Chemical coupling linker and application thereof

Abstract

The invention relates to a chemical coupling connexon and an antibody drug conjugate prepared from the connexon. The invention also relates to application of the antibody drug conjugate in treatment of related diseases such as tumors.

Description

A type of chemical coupling linker and its use

[0001] This application is based on and claims priority to CN application No. 202310181337.6, filed on February 28, 2023, and CN application No. 202311231478.0, filed on September 22, 2023. The disclosed contents of the CN application are hereby introduced as a whole into this application. Technical Field

[0002] The invention relates to a new type of linker for chemical coupling, an antibody-drug conjugate prepared by the linker, and its application in treating tumors and other related diseases. Background Art

[0003] In recent years, antibody-drug conjugates (ADCs) have become a hot topic in precision medicine for diseases such as tumors and autoimmune diseases. ADCs are composed of monoclonal antibodies targeting specific antigens and effector molecules (such as cytotoxic drugs) coupled via a linker. They combine the bioactivity of traditional small molecules with the targeting properties of antibodies.

[0004] Antibody-drug conjugates consist of three parts: antibodies, linkers, and effector molecules. The connection methods between antibodies and linker-effector molecules are mainly divided into non-site-specific conjugation and site-specific conjugation. In the early days, the non-site-specific conjugation method was used, which is to use chemical methods to couple effector molecules to amino acid residues on antibodies. For example, random conjugation is performed through lysine and cysteine ​​residues on antibodies. It does not involve the transformation or modification of antibodies. The number of coupled effector molecules and the coupling sites cannot be determined, and the uniformity is poor. Currently, the commonly used site-specific conjugation often uses specific conjugation to uniformly link effector molecules at specific sites. Antibody-drug conjugates produced by site-specific conjugation can reduce fluctuations in efficacy, pharmacokinetics, and quality control caused by different coupling sites and the number of couplings.

[0005] Currently, common site-specific conjugation methods include THIOMAB technology, non-natural amino acid conjugation technology, glutamine enzymatic conjugation technology, transpeptidase conjugation technology, and ThioBridge technology. Among them, the modification of antibodies through antibody engineering or enzymatic conjugation may have a certain impact on the structural stability of the antibody.

[0006] Patent WO2012007896A1 discloses a polyethylene glycol carboxylic acid fluorophenol ester linker structure, through which a polypeptide molecule is coupled to the K188 site of an antibody. Patent WO2013173392A1 discloses a piperidine amide coupling linker structure, which is linked to an antibody lysine residue via a piperidine linker. Although the coupling methods disclosed in WO2012007896A1 and WO2013173392A1 can achieve coupling at light chain lysine to a certain extent, the uniformity is poor, and the proportion of site-specific coupling needs to be improved.

[0007] The development of site-specific and hydrophilic conjugation linker structures is still of great significance for the research and development of antibody-drug conjugates with good efficacy and safety.

[0008] Summary of the Invention

[0009] The present invention provides a novel linker for chemical conjugation. This linker contains a piperidinesulfonylcarbamate structural fragment that can selectively conjugate with lysine at specific sites in the antibody light chain. The resulting antibody-drug conjugate has good uniformity and a clear anti-tumor effect. The conjugation conditions are mild and the operation is simple.

[0010] Compound

[0011] One aspect of the present invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof, wherein the compound has a structure of Formula I:

[0012] in:

[0013] LG is a leaving group or a reactive group;

[0014] X is selected from -O-, -NR 2 -and-CHR 3 -;R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; the alkyl group is optionally substituted by one or more groups independently selected from amino, alkylamino, nitrogen-containing heterocyclic, sulfonic, carboxylic, quaternary ammonium, hydroxyl and alkoxy groups;

[0015] Each L1 is independently selected from a single bond, C 1-6 Alkylene, -(CH2CH2O) y -、C 2-6 alkenylene, amino acid residues, fragments of polypeptides formed by 2-10 amino acids, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, amino, -CO-NH- and -NH-CO-; said C 1-6Alkylene, amino, C 2-6 Alkenylene, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, -CO-NH- and -NH-CO- are optionally substituted with one or more groups independently selected from sulfonic acid, phosphate, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkynyl and azide;

[0016] Each L2 is independently selected from C 2-6 Alkynyl, tetrazinyl, methyltetrazinyl, trans-cyclooctenyl, benzazacyclooctynyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azido, C 1-6 Alkyl acyl, aldehyde, hydroxylamine, oxime, 4-16 membered heterocyclic group, 5-16 membered heteroaryl;

[0017] Each m is independently an integer from 1 to 10;

[0018] y is an integer from 1 to 20.

[0019] In some embodiments, LG is selected from -OR 1 , hydroxyl, halogen (eg chlorine), C 1-6 Haloalkyl (e.g., chloromethyl), 5-12 membered heteroaryl (e.g., imidazolyl), R 1 Selected from C 1-6 Alkylacyl, maleimido, succinimidyl, sulfosuccinimidyl and phenyl, said phenyl being optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from nitro, cyano, sulfo, sulfonic acid, fluoro and chloro.

[0020] In some embodiments, LG is selected from -OR 1 and imidazolyl, R 1 is selected from succinimidyl, sulfosuccinimidyl and phenyl, said phenyl being optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from nitro, sulfonic acid and fluoro.

[0021] In some embodiments, LG is pentafluorophenoxy.

[0022] In some embodiments, X is selected from -O-, -NR 2 -and-CHR 3 -;R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; said alkyl is optionally substituted by one or more independently selected from amino, C 1-6 Alkylamino, 5-12 membered nitrogen-containing heterocyclic group, sulfonic acid group, carboxylic acid group, quaternary ammonium salt, hydroxyl group and C 1-6 Alkoxy groups are substituted.

[0023] In some embodiments, X is selected from -O-, -NH-, and -CH2-.

[0024] In some embodiments, X is -O-.

[0025] In some embodiments, each L1 is independently selected from a single bond, C 1-6 Alkylene, -(CH2CH2O) y -、C 2-6 alkenylene, amino acid residues, fragments of polypeptides formed by 2-10 amino acids, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, amino, -CO-NH- and -NH-CO-, wherein the C 1-6 Alkylene, amino, 4-16 membered heterocyclyl, 5-16 membered heteroaryl, phenylene, -CO-NH- and -NH-CO- are optionally substituted by one or more independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 Alkynyl and azido groups are substituted.

[0026] In some embodiments, each L1 is independently selected from a single bond, C 1-6 Alkylene, -(CH2CH2O) y -, phenyl, -CO-NH- and -NH-CO-, y is an integer of 1 to 10, for example, an integer of 1 to 5, and m is an integer of 1 to 5.

[0027] In some embodiments, each L1 is independently selected from L1 is a single bond and C 1-4 Alkylene, m is 1 or 2.

[0028] In some embodiments, each L2 is independently selected from C 2-6 Alkynyl, methyl tetrazinyl, trans-cyclooctenyl, benzazacyclooctynyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azido and C 1-6 Carbonyl, m is an integer of 1 to 5.

[0029] In some embodiments, each L2 is independently selected from a single bond and C 2-6 Alkynyl, m is 1.

[0030] In some embodiments, Selected from the following structures:

[0031] In some embodiments, the compound of Formula I is selected from:

[0032] p is 0 or an integer from 1 to 20.

[0033] Another aspect of the present invention provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof, wherein the compound has a structure of Formula II:

[0034] in:

[0035] LG is a leaving group or a reactive group;

[0036] X is selected from -O-, -NR 2 -and-CHR 3 -;R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; the C 1-6 The alkyl group is optionally substituted with one or more groups independently selected from amino, alkylamino, nitrogen-containing heterocyclic, sulfonic, carboxylic, quaternary ammonium salts, hydroxyl and alkoxy groups;

[0037] Each L3 is independently selected from a single bond, C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, amino acid residue, a polypeptide fragment formed by 2-10 amino acids, a 4-16 membered heterocyclylene group, a 5-16 membered heteroarylene group, an amino group, an acyl group, -CO-NH- and -NH-CO-; the C 1-6 Alkylene, oxime, amino, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, -CO-NH- and -NH-CO- are optionally substituted with one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkynyl and azide;

[0038] Each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed by 2-10 amino acids, a glycosyl, a phenylene group, a benzyl, a benzyloxy, a benzyloxycarbonyl, an aminobenzyloxycarbonyl, an acylbenzyloxycarbonyl, a 5-6 membered heteroaryl group, a C 1-6 Alkylene, -(CH2CH2O) y -, acyl and amino; said C 1-6The alkylene and amino groups are optionally substituted with one or more groups independently selected from sulfonic acid, phosphate, carboxylic acid, amide, sulfone, sulfoxide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkylaminoalkyl, alkynyl and azide, and the phenylene, benzyl, benzyloxy, benzyloxycarbonyl, aminobenzyloxycarbonyl, acylbenzyloxycarbonyl groups are optionally substituted with pyranose or furanosyl groups; each D is independently selected from a fragment of an effector molecule, such as a cytotoxin, an immune agonist, a pro-apoptotic agent, a protein degrader or a hormone receptor modulator;

[0039] m is an integer from 1 to 20;

[0040] n is an integer from 1 to 20;

[0041] x is an integer from 1 to 10;

[0042] y is an integer from 1 to 20.

