Chemical bond linkers and their uses

Abstract

The present invention relates to a chemical bond linker and an antibody-drug conjugate prepared by the linker. The present invention further relates to the use of said antibody-drug conjugate in the treatment of related diseases such as tumors.

Description

[Technical Field]

[0001] This application is based on and claims priority from CN Application No. 202310181337.6, filed February 28, 2023, and CN Application No. 202311231478.0, filed September 22, 2023, the disclosures of which are incorporated herein in their entirety.

[0002] The present invention relates to a new type of linker used in chemical conjugation, antibody-drug conjugates prepared with this linker, and their use in the treatment of related diseases such as tumors. [Background technology]

[0003] In recent years, antibody-drug conjugates (ADCs) have attracted attention in precision medicine for tumor and autoimmune diseases. ADCs are composed of a monoclonal antibody that targets a specific antigen and a payload (e.g., a cytotoxic drug) linked together via a linker, and combine the biological activity of conventional small molecules with the targeting properties of antibodies.

[0004] Antibody-drug conjugates consist of three parts: an antibody, a linker, and a payload. Methods for conjugating an antibody to a linker-payload can be broadly divided into non-site-specific conjugation and site-specific conjugation. Non-site-specific conjugation, the first method, uses chemical methods to conjugate a payload to an amino acid residue on the antibody, for example, by randomly linking lysine and cysteine ​​residues on the antibody. The antibody is not modified or altered, and the number and site of conjugation of the payload cannot be determined, resulting in a lack of uniformity. Site-specific conjugation, which is currently commonly used, often achieves uniform conjugation of the payload at a specific site through specific conjugation. Antibody-drug conjugates produced using site-specific conjugation can reduce variations in efficacy, pharmacokinetics, and quality control due to differences in the conjugation site and number of conjugations.

[0005] Currently, common site-specific conjugation methods include THIOMAB technology, unnatural amino acid conjugation technology, glutaminase conjugation technology, transferase conjugation technology, and ThioBridge technology. Among these, antibody engineering and modification using enzyme conjugation may affect the structural stability of the antibody to some extent.

[0006] WO 2012007896A1 discloses a polyethylene glycol carboxylic acid fluorophenyl ester linking structure, which is used to link a polypeptide molecule to the K188 site of an antibody. WO 2013173392A1 discloses a piperidine amide linker structure, which is used to link to a lysine residue of an antibody via a piperidine linker. The linking methods disclosed in WO 2012007896A1 and WO 2013173392A1 can achieve some degree of linkage to light chain lysines, but the uniformity is not very good and there is room for improvement in the rate of site-specific linkage.

[0007] The development of site-specific and hydrophilic linker structures for conjugation remains of great significance for the development of antibody-drug conjugates with good efficacy and safety. Summary of the Invention [Means for solving the problem]

[0008] The present invention provides a new type of linker for chemical conjugation, which contains a piperidine sulfonyl carbamate structural fragment and can selectively bind to a specific lysine residue in the light chain of an antibody, and the resulting antibody-drug conjugate has good homogeneity and a pronounced antitumor effect. The conjugation conditions are mild and the operation is simple.

[0009] compound In one aspect of the present invention, there is provided a compound having the structure of Formula I, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, or isotopically labeled compound thereof. [ka] During the ceremony, LG is a leaving group or a reactive group; X is -O-, -NR 2 -and-CHR 3 - selected from R 2 and R 3 are each independently hydrogen and C 1~6 alkyl groups, which are optionally substituted with one or more groups independently selected from amino groups, alkylamino groups, nitrogen-containing heterocyclic groups, sulfonic acid groups, carboxylic acid groups, quaternary ammonium salts, hydroxyl groups, and alkoxy groups; Each L1 is independently a single bond, C 1~6 Alkylene group, -(CH2CH2O) y -, C 2~6 an alkenylene group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, a phenylene group, an amino group, -CO-NH-, and -NH-CO-; 1~6 Alkylene group, amino group, C 2~6 the alkenylene group, the 4- to 16-membered heterocyclylene group, the 5- to 16-membered heteroarylene group, the phenylene group, -CO-NH- and -NH-CO- are optionally substituted by one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkynyl group and an azide group; Each L2 is independently C 2~6 Alkynyl group, tetrazine group, methyltetrazine group, trans-cyclooctene group, benzazocine group, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azide group, C 1~6 is selected from an alkyl acyl group, a formyl group, a hydroxylamine group, an oxime group, a 4- to 16-membered heterocyclyl group, and a 5- to 16-membered heteroaryl group; each m is independently an integer from 1 to 10; y is an integer from 1 to 20.

[0010] In some embodiments, LG is -OR 1 , hydroxyl group, halogen (e.g., chlorine), C 1~6 R is selected from a halogenated alkyl group (e.g., a halogenated methyl group), a 5- to 12-membered heteroaryl group (e.g., an imidazolyl group), 1 is C 1~6 It is selected from alkyl acyl groups, maleimide groups, succinimide groups, sulfosuccinimide groups, and phenyl groups, the phenyl groups being optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from nitro groups, cyano groups, sulfo groups, sulfonic acid groups, fluorine, and chlorine.

[0011] In some embodiments, LG is -OR 1 and an imidazolyl group, R 1 is selected from a succinimide group, a sulfosuccinimide group, and a phenyl group, said phenyl group optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from a nitro group, a sulfonic acid group, and fluorine.

[0012] In some embodiments, LG is a pentafluorophenoxy group.

[0013] In some embodiments, X is —O—, —NR 2 -and-CHR 3 - selected from R 2 and R 3 are each independently hydrogen and C 1~6 alkyl groups, wherein the alkyl groups are optionally independently selected from amino groups, C 1~6 Alkylamino group, 5-12 membered nitrogen-containing heterocyclic group, sulfonic acid group, carboxylic acid group, quaternary ammonium salt, hydroxyl group and C 1~6 It is substituted with one or more groups selected from alkoxy groups.

[0014] In some embodiments, X is selected from -O-, -NH-, and -CH2-.

[0015] In some embodiments, X is —O—.

[0016] In some embodiments, each L is independently a single bond, C 1~6 Alkylene group, -(CH2CH2O) y -, C 2~6 an alkenylene group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, a phenylene group, an amino group, -CO-NH-, and -NH-CO-; 1~6 The alkylene group, amino group, 4- to 16-membered heterocyclylene group, 5- to 16-membered heteroarylene group, phenylene group, -CO-NH- and -NH-CO- may optionally independently represent a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, C 1~6 Alkyl group, C 1~6 Alkoxy group, C 2~6 Alkoxyalkyl group, C 2~6 It is substituted with one or more groups selected from an alkynyl group and an azide group.

[0017] In some embodiments, each L is independently a single bond, C 1~6 Alkylene group, -(CH2CH2O) y 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.

[0018] In some embodiments, each L is independently a single bond and C 1~4 alkylene groups, and m is 1 or 2.

[0019] In some embodiments, each L2 is independently C 2~6 Alkynyl group, methyltetrazine group, trans-cyclooctene group, benzazocine group, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azide group, and C 1~6carbonyl groups, and m is an integer of 1 to 5.

[0020] In some embodiments, each L2 is independently a single bond and C 2~6 alkynyl groups, and m is 1.

[0021] In some embodiments, [ka] is selected from the following structures: [ka] .

[0022] In some embodiments, the compound of Formula I is selected from: [ka] p is 0 or an integer of 1 to 20.

[0023] In another aspect of the present invention, there is provided a compound having the structure of Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, or isotopically labeled compound thereof. [ka] During the ceremony, LG is a leaving group or a reactive group; X is -O-, -NR 2 -and-CHR 3 - selected from R 2 and R 3 are each independently hydrogen and C 1~6 alkyl group, 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 a single bond, C1~6 Alkylene group, -O-, -(CH2CH2O) y -, an oxime group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, an amino group, an acyl group, -CO-NH-, and -NH-CO-; 1~6 the alkylene group, the oxime group, the amino group, the 4- to 16-membered heterocyclylene group, the 5- to 16-membered heteroarylene group, -CO-NH- and -NH-CO- are optionally substituted with one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkynyl group and an azide group; Each L4 independently represents an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a glycosyl group, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5- to 6-membered heteroarylene group, C 1~6 Alkylene group, -(CH2CH2O) y -, an acyl group, and an amino group; 1~6 the alkylene group and the amino group are optionally substituted by one or more groups independently selected from a sulfonic acid group, a phosphate group, a carboxylic acid group, an amide group, a sulfonyl group, a sulfoxide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkylaminoalkyl group, an alkynyl group, and an azide group; the phenylene group, the benzyl group, the benzyloxy group, the benzyloxycarbonyl group, the aminobenzyloxycarbonyl group, and the acylbenzyloxycarbonyl group are optionally substituted by a pyranosyl or a furanosyl group; each D is independently selected from a fragment of a payload, the payload being, for example, a cytotoxin, an immune agonist, an apoptosis inducer, a proteolysis inducer, or a hormone receptor modulator; 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.

[0024] In some embodiments, LG is -OR 1 , hydroxyl group, halogen (e.g., chlorine), C 1~6 R is selected from a halogenated alkyl group (e.g., a halogenated methyl group), a 5- to 12-membered heteroaryl group (e.g., an imidazolyl group), 1 is C 1~6 It is selected from alkyl acyl groups, maleimide groups, succinimide groups, sulfosuccinimide groups, and phenyl groups, the phenyl groups being optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from nitro groups, cyano groups, sulfo groups, sulfonic acid groups, fluorine, and chlorine.

