Antibody-conjugated drugs of N-oxacycloalkyl-substituted camptothecin derivatives

Abstract

The present invention relates to an antibody-drug conjugate represented by formula (I) having an oxacycloalkyl-substituted camptothecin derivative as a drug toxic moiety, wherein the definitions of each group in the formula are as described in the specification, and the antibody-drug conjugate has a significant antitumor effect. [Formula 1] JPEG2026502963000098.jpg54170

Description

[Technical Field]

[0001] The present invention relates to the field of medicine. Specifically, the present invention provides an antibody-conjugated drug of an N-oxacycloalkyl-substituted camptothecin derivative, its preparation method and its application in the antitumor field. [Background technology]

[0002] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and trunk of the Chinese tree Camptotheca acuminata (Camptotheca / Happy tree). Camptothecin inhibits topoisomerase I, which causes cell death. Due to camptothecin's cytotoxic mechanism and broad-spectrum antitumor activity, considerable effort has been devoted to developing clinical analogs of camptothecin.

[0003] Currently, researchers are conducting extensive research into the use of monoclonal antibodies for targeted delivery of small molecule drugs to tumor cells. Many different drug categories have been evaluated for antibody-mediated delivery, but only a limited number of drug categories have demonstrated sufficient activity and an appropriate toxicity profile as antibody-drug conjugates to support clinical development. One such category that has attracted attention is camptothecin.

[0004] Antibody-drug conjugates (ADCs) are a potent class of drugs effective against abnormal cell proliferation and proliferative diseases (e.g., cancer). Antibody-drug conjugates typically consist of three distinct components: an antibody, a linker, and a cytotoxic moiety (a small molecule drug). ADCs are typically designed by linking a small molecule drug to an antibody via a linker, and the flexibility of the small molecule drug design and the combination of the linker and antigen is crucial. Currently, there is no existing technology for oxacycloalkyl-modified camptothecin and its derivative ADC drugs. Therefore, research into this field of drug technology is of great value for the development of camptothecin and its derivative ADCs. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides antibody-drug conjugates having an oxacycloalkyl-substituted camptothecin derivative as the toxic moiety, which have significant antitumor effects. [Means for solving the problem]

[0006] One aspect of the present invention provides an antibody-drug conjugate shown in formula (I): [ka] (In the formula, m and n1 are each independently selected from 1 or 2; R a is hydrogen and -L 1 -NH-L p -Z-Ab, and R b is hydrogen, -L 2 -NH-L p -Z-Ab and -L 3 -NH-Z-Ab, with the proviso that R a and R b is not hydrogen at the same time, where: L1 is -C(=O)-L 11 -L 12 -L 13 - selected from L 11 , L 12 , and L 13 are each independently selected from —O—, a C1-C3 alkylene group, and a phenyl group; L 2 is -C(=O)-NH-L 21 -CHR 1 - selected from L 21 is selected from -C(=O)- and -NH-C(=O)-; R 1 is selected from hydrogen, deuterium, a C1-C6 alkyl group, and a phenyl-substituted C1-C6 alkyl group; L 3 is -C(=O)-NR 2 -NR 3 -L 31 -L 32 - selected from R 2 and R 3 are each independently selected from hydrogen, deuterium, and a C1-C6 alkyl group; L 31 is selected from -C(=O), -(C1-C3 alkylene)-C(=O), -5- to 8-membered aromatic ring group -C(=O)-, -5- to 8-membered nitrogen heteroaromatic ring group -C(=O)- and -O-(C1-C3 alkylene)-C(=O)-; L 32 is selected from -NH-(C1-C3 alkylene), -N(CH3)2-(C1-C3 alkylene) and -NH-(C1-C3 alkylene)-NH-; L P is a peptide residue consisting of 2 to 7 amino acids, Z is -L z -L j - selected from, where L z is a -C(=O)-C1-C8 alkylene group, -C(=O)-CHO-(CHCHO) 2~5 -CH2CH2NH-, -C(=O)-(CH2CH2O)2~6 -CH2CH2- and C(=O)-(CH2CH2O) 2~6 -CH2CH2NH-, L j is a linker capable of being attached to an antibody, Ab is an antibody.

[0007] In the antibody-drug conjugate of formula (I), the drug-antibody ratio (DAR) is selected from the range of 2 to 8, and may further be 3.5 to 8, 6 to 8, or 6.2 to 7.6, for example, 3.95, 4.05, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

[0008] One aspect of the present invention provides an antibody-drug conjugate shown in formula (IA), (IB) or (IC): [ka] (each group in the formula is defined as above). [ka] (each group in the formula is defined as above). [ka] (n' is selected from 2 to 8, and may further be 3.5 to 8 or 6 to 8 or 6.2 to 7.6, for example, 3.95, 4.05, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6; Each group in the formula is defined as above.

[0009] In some specific embodiments, [ka] The structure represented by the formula: is selected from the structures represented by the formula: [ka]

[0010] In some specific embodiments, L 11 , L 12 , and L 13 are each independently selected from —O—, a methylene group, and a phenyl group.

[0011] In some specific embodiments, L 11 -L 12 -L 13 is selected from -O-CH2-phenyl- and -CH2-O-CH2-.

[0012] In some specific embodiments, L 1 is selected from -C(=O)-O-CH2-phenyl.

[0013] In some specific embodiments, R 1 is selected from hydrogen, deuterium, a C1-C6 alkyl group, and a phenyl-substituted C1-C6 alkyl group.

[0014] In some specific embodiments, R 1 is selected from hydrogen, deuterium, methyl, isopropyl, isobutyl, and phenylmethyl.

[0015] In some specific embodiments, R 1 is hydrogen.

[0016] In some specific embodiments, L 2 is selected from -C(=O)-NH-C(=O)-CH2- and -CH2-NH-NH-C(=O)-CH2-.

[0017] In some specific embodiments, L 2 is -CH2-NH-NH-C(=O)-CH2-.

[0018] In some specific embodiments, R2 and R 3 are each independently selected from hydrogen, deuterium, and a C1-C3 alkyl group.

[0019] In some specific embodiments, R 2 and R 3 are each independently selected from hydrogen, deuterium, and a methyl group.

[0020] In some specific embodiments, R 2 is hydrogen, R 3 is hydrogen.

[0021] In some specific embodiments, L 31 is selected from -C(=O), -methylene-C(=O), -phenyl-C(=O)-, -pyridine-C(=O)-, and -O-CH2-C(=O)-.

[0022] In some specific embodiments, L 32 is selected from -NH-CH2-CH2-, -N(CH3)2-CH2-CH2- and -NH-CH2-NH-.

[0023] In some specific embodiments, L 3 is -C(=O)-NH-NH-phenyl-C(=O)-NH-CH2-CH2-.

[0024] In some specific embodiments, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn).

[0025] In some specific embodiments, L pis selected from peptide residues consisting of 2 to 5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn).

[0026] In some specific embodiments, L p is selected from peptide residues consisting of two or three amino acids selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), and citrulline (Cit).

[0027] In some specific embodiments, L p is selected from Val-Cit, Val-Ala, Gly-Val-Ala, Gly-Val-Ala-Gly, Gly-Lys, Gly-Gly-Lys, Gly-Gly-Lys-Gly, Val-Ala, Ala-Ala-Asn, Gly-Leu, Gly-Gly-Leu, Gly-Gly-Leu-Gly, Gly-Phe, Gly-Gly-Phe, and Gly-Gly-Phe-Gly.

[0028] In some specific embodiments, L p is selected from the structures represented by the formula: [ka] The carbonyl end is linked to -NH- and the other end is linked to Z.