[0043] In some embodiments, LG is selected from -OR 1 , hydroxyl, halogen (eg chlorine), C 1-6 Haloalkyl (e.g., chloromethyl), 5-12 membered heteroaryl (e.g., imidazolyl), R 1 Selected from C 1-6 Alkylacyl, maleimido, succinimidyl, sulfosuccinimidyl and phenyl, said phenyl being optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from nitro, cyano, sulfo, sulfonic acid, fluoro and chloro.

[0044] In some embodiments, LG is selected from -OR 1 and imidazolyl, R 1 is selected from succinimidyl, sulfosuccinimidyl and phenyl, said phenyl being optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from nitro, sulfonic acid and fluoro.

[0045] In some embodiments, LG is pentafluorophenoxy.

[0046] In some embodiments, X is selected from -O-, -NR 2 -and-CHR 3 -;R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; the C 1-6 Alkyl is optionally substituted by one or more independently selected from amino, C 1-6 Alkylamino, 5-12 membered nitrogen-containing heterocyclic group, sulfonic acid group, carboxylic acid group, quaternary ammonium salt, hydroxyl group and C 1-6 Alkoxy groups are substituted.

[0047] In some embodiments, X is selected from -O-, -NH-, and -CH2-.

[0048] In some embodiments, X is -O-.

[0049] In some embodiments, each L3 is independently selected from a single bond, C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, amino acid residue, a polypeptide fragment formed by 2-10 amino acids, a 4-16 membered heterocyclylene group, a 5-16 membered heteroarylene group, an amino group, an acyl group, -CO-NH- and -NH-CO-; the C 1-6 Alkylene, amino, 4-16 membered heterocyclyl, 5-16 membered heteroaryl, -CO-NH- and -NH-CO- are optionally substituted by one or more independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 In the alkynyl and azido groups, y is an integer of 1 to 20, and m is an integer of 1 to 20.

[0050] In some embodiments, each L3 is independently selected from C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO-, the C 1-6 Alkylene, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO- are optionally substituted by one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkynyl and azide, y is an integer from 1 to 20, and m is an integer from 1 to 20.

[0051] In some embodiments, each L3 is independently selected from C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO-, the C 1-6 Alkylene, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO- are optionally substituted by one or more independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 In the alkynyl and azido groups, y is an integer of 1 to 20, and m is an integer of 1 to 20.

[0052] In some embodiments, each L3 is independently selected from C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, 5- to 16-membered heteroarylene, acyl, -CO-NH- and -NH-CO-, y is an integer of 1 to 10, and m is an integer of 1 to 15.

[0053] In some embodiments, each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed by 2-10 amino acids, a saccharide, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5-6 membered heteroaryl group, a C 1-6 Alkylene, -(CH2CH2O) y -, acyl and amino; said C 1-6 The alkylene and amino groups are optionally substituted by one or more groups independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, sulfone groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 1-6 Alkylaminoalkyl, C 2-6 The phenylene, benzyl, benzyloxy, benzyloxycarbonyl, aminobenzyloxycarbonyl, acylbenzyloxycarbonyl groups are optionally substituted with glucuronic acid, galacturonic acid, glucose, galactose or mannose.

[0054] In some embodiments, each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed by 2-10 amino acids, an aminobenzyloxycarbonyl group, a C 1-6 Alkylene and amino, the C 1-6 The alkylene and amino groups are optionally substituted with one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, sulfone, sulfoxide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkylaminoalkyl, alkynyl and azide, and the aminobenzyloxycarbonyl group is optionally substituted with a pyranose or furanosyl group.

[0055] In some embodiments, each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed by 2-10 amino acids, an aminobenzyloxycarbonyl group, a C 1-6 Alkylene and amino, the C 1-6 The alkylene and amino groups are optionally substituted by one or more groups independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, sulfone groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 Alkylaminoalkyl, C 2-6The aminobenzyloxycarbonyl group is optionally substituted with glucuronic acid, galacturonic acid, glucose, galactose or mannose.

[0056] In some embodiments, the amino acid is selected from L-natural amino acids, D-unnatural amino acids, and analogs or derivatives thereof.

[0057] In some embodiments, the amino acid is selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH2CH2 (OCH2CH2) y OCH3), y is an integer from 1 to 20.

[0058] In some embodiments, the polypeptide formed by the 2-10 amino acids is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Val-Arg, Ala-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu -Gly,Gly-Gly-Gly,Gly-Ser-Lys,Gly-Ala-Ala,Glu-Val-Ala,Glu-Val-Cit,Glu-Val-Arg,Ser-Ala-Pro,Val-Leu-Lys,Val- Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, and Gly-Gly-Gly-Gly-Gly.

[0059] In some embodiments, Selected from the following structures:

[0060] p is 0 or an integer from 1 to 20;

[0061] Another aspect of the present invention provides compound fragments Its structure is as follows:

[0062] In some embodiments, Selected from the following structures:

[0063] p is 0 or an integer from 1 to 20.

[0064] In some embodiments, p is 0 or an integer from 1 to 10, or p is 0 or an integer from 1 to 8, or p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0065] In some embodiments, the effector molecule is selected from microtubule inhibitors, such as auristatins, maytansines; DNA intercalators, such as pyrrolobenzodiazepines (PBDs); DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, exitecan) or topoisomerase II inhibitors (such as doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); RNA polymerase inhibitors, such as α-amanitin; and pharmaceutically acceptable salts, esters and analogs of the above agents.

[0066] In some embodiments, the effector molecule is selected from a topoisomerase I inhibitor (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, exitecan), MMAE, and MMAE derivatives.

[0067] In some embodiments, the effector molecule is selected from:

[0068] In some embodiments, D is a structural fragment formed after dehydrogenation of the effector molecule;

[0069] In some embodiments, the effector molecule is linked to L4 via an amino group or a hydroxyl group thereof.

[0070] In some embodiments, D is selected from:

[0071] In some embodiments, the compound of Formula II is selected from:

[0072] p is 0 or an integer from 1 to 20.

[0073] In some embodiments, the compound of Formula II is:

[0074] DL-1:

[0075] DL-2:

[0076] Another aspect of the present invention provides a bioactive conjugate, the structure of which is shown in Formula III:

[0077] in:

[0078] Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein agent (e.g., a sugar, RNA, or DNA)); r is selected from 1-10;

[0079] X, L3, L4, D, m, x and n are as defined above in any one of the compounds of formula II.

[0080] In some embodiments, the target of Ab is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelinreceoptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3 CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1Q A. C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1 C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA, B7H3 and TENB2. .

[0081] In some embodiments, Ab is an antibody, and the bioactive conjugate is an antibody-drug conjugate (ADC).

[0082] In some embodiments, Ab is an antibody, and the antibody is conjugated via the terminal amino group of lysine to form a biologically active conjugate.

[0083] In some embodiments, Ab is an antibody, wherein the light chain of the antibody is linked to the remainder of the conjugate of Formula III via an amide bond formed by the terminal amino group of lysine.

[0084] In some embodiments, Ab is trastuzumab or pertuzumab.

[0085] In some embodiments, Ab is trastuzumab.

[0086] In some embodiments, the bioactive conjugate is selected from:

[0087] In some embodiments, the antibody drug conjugate is:

[0088] Wherein, A1 is trastuzumab, r is 1-10, preferably 1-3, more preferably about 2; preferably, the trastuzumab is connected to the other parts of the biologically active conjugate through the lysine residues thereon.

[0089] Composition

[0090] In another aspect, the present application provides a composition of a bioactive conjugate as described herein, such as an antibody drug conjugate (ADC). Such a composition may comprise a plurality of conjugates as described herein, wherein each conjugate comprises a drug-linker as described herein, wherein r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, r is independently 1, 2, 3, 4 or 5. In other words, each Ab (e.g., antibody) molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (preferably 1, 2, 3, 4 or 5) drug-linkers. Thus, the composition is characterized in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10, preferably in the range of about 1 to about 5. Methods for determining DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

[0091] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10.