[0025] In some embodiments, LG is -OR 1 and an imidazolyl group, R 1 is selected from a succinimide group, a sulfosuccinimide group, and a phenyl group, said phenyl group optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from a nitro group, a sulfonic acid group, and fluorine.

[0026] In some embodiments, LG is a pentafluorophenoxy group.

[0027] In some embodiments, X is —O—, —NR 2 -and-CHR 3 - selected from R 2 and R 3 are each independently hydrogen and C 1~6 alkyl group, 1~6 The alkyl groups may optionally independently be an amino group, C 1~6 Alkylamino group, 5-12 membered nitrogen-containing heterocyclic group, sulfonic acid group, carboxylic acid group, quaternary ammonium salt, hydroxyl group and C 1~6 It is substituted with one or more groups selected from alkoxy groups.

[0028] In some embodiments, X is selected from -O-, -NH-, and -CH2-.

[0029] In some embodiments, X is —O—.

[0030] In some embodiments, each L3 is independently a single bond, C 1~6 Alkylene group, -O-, -(CH2CH2O) y -, an oxime group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, an amino group, an acyl group, -CO-NH-, and -NH-CO-; 1~6 The alkylene group, amino group, 4- to 16-membered heterocyclylene group, 5- to 16-membered heteroarylene group, -CO-NH- and -NH-CO- optionally independently represent a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, C 1~6 Alkyl group, C 1~6 Alkoxy group, C 2~6 Alkoxyalkyl group, C 2~6 It is substituted with one or more groups selected from an alkynyl group and an azide group, y is an integer of 1 to 20, and m is an integer of 1 to 20.

[0031] In some embodiments, each L3 is independently C 1~6 Alkylene group, -O-, -(CH2CH2O) y -, an oxime group, a 5- to 16-membered heteroarylene group, an acyl group, -CO-NH-, and -NH-CO-; 1~6 The alkylene group, oxime group, 5- to 16-membered heteroarylene group, acyl group, -CO-NH-, and -NH-CO- are optionally substituted with one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkynyl group, and an azide group; y is an integer of 1 to 20; and m is an integer of 1 to 20.

[0032] In some embodiments, each L3 is independently C 1~6 Alkylene group, -O-, -(CH2CH2O) y -, an oxime group, a 5- to 16-membered heteroarylene group, an acyl group, -CO-NH-, and -NH-CO-; 1~6 The alkylene group, oxime group, 5- to 16-membered heteroarylene group, acyl group, -CO-NH- and -NH-CO- may optionally independently be a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, C 1~6 Alkyl group, C 1~6 Alkoxy group, C 2~6 Alkoxyalkyl group, C 2~6 It is substituted with one or more groups selected from an alkynyl group and an azide group, y is an integer of 1 to 20, and m is an integer of 1 to 20.

[0033] In some embodiments, each L3 is independently C 1~6 Alkylene group, -O-, -(CH2CH2O) y y is an integer of 1 to 10; and m is an integer of 1 to 15.

[0034] In some embodiments, each L4 independently represents an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, a glycosyl group, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5- to 6-membered heteroarylene group, C 1~6 Alkylene group, -(CH2CH2O) y -, an acyl group, and an amino group; 1~6 The alkylene group and the amino group may optionally be independently selected from sulfonic acid groups, phosphate groups, carboxylic acid groups, amide groups, sulfonyl groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C 1~6 Alkyl group, C 1~6 Alkoxy group, C 2~6 Alkoxyalkyl group, C 1~6Alkylaminoalkyl group, C 2~6 It is substituted with one or more groups selected from an alkynyl group and an azide group, and the phenylene group, benzyl group, benzyloxy group, benzyloxycarbonyl group, aminobenzyloxycarbonyl group, and acylbenzyloxycarbonyl group are optionally substituted with glucuronic acid, galacturonic acid, glucose, galactose, or mannose.

[0035] In some embodiments, each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, an aminobenzyloxycarbonyl group, C 1~6 an alkylene group and an amino group; 1~6 The alkylene group and the amino group are optionally substituted with one or more groups independently selected from sulfonic acid group, phosphoric acid group, carboxylic acid group, amide group, sulfonyl group, sulfoxide group, quaternary ammonium salt, hydroxyl group, alkyl group, alkoxy group, alkoxyalkyl group, alkylaminoalkyl group, alkynyl group, and azide group, and the aminobenzyloxycarbonyl group is optionally substituted with a pyranosyl or furanosyl group.

[0036] In some embodiments, each L4 is independently selected from an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, an aminobenzyloxycarbonyl group, C 1~6 an alkylene group and an amino group; 1~6 The alkylene group and the amino group may optionally be independently selected from sulfonic acid groups, phosphate groups, carboxylic acid groups, amide groups, sulfonyl groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C 1~6 Alkyl group, C 1~6 Alkoxy group, C 2~6 Alkoxyalkyl group, C 2~6 Alkylaminoalkyl group, C 2~6 It is substituted by one or more groups selected from an alkynyl group and an azide group, and the aminobenzyloxycarbonyl group is optionally substituted by glucuronic acid, galacturonic acid, glucose, galactose or mannose.

[0037] In some embodiments, the amino acids are selected from L-form natural amino acids, D-form unnatural amino acids, and analogs or derivatives thereof.

[0038] In some embodiments, the amino acids are 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), and y is an integer of 1 to 20.

[0039] In some embodiments, the polypeptide formed from 2 to 10 amino acids is selected from the group consisting of 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-Ly selected from s-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.

[0040] In some embodiments, [ka] is selected from the following structures: [ka] . p is 0 or an integer of 1 to 20.

[0041] In another embodiment of the invention, a compound fragment having the structure: [ka] To provide: [ka] [ka] [ka] .

[0042] In some embodiments, [ka] is selected from the following structures: [ka] [ka] . p is 0 or an integer of 1 to 20.

[0043] In some embodiments, p is 0 or an integer from 1 to 10; alternatively, p is 0 or an integer from 1 to 8; alternatively, p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0044] In some embodiments, the payload is selected from tubulin inhibitors, such as auristatins, maytansines, DNA intercalators, such as pyrrolobenzodiazepines (PBDs), DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, rubitecan, exatecan, etc.) or topoisomerase II inhibitors (adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide, etc.), RNA polymerase inhibitors, such as α-amanitin, and pharmaceutically acceptable salts, esters, and analogs of the above reagents.

[0045] In some embodiments, the payload is selected from a topoisomerase I inhibitor (camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, rubitecan, exatecan, etc.), MMAE, and MMAE derivatives.

[0046] In some embodiments, the payload is selected from the following: [ka] .

[0047] In some embodiments, D is a structural fragment formed after dehydrogenation of said payload.

[0048] In some embodiments, the payload is attached to L4 via an amino or hydroxyl group on the payload.

[0049] In some embodiments, D is selected from: [ka] .

[0050] In some embodiments, the compound of Formula II is selected from: [ka] [ka] [ka] p is 0 or an integer of 1 to 20.

[0051] In some embodiments, the compound of Formula II is: DL-1: [ka] , DL-2: [ka] .

[0052] In another embodiment of the present invention, there is provided a bioactive conjugate having the structure as shown in Formula III: [ka] During the ceremony, Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein reagent (e.g., a sugar, RNA, or DNA)), and r is selected from 1 to 10; X, L3, L4, D, m, x, and n are as defined in any one of the compounds of Formula II above.

[0053] In some embodiments, the target of the Ab is epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, mesothelin, folate receptor 1, mucin 1, CD138, CD20, CD19, CD30, SLTRK6, nectin 4, tissue factor, mucin 16, endothelin receptor, 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, LY64 , 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, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA, B7H3, and TENB2.

[0054] In some embodiments, the Ab is an antibody and the bioactive conjugate is an antibody drug conjugate (ADC).

[0055] In some embodiments, the Ab is an antibody, which is conjugated via a lysine terminal amino group to form a bioactive conjugate.

[0056] In some embodiments, Ab is an antibody, the light chain of which is linked to the remainder of the Formula III conjugate via a lysine terminal amino group, forming an amide bond.

[0057] In some embodiments, the Ab is trastuzumab or pertuzumab.

[0058] In some embodiments, the Ab is trastuzumab.

[0059] In some embodiments, the bioactive conjugate is selected from the following: [ka] [ka] [ka] .

[0060] In some embodiments, the antibody-drug conjugate is: Trastuzumab-DL-1: [ka] Trastuzumab-DL-2: [ka] wherein A1 is trastuzumab, r is 1 to 10, preferably 1 to 3, and more preferably about 2, and preferably the trastuzumab is linked to the other moiety of the bioactive conjugate via a lysine residue on the trastuzumab.

[0061] composition In another aspect, the present application provides a composition of a bioactive conjugate (e.g., an antibody-drug conjugate (ADC)) described herein. The composition may comprise a plurality of conjugates described herein, each conjugate comprising a drug-linker 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. Therefore, the composition is characterized by a "drug-antibody" ratio (DAR) in the range of about 1 to about 10, preferably about 1 to about 5. Methods for measuring the DAR are well known to those skilled in the art and include methods using reverse-phase chromatography or HPLC-MS.

[0062] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, such as, for example, 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, etc.

[0063] In some embodiments, the DAR of the ADC compositions described herein is about 1 to 10, e.g., about 1.0 to 5.5, about 1.0 to 5.0, about 1.0 to 4.5, about 1.0 to 4.0, about 1.0 to 3.5, about 1.0 to 3.0, about 1.0 to 2.5, about 1.0 to 2.0, about 1.5 to 5.5, about 1.5 to 5.0, or about 1.5 to 4. 5, 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.