[0029] In some specific embodiments, L p is selected from the structures represented by the formula: [ka] The carbonyl end is linked to -NH- and the other end is linked to Z.

[0030] In some specific embodiments, Lj is selected from the structures shown in the following formulas: [ka] [ka] The position indicated by indicates that it is linked to an antibody, [ka] The position indicated by L Z represents that the group is linked to the

[0031] In some specific embodiments, L z is a -C(=O)-C1-C8 alkylene group, -C(=O)-CHO-(CHCHO) 2~5 -CH2CH2NH-, -C(=O)-(CH2CH2O) 2~6 -CH2CH2- and -C(=O)-(CH2CH2O) 2~6 -CH2CH2NH-.

[0032] In some specific embodiments, L z is a -C(=O)-C1-C6 alkylene group, -C(=O)-CHO-(CHCHO) 2~3 -CH2CH2NH-, -C(=O)-(CH2CH2O) 2~4 -CH2CH2- and -C(=O)-(CH2CH2O) 2~4 -CH2CH2NH-.

[0033] In some specific embodiments, L z is selected from —C(═O)—(CH2)5—, —C(═O)—CHO—(CH2CH2O)3—CH2CH2NH—, —C(═O)—(CH2CH2O)2—CH2CH2—, —C(═O)—(CH2CH2O)2—CH2CH2NH— and —C(═O)—(CH2CH2O)4—CH2CH2NH—.

[0034] In some specific embodiments, Z is selected from the group represented by the formula: [ka] [ka] The position indicated by indicates that it is linked to an antibody.

[0035] [ka] The position indicated by L p Group or L 3 represents that the group is linked to the

[0036] In some specific embodiments, the antibody is a tumor-associated antigen antibody.

[0037] In some specific embodiments, the tumor-associated antigen antibody is selected from an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody.

[0038] In some specific embodiments, the antibody-drug conjugate of formula (I) has the structure shown in formula (I-1) or formula (I-1′). [ka] (In the formula, m, n1, L p , Z, and Ab are each defined as in the compound of formula (I), and n' is defined as above.

[0039] In some specific embodiments, the antibody-drug conjugate of Formula (I) has the structure shown in Formula (I-2) or Formula (I-2'), Formula (I-3) or Formula (I-3'). [ka] (In the formula, R 2 , R 3, m, n1, L p , Z, and Ab are each defined as in the compound of formula (I), and n' is defined as above.

[0040] The present invention provides the following antibody-drug conjugates: [ka] [ka] [ka] [ka] (wherein Ab is defined as in the compound of formula (I)).

[0041] In some specific embodiments, the Ab is selected from tumor-associated antigen antibodies, and further selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody, and anti-ROR1 antibody.

[0042] In some specific embodiments, the Ab is the HS627 antibody or the IP140B antibody, wherein the heavy chain amino acid sequence of HS627 is set forth in SEQ ID NO:1 and the light chain amino acid sequence is set forth in SEQ ID NO:2; and the heavy chain amino acid sequence of the IP140B antibody is set forth in SEQ ID NO:3 and the light chain amino acid sequence is set forth in SEQ ID NO:4.

[0043] In one embodiment, n is selected from 2 to 8, and may further be 3.5 to 8, or 6 to 8, or 6.2 to 7.6, for example, 3.95, 4.05, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6.

[0044] The present invention provides the following antibody-drug conjugates: [ka]

[0045] Furthermore, n is selected from 6.2 to 6.4. [ka]

[0046] Furthermore, n is selected from 6.7 to 6.8. [ka]

[0047] Furthermore, n is selected from 3.95 to 7.2, and may be, for example, 3.95 to 4.05 or n=7.1 to 7.2. [ka]

[0048] Furthermore, n is selected from 7.0 to 7.1. [ka]

[0049] Furthermore, n is selected from the range of 6.8 to 7.0. [ka]

[0050] Furthermore, n is selected from the range of 7.4 to 7.5. [ka]

[0051] Furthermore, n is selected from the range of 7.4 to 7.5. [ka]

[0052] Furthermore, n is selected from the range of 7.4 to 7.5. [ka]

[0053] Furthermore, n is selected from the range of 7.3 to 7.5. [ka]

[0054] Furthermore, n is selected from the range of 7.4 to 7.5.

[0055] Here, Ab is defined as in the compound of formula (I).

[0056] In another aspect of the present invention, there is provided a method for producing the above antibody-drug conjugate, the method being selected from the following synthetic routes:

[0057] Synthetic Route 1: A compound of formula (a) is reacted with a compound of formula (b) to obtain a compound of formula (c), and the compound of formula (c) is coupled to an antibody to obtain a compound of formula (I), wherein R a -L 1 -NH-L p -Z-Ab and R b is hydrogen. [ka]

[0058] Synthetic Route 2: The compound of formula (d) is reacted with HO-L z -L j ' to obtain a compound of formula (e), and linking the compound of formula (e) to an antibody to obtain a compound of formula (I), wherein R a -L 1 -NH-L p -Z-Ab and R b is hydrogen. [ka]

[0059] Synthetic Route 3: The compound of formula (f) is z -Lj′ to obtain a compound of formula (g), and linking the compound of formula (g) to an antibody to obtain a compound of formula (I), wherein R a -L 1 -NH-L p -Z-Ab and R b is hydrogen. [ka]

[0060] Synthetic Route 4: The compound of formula (h) is reacted with HO-L z -L j ' to obtain a compound of formula (i), which is then linked to an antibody after removal of the amino-protecting group X from the compound of formula (i), to obtain a compound of formula (I), wherein R a is hydrogen and R b -L 2 -NH-L p -Z-Ab. [ka]

[0061] Synthetic Route 5: The compound of formula (j) is subjected to a condensation reaction with HO-Lz-Lj′ to obtain a compound of formula (k), from which the amino-protecting group X is removed and then linked to an antibody to obtain a compound of formula (I), wherein R a is hydrogen and R b -L 3 -NH-Z-Ab. [ka]

[0062] In the above synthesis route, m, n1, L 1 , L 2, L 3 , L p , L z and Ab are each defined as in the compound of formula (I), L j ' is selected from the group represented by the formula: [ka] X is an amino protecting group.

[0063] In another aspect of the present invention, there is provided a pharmaceutical composition comprising the antibody-drug conjugate or the antibody-drug conjugate produced by the above-described method and a pharmaceutically acceptable carrier.

[0064] In another aspect of the present invention, there is provided use of the above antibody-drug conjugate, the antibody-drug conjugate produced by the above method, or the above pharmaceutical composition in the manufacture of an anti-tumor drug.

[0065] In another aspect of the present invention, there is provided a method for the treatment or therapy of a tumor disease in a patient in need thereof, the method comprising administering to the patient in need thereof the antibody-drug conjugate or pharmaceutical composition described above.

[0066] In some specific embodiments, the amount of the antibody-drug conjugate or pharmaceutical composition administered is a therapeutically effective amount.

[0067] In some specific embodiments, the tumor is selected from a solid tumor.

[0068] In some specific embodiments, the tumor is selected from esophageal cancer, lung cancer, or breast cancer.