[0092] In certain embodiments, the DAR of the ADC compositions described herein is from about 1 to 10, e.g., from about 1.0 to 5.5, from about 1.0 to 5.0, from about 1.0 to 4.5, from about 1.0 to 4.0, from about 1.0 to 3.5, from about 1.0 to 3.0, from about 1.0 to 2.5, from about 1.0 to 2.0, from about 1.5 to 5.5, from about 1.5 to 5.0, from about 1.5 to 4.5, from about 1.5 to 4.0, about 1.5 to 3.5, about 1.5 to 3.0, about 1.5 to 2.5, about 1.5 to 2.0, about 2.0 to 5.5, about 2.0 to 5.0, about 2.0 to 4.5, about 2.0 to 4.0, about 2.0 to 3.5, about 2.0 to 3.0, about 2.0 to 2.5, for example, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5.

[0093] definition

[0094] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of technology or substitutions of equivalent technology that would be apparent to those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

[0095] As used herein, the term "about" refers to a range of 1%-10% around the present value, such as 1%-5%, such as 1%-2.5%, such as 1%, 1.5%, 2% or 2.5%.

[0096] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as C 1-6 Alkylene, methylene, ethylene, propylene or butylene.

[0097] As used herein, the term "alkyl" is defined as a linear or branched saturated aliphatic hydrocarbon. As used herein, the term "C 1-6 "Alkyl" refers to a linear or branched aliphatic hydrocarbon group of 1 to 6 carbon atoms (for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or n-hexyl), which is optionally substituted with 1 or more (such as 1 to 3) suitable substituents (such as halogen) (in this case, the group is referred to as "haloalkyl", for example, C 1-6 haloalkyl) (e.g., CH2F, CHF2, CF3, CCl3, C2F5, C2Cl5, CH2CF3, CH2Cl or -CH2CH2CF3, etc.). The term "C 1-4 "Alkyl" refers to a linear or branched aliphatic hydrocarbon group of 1 to 4 carbon atoms (ie, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl).

[0098] As used herein, the term "alkoxy" is defined as -O-alkyl, wherein alkyl is as defined above. For example, as used herein, the term "C 1-6 "Alkoxy" refers to -OC 1-6 alkyl.

[0099] As used herein, the term "alkoxyalkyl" is defined as an alkyl group substituted with an alkoxy group, said alkyl group being as defined above. For example, as used herein, the term "C 2-6 "Alkoxyalkyl" refers to an alkyl group having 2 to 6 carbon atoms substituted by an alkoxy group.

[0100] As used herein, the term "alkenylene" refers to a divalent hydrocarbon group comprising at least one carbon-carbon double bond, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as C 2-6 alkenylene, vinylene, propenylene or butenylene.

[0101] As used herein, the term "alkenyl" refers to an aliphatic hydrocarbon group comprising at least one carbon-carbon double bond, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as C 2-6 alkenyl, ethenyl, propenyl or butenyl.

[0102] As used herein, the term "alkynylene" refers to a divalent hydrocarbon group including at least one carbon-carbon triple bond, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as ethynylene, propynylene or butynylene.

[0103] As used herein, the term "alkynylene" refers to an aliphatic hydrocarbon group comprising at least one carbon-carbon triple bond, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as C 2-6 alkynyl, ethynyl, propynyl or butynyl.

[0104] As used herein, the term "acyl" refers to -C(=O)-.

[0105] As used herein, the term "alkylacyl" refers to a -C(=O)-alkyl group, wherein the alkyl group is as defined above. For example, as used herein, the term "C 1-6 "Alkylacyl" refers to -C(=O)-C 1-6 alkyl.

[0106] As used herein, the term "sulfo" refers to wherein R is selected from H or alkyl, said alkyl being as defined above.

[0107] As used herein, the term "sulfonic acid group" means

[0108] As used herein, the term "amino" refers to -NH2, -NH- or

[0109] As used herein, the term "alkylamino" refers to -NR a R b , R a and R b are each independently selected from H or an alkyl group as defined above, and R a and R b Not H at the same time. For example, "C 1-6 "Alkylamino" means -NR a R b , R a and Rb Each independently selected from H or C 1-6 alkyl.

[0110] As used herein, the term "carboxylic acid group" refers to -COOH.

[0111] As used herein, the term "phosphate group" refers to

[0112] As used herein, the term "quaternary ammonium salt" refers to -N + R c R d R e , where R c ,R d and R e are each independently selected from alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, or R c ,R d and R e Any two of them together with the nitrogen atom to which they are connected form a heterocyclic group, or R c ,R d and R e The nitrogen atom to which it is attached together forms a heteroaryl group.

[0113] As used herein, the term "amido" refers to a -CO-NR f R g , R f and R g Each is independently selected from H or alkyl as defined above.

[0114] As used herein, the term "azido" refers to

[0115] As used herein, the term "oxime group" refers to where R h is selected from H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0116] As used herein, the term "glycosyl" refers to a group obtained by removing a hemiacetal hydroxyl group of a cyclic monosaccharide or oligosaccharide, including furanosyl and pyranosyl groups, such as glucosyl, galactosyl, mannosyl, and the like.

[0117] As used herein, the term "benzyl" refers to

[0118] As used herein, the term "benzyloxy" refers to

[0119] As used herein, the term "benzyloxycarbonyl" refers to

[0120] As used herein, the term "aminobenzyloxycarbonyl" refers to

[0121] As used herein, the term "acylbenzyloxycarbonyl" refers to

[0122] As used herein, the terms "heterocyclyl" and "heterocycle" refer to a saturated or partially unsaturated (i.e., having one or more double bonds and / or triple bonds within the ring) cyclic group in which at least one ring atom is a heteroatom selected from N, O, and S and the remaining ring atoms are C. For example, a "4-16 membered heterocycle" is a saturated or partially unsaturated cyclic structure having 4-16 (e.g., 5-12, 2, 3, 4, 5, 6, 7, 8, or 9) ring atoms, in which at least one ring atom (e.g., 1, 2, 3, or 4) is a heteroatom selected from N, O, and S. A "nitrogen-containing heterocycle" is a heterocycle in which at least one of the ring atoms is N. Examples of heterocyclic groups include, but are not limited to, oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolinyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, or trithianyl. The heterocyclic group may be optionally substituted with one or more (e.g., 1, 2, 3, or 4) suitable substituents, and may optionally form a ring structure with one or more aromatic rings or heteroaromatic rings.

[0123] As used herein, the term "aromatic ring" or "aryl" refers to a monocyclic or polycyclic aromatic ring system having, for example, 6, 8, 9, 10, 11, 12, 13 or 14 ring-forming carbon atoms, for example, a benzene ring or a naphthalene ring.

[0124] As used herein, the term "aromatic heterocycle" or "heteroaryl" refers to a monocyclic or polycyclic aromatic ring system having, for example, 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms, and which contains at least one heteroatom which may be identical or different (the heteroatom being, for example, oxygen, nitrogen or sulfur) and, in each case, may additionally be benzo-fused. For example, 5-16 membered heteroaryl, 5-12 membered heteroaryl.

[0125] As used herein, the term "halogen" includes F, Cl, Br or I.

[0126] As used herein, the term "amino acid residue" refers to an amino acid unit in a polypeptide, ie, the portion remaining after the amino acids linked by peptide bonds have lost water.

[0127] The term "substituted" means that one or more (e.g., one, two, three, or four) hydrogen atoms on the designated atom are replaced with a group selected from the indicated group, provided that the designated atom's normal valence is not exceeded in the current context and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0128] If a substituent is described as being "optionally substituted with," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or collectively, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the listed substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected optional substituent.

[0129] If a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0130] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10, where reasonable.

[0131] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0132] When a bond to a substituent is shown to pass through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

[0133] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( 2 H), tritium ( 3 H)); carbon isotopes (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g.13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S).

[0134] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0135] In this article, solid lines can be used Solid wedge or virtual wedge The carbon-carbon bonds of the compounds of the present invention are depicted. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the indicated stereoisomers exist. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0136] The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

[0137] It should also be understood that certain compounds of the present invention may be used therapeutically in free form or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites, or prodrugs that, upon administration to a patient in need thereof, are capable of directly or indirectly providing a compound of the present invention or a metabolite or residue thereof. Therefore, when reference is made herein to a "compound of the present invention," such various derivative forms of the compound are also intended to be encompassed.

[0138] Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.

[0139] Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include aspartate, benzoate, bicarbonate / carbonate, glucoheptonate, gluconate, nitrate, orotate, palmitate and other similar salts.

[0140] Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts and other similar salts.

[0141] For a review of suitable salts see Stahl and Wermuth, “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

[0142] As used herein, the term "ester" refers to esters derived from the compounds of the general formulae herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in the form of free acid or alcohol). The compounds of the present invention themselves may also be esters.