[0064] definition Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. The technical meanings used herein refer to techniques commonly understood in the art and include modifications of techniques or equivalent technical substitutions that are obvious to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

[0065] As used herein, the term "about" refers to a range of 1% to 10% above and below the value, such as 1% to 5%, further such as 1% to 2.5%, further such as 1%, 1.5%, 2% or 2.5%.

[0066] As used herein, the term "alkylene group" 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 It represents an alkylene group, a methylidene group, an ethylidene group, a propylidene group, or a butylidene group.

[0067] As used herein, the term "alkyl group" is defined as a straight-chain or branched saturated aliphatic hydrocarbon. 1~6 The term "alkyl group" refers to a straight or branched chain aliphatic hydrocarbon group having from 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl), optionally substituted with one or more (e.g., 1 to 3) suitable substituents (e.g., halogen) (wherein the group is also referred to as a "halogenated alkyl group", e.g., C 1~6 (Also called halogenated alkyl groups) (e.g., CH2F, CHF2, CF3, CCl3, C2F5, C2Cl5, CH2CF3, CH2Cl, or -CH2CH2CF3, etc.). 1~4The term "alkyl group" refers to a straight or branched chain aliphatic hydrocarbon group having 1 to 4 carbon atoms (i.e., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl).

[0068] The term "alkoxy group" as used herein is defined as an -O-alkyl group, wherein said alkyl group is as defined above. For example, as used herein, "C 1~6 The term "alkoxy group" refers to -OC 1~6 Refers to an alkyl group.

[0069] The term "alkoxyalkyl group" as used herein is defined as an alkyl group substituted with an alkoxy group, said alkyl group being as defined above. For example, as used herein, "C 2~6 The term "alkoxyalkyl group" refers to an alkyl group of 2 to 6 carbon atoms substituted with an alkoxy group.

[0070] As used herein, the term "alkenylene group" refers to a divalent hydrocarbon group containing at least one carbon-carbon double bond, preferably having 2, 3, 4, 5, or 6 carbon atoms, e.g., C 2~6 It is an alkenylene group, a vinylene group, a propenylene group or a butenylene group.

[0071] As used herein, the term "alkenyl group" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, preferably having 2, 3, 4, 5 or 6 carbon atoms, e.g., C 2~6 It is an alkenyl group, a vinyl group, a propenyl group or a butenyl group.

[0072] As used herein, the term "alkynylene group" refers to a divalent hydrocarbon group containing at least one carbon-carbon triple bond, preferably having 2, 3, 4, 5, or 6 carbon atoms, such as an ethynylene group, a propynylene group, or a butynylene group.

[0073] As used herein, the term "alkynylene group" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as C 2~6 It is an alkynyl group, an ethynyl group, a propynyl group or a butynyl group.

[0074] The term "acyl group" as used herein refers to -C(=O)-.

[0075] As used herein, the term "alkyl acyl group" refers to a -C(=O)-alkyl group, wherein said alkyl group is as defined above. For example, as used herein, "C 1~6 The term "alkyl acyl group" refers to a group consisting of -C(=O)-C 1~6 Refers to an alkyl group.

[0076] As used herein, the term "sulfo group" means: [ka] wherein R is selected from H or an alkyl group, said alkyl group being as defined above.

[0077] As used herein, the term "sulfonic acid group" means: [ka] Represents.

[0078] As used herein, the term "amino group" means -NH, -NH- or [ka] Represents.

[0079] As used herein, the term "alkylamino group" refers to an -NR a R b represents R a and R bare each independently selected from H or an alkyl group as defined above, and R a and R b is not H at the same time. For example, "C 1~6 The alkylamino group is -NR a R b represents R a and R b are each independently H or C 1~6 The alkyl group is selected from the group consisting of:

[0080] As used herein, the term "carboxylic acid group" refers to -COOH.

[0081] As used herein, the term "phosphate group" means [ka] Represents.

[0082] As used herein, the term "quaternary ammonium salt" means a -N + R c R d R e wherein R c , R d and R e are each independently selected from an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group, or R c , R d and R e any two of these together with the N atom to which they are attached form a heterocyclyl group, or R c , R d and R e together with the N atom to which it is attached form a heteroaryl group.

[0083] As used herein, the term "amide group" refers to a group represented by -CO-NR f R g represents R f and R g are each independently selected from H or an alkyl group as defined above.

[0084] As used herein, the term "azido group" refers to -N=N + =N - Represents.

[0085] As used herein, the term "oxime group" means: [ka] or [ka] wherein R h is selected from H, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group.

[0086] As used herein, the term "glycosyl group" refers to a group obtained by removing the hemiacetal hydroxyl group of a cyclic monosaccharide or oligosaccharide, and includes furanosyl and pyranosyl groups, such as glucosyl, galactosyl, and mannosyl groups.

[0087] As used herein, the term "benzyl group" means [ka] Represents.

[0088] As used herein, the term "benzyloxy group" means [ka] Represents.

[0089] As used herein, the term "benzyloxycarbonyl group" means [ka] Represents.

[0090] As used herein, the term "aminobenzyloxycarbonyl group" means [ka] Represents.

[0091] As used herein, the term "acylbenzyloxycarbonyl group" means [ka] Represents.

[0092] As used herein, the terms "heterocyclyl group" and "heterocycle" refer to saturated or partially unsaturated cyclic groups (i.e., having one or more double and / or triple bonds within the ring) in which at least one ring atom is selected from N, O, and S heteroatoms and the remaining ring atoms are C. For example, a "4- to 16-membered heterocyclic ring (group)" is a saturated or partially unsaturated cyclic structure having 4 to 16 (e.g., 5 to 12, 2, 3, 4, 5, 6, 7, 8, or 9) ring atoms, at least one of which (e.g., 1, 2, 3, or 4) is selected from N, O, and S heteroatoms. A "nitrogen-containing heterocyclic ring (group)" is a heterocyclic ring (group) in which at least one of the ring atoms is N. Examples of heterocyclyl groups include, but are not limited to, oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolinyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, and trithianyl. The heterocyclyl group may be optionally substituted with one or more (e.g., 1, 2, 3, or 4) suitable substituents, and may optionally form a fused ring structure with one or more aromatic or heteroaromatic rings.

[0093] As used herein, the term "aromatic ring" or "aryl group" refers to a monocyclic or polycyclic aromatic ring system, e.g., having 6, 8, 9, 10, 11, 12, 13, or 14 carbon atoms comprising the ring, e.g., a benzene ring or a naphthalene ring.

[0094] The term "aromatic heterocycle" or "heteroaryl group" as used herein 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, 2, 3, 4, 5, 6, 9, or 10 carbon atoms, and containing at least one heteroatom which may be the same or different (e.g., oxygen, nitrogen, or sulfur), and which, in each case, may be benzo-fused, e.g., a 5- to 16-membered heteroaryl group, a 5- to 12-membered heteroaryl group.

[0095] As used herein, the term "halogen" includes F, Cl, Br, or I.

[0096] As used herein, the term "amino acid residue" refers to an amino acid unit in a polypeptide, i.e., the remainder of amino acids joined by peptide bonds, minus water.

[0097] The term "substitution" refers to the selective replacement of one or more (e.g., 1, 2, 3, or 4) hydrogens on the specified atom from a specified group, provided that the normal valence of the specified atom in its current context is not exceeded and that said substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0098] When a substituent is described as "optionally substituted by ...," the substituent can be (1) unsubstituted or (2) substituted. When a carbon of a substituent is described as being optionally substituted by one or more of a list of substituents, one or more hydrogens on the carbon (to the extent that there are any hydrogens) can be replaced, singly and / or together, with any independently selected substituent. When a nitrogen of a substituent is described as being optionally substituted by one or more of a list of substituents, one or more hydrogens on the nitrogen (to the extent that there are any hydrogens) can be replaced, each with any independently selected substituent.

[0099] When substituents are described as being "independently" selected from a group of groups, each substituent is selected independently of the others. Thus, each substituent can be the same as or different from another (other) substituent.

[0100] As used herein, the term "one or more" refers to one or more, under reasonable circumstances, such as two, three, four, five or ten.

[0101] Unless otherwise specified, the point of attachment of substituents as used herein may be at any suitable position on the substituent.

[0102] When a bond of a substituent passes through a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the substitutable ring.

[0103] The present invention further includes all pharmaceutically acceptable isotopically labeled compounds that 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 a different atomic mass or mass number from that which predominates in nature. Examples of isotopes suitable for inclusion in compounds of the present invention include isotopes of hydrogen (e.g., deuterium ( 2 H), tritium ( 3 H), isotopes of carbon (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 (e.g. 123 I and 125 I), nitrogen isotopes (e.g. 13 N and 15 N), isotopes of oxygen (e.g. 15 O. 17 O and 18 O), isotopes of phosphorus (e.g. 32 P), and sulfur isotopes (e.g. 35 S) and others.