[0069] In some specific embodiments, the lung cancer is non-small cell lung cancer. [Brief explanation of the drawings]

[0070] [Figure 1]1 is a graph showing the tumor growth curve of the NCI-H292 human lung cancer CDX model with in vivo inhibition by ADC4 in Test Example 2. [Figure 2] 1 shows photographs of tumors after dissection in an NCI-H292 human lung cancer CDX model with in vivo inhibition by ADC4 in Test Example 2. [Figure 3] 1 is a graph showing the tumor growth curve of the NCI-H1975 human lung cancer CDX model with in vivo inhibition by ADC4 in Test Example 2. [Figure 4] 1 shows photographs of tumors after dissection in an NCI-H1975 human lung cancer CDX model with in vivo inhibition by ADC4 in Test Example 2. [Figure 5] 1 is a graph showing tumor growth curves in the NCI-H1975 human lung cancer CDX model with in vivo inhibition by ADC6, 8, and 22 in Test Example 2. [Figure 6] 1 shows photographs of tumors after dissection in an NCI-H1975 human lung cancer CDX model with in vivo inhibition by ADC6, 8, and 22 in Test Example 2. [Figure 7] 1 is a graph showing tumor growth curves in the NCI-H1975 human lung cancer CDX model due to in vivo inhibition by ADC14, 16, and 12 in Test Example 2. [Figure 8] 1 shows photographs of tumors after dissection in an NCI-H1975 human lung cancer CDX model with in vivo inhibition by ADC14, 16, and 12 in Test Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0071] I. Definition: In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the relevant terms and experimental procedures used herein are terms and routine steps that are widely used in the corresponding fields.In addition, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0072] As used herein, unless otherwise specified, the terms "comprise," "include," "have," and "contain," including their grammatical equivalents, are generally to be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps.

[0073] The compounds of the present disclosure may be asymmetric, e.g., may have one or more stereoisomers. Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are included. The stereoisomers include geometric isomers (e.g., cis- and trans-configurations) and optical isomers (e.g., enantiomers), and are therapeutic agents consisting of individual compounds, racemates, racemic mixtures, and pharmaceutically acceptable salts thereof. Compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all within the scope of the present invention.

[0074] Numerical ranges herein refer to individual integers within the specified range, for example, "C1-C6" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0075] When any variable (e.g., Rn) occurs more than one time in any composition or structure of a compound, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1 to 5 R, then that group is optionally substituted with up to 5 R, and each occurrence of R is an independent option. Further, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0076] The term "C1-C6 alkyl group" refers to a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched-chain isomers thereof.

[0077] [ka] indicates the point of attachment of a chemical bond. 11 -L 12 -L 12 -or-L z -L j -) represents a linking of the corresponding groups in left to right order unless otherwise specified. For example, -L 12 is C1-C2 alkylene-O-, this means that the left side of C1-C2 alkylene-O- is -L 11 -L on the right 13 - indicates that it will be linked to.

[0078] As will be understood by those skilled in the art, in the antibody-drug conjugates of the present application, for example, in formulae (I), (I-1), (I-2), and (I-3), the "-" between the drug-linker fragment and the antibody is intended to represent the linkage between the antibody and the fragment, and is not intended to limit the linkage of one antibody to only one drug-linker fragment. As is well known in the art, one antibody can have multiple intermolecular disulfide bonds, and therefore one antibody can have one or more drugs linked to it.

[0079] Drug or pharmaceutical composition The drugs or pharmaceutical compositions of the present invention can be administered orally, topically, parenterally, or mucosally (e.g., sublingually, by inhalation, or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. Oral administration is generally preferred. The active agent can be administered orally in the form of capsules, tablets, or the like.

[0080] The term "treatment" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

[0081] The use of the term "inhibition" refers to a control. Those skilled in the art can easily determine the appropriate control for each experiment. For example, the reduction in the response in subjects or cells treated with a compound is compared with the response in subjects or cells not treated with a compound.

[0082] The term "patient" refers to an animal, preferably a mammal, and more preferably a human.

[0083] The term "pharmaceutical composition" means a composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the group consisting of, but not limited to, carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersing agents, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, and suspension aids, depending on the nature of the administration method and dosage form.

[0084] The term "effective amount" or "therapeutically effective amount" refers to a nontoxic but sufficient amount of a drug or agent to achieve a desired effect. In embodiments of the present invention, when treating a patient according to the present invention, the amount of a given drug to be administered will depend on many factors, such as the specific administration protocol, the type and severity of the disease or condition, and the specific characteristics (e.g., body weight) of the subject or host requiring treatment. However, dosages can be routinely determined by methods known in the art depending on the specific circumstances, including, for example, the specific drug employed, the route of administration, the condition being treated, and the subject or host being treated. Generally, dosages used in adult treatment typically range from 0.02 to 5000 mg / day, e.g., about 1 to 1500 mg / day. This required dose can conveniently be expressed as a single dose or as divided doses administered simultaneously (or shortly thereafter) or at appropriate intervals, e.g., two, three, four, or more divided doses per day. Although the above dosage ranges are given, it will be understood by those skilled in the art that the specific effective amount may be appropriately adjusted depending on the condition of the patient and in conjunction with the diagnosis of a physician.

[0085] The term "antibody-drug conjugate (ADC)" refers to a monoclonal antibody to which a biologically active small molecule drug is linked via a chemical bond, and the monoclonal antibody functions as a carrier for delivering the small molecule drug to target cells. In this specification, the meaning of "antibody-conjugated drug" is the same as that of antibody-drug conjugate.

[0086] The term "drug-to-antibody ratio (DAR)" refers to the average number of active ingredient molecules (anti-tumor compound or drug) bound to a single monoclonal antibody.

[0087] Abbreviation: Fmoc: 9-fluorenylmethoxycarbonyl group The amino acid constituting the peptide residue LP is Val: valine, and its structural formula is [ka] and Ala: Alanine, its structural formula is [ka] and Gly: Glycine, whose structural formula is [ka] and Phe: Phenylalanine, its structural formula is [ka] and L P can be obtained by amino acid condensation methods known in the art. PABC: [ka] ha: [ka] m: [ka]

[0088] In the present invention, a linker L for linking antibodies j can be obtained by linking by methods known in the art. For example, L j The structure of [ka] (or [ka] ), then it is [ka] (or [ka] ) with a reactive group such as thiol on the antibody, and the present invention is not particularly limited thereto.

[0089] II. Working Examples The antibodies used to produce the ADC in this example are not limited to pertuzumab and IP140B antibodies. The heavy chain amino acid sequence of pertuzumab (HS627, PERTUZUMAB) is as follows (SEQ ID NO: 1): [ka]

[0090] The light chain amino acid sequence is as follows (SEQ ID NO:2): [ka]

[0091] The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO:3): [ka]

[0092] The light chain amino acid sequence is as follows (SEQ ID NO:4): [ka]

[0093] Unless otherwise specified, the materials and equipment used in specific embodiments of the present invention are known products and can be obtained commercially.

[0094] Intermediate HX-1a: 7-(2-(tetrahydropyran-4-)amino)ethyl-10,11-methylenedioxycamptothecin [ka] 4-Aminotetrahydropyran (0.75 g, 7.4 mmol), concentrated hydrochloric acid (0.7 mL, 7.5 mmol), and DMSO (15 mL) were heated to 120°C in an oil bath, 7-methyl-10,11-methylenedioxycamptothecin (0.5 g, 1.2 mmol) was added, and the temperature was raised to 130-140°C and the reaction was carried out for 1 hour. Methanol was then added, and the mixture was subjected to suction filtration. The filtrate was concentrated and then purified by silica gel column chromatography to obtain the title compound (0.41 g, yield 63%, HPLC 96%) as a gray solid. 1 H NMR(500MHz,DMSO-d6)δ7.59(s,1H),7.47-7.41(m,1H),7.25-7.14(m,1H),6.54(s,1H),6.27(t,J=9.9Hz,2H),5.77(s,1H),5.44(d,J=16.5Hz,2 H),5.23-5.11(m,2H),3.83(d,J=9.5Hz,2H),3.23(t,J=24.9Hz,4H),2. 93(d,J=32.7Hz,3H),1.99-1.72(m,4H),1.33(s,2H),0.96-0.81(m,3H); 13 CNMR(126MHz,DMSO)δ173.04,157.26,151.30,150.58,149.68,149.42,147.49,146.73,146.68,128.52,124. 79,118.49,105.90,103.14,99.83,96.39,72.82,66.05,65.69,55.41,53.65,50.31,30.66,8.27;LC-MS(M+H) + 520.11 (theoretical value 519.20).