[0143] The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0144] Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; AR Katritzky and AJ Boulton, Eds., Academic Press; and GWH Cheeseman and ESGWerstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0145] Also included within the scope of the present invention are metabolites of the compounds of the invention, i.e., substances formed in vivo upon administration of the compounds of the invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0146] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are certain derivatives of the compounds of the present invention that may themselves have little or no pharmacological activity and can be converted into compounds of the present invention having the desired activity by, for example, hydrolytic cleavage when administered to the body or thereon. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted into the desired therapeutically active compounds in vivo. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Volume 14, ACS Symposium Series (T. Higuchi and V. Stella). Prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties" (e.g., as described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).

[0147] The present invention also encompasses compounds of the present invention that contain protecting groups. During any process for preparing the compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved using conventional protecting groups, for example, those described in TW Greene & PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0148] In addition, the compounds of the present invention can also be prepared in a variety of ways known to those skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below and synthetic methods known in the field of synthetic organic chemistry or variations thereof known to those skilled in the art. Preferred methods include, but are not limited to, those described above. The reaction can be carried out in a solvent or solvent mixture suitable for the reagents and materials used and suitable for achieving the transformation. Those skilled in the art of organic synthesis will understand that the functional groups present on the molecule should be consistent with the proposed transformation. This will sometimes require the following judgment: modifying the order of the synthesis steps or selecting another specific method route relative to one method route to obtain the desired compound of the present invention.

[0149] It will also be appreciated that another major consideration in designing any synthetic route in this area is the proper selection of protecting groups for use in protecting the reactive functional groups present in the compounds described herein. An authoritative description of many alternatives to the trained eye is provided by Greene et al. (Protective Groups in Organic Synthesis, 4th edition, Wiley-Interscience (2006)).

[0150] Unless otherwise indicated, the substituents of the compounds in the above routes are as defined herein. Those skilled in the art will appreciate that one or more steps in the above routes may be omitted depending on the desired product structure. Those skilled in the art may also adjust the order of the reaction steps as needed.

[0151] Pharmaceutical composition

[0152] The present invention also provides a pharmaceutical composition comprising the bioactive conjugate of the present invention and one or more pharmaceutically acceptable carriers.

[0153] The pharmaceutical excipients mentioned in this article refer to the excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances other than active ingredients that have been reasonably evaluated for safety and are included in pharmaceutical preparations.

[0154] The pharmaceutical composition can be administered in any form, as long as it prevents, alleviates, prevents or cures the symptoms of a human or animal patient. For example, it can be prepared into various suitable dosage forms according to the administration route.

[0155] The present application also provides a drug kit product, which contains the bioactive conjugate or the pharmaceutical composition of the present invention, and optional drug instructions.

[0156] Treatment methods and uses

[0157] Another aspect of the present application provides use of the bioactive conjugate in preparing a medicament for preventing or treating tumor diseases.

[0158] Another aspect of the present application provides the bioactive conjugate for use in preventing or treating tumor diseases.

[0159] Another aspect of the present application provides a method for preventing or treating tumor diseases, comprising administering an effective amount of the bioactive conjugate or a pharmaceutical composition comprising the bioactive conjugate to a subject in need thereof.

[0160] In one embodiment of the present invention, the tumor disease is a solid tumor or a hematological malignancy; for example, selected from colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, specifically lung adenocarcinoma), lymphoma.

[0161] As used herein, the term "effective amount" refers to that amount of the conjugate which, when administered, will relieve to some extent one or more symptoms of the condition being treated.

[0162] As used herein, unless otherwise indicated, the term "treat," ...

[0163] As used herein, "individual" or "subject" includes humans and non-human animals. Exemplary human individuals include human individuals suffering from a disease (e.g., a disease described herein) (referred to as a patient) or normal individuals. "Non-human animals" herein include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0164] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0165] Figure 1 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the JIMT-1 cell subcutaneous tumor-bearing mouse model.

[0166] Figure 2 shows the changes in body weight of mice in each group in the human breast cancer cell JIMT-1CDX model.

[0167] Figure 3 shows the efficacy of anti-human HER2 antibody-drug conjugate ADC in the NCI-N87 cell subcutaneous tumor-bearing mouse model.

[0168] Figure 4. Changes in body weight of mice in each group in the human gastric cancer cell NCI-N87CDX model. DETAILED DESCRIPTION

[0169] The following will clearly and completely describe the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all. The following description of at least one exemplary embodiment is actually only illustrative and in no way limits the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0170] The present invention will be described in detail below with reference to Examples and Test Examples. However, these examples are not intended to limit the scope of the present invention, and variations are possible without departing from the scope of the present invention.

[0171] As used herein, the abbreviations have the following meanings:

[0172] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0173] Nuclear magnetic resonance (NMR) 1 H NMR was measured using a Bruker 400 MHz nuclear magnetic resonance spectrometer; deuterated chloroform (CDCl 3 ) was used; and tetramethylsilane (TMS) was used as the internal standard.

[0174] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0175] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, CDCl3: deuterated chloroform. δ values ​​are expressed in ppm.

[0176] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0177] Example 1 (6S,9S)-amino-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-9-isopropyl-1,8,11-trioxy-14,17-dioxa-2,7,10-triaza-eicosanoic acid perfluorophenol ester (DL-A)

[0178] Step 1:

[0179] VC-MMAE (100 mg, 0.089 mmol), diethylene glycol-bis(2-amino acid) (19 mg, 0.089 mmol), and HATU (34 mg, 0.089 mmol) were dissolved in DMF (3 mL). Diisopropylethylamine (35 mg, 0.267 mmol) was added dropwise and stirred for 1 hour. LC-MS monitoring confirmed the complete reaction. 0.1 mL of water was added to the reaction system, and DL-A-1 (30 mg) was obtained after separation and purification by HPLC (conditions as follows).

[0180] The structural characterization data are as follows:

[0181] ESI-MS (m / z): 1312.6 [M+1] + .

[0182] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0183] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0184] Step 2:

[0185] DL-A-1 (30 mg, 0.023 mmol), pentafluorophenol (8 mg, 0.046 mmol), and EDCI (34 mg, 0.114 mmol) were dissolved in DMF (1 mL) and stirred for 4 hours. LC-MS monitoring confirmed the complete reaction. 0.1 mL of water was added to the reaction system, and DL-A (3.6 mg) was obtained after purification by HPLC (conditions as follows).

[0186] The structural characterization data are as follows:

[0187] ESI-MS (m / z): 1478.7 [M+1] + .

[0188] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0189] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0190] Example 2 1-(6-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1 (4-amino)-1-oxo-5-ureidopentan-2-yl) (5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl) (phenyl) (amino)-1-oxo-5-ureidopentan-2-yl) (amino)-3-methyl-1-oxobutan-2-yl) (amino)-6-oxohexanoyl) piperidine-4-carboxylic acid pentafluorophenol ester (DL-B)

[0191] Step 1:

[0192] Methyl 4-piperidinic acid ester (DL-B-1, 500 mg, 3.49 mmol) and 6-(tert-butoxy)-6-oxohexanoic acid (706 mg, 3.49 mmol) were dissolved in DMF (2.00 mL). HATU (1.46 g, 3.84 mmol) and DIPEA (1.35 g, 10.48 mmol, 1.82 mL) were added, and the mixture was heated to 40°C and reacted for 18 hours. Water (30.0 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 10 / 1), and then concentrated again to obtain compound DL-B-2 (500 mg, 1.53 mmol).

[0193] The structural characterization data are as follows:

[0194] ESI-MS (m / z): 328.5 [M+H] + .

[0195] Step 2:

[0196] Dissolve DL-B-2 (500 mg, 1.53 mmol) in dichloromethane (5.00 mL), add trifluoroacetic acid (1.54 g, 13.4 mmol, 1.00 mL), and react at 25°C for 1 hour. The reaction solution is directly concentrated to obtain the crude trifluoroacetate salt of DL-B-3 (550 mg, 1.43 mmol).

[0197] The structural characterization data are as follows:

[0198] ESI-MS (m / z): 271.9 [M+H] + .

[0199] Step 3:

[0200] VC-MMAE (60.0 mg, 53.4 μmol) and DL-B-3 (20.5 mg, 53.4 μmol, trifluoroacetate) were dissolved in DMF (10.0 mL). EDCI (37.5 mg, 195 μmol), HOBt (37.5 mg, 277 μmol), and DIPEA (34.5 mg, 267 μmol, 46.5 μL) were added sequentially, and the mixture was stirred at 25°C for 1 hour. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (20.0 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to obtain compound DL-B-4 (10.0 mg, 7.26 μmol).

[0201] The structural characterization data are as follows:

[0202] ESI-MS (m / z): 1377.0 [M+H] + .

