 2.0, 3.87, 6.70 or 6.74; (11) Ab is trastuzumab, trastuzumab with N297A variant, trastuzumab with N297Q variant, or trastuzumab with LPETG tag at the C-terminus of the heavy chain.

4. The drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug conjugate as shown in formula I is a drug conjugate as shown in formula I-1, Where Z is a chemical bond or The a end is connected to Ab; Y is The g end is connected to Z; W1 and W2 are independently a chemical bond or The e-terminal is connected to L1 or L3; L1 and L3 are independently The h end is connected to L2 or L4; L2 and L4 are independently Terminal f is connected to D1 or D2; n1 is any integer between 1 and 6; n11 is any integer between 8 and 16; D1 and D2 are different and are independently q is any value between 1 and 8; Ab is a HER2 antibody, an antigen-binding fragment of a HER2 antibody, or a HER2 antibody with a transpeptidase tag at the C-terminus of the heavy chain.

5. The drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug conjugate as shown in formula I is a drug conjugate as shown in formula I-2 or I-3, wherein Z, Y, W1, W2, L1, L3, L2, L4, D1, D2, q, Ab, n3, n4, n5, n6, n7, n8, n9 and n10 are as described in any one of claims 1-3.

6. The drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug conjugate as shown in formula I is any of the following structures: Among them, Her2(N297A) is trastuzumab with N297A variant, Her2(N297Q) is trastuzumab with N297Q variant, and Her2(HC)-LPET is trastuzumab with a transpeptidase tag at the C-terminus of the heavy chain.

7. The drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: The drug conjugate as shown in formula I is any of the following structures: Among them, Her2(HC)-LPET is trastuzumab with a transpeptidase tag at the C-terminus of the heavy chain, Her2(N297A) is trastuzumab with N297A variant, and Her2(N297Q) is trastuzumab with N297Q variant.

8. A pharmaceutical composition comprising the drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, and at least one pharmaceutical excipient.

9. Use of a drug conjugate as shown in formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 8, in the preparation of a medicament for treating and / or preventing a disease associated with abnormal cell activity; the disease associated with abnormal cell activity is preferably lung cancer, urethral cancer, colorectal cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

10. A drug linker as shown in formula II or a pharmaceutically acceptable salt thereof, in, The definitions of U, X, W1, W2, L1, L2, L3, L4, D1, D2 and m are as described in any one of claims 1 to 7; Y1 is y1 and y2 are independently any integer between 8 and 16, and y3 is independently any integer between 0 and 6.

11. The drug linker of formula II or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that: The drug linker as shown in formula II is a drug linker as shown in formula II-1, II-2 or II-3, Wherein, the definitions of Y1, W1, W2, L1, L2, L3, L4, D1, and D2 are as described in any one of claims 1-7.

12. The drug linker of formula II or a pharmaceutically acceptable salt thereof according to claim 10, characterized in that: The drug linker as shown in Formula II is any of the following structures:

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