[0095] Intermediate HX-6a: 7-(2-(N-(S)-tetrahydrofuran-3-amino))ethyl-10,11-methylenedioxycamptothecin [ka] (S)-3-aminotetrahydrofuran (64.31 mg, 0.738 mmol), concentrated hydrochloric acid (0.05 mL, 0.6 mmol), DMSO (1.5 mL), and 7-methyl-10,11-methylenedioxycamptothecin (50 mg, 0.12 mmol) were added to a reaction flask, and the mixture was heated to 120-130°C with stirring and reacted for 1 hour. After that, the mixture was cooled to room temperature, and methyl tert-butyl ether was added. The mixture was filtered to precipitate a solid, which was then purified by silica gel column chromatography to obtain the title compound (18 mg, yield 33%, HPLC 95.9%). 1 H NMR(500MHz,DMSO-d6)δ7.61(d,J=3.4Hz,1H),7.47(d,J=3.9Hz,1H),7.22(d,J=4.2Hz,1H),6.50(s,1H),6.29(d,J=4.0Hz,2H),5.42 (d,J=4.0Hz,2H),5.21(d,J=3.2Hz,2H),3.87-3.58(m,4H),3.21(s,2H),2.85(td,J=11.2,10.7,5.8Hz,2H),1.90(ddt,J=29.9,10.7 ,6.2Hz,3H),1.68-1.60(m,1H),1.08-0.76(m,3H);13CNMR(126MHz,DMSO)δ173.0,157.3,151.3,150.6,149.7,149.4,147.6,146.9, LC-MS(M+H) + 506.31 (theoretical value 505.18).

[0096] Intermediate HX-8a: 7-(2-(N-(R)-tetrahydrofuran-3-amino))ethyl-10,11-methylenedioxycamptothecin [ka] (R)-3-aminotetrahydrofuran (64.31 mg, 0.738 mmol), concentrated hydrochloric acid (0.05 mL, 0.6 mmol), DMSO (1.5 mL), and 7-methyl-10,11-methylenedioxycamptothecin (50 mg, 0.12 mmol) were added to a reaction flask, and the mixture was heated to 120°C to 130°C with stirring and reacted for 1 hour. After cooling to room temperature, methyl tert-butyl ether was added, and the mixture was filtered to precipitate a solid. The solid was purified by silica gel column chromatography to obtain the title compound (16.2 mg, yield 32.3%, HPLC 96.2%). 1 H NMR(500MHz,DMSO-d6)δ7.61(s,1H),7.47(s,1H),7.22(s,1H),6.50(s,1H),6.28(s,2H),5.42(s,2H),5.21(s,2H),4.21- 3.53(m,4H),3.21(t,J=7.6Hz,2H),2.84(q,J=8.7,8.1Hz,2H),1.90(ddt,J=31.9,16.2,7.1Hz,3H),1.63(dd,J=12.3,6.1 Hz,1H),0.88(t,J=7.3Hz,3H);13CNMR(126MHz,DMSO)δ173.0,157.3,151.3,150.6,149.7,149.4,147.5,146.8,141.2,12 8.6,124.9,118.4,105.9,103.1,99.9,96.4,72.9,66.9,65.7,58.4,55.4,50.4,47.6,32.6,30.7,30.6,8.3;LC-MS(M+H) + 506.34 (theoretical value 505.18).

[0097] Example 1: 7-[N-(HS627-mc-Val-Cit-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (1) [ka] 0.2 mL of NMP was added to the reaction flask, followed by the addition of compounds mc-VC-PAB-PNP (20 mg, 0.027 mmol), DIPEA (17.52 mg, 0.135 mmol), HX-1a (14.08 mg, 0.027 mmol), and HOBt (7.33 mg, 0.054 mmol). The mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 10% to 55% acetonitrile in water, 50 min), and lyophilized to obtain the yellow solid product HX-1 (20.2 mg, 66% yield). LCMS: (M+1) + 1118.31 (theoretical value: 1117.48).

[0098] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0099] Compound HX-1 (0.89 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugated drug ADC 1 (3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.20.

[0100] Example 2: 7-[N-(IP140B-mc-Val-Cit-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (2) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0101] Compound HX-1 (0.89 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain the antibody conjugate drug ADC 2 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.40.

[0102] Example 3: 7-[N-(HS627-mPEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (3) [ka] To a 10 mL single-neck flask, HX-1a (40 mg, 0.077 mmol), 0.4 mL of NMP, DIPEA (50 mg, 0.38 mmol), Fmoc-GK-PAB-PNP (73 mg, 0.077 mmol), and HOBt (10.4 mg, 0.077 mmol) were added sequentially and stirred at room temperature for 0.5 hours to give HX-2a. 0.4 mL of piperidine (V:V = 10%) was added and the reaction was allowed to proceed at room temperature for 0.5 hours. 30 mL of methyl tert-butyl ether was added to the reaction mixture, which was then centrifuged. The supernatant was removed, and the solvent was removed under reduced pressure. The resulting solid product, HX-2b (69 mg, 77% yield), was used directly in the next reaction. LCMS: (M+1) +1096.45 (theoretical value: 1095.47).

[0103] To a 10 mL single-neck flask, DCM (2 mL), HX-2b (69 mg, 0.063 mmol), and HX-2c (27 mg, 0.075 mmol) were added sequentially and stirred for 2 minutes. After that, TEA (6.40 mg, 0.063 mmol) was added and the reaction was continued with stirring at room temperature for 15 minutes. The reaction mixture was purified by 3 g silica gel column chromatography to obtain intermediate HX-2d (72 mg, 85% yield). LCMS: (M+1) + 1335.48 (theoretical value: 1334.55).

[0104] DCM (1 mL), HX-2d (72 mg, 0.053 mmol), and TFA (0.1 mL) were added sequentially to a 10 mL single-neck flask and stirred at room temperature for 20 minutes. The reaction mixture was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-2 (29 mg, 50% yield). LCMS: (M+1) + 1093.16 (theoretical value: 1092.44).

[0105] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0106] Compound HX-2 (0.87 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 3 (2.9 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.80.

[0107] Example 4: 7-[N-(IP140B-mPEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (4) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0108] Compound HX-2 (0.87 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 4 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain the antibody conjugate drug ADC 4 (3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.70.

[0109] Example 5: 7-[N-(HS627-AcPEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (5) [ka] To a 50 mL single-neck flask, DCM (20 mL), 3-[2-(2-aminoethoxy)ethoxy]propionic acid (2 g, 11.3 mmol), and bromoacetyl bromide (2.27 g, 11.3 mmol) were added sequentially and stirred for 30 minutes. The reaction mixture was extracted with 10 mL of saturated sodium chloride solution, and the DCM phase was dried over anhydrous magnesium sulfate to give intermediate HX-3b (3.1 g, 89% yield). LCMS: (M+1) + 298.00 (theoretical value: 297.02).

[0110] To a 10 mL single-neck flask, DCM (1 mL), HX-2b (50 mg, 0.045 mmol), HX-3b (13.6 mg, 0.045 mmol), and DIC (5.75 mg, 0.045 mmol) were added sequentially and stirred at room temperature for 60 min. 0.1 mL of TFA was added to the reaction mixture and allowed to react for another 30 min. Finally, the reaction mixture was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile and then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, 30 min), and lyophilized to give the white solid product HX-3 (16 mg, 31% yield). LCMS: (M+1) +1 113.24 (theoretical value: 1132.38).