[0203] Step 4:

[0204] DL-B-4 (10.0 mg, 7.26 μmol) was dissolved in a mixed solvent of tetrahydrofuran (0.50 mL), methanol (0.50 mL), and water (0.50 mL). Lithium hydroxide (347 μg, 14.5 μmol) was added, and the mixture was stirred at 25°C for 2 hours. The pH of the reaction solution was adjusted to 5-6 with 1N dilute hydrochloric acid. Solids precipitated and were filtered. The filter cake was dried under vacuum to obtain crude compound DL-B-5 (10.0 mg).

[0205] The structural characterization data are as follows:

[0206] ESI-MS (m / z): 1363.0 [M+H] + .

[0207] Step 5:

[0208] DL-B-5 (10.0 mg, 7.34 μmol) and pentafluorophenol (2.03 mg, 11.01 μmol) were dissolved in dichloromethane (2.50 mL). EDCI (6.33 mg, 33.0 μmol) was added and stirred at 30°C for 1 hour. Water (10.0 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (10.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After separation and purification by HPLC, the product was freeze-dried to obtain compound DL-B (2.88 mg, 1.82 μmol).

[0209] The structural characterization data are as follows:

[0210] ESI-MS (m / z): 1529.6 [M+H] + .

[0211] The separation and purification methods are as follows:

[0212] Chromatographic column: Phenomenex luna C18 (10μm*25mm*150mm)

[0213] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0214] Example 3 1-(N-((6S,9S)-14-amino-9-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine- (1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-6-isopropyl-4,7,14-trioxo-2-oxa-5,8,13-triazatetradecyl)aminosulfonyl)piperidine-4-carboxylic acid pentafluorophenol ester (DL-1)

[0215] Step 1:

[0216] Chlorosulfonyl isocyanate (500 mg, 3.53 mmol, 307 μL) was dissolved in dichloromethane (10.0 mL) at 0°C. Benzyl glycolate (533 mg, 3.21 mmol, 455 μL) was added and stirred for 1 hour. A solution of methyl 4-piperidinylcarboxylate DL-B-1 (459 mg, 3.21 mmol) and triethylamine (974 mg, 9.63 mmol, 1.34 mL) in dichloromethane (5.00 mL) was then added to the reaction mixture. The temperature was raised to 25°C and stirring was continued for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The combined organic phases were washed with cooled 1N dilute hydrochloric acid (20.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude DL-1-1 (1.30 g, 3.14 mmol), which was used directly in the next step without purification.

[0217] The structural characterization data are as follows:

[0218] ESI-MS (m / z): 414.9 [M+H]+ .

[0219] Step 2:

[0220] Under a nitrogen atmosphere, to a solution of DL-1-1 (700 mg, 1.69 mmol) in methanol (20.0 mL) was added Pd / C (0.70 g, 10%). The atmosphere was replaced with hydrogen three times and then reacted at 25°C for 3 hours (15 PSI). The reaction mixture was filtered, and the filter cake was rinsed with methanol three times (100 mL x 3). The filtrate was concentrated to obtain crude DL-1-2 (590 mg).

[0221] Step 3:

[0222] VC-MMAE (60 mg, 53.4 μmol) and DL-1-2 (17.3 mg, 53.4 μmol) were dissolved in DMF (10.0 mL). HOBt (36.0 mg, 267 μmol), DIPEA (34.5 mg, 267 μmol, 46.5 μL), and EDCI (30.7 mg, 160 μmol) were added sequentially, and the mixture was reacted at 25°C for 2 hours. Water (20.0 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude DL-1-3 (60.0 mg), which was used directly in the next step without purification.

[0223] The structural characterization data are as follows:

[0224] ESI-MS (m / z): 715.4 [M / 2+H] + .

[0225] Step 4:

[0226] DL-1-3 (50.0 mg, 34.9 μmol) was dissolved in tetrahydrofuran (2.50 mL), water (1.00 mL), and MeOH (1.00 mL). Lithium hydroxide monohydrate (2.94 mg, 69.9 μmol) was added and stirred at 30°C for 1 hour. The reaction solution was adjusted to pH 6 with 1N dilute hydrochloric acid and then concentrated to obtain crude DL-1-4 (50.0 mg), which was used in the next step without purification.

[0227] The structural characterization data are as follows:

[0228] ESI-MS (m / z): 1416.6 [M+H] + .

[0229] Step 5:

[0230] DL-1-4 (50.0 mg, 35.3 μmol) and pentafluorophenol (13.0 mg, 70.6 μmol) were dissolved in DCM (10.0 mL). EDCI (30.4 mg, 158 μmol) was added and stirred at 30°C for 2 hours. The reaction solution was concentrated at 25°C to obtain the crude product, which was purified by HPLC and freeze-dried to obtain DL-1 (4.70 mg, 2.86 μmol).

[0231] The structural characterization data are as follows:

[0232] ESI-MS (m / z): 1582.1 [M+H] + .

[0233] The separation and purification methods are as follows:

[0234] Chromatographic column: Phenomenex luna C18 (10μm*25mm*150mm)

[0235] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0236] Example 4 1-(N-((2-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine- (1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-carboxylic acid pentafluorophenol ester (DL-2)

[0237] Step 1:

[0238] (((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine-L-alanine (DL-2-1, 1.00 g, 2.44 mmol) and 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline (1.16 g, 4.87 mmol) were dissolved in dichloromethane (10.0 mL) and methanol (5.00 mL). EEDQ (1.20 g, 4.87 mmol) was added, and the mixture was stirred at 25°C for 5 hours. The reaction solution was concentrated to obtain a crude product, which was purified on a silica gel column (petroleum ether / ethyl acetate = 100 / 1 to 0 / 100) and concentrated again to obtain compound DL-2-2 (1.51 g, 2.37 mmol), which was used directly in the next step without purification.

[0239] Its structural characterization data are as follows:

[0240] ESI-MS (m / z): 630.3 [M+H] + .

[0241] Step 2:

[0242] (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (DL-2-2, 1.45 g, 2.30 mmol) was dissolved in DMF (10.0 mL). DBU (350 mg, 2.30 mmol, 347 μL) was added and stirred at 25°C for 1 hour. The reaction mixture was used directly in the next step without further treatment.

[0243] Its structural characterization data are as follows:

[0244] ESI-MS (m / z): 408.2 [M+H] + .

[0245] Step 3:

[0246] 2-((((4-(Methoxycarbonyl)piperidin-1-yl)sulfonyl)carbamoyl)oxy)acetic acid (795 mg, 2.45 mmol) was dissolved in DMF (5.0 mL) and added to the reaction mixture from the previous step. HOBt (1.66 g, 12.2 mmol), EDCI (1.41 g, 7.36 mmol), and DIPEA (1.59 g, 12.2 mmol, 2.14 mL) were then added, and the mixture was stirred at 25°C for 2 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 97 / 3) and concentrated again to obtain compound DL-2-4 (1.25 g, 1.57 mmol).

[0247] Its structural characterization data are as follows:

[0248] ESI-MS (m / z): 736.2 [M+Na] + .

[0249] Step 4:

[0250] Methyl 1-(N-((2-(((S)-1-(((S)-((4-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (DL-2-4, 1.15 g, 1.61 mmol) was dissolved in tetrahydrofuran (12.0 mL), TBAF (1 M, 4.83 mL) was added, and the mixture was stirred at 25°C for 1 hour. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column (dichloromethane / methanol = 100 / 1 to 95 / 5) and concentrated again to obtain compound DL-2-5 (900 mg, 1.38 mmol).

[0251] Its structural characterization data are as follows:

[0252] ESI-MS (m / z): 600.1 [M+H] + .

[0253] Step 5:

[0254] Methyl 1-(N-((2-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (DL-2-5, 850 mg, 1.42 mmol) and p-nitrophenyl chloroformate (428 mg, 2.13 mmol) were dissolved in dichloromethane (10 mL). Trifluoroacetic acid (430 mg, 4.25 mmol, 591 μL) was added, and the mixture was stirred at 25°C for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column (dichloromethane / methanol = 100 / 1 to 94 / 6) and concentrated again to obtain compound DL-2-6 (450 mg, 508.99 μmol).

[0255] Its structural characterization data are as follows:

[0256] ESI-MS (m / z): 765.2 [M+H] + .

[0257] Step 6:

[0258] Methyl 1-(N-((2-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobutan-2-yl)amino)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (DL-2-6, 400 mg, 523 μmol) and MMAE (268 mg, 373 μmol) were dissolved in DMF (6.00 mL). HOBt (25.2 mg, 186 μmol) and DIPEA (169 mg, 1.31 mmol, 227 μL) were added, and the mixture was stirred at 25°C for 10 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column (dichloromethane / methanol = 100 / 1 to 94 / 6) and concentrated again to obtain compound DL-2-7 (650 mg).