[0104] The term "stereoisomer" refers to an isomer formed by at least one center of asymmetry. In compounds with one or more (e.g., 1, 2, 3, or 4) centers of asymmetry, racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers can be produced. Certain individual molecules may exist in geometric isomeric (cis / trans) forms. Similarly, compounds of the present invention may exist as mixtures of two or more rapidly equilibrating structurally distinct forms (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. It should be understood that the scope of the present application includes all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0105] In this specification, a solid line [ka] , solid wedge line [ka] or wedge-shaped dashed line [ka] The carbon-carbon bonds of the compounds of the present invention can be depicted using the following symbols: When a solid line is used to depict a bond leading to an asymmetric carbon atom, all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). When a solid or dashed wedge line is used to depict a bond leading to an asymmetric carbon atom, the depicted stereoisomer is present. When present in a racemic mixture, the solid or dashed wedge line is used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, it is intended that the compounds of the present invention may exist in the form of stereoisomers, including cis-trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present invention can exhibit one or more types of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0106] The present invention includes all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of multiple polymorphs in any proportions.

[0107] It should be further understood that some compounds of the present invention may exist in free form and be used in therapy, or, where appropriate, may exist in the form of a pharmaceutically acceptable derivative thereof. In the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, a pharmaceutically acceptable salt, ester, solvate, N-oxide, metabolite, or prodrug, which, upon administration to a patient in need thereof, can directly or indirectly provide a compound of the present invention or a metabolite or residue thereof. Therefore, when referring to a "compound of the present invention" in this specification, it is intended to include the above-mentioned various derivative forms of the compound.

[0108] Pharmaceutically acceptable salts of the compounds of the present invention include the acid addition and base salts thereof.

[0109] Suitable acid addition salts are formed with acids that form pharmaceutically acceptable salts, examples of which include aspartate, benzoate, bicarbonate / carbonate, glucoheptonate, gluconate, nitrate, orotate, palmitate and other similar salts.

[0110] Suitable base addition salts are formed with bases which form pharmaceutically acceptable salts, examples include aluminum salts, arginine salts, choline salts, magnesium salts and other similar salts.

[0111] For a description of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," Wiley-VCH, 2002. The methods used to prepare pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

[0112] The term "ester" as used herein refers to an ester derived from a compound of each general formula in this application, and includes physiologically hydrolyzable esters (a compound of the present invention that is hydrolyzed under physiological conditions to release a free acid or alcohol). The compound of the present invention itself may be an ester.

[0113] The compounds of the present invention may exist in the form of solvates, preferably hydrates, which contain polar solvents that are structural elements of the crystalline lattice of said compounds, in particular water, methanol or ethanol. The amount of polar solvent, in particular water, can be present in a stoichiometric or non-stoichiometric ratio.

[0114] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form N-oxides, as the nitrogen must be oxidized to the oxide by an available lone electron pair. Those skilled in the art will be able to identify nitrogen-containing heterocycles capable of forming N-oxides. Those skilled in the art will also recognize that N-oxides can be formed with tertiary amines. Synthetic methods used to prepare 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 peracids such as peracetic acid and metachloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydrogen peroxides such as tert-butyl hydrogen peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods used for the preparation of N-oxides have already been extensively described and explained in the literature, see, for example, T.L.G. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750; A.R.Katritzky and A.J.Boulton, Eds., Academic Press, and G.W.H. Cheeseman and E.S.G. Werstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R.Katritzky and A.J.Boulton, Eds., Academic Press.

[0115] Also included within the scope of this invention are metabolites of the compounds of this invention, i.e., substances formed in the body upon administration of a compound of this invention. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic degradation, etc. of the administered compound. Thus, the invention includes metabolites of the compounds of this invention, including compounds obtained by any process comprising contacting a compound of this invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0116] The present invention further includes within its scope prodrugs of the compounds of the present invention, which are derivatives of the compounds of the present invention that themselves have relatively little or no pharmacological activity and can be converted, for example, by hydrolysis, into compounds of the present invention having the desired activity when administered to or on the body. Typically, such prodrugs are functional derivatives of the compounds that are easily converted into the desired therapeutically active compound in the body. For further information on the use of prodrugs, see "Prodrugs as Novel Delivery Systems," Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella). Prodrugs of the present invention may be prepared, for example, by substituting certain moieties known to those skilled in the art as "promoieties" (e.g., as described in "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)) for appropriate functional groups present in the compounds of the present invention.

[0117] The present invention further includes compounds of the present invention that contain protecting groups. In any process for preparing compounds of the present invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any relevant molecule, thereby forming a form of chemical protection for the compounds of the present invention. This can be achieved, for example, by conventional protecting groups, such as those described in TW Greene & PG M Butts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. The protecting groups can be removed at an appropriate subsequent step using methods known in the art.

[0118] The compounds of the present invention can also be prepared by various methods 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 art of synthetic organic chemistry, or variations thereof understood by those skilled in the art. Preferred methods include, but are not limited to, those described above. The reactions can be carried out in a solvent or solvent mixture appropriate to the reagents and materials used and suitable for effecting the transformation. Those skilled in the art of organic synthesis should understand that the functional groups present on the molecule should be consistent with the proposed transformation. This may require the determination to modify the order of synthetic steps or to select one particular methodology route over another to obtain the required compound of the present invention.

[0119] Furthermore, it should be recognized in the art that another major consideration when designing any synthetic route is the correct selection of protecting groups to protect reactive functional groups present in the compounds of the present invention. An authoritative account describing many alternatives for those skilled in the art is Greene et al. (Protective Groups in Organic Synthesis, 4th ed., Wiley-Interscience (2006)).

[0120] Unless otherwise specified, the substituents of the compounds in the above pathways are as defined in the present invention. Those skilled in the art will understand that one or more steps in the above pathways may be omitted based on the structure of the product desired to be obtained. Those skilled in the art may also adjust the order of reaction steps as needed.

[0121] Pharmaceutical Composition The present invention further provides a pharmaceutical composition comprising the bioactive conjugate of the present invention and one or more pharmaceutically acceptable carriers.

[0122] Pharmaceutical additives as used herein refer to excipients and additives used in the manufacture of pharmaceuticals and compounded formulations, and refer to substances other than active ingredients that have been reasonably evaluated for safety and are included in pharmaceutical preparations.

[0123] The pharmaceutical composition may be administered in any manner that achieves the prevention, alleviation, prevention or cure of symptoms in a human or animal patient, including in a variety of suitable dosage forms depending on the route of administration.

[0124] The present application further provides a kit containing the bioactive conjugate or pharmaceutical composition of the present invention and an optional package insert.

[0125] Treatment methods and uses Another aspect of the present application provides the use of said bioactive conjugate in the manufacture of a medicament for the prevention or treatment of a tumor disease.

[0126] Another aspect of the present application provides the bioactive conjugate for use in the prevention or treatment of a tumor disease.

[0127] Another aspect of the present application provides a method for preventing or treating a tumor disease, comprising administering to a subject in need thereof an effective amount of the bioactive conjugate or a pharmaceutical composition comprising the bioactive conjugate.

[0128] In one embodiment of the present invention, the tumor disease is a solid tumor or a hematopoietic malignancy, for example, selected from colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), and lymphoma.

[0129] As used herein, the term "effective amount" refers to that amount of the conjugate which, upon administration, ameliorates to some extent one or more of the symptoms of the condition being treated.

[0130] Unless otherwise stated, the term "treatment" as used herein refers to halting the progression of or alleviating the disease or condition to which it applies or one or more symptoms of such disease or condition.

[0131] As used herein, an "individual" or "subject" includes a human or non-human animal. By way of example, a human individual includes a human individual (referred to as a patient) suffering from a disease (e.g., a disease described herein) or a healthy individual. A "non-human animal" in the present invention includes all vertebrates, including non-mammalian animals (e.g., birds, amphibians, reptiles) and mammals, e.g., non-human primates, and farm and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0132] The drawings described herein are provided for a further understanding of the present invention and constitute a part of this application, but the schematic examples of the present invention and their explanations are used to interpret the present invention and do not constitute an unduly limiting embodiment of the present invention. [Brief explanation of the drawings]

[0133] [Figure 1] These are the results of the efficacy of an anti-human HER2 antibody-drug conjugate ADC against a JIMT-1 cell subcutaneous tumor-bearing mouse model. [Figure 2] This shows the changes in body weight of mice in each group in the human breast cancer cell JIMT-1 CDX model. [Figure 3] These are the results of the efficacy of an anti-human HER2 antibody-drug conjugate ADC in a subcutaneous NCI-N87 cell tumor-bearing mouse model. [Figure 4] This shows the weight change of mice in each group in the human gastric cancer cell NCI-N87 CDX model. DETAILED DESCRIPTION OF THE INVENTION

[0134] The following description of the embodiments of the present invention will be made clearly and completely in conjunction with the drawings. However, it is clear that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and does not limit the present invention and its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without the need for creative labor are within the scope of protection of the present invention.

[0135] The present invention will be described in detail below with reference to examples and test examples, but these do not limit the scope of the present invention and can be modified within the scope of the present invention.

[0136] The abbreviations used herein have the following meanings:

[0137] [Table 1]

[0138] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 1 H NMR) or mass spectrometry (MS).

[0139] Nuclear magnetic resonance ( 1 The H NMR measurement equipment used was a Bruker 400 MHz nuclear magnetic resonance equipment, deuterated chloroform (CDCl3), and tetramethylsilane (TMS) was used as the internal standard substance.

[0140] The abbreviations used in the examples for nuclear magnetic resonance (NMR) spectra are as follows: 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.

[0141] The mass spectrometry (MS) measurement device used was an Agilent (ESI) mass spectrometer, model Agilent 6120B. [Example]

[0142] 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-phenylprop-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 perfluorophenyl ester (DL-A) [ka] Step 1: VC-MMAE (100 mg, 0.089 mmol), Bis-PEG2-acid (19 mg, 0.089 mmol), and HATU (34 mg, 0.089 mmol) were dissolved in DMF (3 mL), and diisopropylethylamine (35 mg, 0.267 mmol) was added dropwise. The reaction was stirred for 1 hour. LC-MS was used to monitor the complete reaction of the raw materials. After adding 0.1 mL of water, the reaction mixture was purified by high-performance liquid chromatography (conditions are as follows) to obtain DL-A-1 (30 mg).