[0111] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0112] Compound HX-3 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 5 (3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.2.

[0113] Example 6: 7-[N-(IP140B-AcPEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (6) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0114] Compound HX-3 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 6 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 6 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.10.

[0115] Example 7: 7-[N-(HS627-AcPEG4-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (7) [ka] To a 50 mL single-neck flask, DCM (30 mL), 1-amino-3,6,9,12-tetraoxopentadecanoic acid (2 g, 7.5 mmol), and bromoacetyl bromide (1.51 g, 7.5 mmol) were added sequentially and stirred for 30 minutes. The reaction mixture was extracted with 15 mL of saturated sodium chloride solution, and the DCM phase was dried over anhydrous magnesium sulfate to give intermediate HX-4a (2.5 g, 85% yield). LCMS: (M+1) + 386.24 (theoretical value: 385.07).

[0116] To a 10 mL single-neck flask, DCM (1 mL), HX-2b (50 mg, 0.045 mmol), HX-3b (17.4 mg, 0.045 mmol), and DIC (5.75 mg, 0.045 mmol) were added sequentially and stirred at room temperature for 60 min. 0.1 mL of TFA was added to the reaction mixture and allowed to react for another 30 min. Finally, the reaction mixture was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile and then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, 30 min), and lyophilized to give the white solid product HX-4 (16 mg, 31% yield). LCMS: (M+1). + 1223.05 (theoretical value: 1222.15).

[0117] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0118] Compound HX-4 (0.98 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 7 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.1.

[0119] Example 8: 7-[N-(IP140B-AcPEG4-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (8) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0120] Compound HX-4 (0.98 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 8 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.0.

[0121] Example 9: 7-[N-(HS627-Qx-PEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (9) [ka] In a 250 mL single-neck flask, 3,4-diaminobenzoic acid (5 g, 0.032 mol) was dispersed in 65 mL of ethanol, and methyl bromopropionate (5.95 g, 0.032 mol) was added. The mixture was stirred at room temperature for 1 hour, and the reaction mixture was filtered and dried. The mixture was then purified by silica gel column chromatography to give a yellow solid product, HX-5a (8 g, 83% yield). LCMS: (M+1) + 283.09 (theoretical value: 281.96).

[0122] To a 50 mL single-neck flask, DCM (10 mL), HX-5a (1 g, 3.53 mmol), and DCC (0.36 g, 1.76 mmol) were added sequentially, and the reaction was stirred at room temperature for 60 minutes. The reaction was filtered to remove DCU, and intermediate HX-5b (0.96 g, 100% yield) was obtained, which was directly used in the next step. LCMS: (M+2) + 548.15 (theoretical value: 545.92).

[0123] DCM (10 mL), HX-5b (0.96 g, 1.76 mmol), 3-[2-(2-aminoethoxy)ethoxy]propionic acid (0.31 g, 1.76 mmol), and TEA (0.17 g, 1.76 mmol) were added sequentially to a 50 mL single-neck reaction flask and stirred at room temperature for 60 minutes. The reaction mixture was then loaded onto a 40 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 0% to 40% acetonitrile in water, 30 min), and lyophilized to give the pink solid product HX-5c (1.21 g, 80% yield). LCMS: (M+1) + 442.27 (theoretical value: 441.05).

[0124] To a 10 mL single-neck flask, DCM (1 mL), HX-2b (50 mg, 0.045 mmol), HX-5c (19.89 mg, 0.045 mmol), and DIC (5.75 mg, 0.045 mmol) were added sequentially and allowed to react at room temperature for 90 minutes. After confirming complete reaction of the starting materials by HPLC, 0.1 mL of TFA was added to the reaction mixture and allowed to react for another 30 minutes. Finally, the reaction mixture was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 15% to 40% acetonitrile in water, 30 min), and lyophilized to obtain the white solid product HX-5 (16 mg, 31% yield). LCMS: (M+1) + 1277.31 (theoretical value: 1276.46).

[0125] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0126] Compound HX-5 (1 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 9 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 6.8.

[0127] Example 10: 7-[N-(IP140B-Qx-PEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (10) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0128] Compound HX-5 (1 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 10 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.0.

[0129] Example 11: 7-[N-(HS627-Qx-PEG2-Gly-Lys-PABC),N-((R)-tetrahydropyran-3-)]aminoethyl-10,11-methylenedioxycamptothecin (11) [ka] To a 10 mL single-neck flask, HX-6a (50 mg, 0.099 mmol), 1 mL NMP, DIPEA (64 mg, 0.49 mmol), Fmoc-GK-PAB-PNP (93 mg, 0.099 mmol), and HOBt (13.3 mg, 0.099 mmol) were added sequentially, and the mixture was stirred at room temperature for 0.5 h to give HX-6b, which was directly used in the next step without further purification. LCMS: (M+1) + 1304.47 (theoretical value: 1303.53).

[0130] To the HX-6b reaction mixture, 0.1 mL of piperidine (V:V = 10%) was added and the mixture was allowed to react at room temperature for 0.5 hours. 20 mL of methyl tert-butyl ether was added to the reaction mixture, the mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure. The resulting solid product, HX-6c (61 mg, 56% yield), was used directly in the next reaction. LCMS: (M+1) + 1082.22 (theoretical value: 1081.46).

[0131] To a 10 mL single-neck flask, DCM (1 mL), HX-6c (61 mg, 0.056 mmol), HX-5c (24.7 mg, 0.056 mmol), and DIC (7.05 mg, 0.056 mmol) were added sequentially and stirred at room temperature for 90 minutes. 0.1 mL of TFA was added to the reaction mixture and the mixture was allowed to react for another 30 minutes. Finally, the reaction mixture was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the white solid product HX-6 (21 mg, 29% yield). LCMS: (M+1) + 1264.15 (theoretical value: 1262.39).

[0132] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0133] Compound HX-6 (1 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 11 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.4.

[0134] Example 12: 7-[N-(IP140B-Qx-PEG2-Gly-Lys-PABC),N-((R)-tetrahydropyran-3-)]aminoethyl-10,11-methylenedioxycamptothecin (12) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0135] Compound HX-6 (1 mg, 0.8 mmol) was dissolved in 0.1 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7, using a NAP-5 gel column (Cytiva), to obtain antibody conjugate drug ADC 12 (3.4 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0136] Example 13: 7-[N-(HS627-Ac-PEG2-Gly-Lys-PABC),N-((R)-tetrahydropyran-3-)]aminoethyl-10,11-methylenedioxycamptothecin (13) [ka] To a 10 mL single-neck flask, HX-6a (50 mg, 0.046 mmol), 1 mL DMF, Fmoc-NH-PEG-CHCHCOOH (18 mg, 0.046 mmol), and HATU (17.57 mg, 0.046 mmol) were added sequentially, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by silica gel column chromatography to give yellow powder product HX-7a (55 mg, 82% yield). LCMS: (M+1) + 1463.51 (theoretical value: 1462.62).

[0137] HX-7a (55 mg, 0.037 mmol) was dissolved in 0.2 mL of DMF, 0.2 mL of piperidine (V:V = 10%) was added, and the mixture was allowed to react at room temperature for 0.5 hours. 30 mL of methyl tert-butyl ether was added to the reaction mixture, the mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure. The resulting solid product HX-7b (35 mg, 76% yield) was used directly in the next reaction. LCMS: (M+1) + 1241.41 (theoretical value: 1240.55).