[0259] Its structural characterization data are as follows:

[0260] ESI-MS (m / z): 1343.9 [M+H] + .

[0261] Step 7:

[0262] 1-(N-((2-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S,sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxo-4,7,10-triazatetradecyl)phenyl)amino)-1-oxopropan-2-yl) Methyl 1-amino-3-methyl-1-oxobutan-2-yl-amino-2-oxoethoxy-carbonyl-sulfamoyl-piperidine-4-carboxylate (DL-2-7, 600 mg, 446 μmol) was dissolved in a mixture of tetrahydrofuran (2.00 mL), methanol (2.00 mL), and water (2.00 mL). Lithium hydroxide monohydrate (37.4 mg, 893 μmol) was added, and the mixture was heated to 45°C and stirred for 2 hours. The reaction mixture was adjusted to pH 6 with aqueous citric acid solution and filtered. The filter cake was dried under vacuum to obtain compound DL-2-8 (530 mg), which was used directly in the next step without purification.

[0263] Its structural characterization data are as follows:

[0264] ESI-MS (m / z): 665.3 [M / 2+H] + .

[0265] Step 8:

[0266] 1-(N-((2-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S,sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxo-4,7,10-triazatetradecyl)phenyl)amino)- ... A mixture of DL-2-8 (500 mg, 376 μmol) and pentafluorophenol (138 mg, 752 μmol) was dissolved in dichloromethane (10.0 mL). EDCI (324 mg, 1.69 mmol) was added and stirred at 25°C for 1 hour. The reaction mixture was concentrated at 30°C to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain the title compound (18.5 mg, 8.45 μmol).

[0267] Its structural characterization data are as follows:

[0268] ESI-MS (m / z): 1496.2 [M+H] + .

[0269] The purification and separation methods are as follows:

[0270] Chromatographic column: Phenomenex luna C18 (10μm*25mm*150mm)

[0271] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0272] Preparation Example (Preparation of Antibody Drug Conjugates)

[0273] Preparation of Trastuzumab-DL-A (target DAR 2)

[0274] 0.31 mL of trastuzumab (16.2 mg / mL) was adjusted to pH 7.40 with 1 M Na₂HPO₄ solution. 17.77 μL of DL-A (10 mM, equivalent to 5 times the amount of antibody) dissolved in dimethyl sulfoxide was added and mixed. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-A). The average loading (DAR value) determined by mass spectrometry was 1.67, of which DAR2 accounted for 36.11%.

[0275] Table 1: Measured molecular weight and DAR calculation of Trastuzumab-DL-A

[0276] Preparation of Trastuzumab-DL-B (target DAR 2)

[0277] 0.31 mL of trastuzumab (16.2 mg / mL) was adjusted to pH 7.40 with 1 M Na₂HPO₄ solution. A solution of DL-B (17.24 μL, 10 mM, equivalent to 5 times the amount of antibody) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. Afterwards, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-B). The average loading (DAR value) determined by mass spectrometry was 1.67, of which DAR2 accounted for 44.88%.

[0278] Table 2: Measured molecular weight and DAR calculation of Trastuzumab-DL-B

[0279] Preparation of Trastuzumab-DL-1 (target DAR 2)

[0280] 0.31 mL of trastuzumab (16.2 mg / mL) was adjusted to pH 7.40 with 1 M Na₂HPO₄ solution. A solution of DL-1 (18.15 μL, 10 mM, equivalent to 5 times the amount of antibody) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-1). The average loading (DAR value) determined by mass spectrometry was 2.28, of which DAR2 accounted for 60.28%.

[0281] Table 3: Measured molecular weight and DAR calculation of Trastuzumab-DL-1

[0282] Preparation of Trastuzumab-DL-2 (target DAR 2)

[0283] 2.16 mL of trastuzumab (16.2 mg / mL) was adjusted to pH 7.40 with 1 M Na₂HPO₄ solution. A solution of DL-2 (234 μL, 10 mM, equivalent to 5 times the amount of antibody) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 18 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-DL-2). The average loading (DAR value) determined by mass spectrometry was 2.20.

[0284] Table 4: Measured molecular weight and DAR calculation of Trastuzumab-DL-2

[0285] Based on the loading (DAR value) and DAR2 ratio of the above coupling examples, it is shown that the chemical coupling linker of the present application, especially the piperidinesulfonylcarbamate structure, is beneficial for improving the coupling efficiency and obtaining a more uniform antibody drug conjugate with a higher DAR2 ratio.

[0286] Test Example 1: Detection of in vitro cell viability of antibody-drug conjugates

[0287] 1. Inhibitory effect of antibody-drug conjugates on HT-29 cell proliferation

[0288] (1) Cell plating: First, culture HT-29 tumor cells in the appropriate culture medium, digest the cells with trypsin, centrifuge, resuspend the cells, count the cells, and adjust the cells to an appropriate concentration for plating. The sources of tumor cells are shown in Table 5.

[0289] Table 5: Tumor cell sources

[0290] Co-incubation of the ADC of the present application and tumor cells: After the cells adhere to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (ADC of the present application) is added to the above-mentioned plate wells and incubated for 96 hours.

[0291] In vitro cell activity assay: After incubation, add Cell Counting-Lite TM 2.0 reagent (Vazyme / Novozyme) 50μL, shake and mix in the dark, react for 10 minutes and then detect, read with a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM Obtain background RLU, Cell Counting-Lite of culture medium containing cells TM Obtain vehicle RLU. Cell inhibition rate = 1-(sample RLU-background RLU) / (vehicle RLU-background RLU) × 100%, and calculate the half-maximal inhibitory concentration (IC) of the compound according to the four-parameter model fitting curve. 50 ). RLU (relative light unit): relative light unit.

[0292] (2) Data results: The test results are shown in Table 6.

[0293] Table 6: Antibody drug conjugates killing results on HT-29 cell line (96 hours)

[0294] In vitro cell viability (inhibitory effect) tests of antibody-drug conjugates (ADCs) demonstrated that the ADCs conjugated using the novel chemical linker exhibited significant tumor cell cytotoxicity. Compared to control ADCs (Trastuzumab-DL-A and Trastuzumab-DL-B), the ADC disclosed in this application (Trastuzumab-DL-1) exhibited superior cytotoxicity, demonstrating that the ADCs conjugated using the novel chemical linker are effective in inhibiting tumor growth.

[0295] Test Example 2: Evaluation of the Antibody Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0296] The preparations containing the ADC of the present invention were administered via tail vein injection to a CDX mouse model subcutaneously transplanted with human breast cancer cells JIMT-1. The tumor volume and animal body weight changes were measured once a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0297] Test drug

[0298] Drug Name, Source, and Preparation: Take an appropriate amount of the ADC of the present invention at a dosing volume of 3 mg / kg and dilute the stock solution with 0.9% NaCl injection to a dosing solution. Use 0.9% NaCl injection as a vehicle control.

[0299] Experimental animals and cell lines

[0300] NOD SCID mice (Chengdu Yaokang Biotechnology Co., Ltd.)

[0301] Human breast cancer cells JIMT-1 (Nanjing Kebai Biotechnology Co., Ltd.)

[0302] Experimental grouping and evaluation methods

[0303] The average tumor volume was 100-150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). Groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and the ADC of the present invention, as well as the dosing frequency described in the specific examples. Administration was via tail vein injection at a volume of 10 ml / kg. Tumor diameter was measured with a vernier caliper once weekly after administration, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0304] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0305] VT end > VT0, TGI (%) = [1-(VT end - VT0) / (VC end - VC0)] * 100% or VT end ≤ VT0, TGI (%) = [1-(VT end - VT0) / VT0] * 100%.

[0306] VTend: mean tumor volume at the end of the treatment group experiment

[0307] VT0: mean tumor volume at the start of drug administration in the treatment group

[0308] VC end: mean tumor volume of negative control group at the end of the experiment

[0309] VC0: Mean tumor volume of the negative control group at the start of drug administration

[0310] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0311] T / C = (VT end / VT0) / (VC end / VC0).

[0312] (1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the JIMT-1 model

[0313] JIMT-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. JIMT-1 cells were harvested during the exponential growth phase, resuspended in a 1:1 (v / v) mixture of Matrigel and PBS to a suitable concentration, and inoculated subcutaneously into female NOD SCID mice to establish a breast cancer model. The average tumor volume was approximately 150 mm. 3 At about 14 days, the mice were randomly divided into groups according to the size of the tumor, namely: vehicle control group (i.e., negative control, vehicle group), trastuzumab-DL-A, trastuzumab-DL-B, trastuzumab-DL-1, and trastuzumab-DL-2 groups of the present invention. Each group was injected with the tail vein (iv) and the drug was administered on Day 0, for a total of 1 dose. After administration, the body weight of the mice was measured once a week, and the long and short diameters of the tumor were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0314] The ADC of this invention demonstrated significant tumor growth inhibition in the JIMT-1 human breast cancer xenograft model. Compared with the vehicle group, the tumor growth inhibition rates (TGI) of the trastuzumab-DL-A, trastuzumab-DL-B, trastuzumab-DL-1, and trastuzumab-DL-2 groups were 38.54%, 15.04%, 75.08%, and 80.90%, respectively. On Day 21, there were no animal deaths or significant weight loss in any treatment group, and no significant drug toxicity was observed. The ADC of this invention was well tolerated by mice during treatment. Specific results are shown in Table 7 and Figures 1 and 2.