[0143] The structural property data were as follows: ESI-MS(m / z): 1312.6[M+1] + . Column: Waters XBridge Prep C18OBD (5 μm * 19 mm * 150 mm)

[0144] [Table 2]

[0145] Step 2: 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 reacted with stirring for 4 hours. LC-MS was used to monitor the complete reaction of the raw materials. After adding 0.1 mL of water to the reaction mixture, the mixture was purified by high-performance liquid chromatography (conditions are as follows) to obtain DL-A (3.6 mg).

[0146] The structural property data were as follows: ESI-MS(m / z):1478.7[M+1] + . Column: Waters XBridge Prep C18OBD (5 μm * 19 mm * 150 mm)

[0147] [Table 3]

[0148] 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-phenylprop-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)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-6-oxohexanoyl)piperidine-4-formic acid pentafluorophenyl ester (DL-B) [ka] Step 1: Methyl 4-piperidineformate (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), and HATU (1.46 g, 3.84 mmol) and DIPEA (1.35 g, 10.48 mmol, 1.82 mL) were added. The mixture was then heated to 40 °C and reacted for 18 h. Water (30 mL) was added to the reaction mixture, which was then extracted three times with dichloromethane (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The mixture was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 10 / 1) and concentrated again to give compound DL-B-2 (500 mg, 1.53 mmol).

[0149] The structural property data were as follows: ESI-MS(m / z):328.5[M+H] + .

[0150] Step 2: DL-B-2 (500 mg, 1.53 mmol) was dissolved in dichloromethane (5.00 mL), and trifluoroacetic acid (1.54 g, 13.4 mmol, 1.00 mL) was added, followed by reaction for 1 hour at 25° C. The reaction solution was concentrated as is to obtain a crude product of the trifluoroacetate salt of DL-B-3 (550 mg, 1.43 mmol).

[0151] The structural property data were as follows: ESI-MS(m / z):271.9[M+H] + .

[0152] Step 3: VC-MMAE (60.0 mg, 53.4 μmol) and DL-B-3 (20.5 mg, 53.4 μmol, trifluoroacetate salt) 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 in this order, followed by stirring at 25°C for 1 hour. Water (20.0 mL) was added to the reaction solution, which was then 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 give the crude product. The mixture was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 10 / 1) and then concentrated again to give compound DL-B-4 (10.0 mg, 7.26 μmol).

[0153] The structural property data were as follows: ESI-MS(m / z): 1377.0 [M + H] + .

[0154] Step 4: 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), and lithium hydroxide (347 μg, 14.5 μmol) was added, followed by stirring at 25° C. for 2 hours. The pH of the reaction solution was adjusted to 5-6 with a 1N diluted hydrochloric acid solution, and the solid was precipitated and filtered. The filter cake was dried under vacuum to obtain crude compound DL-B-5 (10.0 mg).

[0155] The structural property data were as follows: ESI-MS(m / z): 1363.0 [M + H] + .

[0156] Step 5: 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 the mixture was stirred at 30 °C for 1 hour. Water (10.0 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (10.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After separation and purification by high-performance liquid chromatography, the product was lyophilized to obtain compound DL-B (2.88 mg, 1.82 μmol).

[0157] The structural property data were as follows: ESI-MS(m / z):1529.6[M+H] + .

[0158] The separation and purification method was as follows. Column: Phenomenex luna C18 (10 μm × 25 mm × 150 mm)

[0159] [Table 4]

[0160] 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-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl (ethyl)-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-formic acid pentafluorophenyl ester (DL-1) [ka] Step 1: Chlorosulfonyl isocyanate (500 mg, 3.53 mmol, 307 μL) was dissolved in dichloromethane (10.0 mL) at 0°C, and benzyl glycolate (533 mg, 3.21 mmol, 455 μL) was added, followed by stirring for 1 hour. Next, a solution of methyl 4-piperidineformate DL-B-1 (459 mg, 3.21 mmol) and triethylamine (974 mg, 9.63 mmol, 1.34 mL) in dichloromethane (5.00 mL) was added to the reaction solution, and the mixture was heated to 25°C and stirred for 1 hour. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50.0 mL × 3). The organic phases were combined and washed with cold 1N dilute hydrochloric acid (20.0 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated to give crude DL-1-1 (1.30 g, 3.14 mmol), which was used directly in the next step without purification.

[0161] The structural property data were as follows: ESI-MS(m / z):414.9[M+H] + .

[0162] Step 2: Under a nitrogen atmosphere, Pd / C (0.70 g, 10%) was added to a solution of DL-1-1 (700 mg, 1.69 mmol) in methanol (20.0 mL). The mixture was purged with hydrogen gas three times and then reacted at 25°C for 3 hours (15 PSI). The reaction mixture was filtered, and the filter cake was washed three times with methanol (100 mL x 3). The filtrate was concentrated to give crude DL-1-2 (590 mg).

[0163] Step 3: VC-MMAE (60 mg, 53.4 μmol) and DL-1-2 (17.3 mg, 53.4 μmol) were dissolved in DMF (10.0 mL), and HOBt (36.0 mg, 267 μmol), DIPEA (34.5 mg, 267 μmol, 46.5 μL), and EDCI (30.7 mg, 160 μmol) were added in this order. The mixture was then reacted at 25 °C for 2 h. Water (20.0 mL) was added to the reaction mixture, and the mixture was extracted three times with dichloromethane (20.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude DL-1-3 (60.0 mg), which was used directly in the next step without further purification.

[0164] The structural property data were as follows: ESI-MS(m / z):715.4[M / 2+H] + .

[0165] Step 4: 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), and lithium hydroxide monohydrate (2.94 mg, 69.9 μmol) was added, followed by stirring for 1 hour at 30° C. The reaction mixture was adjusted to pH 6 with 1 N dilute aqueous hydrochloric acid and then concentrated to give crude DL-1-4 (50.0 mg), which was used directly in the next step without further purification.

[0166] The structural property data were as follows: ESI-MS(m / z):1416.6[M+H] + .

[0167] Step 5: 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 the mixture was stirred at 30°C for 2 hours. The reaction mixture was concentrated at 25°C to obtain a crude product. The product was purified by high-performance liquid chromatography and then lyophilized to obtain DL-1 (4.70 mg, 2.86 μmol).

[0168] The structural property data were as follows: ESI-MS(m / z):1582.1[M+H] + .

[0169] The separation and purification method was as follows. Column: Phenomenex luna C18 (10 μm × 25 mm × 150 mm)

[0170] [Table 5]

[0171] 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-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)- 5,8-Diisopropyl-4,10-dimethyl-3,6,9-trioxy-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethoxy)carbonyl)aminosulfonyl)piperidine-4-formic acid pentafluorophenyl ester (DL-2) [ka] Step 1: (((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), and EEDQ (1.20 g, 4.87 mmol) was added. The mixture was stirred at 25 °C for 5 hours. The reaction mixture was concentrated to give the crude product. The mixture was purified on a silica gel column (petroleum ether / ethyl acetate = 100 / 1 to 0 / 100) and then concentrated again to give compound DL-2-2 (1.51 g, 2.37 mmol), which was used directly in the next step without further purification.

[0172] Its structural property data were as follows: ESI-MS(m / z):630.3[M+H] + .

[0173] Step 2: (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-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 the mixture was stirred at 25° C. for 1 hour. The reaction mixture was used directly in the next step without further treatment.

[0174] Its structural property data were as follows: ESI-MS(m / z):408.2[M+H] + .

[0175] Step 3: 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 solution 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 added, and the mixture was stirred at 25 °C for 2 hours. Water (100 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The mixture was purified using a silica gel column (dichloromethane / methanol = 100 / 1 to 97 / 3) and then concentrated again to obtain compound DL-2-4 (1.25 g, 1.57 mmol).

[0176] Its structural property data were as follows: ESI-MS (m / z): 736.2 [M + Na] + .

[0177] Step 4: Methyl 1-(N-((2-(((S)-1-(((S)-((4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)amine)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (DL-2-4, 1.15 g, 1.61 mmol) was dissolved in tetrahydrofuran (12.0 mL), and TBAF (1 M, 4.83 mL) was added, followed by stirring 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×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. After purification with a silica gel column (dichloromethane / methanol = 100 / 1 to 95 / 5), the mixture was concentrated again to obtain compound DL-2-5 (900 mg, 1.38 mmol).

[0178] Its structural property data were as follows: ESI-MS (m / z): 600.1 [M + H] + .

[0179] Step 5: Methyl 1-(N-((2-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)amine)-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), and trifluoroacetic acid (430 mg, 4.25 mmol, 591 μL) was added, followed by stirring at 25° C. for 1 hour. Water (100 mL) was added to the reaction mixture, which was then extracted three times with dichloromethane (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. After purification with a silica gel column (dichloromethane / methanol = 100 / 1 to 94 / 6), the mixture was concentrated again to obtain compound DL-2-6 (450 mg, 508.99 mmol).

[0180] Its structural property data were as follows: ESI-MS(m / z):765.2[M+H] + .