[0138] To a 10 mL single-neck flask, DCM (1 mL), HX-7b (35 mg, 0.028 mmol), bromoacetic acid (24.7 mg, 0.056 mmol), and DIC (7.05 mg, 0.056 mmol) were added sequentially and stirred at room temperature for 90 minutes. 0.1 mL of TFA was added to the reaction mixture and the mixture was allowed to react for another 30 minutes. Finally, the reaction mixture was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the yellow solid product HX-7 (16 mg, 57% yield). LCMS: (M+1) +1119.24 (theoretical value: 1118.36).

[0139] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0140] Compound HX-7 (0.9 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 13 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0141] Example 14: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC),N-((R)-tetrahydropyran-3-)]aminoethyl-10,11-methylenedioxycamptothecin (14) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0142] Compound HX-7 (0.9 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 14 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 14 (3.0 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.4.

[0143] Example 15: 7-[N-(HS627-Ac-PEG2-Gly-Lys-PABC),N-((S)-tetrahydropyran-3-)]aminoethyl-10,11-methylenedioxycamptothecin (15) [ka] To a 10 mL single-neck flask, HX-8a (60 mg, 0.118 mmol), 1 mL NMP, DIPEA (46 mg, 356 mmol), Fmoc-GK-PAB-PNP (80 mg, 0.118 mmol), and HOBt (16 mg, 0.118 mmol) were added sequentially, and the mixture was stirred at room temperature for 4 hours. The resulting HX-8b reaction mixture was then directly added to the next step. LCMS: (M+1) + 1304.45 (theoretical value: 1303.53).

[0144] To the HX-8b reaction mixture, 0.1 mL of piperidine (V:V = 10%) was added and the mixture was allowed to react at room temperature for 0.5 hours. 20 mL of methyl tert-butyl ether was added to the reaction mixture, the mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure. The resulting solid HX-8c (90 mg, 70% yield) was used directly in the next reaction. LCMS: (M+1) + 1082.22 (theoretical value: 1081.46).

[0145] To a 10 mL single-neck flask, DCM (1 mL), HX-8c (90 mg, 0.083 mmol), HX-3b (24 mg, 0.083 mmol), and DIC (10.45 mg, 0.083 mmol) were added sequentially and stirred at room temperature for 90 min. 0.1 mL of TFA was added to the reaction mixture and allowed to react for another 30 min. Finally, the reaction mixture was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The mixture was then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 min), and lyophilized to give the yellow solid product HX-8 (32 mg, 34% yield). LCMS: (M+1) + 1119.23 (theoretical value: 1118.36).

[0146] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0147] Compound HX-8 (0.9 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system. After thorough mixing, the mixture was allowed to react on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 15 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.4.

[0148] Example 16: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC),N-((S)-tetrahydropyran-3-)]aminoethyl-10,11-methylenedioxycamptothecin (16) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0149] Compound HX-8 (0.9 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 16 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0150] Example 17: 20-O-(HS627-Ac-PEG2-Gly-Val-Ala-Gly-NHNHC)-7-N-(tetrahydropyran-4-)aminoethyl-10,11-methylenedioxycamptothecin (17) [ka] To a 50 mL single-neck flask, HX-1a (200 mg, 0.385 mmol) and 3 mL of dichloromethane were added, followed by Boc anhydride (92.4 mg, 0.423 mmol) and TEA (85.7 mg, 0.2846 mmol), and the mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure, and the resulting mixture was purified by silica gel column chromatography to give a pale yellow solid, HX-26a (223.5 mg, 93.7% yield). LCMS: (M+1) + 620.26 (theoretical value: 619.67).

[0151] HX-26a (85 mg, 0.137 mmol), DMAP (83.8 mg, 0.686 mmol), and 2 mL of dichloromethane were added to a 50 mL single-neck flask in this order, and the mixture was stirred at 0°C for 5 minutes to react. 2 mL of dichloromethane and triphosgene (32.6 mg, 0.11 mmol) were added, and the mixture was allowed to react at room temperature for 2 hours. The resulting clear yellow solution of HX-26b was used directly in the next reaction.

[0152] To the clear yellow solution of HX-26b, HX-26c (69.8 mg, 0.274 mmol) was added directly, and the mixture was stirred at room temperature for 3 hours. The resulting mixture was purified by gel column chromatography to give a pale yellow solid, HX-26d (118.8 mg, 96.2% yield). LCMS: (M+1) + 900.34 (theoretical value: 899.95).

[0153] HX-26d (100 mg, 0.11 mmol) was added to a 10 mL single-neck flask, and 1 mL of DMF was added to dissolve it. Then piperidine (94.6 mg, 1.1 mmol) was added and the mixture was allowed to react at room temperature for 0.5 hours. The reaction mixture was added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-26e (70.5 mg, 93.2% yield). LCMS: (M+1) + 678.27 (theoretical value: 677.71).

[0154] HX-26e (70 mg, 0.103 mmol) was added to DMF (2 mL), HATU (47.2 mg, 0.124 mmol), DIPEA (16.02 mg, 0.124 mmol), and Fmoc-Gly-Val-Ala-Gly-OH (54.08 mg, 0.103 mmol). The mixture was allowed to react at room temperature for 2 hours, diluted with 200 mL of DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to give a yellow solid, HX-26f (101.3 mg, 82.9% yield). LCMS: (M+1) + 1082.49 (theoretical value: 1183.28).

[0155] HX-26f (101 mg, 0.085 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. Then piperidine (72.7 mg, 0.85 mmol) was added and the mixture was allowed to react at room temperature for 0.5 hours. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-26g (79.8 mg, 97.3% yield). LCMS: (M+1) + 961.04 (theoretical value: 960.42).

[0156] To a 10 mL single-neck flask, DCM (2 mL), HX-26g (70 mg, 0.073 mmol), HX-3b (26 mg, 0.087 mmol), and DIC (11.02 mg, 0.087 mmol) were added sequentially and stirred at room temperature for 30 minutes. After confirming complete reaction of the starting materials by HPLC, a clear yellow solution of HX-26h was obtained. 200 μL of trifluoroacetic acid was added and stirred at room temperature for 30 minutes. The reaction solution was loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase) and eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes). The yellow solid product HX-26 (37.5 mg, 45% yield) was obtained after lyophilization. LCMS: (M+1) + 1142.03 (theoretical value: 1140.38).

[0157] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0158] Compound HX-26 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, and mixed well. The mixture was then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 17 (3.2 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.3.

[0159] Example 18: 20-O-(IP140B-AcPEG2-Gly-Val-Ala-Gly-NHNHC)-7-N-(tetrahydropyran-4-)aminoethyl-10,11-methylenedioxycamptothecin (18) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0160] Compound HX-26 (0.91 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody conjugate drug ADC 18 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0161] Example 19: 20-O-(HS627-Ac-PEG4-daP-NHNHC)-7-N-(tetrahydropyran-4-)aminoethyl-10,11-methylenedioxycamptothecin (19) [ka] To the clear yellow solution of HX-28b prepared according to the method described above, HX-27a (80.8 mg, 0.274 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The resulting mixture was purified by gel column chromatography to give a pale yellow solid, HX-27b (140.3 mg, 96.3% yield). LCMS: (M+1) + 1062.15 (theoretical value: 1061.42).

[0162] HX-27b (140.3 mg, 0.132 mmol) was added to a 10 mL single-neck flask and dissolved in 1 mL of DMF. Then piperidine (112.6 mg, 1.32 mmol) was added and the mixture was allowed to react at room temperature for 0.5 hours. The reaction mixture was then added dropwise to 20 mL of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the mixture was dried to give HX-27c (89.5 mg, 80.6% yield). LCMS: (M+1) + 840.35 (theoretical value: 839.90).