[0315] Table 7 Human breast cancer cell JIMT-1CDX model

[0316] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate, the same below.

[0317] Test Example 3: Evaluation of the Antibody-Drug Conjugate's Inhibitory Effect on Tumor Growth in a Mouse Subcutaneous Xenograft Tumor Model

[0318] The preparations containing the ADC of the present invention were administered via tail vein injection to CDX mouse models subcutaneously transplanted with human gastric cancer cells NCI-N87. The tumor volume and animal body weight changes were measured twice a week, and the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice was calculated.

[0319] Test drug

[0320] Drug Name, Source, and Preparation: Take an appropriate amount of the ADC of the present invention and dilute the stock solution to a 1 mg / kg dosing volume with 0.9% NaCl injection to obtain a dosing solution. Use 0.9% NaCl injection as a vehicle control.

[0321] Experimental animals and cell lines

[0322] Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2020-0034, animal certificate number: 511214900025102)

[0323] Human gastric cancer cells NCI-N87 (ATCC)

[0324] Experimental grouping and evaluation methods

[0325] The average tumor volume was about 150 mm. 3 Tumor-bearing mice were randomly divided into groups (the number of groups was determined based on the sample size). The groups were administered 0.9% NaCl injection (hereinafter referred to as vehicle control) and the ADC of the present invention. The dosing frequency was as described in the specific examples. The administration method was tail vein injection, and the administration volume was 10 ml / kg. Tumor diameter was measured twice weekly with a vernier caliper, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0326] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0327] VT end > VT0, TGI (%) = [1-(VT end - VT0) / (VC end - VC0)] * 100% or VT end ≤ VT0, TGI (%) = [1-(VT end - VT0) / VT0] * 100%.

[0328] VTend: mean tumor volume at the end of the treatment group experiment

[0329] VT0: Mean tumor volume at the start of drug administration in the treatment group

[0330] VC end: mean tumor volume of negative control group at the end of the experiment

[0331] VC0: Mean tumor volume of the negative control group at the start of drug administration

[0332] The following formula was used to calculate the tumor relative proliferation rate T / C (%), which was used to evaluate the tumor inhibition efficacy of the ADC of the present invention:

[0333] T / C = (VT end / VT0) / (VC end / VC0).

[0334] (1) Efficacy testing of anti-human Her2 antibody-drug conjugates in the NCI-N87 model

[0335] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were collected during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer model. The average tumor volume was approximately 150 mm. 3 At approximately 14 days, the mice were randomly divided into the following groups according to tumor size: vehicle control group (i.e., negative control, vehicle group), 1 mg / kg trastuzumab-DL-1 group of the present invention, and 3 trastuzumab-DL-2 groups. The mice were injected intravenously (iv) on Day 0, Day 7, and Day 14, for a total of 3 doses. After administration, the mice were weighed twice a week and the long and short diameters of the tumors were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0336] The ADC of this invention demonstrated significant tumor growth inhibition in the NCI-N87 gastric cancer xenograft model. Compared to the vehicle group, the tumor growth inhibition rates (TGI) of the 1 mg / kg trastuzumab-DL-1 and 1 mg / kg trastuzumab-DL-2 groups were 66.23% and 63.87% respectively. No animals died or experienced significant weight loss on Day 20, and no significant drug toxicity was observed. The ADC of this invention was well tolerated by mice during treatment. Specific results are shown in Table 8 and Figures 3 and 4.

[0337] Table 8 Human gastric cancer cell NCI-N87CDX model

[0338] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0339] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) is incorporated herein by reference in its entirety.

Claims

1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or isotope-labeled compound thereof, wherein the compound has a structure of Formula I: in: LG is a leaving group or a reactive group; X is selected from -O-, -NR 2 -and-CHR 3 -; R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; the C 1-6 The alkyl group is optionally substituted with one or more groups independently selected from an amino group, an alkylamino group, a nitrogen-containing heterocyclic group, a sulfonic acid group, a carboxylic acid group, a quaternary ammonium salt, a hydroxyl group, and an alkoxy group; Each L1 is independently selected from a single bond, C 1-6 Alkylene, -(CH2CH2O) y -、C 2-6 alkenylene, amino acid residues, fragments of polypeptides formed by 2-10 amino acids, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, amino, -CO-NH- and -NH-CO-; the C 1-6 Alkylene, amino, C 2-6 Alkenylene, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, -CO-NH- and -NH-CO- are optionally substituted with one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkynyl and azido; Each L2 is independently selected from C 2-6 Alkynyl, tetrazinyl, methyltetrazinyl, trans-cyclooctenyl, benzazacyclooctinyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azido, C 1-6 Alkyl acyl, aldehyde, hydroxylamine, oxime, 4-16 membered heterocyclic group, 5-16 membered heteroaryl group; Each m is independently an integer from 1 to 10; y is an integer from 1 to 20.

2. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or isotope-labeled compound thereof, wherein the compound has a structure of Formula II: in: LG is a leaving group or a reactive group; X is selected from -O-, -NR 2 -and-CHR 3 -; R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; the C 1-6 The alkyl group is optionally substituted with one or more groups independently selected from an amino group, an alkylamino group, a nitrogen-containing heterocyclic group, a sulfonic acid group, a carboxylic acid group, a quaternary ammonium salt, a hydroxyl group, and an alkoxy group; Each L3 is independently selected from a single bond, C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, amino acid residues, fragments of polypeptides formed by 2-10 amino acids, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, amino, acyl, -CO-NH- and -NH-CO-; the C 1-6 Alkylene, oxime, amino, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, -CO-NH- and -NH-CO- are optionally substituted with one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkynyl and azido; Each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed by 2-10 amino acids, a glycosyl, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5-6 membered heteroarylene group, a C 1-6 Alkylene, -(CH2CH2O) y -, acyl and amino; said C 1-6 The alkylene and amino groups are optionally substituted with one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, sulfone, sulfoxide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkylaminoalkyl, alkynyl and azido, and the phenylene, benzyl, benzyloxy, benzyloxycarbonyl, aminobenzyloxycarbonyl, acylbenzyloxycarbonyl groups are optionally substituted with pyranose or furanosyl groups; Each D is independently selected from a fragment of an effector molecule, such as a cytotoxin, an immune agonist, a pro-apoptotic agent, a protein degrader or a hormone receptor modulator; preferably, the effector molecule is connected to L4 via an amino group or a hydroxyl group thereon; m is an integer from 1 to 20; n is an integer from 1 to 20; x is an integer from 1 to 10; y is an integer from 1 to 20.

3. The compound according to claim 1, wherein LG Selected from -OR 1 , hydroxyl, halogen (e.g. chlorine), C 1-6 haloalkyl (e.g. chloromethyl), 5-12 membered heteroaryl (e.g. imidazolyl), R 1 Selected from C 1-6 alkylacyl, maleimido, succinimidyl, sulfosuccinimidyl and phenyl, the phenyl group is optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from nitro, cyano, sulfo, sulfonic acid, fluorine and chlorine; preferably, LG is selected from -OR 1 and imidazolyl, R 1 is selected from succinimidyl, sulfosuccinimidyl and phenyl, the phenyl being optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from nitro, sulfonic acid and fluorine; preferably, LG is pentafluorophenoxy; Preferably, each L1 is independently selected from a single bond, C 1-6 Alkylene, -(CH2CH2O) y -、C 2-6 alkenylene, amino acid residues, polypeptide fragments formed by 2-10 amino acids, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, amino group, -CO-NH- and -NH-CO-, wherein the C 1-6 Alkylene, amino, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, phenylene, -CO-NH- and -NH-CO- are optionally substituted by one or more independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 Preferably, each L1 is independently selected from a single bond, C 1-6 Alkylene, -(CH2CH2O) y -, phenyl, -CO-NH- and -NH-CO-, y is an integer from 1 to 10, such as an integer from 1 to 5, and m is an integer from 1 to 5; preferably, each L1 is independently selected from L1 is a single bond and C 1-4 Alkylene, m is 1 or 2; Preferably, each L2 is independently selected from C 2-6 Alkynyl, methyl tetrazinyl, trans-cyclooctenyl, benzazacyclooctinyl, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azido and C 1-6 Alkyl acyl, m is an integer from 1 to 5; preferably, each L2 is independently selected from a single bond and C 2-6 Alkynyl, m is 1.