[0181] Step 6: Methyl 1-(N-((2-(((S)-3-methyl-1-(((S)-1-((4-(((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxoprop-2-yl)amino)-1-oxobut-2-yl)amine)-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 mixture, and the mixture was extracted three times with dichloromethane (50.0 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified on a silica gel column (dichloromethane / methanol = 100 / 1 to 94 / 6) and then concentrated again to give compound DL-2-7 (650 mg).

[0182] Its structural property data were as follows: ESI-MS(m / z):1343.9[M+H] + .

[0183] Step 7: 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-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-2-oxyethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxy-2,13-dioxy-4,7,10-triazatetradecyl)phenyl)amino)-1-oxoprop-2-yl)amino)-3-methyl- Methyl 1-oxobut-2-yl)amino)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylate (DL-2-7, 600 mg, 446 μmol) was dissolved in a mixed solvent of tetrahydrofuran (2.00 mL), methanol (2.00 mL), and water (2.00 mL), and lithium hydroxide monohydrate (37.4 mg, 893 μmol) was added. The mixture was heated to 45 °C and stirred for 2 hours. The pH of the reaction mixture was adjusted to 6 with aqueous citric acid, filtered, and the filter cake was dried under vacuum to obtain compound DL-2-8 (530 mg), which was used directly in the next step without further purification.

[0184] Its structural property data were as follows: ESI-MS(m / z):665.3[M / 2+H] + .

[0185] Step 8: 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-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-2-oxyethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxy-2,13-dioxy-4,7,10-triazatetradecyl)phenyl)amino)-1-oxoprop-2-yl)a (amino)-3-methyl-1-oxobut-2-yl)amino)-2-oxoethoxy)carbonyl)sulfamoyl)piperidine-4-carboxylic acid (DL-2-8, 500 mg, 376 μmol) and pentafluorophenol (138 mg, 752 μmol) were dissolved in dichloromethane (10.0 mL), EDCI (324 mg, 1.69 mmol) was added, and the mixture was stirred at 25°C for 1 hour. The reaction mixture was concentrated at 30°C to obtain the crude product. The product was purified by preparative high-performance liquid chromatography and then lyophilized to obtain the title compound (18.5 mg, 8.45 μmol).

[0186] Its structural property data were as follows: ESI-MS(m / z):1496.2[M+H] + .

[0187] The purification and separation methods were as follows. Column: Phenomenex luna C18 (10 μm × 25 mm × 150 mm)

[0188] [Table 6]

[0189] Preparation example (preparation of antibody-drug conjugate) Preparation of Trastuzumab-DL-A (target DAR 2) [ka] Trastuzumab (16.2 mg / mL, 0.31 mL) was taken, the pH adjusted to 7.40 with 1 M NaHPO, and DL-A (17.77 μL, 10 mM, equivalent to 5x the antibody mass) dissolved in dimethyl sulfoxide was added. The mixture was mixed evenly and allowed to stand at room temperature for 18 hours. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), yielding the antibody-drug conjugate (Trastuzumab-DL-A). The average payload (DAR) measured by mass spectrometry was 1.67, of which DAR2 accounted for 36.11%.

[0190] [Table 7]

[0191] Preparation of Trastuzumab-DL-B (target DAR 2) [ka] Trastuzumab (16.2 mg / mL, 0.31 mL) was taken, the pH adjusted to 7.40 with 1 M NaHPO solution, and DL-B (17.24 μL, 10 mM, equivalent to 5x the antibody mass) dissolved in dimethyl sulfoxide was added. The mixture was mixed evenly and allowed to stand at room temperature for 18 hours. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), yielding the antibody-drug conjugate (Trastuzumab-DL-B). The average payload (DAR) measured by mass spectrometry was 1.67, of which DAR2 accounted for 44.88%.

[0192] [Table 8]

[0193] Preparation of Trastuzumab-DL-1 (target DAR 2) [ka] Trastuzumab (16.2 mg / mL, 0.31 mL) was taken, the pH adjusted to 7.40 with 1 M NaHPO, and DL-1 (18.15 μL, 10 mM, equivalent to 5x the antibody mass) dissolved in dimethyl sulfoxide was added. The mixture was mixed evenly and allowed to stand at room temperature for 18 hours. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), yielding the antibody-drug conjugate (Trastuzumab-DL-1). The average payload (DAR) measured by mass spectrometry was 2.28, of which DAR2 accounted for 60.28%.

[0194] [Table 9]

[0195] Preparation of Trastuzumab-DL-2 (target DAR 2) [ka] 2.16 mL of trastuzumab (16.2 mg / mL) was taken, the pH adjusted to 7.40 with 1 M NaHPO, and 234 μL of DL-2 (10 mM, equivalent to 5x the antibody mass) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 18 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva), yielding the antibody-drug conjugate (Trastuzumab-DL-2). The average payload (DAR) measured by mass spectrometry was 2.20.

[0196] [Table 10]

[0197] The payloads (DAR values) and DAR2 ratios in the above conjugation examples demonstrate that the chemical linker of the present application, particularly the piperidine sulfonyl carbamate structure, is advantageous in improving conjugation efficiency and results in a more uniform antibody-drug conjugate with a higher DAR2 ratio.

[0198] Test Example 1 Measurement of in vitro cellular activity of antibody-drug conjugates 1. Antiproliferative effect of antibody-drug conjugates on HT-29 cells (1) Cell inoculation: First, HT-29 tumor cells were cultured in the appropriate medium, digested with pancreatin, centrifuged, resuspended, counted, and adjusted to a concentration suitable for inoculation onto plates. See Table 5 for tumor cell sources.

[0199] [Table 11]

[0200] Co-incubation of the ADC of the present application and tumor cells: After the cells attached, the medium in the cells was removed, and diluted bioactive molecules (ADC of the present application) were added to the wells of the plate and incubated for 96 hours.

[0201] Measurement of in vitro cell activity: After incubation, add Cell Counting-Lite to each well. TM 50 μL of 2.0 Reagent (Vazyme) was added and mixed evenly by shaking in the dark. After 10 minutes of reaction, the cells were detectable and read using a microplate reader (manufacturer: BMG, model: PHERAStar-FS). TM Obtain background RLU using Cell Counting-Lite™. TM The solvent RLU was obtained by the following formula: Cell inhibition rate = 1 - (sample RLU - background RLU) / (solvent RLU - background RLU) x 100%. The curve was fitted using a four-parameter model to determine the half inhibitory concentration (IC 50 ) was calculated. RLU (relative light unit): Relative light unit.

[0202] (2) Data results: The measurement results are shown in Table 6.

[0203] [Table 12]

[0204] Measurement of antibody-drug conjugate (ADC) activity in vitro (inhibitory effect) demonstrated that ADC molecules obtained by conjugation using the new chemical linker exhibited significant tumor cell killing activity. Compared with control ADCs (Trastuzumab-DL-A and Trastuzumab-DL-B), the ADC disclosed herein (Trastuzumab-DL-1) exhibited superior cell killing activity, demonstrating that ADC molecules obtained by conjugation using the new chemical linker are effective in inhibiting tumor growth.

[0205] Test Example 2: Evaluation of the tumor growth inhibitory effect of antibody-drug conjugates on a mouse subcutaneously transplanted tumor model Formulations containing the ADC of the present invention were administered via tail vein injection to mouse CDX models subcutaneously implanted with human breast cancer cells JIMT-1, and changes in tumor volume and animal body weight were measured once a week to calculate the tumor-suppressing therapeutic effect of the ADC of the present invention on cancer-bearing mice.

[0206] Test drug Drug name, source, and manufacturing method: An appropriate amount of the ADC of the present invention was taken at a dose of 3 mg / kg. The mother solution was diluted with 0.9% NaCl injection to form the administration solution, and 0.9% NaCl injection was used as the solvent control (vehicle).

[0207] Experimental animals and cell lines NOD SCID Mouse (Chengdu Pharmacological Biotechnology Co., Ltd.) Human breast cancer cell JIMT-1 (Nanjing Kebei Biotechnology Co., Ltd.)

[0208] Experiment allocation and evaluation method Average tumor volume is 100-150mm 3Tumor-bearing mice were selected and randomly assigned (the number of assignments was determined based on the number of samples). Each group was administered with 0.9% NaCl injection (hereinafter referred to as the solvent control, vehicle). For the ADC of the present invention and its administration frequency, see the specific examples. The administration method was tail vein injection, and the dose was 10 ml / kg. After administration, the diameter of the tumor was measured once a week with a vernier caliper, and the tumor volume was calculated using the following formula: V=0.5a×b 2 In the formula, a and b represent the long and short diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0209] The tumor growth inhibition rate TGI (%) was calculated using the following formula and used to evaluate the tumor-inhibitory effect of the ADC of the present invention. VT end >VT0, TGI(%)=[1-(VT end -VT0) / (VC end -VC0)]*100% or VT end ≦VT0, TGI(%)=[1-(VT end -VT0) / VT0]*100%. In the formula, VT end : Mean tumor volume at the end of the experiment for each treatment group VT0: Mean tumor volume at the start of treatment VC end : Mean tumor volume at the end of the experiment in the negative control group VC0: Mean tumor volume at the start of administration in the negative control group

[0210] The relative proliferation rate T / C (%) was calculated using the following formula and used to evaluate the tumor-suppressing effect of the ADC of the present invention. T / C=(VT end / VT0) / (VC end / VC0).