[0163] To a 10 mL single-neck flask, DCM (2 mL), HX-27c (70 mg, 0.083 mmol), HX-4a (38.6 mg, 0.1 mmol), and DIC (12.6 mg, 0.1 mmol) were added sequentially and stirred at room temperature for 30 minutes to give a clear yellow solution of HX-27d. 200 μL of trifluoroacetic acid was added and stirred at room temperature for 30 minutes. The reaction solution was then loaded onto a 25 g C18 precolumn (first equilibrated with acetonitrile, then with water, containing 0.1% TFA in the aqueous phase). The solution was eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, 30 minutes), and lyophilized to give the yellow solid product HX-27 (43.9 mg, 47.9% yield). LCMS: (M+1) + 1093.98 (theoretical value: 1092.38).

[0164] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0165] Compound HX-27 (0.87 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system. After thorough mixing, the mixture was allowed to react on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer solution was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 19 (3.1 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.5.

[0166] Example 20: 20-O-(IP140B-Ac-PEG4-daP-NHNHC)-7-N-(tetrahydropyran-4-)aminoethyl-10,11-methylenedioxycamptothecin (20) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0167] Compound HX-27 (0.87 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution, mixed well, and then reacted on a rotary turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain the antibody conjugate drug ADC 20 (3.3 mg / mL, 2 mL). Mean value calculated by UV-HPLC: n = 7.4.

[0168] Example 21: 7-[N-(HS627-AcPEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (21) [ka] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.016 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0169] Compound HX-3 (0.42 mg, 0.367 mmol) was dissolved in 0.04 mL of DMA and added to the above solution. After thorough mixing, the mixture was allowed to react on a rotating turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g / L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain ADC 21 (3.2 mg / mL, 2 mL). Average value calculated by UV-HPLC: n=4.05

[0170] Example 22: 7-[N-(IP140B-AcPEG2-Gly-Lys-PABC),N-(tetrahydropyran-4-)]aminoethyl-10,11-methylenedioxycamptothecin (22) [ka] IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 using 1 M NaHPO solution. Then, 0.1 M ethylenediaminetetraacetic acid disodium solution (25 μL) was added, followed by the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.016 mL). The mixture was allowed to react on a rotating turntable at room temperature (25°C) for 90 minutes.

[0171] Compound HX-3 (0.42 mg, 0.367 mmol) was dissolved in 0.04 mL of DMA and added to the above solution. After mixing well, the mixture was allowed to react on a rotating turntable at room temperature for 2 hours. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Twenty-nine 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain ADC 22 (3.1 mg / mL, 2 mL). Average value calculated by UV-HPLC: n=3.95

[0172] Test Example 1: In vitro tumor cell proliferation inhibitory activity of ADC Test method for in vitro inhibitory activity of ADC: Human esophageal cancer cells OE-33, human lung cancer cells NCI-H1975, and human breast cancer cells MDA-MB-231 used for activity detection were cultured in RPMI1640 (Cellmax), RPMI1640 (Cellmax), and DMEM (Cellmax) media containing 10% fetal bovine serum until exponential growth phase. After trypsin digestion, the supernatant was discarded by centrifugation, and 3 × 10 cells were cultured in the media. 4 cells / mL, 0.5×10 4 cells / mL, 1.5×10 4The ADCs were diluted to 1000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, and 0.026 nM in medium. 100 μL of the diluted ADCs were added to each well of a 96-well cell culture plate. Each well was replicated for each concentration. For negative and blank controls, 100 μL of culture medium was added per well. After incubation, the plates were placed in a 37°C, 5% CO2 incubator for 6 days. After incubation, the cell culture plate was removed, the medium in the plate was aspirated and removed with a pipette, and then 100 μL of medium containing 10% CCK-8 was added per well and incubated at 37°C for 3 hours. After incubation, the culture plate was removed, shielded from light, and placed on an enzyme plate. The absorbance was measured using a reference wavelength of 630 nm and a measurement wavelength of 450 nm. IC 50 was calculated from the absorbance values ​​using a four-parameter regression in GraphPad (Table 1). The ADC drug corresponding to Dxd was used as a positive control drug and has the following structure: [ka] (where Ab is IP140B antibody.) [Table 1] I C 50 For values, "++++" is IC 50 <5nM, and "++++" indicates IC 50 indicates that the IC is between 10 and 50 nM, and "++" indicates that the IC 50 indicates that the IC is between 50 and 500 nM, and "+" indicates that the IC 50 Indicates >500nM.

[0173] The ADC compounds in the examples of the present invention have excellent inhibitory activity against NCI-H1975, MDA-MB-231, and OE-33 cells, and the IC50 All of the values ​​were below 250 nM, and some ADC compounds, such as ADC2, 3, 4, 10, 12, 14, 18, and 20, had IC values ​​of inhibitory activity against these cancer cells. 50 The IC values ​​were even lower than 50 nM, and some ADC compounds, such as ADC4, 10, 18, and 20, had IC values ​​of inhibitory activity against these cancer cells. 50 The value is even less than 10 nM.

[0174] Test Example 2: In vivo tumor growth inhibitory activity of ADC Test method for in vitro inhibitory activity of ADC: Human lung cancer cells NCI-H292 and NCI-H1975 were cultured in vitro as monolayers. When the cell density reached 80-90%, they were digested with trypsin-EDTA, centrifuged, and the supernatant discarded. The cells were resuspended in PBS and adjusted to the appropriate concentration. NCI-H292 and NCI-H1975 cells (2-10 × 10) were cultured in vitro. 6 BALB / c nude mice were subcutaneously inoculated with 0.1 mL of cells. The animals and the growth of the transplanted tumor were monitored periodically. The tumor volume was measured to be between 100 and 200 mm. 3 Once the tumors reached a certain size, they were randomly divided into two groups based on tumor volume and body weight: a vehicle control group (physiological saline) and an ADC-treated group (dissolved in physiological saline), with six animals in each group. The tumors were administered intravenously. The administration frequency was Q4D, with a total of two doses (the first dose was administered on Day 1, and the second on Day 5). See Table 2 for experimental grouping and administration settings. The tumor's major axis (a) and minor axis (b) were measured twice weekly using calipers, along with the mouse's body weight. Tumor volume (V) was calculated using the following formula: V = 1 / 2 × a × b. 2 (mm 3), where a and b represent the length and width of the tumor, respectively. A growth curve was then created, and the tumors were finally excised and weighed. Statistical analysis was performed using GraphPad Prism software using the tumor volume and weight data of the tumor-bearing mice at the end of the experiment to obtain the results of the antitumor effect. In the figure, "" in the control group indicates that there was no result for that group or that the tumor status was not measured. In the experimental group, "no tumor" in the corresponding group, "tumor shrinkage to 0," and "tumor disappearance to 0" indicate that no tumor was found in the corresponding dissected animal. [Table 2-1] [Table 2-2]

[0175] The foregoing description of specific exemplary embodiments of the present invention has been presented for purposes of explanation and illustration. It is not intended that these descriptions be construed as limiting the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary examples is to explain certain principles of the present invention and their practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention, as well as various alternatives and modifications. It is intended that the scope of the present invention be limited by the claims and their equivalents.