4. The compound according to claim 1, wherein Selected from the following structures: Best 5. The compound of formula I according to claim 1, which is selected from the following structures: p is 0 or an integer of 1-20.

6. The compound according to claim 2, wherein LG Selected from -OR 1 , hydroxyl, halogen (e.g. chlorine), C 1-6 haloalkyl (e.g. chloromethyl), 5-12 membered heteroaryl (e.g. imidazolyl), R 1 Selected from C 1-6 alkylacyl, maleimido, succinimidyl, sulfosuccinimidyl and phenyl, the phenyl group is optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from nitro, cyano, sulfo, sulfonic acid, fluorine and chlorine; preferably, LG is selected from -OR 1 and imidazolyl, R 1 is selected from succinimidyl, sulfosuccinimidyl and phenyl, the phenyl being optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from nitro, sulfonic acid and fluorine; preferably, LG is pentafluorophenoxy; Preferably, X is selected from -O-, -NR 2 -and-CHR 3 -; R 2 and R 3 are each independently selected from hydrogen and C 1-6 Alkyl; the C 1-6 The alkyl group is optionally substituted with one or more independently selected from amino, C 1-6 Alkylamino, 5-12 membered nitrogen-containing heterocyclic group, sulfonic acid group, carboxylic acid group, quaternary ammonium salt, hydroxyl group and C 1-6 Alkoxy group substitution; X is selected from -O-, -NH- and -CH2-; preferably, X is -O-; Preferably, each L3 is independently selected from a single bond, C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, amino acid residues, fragments of polypeptides formed by 2-10 amino acids, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, amino, acyl, -CO-NH- and -NH-CO-; the C 1-6 Alkylene, amino, 4-16 membered heterocyclylene, 5-16 membered heteroarylene, -CO-NH- and -NH-CO- are optionally substituted by one or more independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 substituted with alkynyl and azido groups, y is an integer from 1 to 20, and m is an integer from 1 to 20; preferably, each L3 is independently selected from C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO-, the C 1-6 Alkylene, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO- are optionally substituted by one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkynyl and azido, y is an integer of 1 to 20, m is an integer of 1 to 20; preferably, each L3 is independently selected from C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO-, the C 1-6 Alkylene, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO- are optionally substituted by one or more independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, quaternary ammonium salt, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 substituted with alkynyl and azido groups, y is an integer from 1 to 20, and m is an integer from 1 to 20; preferably, each L3 is independently selected from C 1-6 Alkylene, -O-, -(CH2CH2O) y -, oxime, 5-16 membered heteroarylene, acyl, -CO-NH- and -NH-CO-, y is an integer from 1 to 10, and m is an integer from 1 to 15; Each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed by 2-10 amino acids, a glycosyl, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5-6 membered heteroarylene group, a C 1-6 Alkylene, -(CH2CH2O) y -, acyl and amino; said C 1-6 The alkylene and amino groups are optionally substituted by one or more groups independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, sulfone groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 1-6 Alkylamine, C 2-6 The phenylene, benzyl, benzyloxy, benzyloxycarbonyl, aminobenzyloxycarbonyl, acylbenzyloxycarbonyl are optionally substituted with glucuronic acid, galacturonic acid, glucose, galactose or mannose; preferably, each L4 is independently selected from an amino acid residue, a polypeptide fragment formed by 2-10 amino acids, an aminobenzyloxycarbonyl, a C 1-6 Alkylene and amino, the C 1-6 The alkylene group and the amino group are optionally substituted by one or more groups independently selected from sulfonic acid, phosphoric acid, carboxylic acid, amide, sulfone, sulfoxide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkylaminoalkyl, alkynyl and azido, and the aminobenzyloxycarbonyl group is optionally substituted by pyranose or furanose; preferably, each L4 is independently selected from amino acid residues, fragments of polypeptides formed by 2-10 amino acids, aminobenzyloxycarbonyl, C 1-6 Alkylene and amino, the C 1-6 The alkylene and amino groups are optionally substituted by one or more groups independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, sulfone groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkoxyalkyl, C 2-6 Alkylamine, C 2-6 The aminobenzyloxycarbonyl group is optionally substituted with glucuronic acid, galacturonic acid, glucose, galactose or mannose; Preferably, the amino acid is selected from L-type natural amino acids, D-type non-natural amino acids and their analogs or derivatives; preferably, the amino acid is selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH2CH2 (OCH2CH2) y OCH3), y is an integer from 1 to 20; preferably, the polypeptide formed by the 2-10 amino acids is selected from Ala-Ala, Ala-Lys, Ala- Lys(Ac),Ala-Pro,Gly-Glu,Gly-Gly,Phe-Lys,Phe-Lys(Ac),Val-Ala,Val-Lys,Val-Lys(Ac),Val-Cit,Val-A rg,Ala-Ala-Ala,Ala-Ala-Asn,Ala-Ala-Gly,Leu-Ala-Glu,Gly-Gly-Arg,Gly-Glu-Gly,Gly-Gly-Gly,Gly-Se r-Lys,Gly-Ala-Ala,Glu-Val-Ala,Glu-Val-Cit,Glu-Val-Arg,Ser-Ala-Pro,Val-Leu-Lys,Val-Lys-Ala,Val -Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, and Gly-Gly-Gly-Gly-Gly.

7. The compound according to claim 2 or 6, wherein Selected from the following structures: p is 0 or an integer of 1-20.

8. The compound according to claim 2 or 6, wherein Selected from the following structures:

9. The compound according to any one of claims 2, 6 to 8, Selected from the following structures: p is 0 or an integer of 1-20.

10. The compound according to claim 2, wherein The effector molecule is selected from microtubule inhibitors, such as auristatins, maytansines; DNA intercalators, such as pyrrolobenzodiazepines (PBD); DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, exitecan) or topoisomerase II inhibitors (such as doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); RNA polymerase inhibitors, such as α-amanitin; and pharmaceutically acceptable salts, esters and analogs of the above agents; Preferably, the effector molecule is selected from topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-amino Camptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, exitecan), MMAE and MMAE derivatives; Preferably, the effector molecule is selected from: Preferably, D is selected from:

11. The compound of formula II according to claim 2, which is selected from the following structures: ; p is 0 or an integer from 1 to 20.

12. A compound of formula II according to any one of claims 2, 6-11, which has the following structure:

13. A biologically active conjugate, the structure of which is shown in Formula III: in: Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein agent (e.g., a sugar, RNA, or DNA)); r is selected from 1-10; preferably, Ab is trastuzumab or pertuzumab; preferably, Ab is trastuzumab; X, L3, L4, D, m, n and x are as defined in any one of claims 2, 6-10.

14. The bioactive conjugate according to claim 13, which is selected from: r is 1-10, preferably 1-3, more preferably about 2; p is 0 or an integer of 1 to 20, preferably 0 or an integer of 1 to 10; Preferably, Ab is an antibody, wherein the light chain of the antibody is linked to the remaining part of the compound of formula III through an amide bond formed by the terminal amino group of lysine.

15. The bioactive conjugate according to claim 13 or 14, which has the following structure: Trastuzumab-DL-1: Trastuzumab-DL-2: in, A1 is trastuzumab, r is 1-10, preferably 1-3, more preferably about 2; preferably, the trastuzumab is connected to the other parts of the biologically active conjugate through the lysine residues thereon.

16. A composition comprising one or more bioactive conjugates according to any one of claims 13 to 15; Preferably, the composition has a DAR of about 1 to 10, preferably about 1 to 5, further preferably about 2.0 to 2.5, such as 2.0, 2.1, 2.2, 2.3, 2.4 or 2.

5.

17. A pharmaceutical composition comprising the bioactive conjugate according to any one of claims 13 to 15, and one or more pharmaceutically acceptable carriers.

18. A drug kit product, comprising the bioactive conjugate according to any one of claims 13 to 15, the composition according to claim 16, or the pharmaceutical composition according to claim 17, and optional drug instructions.

19. Use of the bioactive conjugate according to any one of claims 13 to 15, the composition according to claim 16, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for preventing or treating tumors or autoimmune diseases.

20. The bioactive conjugate according to any one of claims 13 to 15, the composition according to claim 16, or the pharmaceutical composition according to claim 17, which is used for preventing or treating tumors or autoimmune diseases.

21. A method for preventing or treating a tumor disease, comprising administering an effective amount of the bioactive conjugate according to any one of claims 13 to 15, the composition according to claim 16, or the pharmaceutical composition according to claim 17 to a subject in need thereof.

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