[0211] (1) Efficacy of anti-human Her2 antibody-drug conjugates in the JIMT-1 model JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C under 5% CO2. Exponentially growing JIMT-1 cells were collected, resuspended in Matrigel:PBS = 1:1 (v / v) to an appropriate concentration, and subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. The average tumor volume was approximately 150 mm. 3 At about this time, the mice were randomly assigned based on tumor size and assigned, in order, to a solvent 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 administered via tail vein injection (iv) on Day 0, for a total of one administration. After administration, the mice were weighed once a week, and the long and short diameters of the tumor were measured with calipers, and the tumor volume was calculated using the following formula: V=0.5a×b 2 In the formula, a and b represent the long and short diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0212] The ADC of the present invention exhibited significant tumor growth inhibition in a 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. By day 21, there were no deaths or significant weight loss in any of the animals in each treatment group, and no significant drug toxicity was observed. The ADC of the present invention was well tolerated by mice during the treatment period. See Table 7 and Figures 1 and 2 for specific results.

[0213] [Table 13]

[0214] Test Example 3 Evaluation of the tumor growth inhibitory effect of antibody-drug conjugates on a mouse subcutaneously transplanted tumor model Formulations containing the ADC of the present invention were administered via tail vein injection to mouse CDX models subcutaneously implanted with human gastric cancer cells NCI-N87, and changes in tumor volume and animal body weight were measured twice a week to calculate the tumor-suppressing therapeutic effect of the ADC of the present invention on cancer-bearing mice.

[0215] Test drug Drug name, source, and manufacturing method: An appropriate amount of the ADC of the present invention was taken at a dose of 1 mg / kg. The mother solution was diluted with 0.9% NaCl injection to prepare the administration solution, and 0.9% NaCl injection was used as the solvent control (vehicle).

[0216] Experimental animals and cell lines Balb / c Nude Mouse (Chengdu Yakang Biological Technology Co., Ltd., Production permit number: SCXK (River) 2020-0034, Animal certificate number: 511214900025102) Human gastric cancer cell line NCI-N87 (ATCC)

[0217] Experiment allocation and evaluation method The average tumor volume was approximately 150 mm 3 Tumor-bearing mice were selected and randomly assigned (the number of assignments was determined based on the number of samples). Each group was administered 0.9% NaCl injection (hereinafter referred to as the solvent control, vehicle). For the ADC of the present invention and the frequency of administration, see the specific examples. The administration method was tail vein injection, and the dose was 10 ml / kg. After administration, the diameter of the tumor was measured twice a week with a vernier caliper, and the tumor volume was calculated using the following formula: V=0.5a×b 2 In the formula, a and b represent the long and short diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0218] The tumor growth inhibition rate TGI (%) was calculated using the following formula and used to evaluate the tumor-inhibitory effect of the ADC of the present invention. VT end >VT0, TGI(%)=[1-(VT end -VT0) / (VC end -VC0)]*100% or VT end ≦VT0, TGI(%)=[1-(VT end-VT0) / VT0]*100%. In the formula, VT end : Mean tumor volume at the end of the experiment for each treatment group VT0: Mean tumor volume at the start of treatment VC end : Mean tumor volume at the end of the experiment in the negative control group VC0: Mean tumor volume at the start of administration in the negative control group

[0219] The relative proliferation rate T / C (%) was calculated using the following formula and used to evaluate the tumor-suppressing effect of the ADC of the present invention. T / C=(VT end / VT0) / (VC end / VC0).

[0220] (1) Efficacy of anti-human Her2 antibody-drug conjugates in the NCI-N87 model NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C under 5% CO2. Exponentially growing NCI-N87 cells were harvested, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c-nu mice to establish a gastric cancer model. The average tumor volume was approximately 150 mm. 3 At approximately 1 mg / kg, mice were randomly assigned based on tumor size and assigned to a solvent control group (i.e., negative control, vehicle group), a 1 mg / kg Trastuzumab-DL-1 group of the present invention, and a Trastuzumab-DL-2 group. The mice were administered the drug via tail vein injection (iv) on days 0, 7, and 14, for a total of three doses. After administration, the mice were weighed twice a week, and the long and short diameters of the tumors were measured with calipers. The tumor volume was calculated using the following formula: V=0.5a×b 2 In the formula, a and b represent the long and short diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0221] The ADC of the present invention exhibited significant tumor growth inhibition in an NCI-N87 gastric cancer xenograft model. Compared to the vehicle group, the tumor growth inhibition rates (TGI) of the Trastuzumab-DL-11 mg / kg group and the Trastuzumab-DL-2 1 mg / kg group were 66.23% and 63.87%, respectively. By Day 20, no animals in either treatment group died or experienced significant weight loss, and no significant drug toxicity was observed. The ADC of the present invention was well tolerated by mice during the treatment period. See Table 8 and Figures 3 and 4 for specific results.

[0222] [Table 14]

[0223] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art in light of the above description. Such modifications are 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 having the structure of Formula I, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or isotopically labeled compound thereof: 【Chemistry 1】 (In the formula, LG is a leaving group or a reactive group; X is —O—, —NR ２ - and -CHR ３ -, and R ２ and R ３ are each independently hydrogen and C １～６ alkyl group, 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 L １ are independently a single bond, C １～６ Alkylene group, -(CH ２ CH ２ O) ｙ -, C ２～６ an alkenylene group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, a phenylene group, an amino group, —CO—NH—, and —NH—CO—; １～６ Alkylene group, amino group, C ２～６ the alkenylene group, the 4- to 16-membered heterocyclylene group, the 5- to 16-membered heteroarylene group, the phenylene group, —CO—NH—, and —NH—CO— are optionally substituted by one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkynyl group, and an azide group; Each L ２ is independently C ２～６ Alkynyl group, tetrazine group, methyltetrazine group, trans-cyclooctene group, benzazocine group, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azide group, C １～６ selected from alkyl acyl groups, formyl groups, hydroxylamine groups, oxime groups, 4- to 16-membered heterocyclyl groups, and 5- to 16-membered heteroaryl groups; each m is independently an integer from 1 to 10; and y is an integer from 1 to 20.

2. A compound having the structure of Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or isotopically labeled compound thereof: 【Chemistry 2】 (In the formula, LG is a leaving group or a reactive group; X is —O—, —NR ２ - and -CHR ３ -, and R ２ and R ３ are each independently hydrogen and C １～６ alkyl group, 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 L ３ are independently a single bond, C １～６ Alkylene group, —O—, —(CH ２ CH ２ O) ｙ -, an oxime group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, an amino group, an acyl group, -CO-NH-, and -NH-CO-; １～６ the alkylene group, the oxime group, the amino group, the 4- to 16-membered heterocyclylene group, the 5- to 16-membered heteroarylene group, -CO-NH- and -NH-CO- are optionally substituted with one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkynyl group and an azide group; Each L ４ are independently an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, a glycosyl group, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5- to 6-membered heteroarylene group, C １～６ Alkylene group, -(CH ２ CH ２ O) ｙ -, an acyl group, and an amino group, １～６ the alkylene group and the amino group are optionally substituted with one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a sulfonyl group, a sulfoxide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkylaminoalkyl group, an alkynyl group, and an azide group; and the phenylene group, benzyl group, benzyloxy group, benzyloxycarbonyl group, aminobenzyloxycarbonyl group, and acylbenzyloxycarbonyl group are optionally substituted with a pyranosyl or furanosyl group; Each D is independently selected from a fragment of a payload, e.g., a cytotoxin, an immune agonist, an apoptosis inducer, a proteolysis inducer, or a hormone receptor modulator, and preferably, the payload is linked to L via an amino or hydroxyl group on the payload. ４ Combined with m is an integer from 1 to 20, n is an integer from 1 to 20, x is an integer from 1 to 10, and y is an integer from 1 to 20.

3. LG is -OR １ , hydroxyl group, halogen (e.g., chlorine), C １～６ R is selected from halogenated alkyl groups (e.g., halogenated methyl groups), 5- to 12-membered heteroaryl groups (e.g., imidazolyl groups), １ is C １～６ and LG is selected from alkyl acyl groups, maleimide groups, succinimide groups, sulfosuccinimide groups, and phenyl groups, wherein the phenyl groups are optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from nitro groups, cyano groups, sulfo groups, sulfonic acid groups, fluorine, and chlorine; preferably, LG is -OR １ and an imidazolyl group, R １ is selected from a succinimide group, a sulfosuccinimide group, and a phenyl group, said phenyl group being optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from a nitro group, a sulfonic acid group, and fluorine, preferably LG is a pentafluorophenoxy group; Preferably, each L １ are independently a single bond, C １～６ Alkylene group, -(CH ２ CH ２ O) ｙ -, C ２～６ an alkenylene group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, a phenylene group, an amino group, —CO—NH—, and —NH—CO—; １～６ The alkylene group, amino group, 4- to 16-membered heterocyclylene group, 5- to 16-membered heteroarylene group, phenylene group, —CO—NH— and —NH—CO— optionally independently represent a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, C １～６ Alkyl group, C １～６ Alkoxy group, C ２～６ Alkoxyalkyl group, C ２～６ substituted by one or more groups selected from alkynyl groups and azido groups, and preferably each L １ are independently a single bond, C １～６ Alkylene group, -(CH ２ CH ２ O) ｙ -, a phenyl group, -CO-NH-, and -NH-CO-, y is an integer from 1 to 10, for example, an integer from 1 to 5, and m is an integer from 1 to 5, and preferably, each L １ are independently a single bond and C １～４ alkylene groups, m is 1 or 2; Preferably, each L ２ is independently C ２～６ Alkynyl group, methyltetrazine group, trans-cyclooctene group, benzazocine group, (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl, azide group, and C １～６ alkyl acyl groups, m is an integer from 1 to 5, and preferably each L ２ are independently a single bond and C ２～６ alkynyl groups and m is 1. 【Request Item 4】 【Chemistry 3】 has the following structure: 【Chemistry 4】 is selected from Preferably 【Transformation 5】 2. The compound of claim 1, wherein:

5. The following structure: 【Transformation 6】 is selected from 2. The compound of formula I according to claim 1, wherein p is 0 or an integer from 1 to 20.