Claims

1. An antibody-drug conjugate as shown in formula (I): 【Chemistry 1】 (In the formula, m, n １ are each independently selected from 1 or 2; R ａ is hydrogen and -L １ -NH-L ｐ -Z-Ab, and R ｂ is hydrogen, -L ２ -NH-L ｐ -Z-Ab and -L ３ -NH-Z-Ab, with the proviso that R ａ and R ｂ is not hydrogen at the same time, where: L １ is -C(=O)-L １１ -L １２ -L １３ - is selected from, L 11 , L １２ , and L １３ are each independently —O—, C １ ~C ３ selected from alkylene groups and phenyl groups; L ２ is -C(=O)-NH-L ２１ -CHR １ - is selected from, L ２１ is selected from —C(═O)— and —NH—C(═O)—; R １ is hydrogen, deuterium, C １ ~C ６ Alkyl and phenyl substituted C １ ~C ６ alkyl groups, L ３ is -C(=O)-NR ２ -NR ３ -L ３１ -L ３２ - is selected from, R ２ and R ３ are each independently hydrogen, deuterium, and C １ ~C ６ alkyl groups, L ３１ is -C(=O), -(C １ ~C ３ alkylene)-C(=O), -5- to 8-membered aromatic ring group -C(=O)-, -5- to 8-membered nitrogen heteroaromatic ring group -C(=O)-, and -O-(C １ ~C ３ alkylene)-C(═O)—; L ３２ is -NH-(C １ ~C ３ alkylene), -N(CH ３ ) ２ -(C １ ~C ３ alkylene) and —NH—(C １ ~C ３ alkylene)-NH—; L Ｐ is a peptide residue consisting of 2 to 7 amino acids, Z is -L ｚ -L ｊ -, where L ｚ is -C(=O)-C １ ~C ８ Alkylene group, —C(═O)—CH ２ O-(CH ２ CH ２ O) ２～５ -CH ２ CH ２ NH-, -C(=O)-(CH ２ CH ２ O) ２～６ -CH ２ CH ２ - and C(=O)-(CH ２ CH ２ O) ２～６ -CH ２ CH ２ NH—, and L ｊ is a linker capable of being attached to an antibody, Ab is an antibody. 【Request Item 2】 【Chemistry 2】 is selected from the structures represented by the formula: 【Transformation 3】 Preferably, L １１ , L １２ , and L １３ are each independently selected from —O—, a methylene group, and a phenyl group; Preferably, L １１ -L １２ -L １３ is -O-CH ２ -phenyl- and -CH ２ -O-CH ２ - is selected from, Preferably, L １ is -C(=O)-O-CH ２ - phenyl groups, The antibody-drug conjugate of claim 1.

3. R １ is hydrogen, deuterium, C １ ~C ６ Alkyl and phenyl substituted C １ ~C ６ alkyl groups, Preferably, R １ is selected from hydrogen, deuterium, methyl, isopropyl, isobutyl, and phenylmethyl; Preferably, R １ is hydrogen, Preferably, L ２ is -C(=O)-NH-C(=O)-CH ２ - and -CH ２ -NH-NH-C(=O)-CH ２ - is selected from, Preferably, L ２ is -CH ２ -NH-NH-C(=O)-CH ２ -is, The antibody-drug conjugate according to claim 1 or 2.

4. R ２ and R ３ are each independently hydrogen, deuterium, and C １ ~C ３ alkyl groups, Preferably, R ２ and R ３ are each independently selected from hydrogen, deuterium, and a methyl group; Preferably, R ２ is hydrogen, R ３ is hydrogen, Preferably, L ３１ is —C(═O), -methylene-C(═O), -phenyl-C(═O)—, -pyridyl-C(═O)— and —O—CH ２ —C(═O)—; Preferably, L ３２ is -NH-CH ２ -CH ２ -, -N(CH ３ ) ２ -CH ２ -CH ２ - and -NH-CH ２ -NH-; Preferably, L ３ is -C(=O)-NH-NH-phenyl-C(=O)-NH-CH ２ -CH ２ -is, The antibody-drug conjugate according to any one of claims 1 to 3.

5. the amino acids are selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn); Preferably, L ｐ is selected from peptide residues consisting of 2 to 5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), and aspartic acid (Asn); Preferably, L ｐ is selected from peptide residues consisting of two or three amino acids selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), and citrulline (Cit); Preferably, L ｐ is selected from Val-Cit, Val-Ala, Gly-Val-Ala, Gly-Val-Ala-Gly, Gly-Lys, Gly-Gly-Lys, Gly-Gly-Lys-Gly, Val-Ala, Ala-Ala-Asn, Gly-Leu, Gly-Gly-Leu, Gly-Gly-Leu-Gly, Gly-Phe, Gly-Gly-Phe and Gly-Gly-Phe-Gly; Preferably, L ｐ is selected from the group represented by the formula: 【Chemistry 4】 The carbonyl group end is linked to —NH—, and the other end is linked to Z; Preferably, L ｊ is selected from the group represented by the formula: 【Transformation 5】 【Transformation 6】 The position indicated by indicates that it is linked to an antibody, 【Transformation 7】 The position indicated by L Ｚ represents being linked to a group, Preferably, L ｚ is -C(=O)-C １ ~C ８ Alkylene group, —C(═O)—CH ２ O-(CH ２ CH ２ O) ２～５ -CH ２ CH ２ NH-, -C(=O)-(CH ２ CH ２ O) ２～６ -CH ２ CH ２ - and -C(=O)-(CH ２ CH ２ O) ２～６ -CH ２ CH ２ NH-; Preferably, L ｚ is -C(=O)-C １ ~C ６ Alkylene group, —C(═O)—CH ２ O-(CH ２ CH ２ O) ２～３ -CH ２ CH ２ NH-, -C(=O)-(CH ２ CH ２ O) ２～４ -CH ２ CH ２ - and -C(=O)-(CH ２ CH ２ O) ２～４ -CH ２ CH ２ NH-; Preferably, L ｚ is -C(=O)-(CH ２ ) ５ -, -C(=O)-CH ２ O-(CH ２ CH ２ O) ３ -CH ２ CH ２ NH-, -C(=O)-(CH ２ CH ２ O) ２ -CH ２ CH ２ -, -C(=O)-(CH ２ CH ２ O) ２ -CH ２ CH ２ NH— and —C(═O)—(CH ２ CH ２ O) ４ -CH ２ CH ２ NH-; Preferably, Z is selected from the group represented by the formula: 【Transformation 8】 【Chemistry 9】 The position indicated by indicates that it is linked to an antibody, 【Chemistry 10】 The position indicated by L ｐ group or L ３ represents a bond to the group, The antibody-drug conjugate according to any one of claims 1 to 4.

6. the antibody is a tumor-associated antigen antibody, Preferably, the tumor-associated antigen antibody is selected from an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody. The antibody-drug conjugate according to any one of claims 1 to 5.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein the antibody-drug conjugate represented by formula (I) has a structure represented by formula (I-1), formula (I-2), or formula (I-3). 【Chemistry 11】 (In the formula, R ２ , R ３ , m, n １ , L ｐ , Z, and Ab are each defined as in the compounds of formula (I) at each occurrence.

8. An antibody-drug conjugate comprising: 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 wherein A b is defined as in the compound of formula (I); Preferably, the Ab is an HS627 antibody or an IP140B antibody, wherein the heavy chain amino acid sequence of the HS627 is shown in SEQ ID NO: 1 and the light chain amino acid sequence is shown in SEQ ID NO: 2; and the heavy chain amino acid sequence of the IP140B antibody is shown in SEQ ID NO: 3 and the light chain amino acid sequence is shown in SEQ ID NO:

4. Antibody-drug conjugates.

9. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. Use of the antibody-drug conjugate of any one of claims 1 to 8 or the pharmaceutical composition of claim 9 in the preparation of an antitumor drug, Preferably, the tumor is selected from solid tumors, more preferably selected from esophageal cancer, lung cancer or breast cancer.

11. A method for treating a tumor disease, comprising the step of administering to a patient in need thereof the antibody-drug conjugate of any one of claims 1 to 8 or the pharmaceutical composition of claim 9.

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