6. LG is -OR １ , hydroxyl group, halogen (e.g., chlorine), C １～６ R is selected from halogenated alkyl groups (e.g., halogenated methyl groups), 5- to 12-membered heteroaryl groups (e.g., imidazolyl groups), １ is C １～６ and LG is selected from alkyl acyl groups, maleimide groups, succinimide groups, sulfosuccinimide groups, and phenyl groups, wherein the phenyl groups are optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from nitro groups, cyano groups, sulfo groups, sulfonic acid groups, fluorine, and chlorine; preferably, LG is -OR １ and an imidazolyl group, R １ is selected from a succinimide group, a sulfosuccinimide group, and a phenyl group, said phenyl group being optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from a nitro group, a sulfonic acid group, and fluorine, preferably LG is a pentafluorophenoxy group; Preferably, X is —O—, —NR ２ - and -CHR ３ -, and R ２ and R ３ are each independently hydrogen and C １～６ alkyl group, １～６ The alkyl groups may optionally independently be selected from the group consisting of an amino group, a C １～６ Alkylamino group, 5- to 12-membered nitrogen-containing heterocyclic group, sulfonic acid group, carboxylic acid group, quaternary ammonium salt, hydroxyl group and C １～６ alkoxy groups, and X is -O-, -NH-, and -CH ２ -, preferably X is -O-, Preferably, each L ３ are independently a single bond, C １～６ Alkylene group, —O—, —(CH ２ CH ２ O) ｙ -, an oxime group, an amino acid residue, a polypeptide fragment formed from 2 to 10 amino acids, a 4- to 16-membered heterocyclylene group, a 5- to 16-membered heteroarylene group, an amino group, an acyl group, -CO-NH-, and -NH-CO-; １～６ The alkylene group, amino group, 4- to 16-membered heterocyclylene group, 5- to 16-membered heteroarylene group, —CO—NH— and —NH—CO— optionally independently represent a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, C １～６ Alkyl group, C １～６ Alkoxy group, C ２～６ Alkoxyalkyl group, C ２～６ substituted by one or more groups selected from an alkynyl group and an azide group, y is an integer from 1 to 20, m is an integer from 1 to 20, and preferably each L ３ is independently C １～６ Alkylene group, —O—, —(CH ２ CH ２ O) ｙ -, an oxime group, a 5- to 16-membered heteroarylene group, an acyl group, -CO-NH-, and -NH-CO-; １～６ The alkylene group, the oxime group, the 5- to 16-membered heteroarylene group, the acyl group, —CO—NH— and —NH—CO— are optionally substituted with one or more groups independently selected from a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, an amide group, a quaternary ammonium salt, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, an alkynyl group, and an azide group; y is an integer from 1 to 20; m is an integer from 1 to 20; and preferably, each L ３ is independently C １～６ Alkylene group, —O—, —(CH ２ CH ２ O) ｙ -, an oxime group, a 5- to 16-membered heteroarylene group, an acyl group, -CO-NH-, and -NH-CO-; １～６ The alkylene group, the oxime group, the 5- to 16-membered heteroarylene group, the acyl group, —CO—NH— and —NH—CO— are optionally independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, quaternary ammonium salts, hydroxyl groups, C １～６ Alkyl group, C １～６ Alkoxy group, C ２～６ Alkoxyalkyl group, C ２～６ substituted by one or more groups selected from an alkynyl group and an azide group, y is an integer from 1 to 20, m is an integer from 1 to 20, and preferably each L ３ is independently C １～６ Alkylene group, —O—, —(CH ２ CH ２ O) ｙ -, an oxime group, a 5- to 16-membered heteroarylene group, an acyl group, -CO-NH-, and -NH-CO-, y is an integer of 1 to 10, and m is an integer of 1 to 15; Each L ４ are independently an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, a glycosyl group, a phenylene group, a benzyl group, a benzyloxy group, a benzyloxycarbonyl group, an aminobenzyloxycarbonyl group, an acylbenzyloxycarbonyl group, a 5- to 6-membered heteroarylene group, C １～６ Alkylene group, -(CH ２ CH ２ O) ｙ -, an acyl group, and an amino group, １～６ The alkylene group and the amino group may optionally be independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, sulfonyl groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C １～６ Alkyl group, C １～６ Alkoxy group, C ２～６ Alkoxyalkyl group, C １～６ Alkylaminoalkyl group, C ２～６ and each L is substituted by one or more groups selected from an alkynyl group and an azide group, and the phenylene group, benzyl group, benzyloxy group, benzyloxycarbonyl group, aminobenzyloxycarbonyl group, acylbenzyloxycarbonyl group is optionally substituted by glucuronic acid, galacturonic acid, glucose, galactose or mannose, and preferably ４ are independently an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, an aminobenzyloxycarbonyl group, C １～６ an alkylene group and an amino group; １～６ The alkylene and amino groups are optionally substituted with one or more groups independently selected from sulfonic acid, phosphate, carboxylic acid, amide, sulfonyl, sulfoxide, quaternary ammonium salt, hydroxyl, alkyl, alkoxy, alkoxyalkyl, alkylaminoalkyl, alkynyl, and azide groups, and the aminobenzyloxycarbonyl groups are optionally substituted with pyranosyl or furanosyl groups, preferably each L ４ are independently an amino acid residue, a fragment of a polypeptide formed from 2 to 10 amino acids, an aminobenzyloxycarbonyl group, C １～６ an alkylene group and an amino group; １～６ The alkylene group and the amino group may optionally be independently selected from sulfonic acid groups, phosphoric acid groups, carboxylic acid groups, amide groups, sulfonyl groups, sulfoxide groups, quaternary ammonium salts, hydroxyl groups, C １～６ Alkyl group, C １～６ Alkoxy group, C ２～６ Alkoxyalkyl group, C ２～６ Alkylaminoalkyl group, C ２～６ substituted by one or more groups selected from an alkynyl group and an azido group, wherein the aminobenzyloxycarbonyl group is optionally substituted by glucuronic acid, galacturonic acid, glucose, galactose, or mannose; Preferably, the amino acids are selected from L-type natural amino acids, D-type unnatural amino acids and analogs or derivatives thereof, and preferably, the amino acids are selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH ２ CH ２ (OCH ２ CH ２ ) ｙ OCH ３ ), and y is an integer of 1 to 20. Preferably, the polypeptide formed from 2 to 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 3. The compound of claim 2, wherein the amino acid residue is selected from: 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. 【Request Item 7】 【Chemistry 7】 has the following structure: 【Transformation 8】 is selected from The compound according to claim 2 or 6, wherein p is 0 or an integer from 1 to 20. [Request Item 8] [Chemistry 9] has the following structure: 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 7. The compound of claim 2 or 6, selected from: 【Request Item 9】 【Chemistry 13】 has the following structure: 【Chemistry 14】 【Chemistry 15】 is selected from The compound according to any one of claims 2, 6 to 8, wherein p is 0 or an integer from 1 to 20.

10. the payload is selected from tubulin inhibitors, such as auristatins, maytansines, DNA intercalators, such as pyrrolobenzodiazepines (PBDs), DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, rubitecan, exatecan, etc.) or topoisomerase II inhibitors (adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide, etc.), RNA polymerase inhibitors, such as α-amanitin, and pharmaceutically acceptable salts, esters, and analogs of the above reagents; Preferably, the payload is selected from topoisomerase I inhibitors (camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, rubitecan, exatecan, etc.), MMAE and MMAE derivatives; Preferably, the payload comprises: 【Chemistry 16】 is selected from Preferably, D is 【Chemistry 17】 3. The compound of claim 2, selected from:

11. The following structure: [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 and p is 0 or an integer from 1 to 20.

12. The following structure: DL-1: 【Chemistry 21】 DL-2: 【Chemistry 22】 12. The compound of formula II according to any one of claims 2, 6 to 11, wherein

13. The structure is of Formula III: 【Chemistry 23】 (In the formula, Ab is a targeting moiety (e.g., a small molecule ligand, a protein (e.g., an antibody), a polypeptide, a non-protein reagent (e.g., a sugar, RNA, or DNA)), r is selected from 1 to 10, preferably Ab is trastuzumab or pertuzumab, preferably Ab is trastuzumab; X, L ３ , L ４ , D, m, n and x are defined in any one of claims 2, 6 to 10. The bioactive complex shown in

14. below: 【Chemistry 24】 【Chemistry 25】 (r is 1 to 10, preferably 1 to 3, more preferably about 2; p is 0 or an integer from 1 to 20, preferably 0 or an integer from 1 to 10; Preferably, Ab is an antibody, the light chain of which is linked to the remainder of the compound of Formula III via a lysine terminal amino group, forming an amide bond.

14. The bioactive conjugate of claim 13, selected from:

15. The following structure: Trastuzumab-DL-1: 【Chemistry 26】 Trastuzumab-DL-2: 【Chemistry 27】 (In the formula, A １ is trastuzumab, r is 1-10, preferably 1-3, and more preferably about 2, and preferably said trastuzumab is attached to the other moiety of said bioactive conjugate via a lysine residue on trastuzumab.

15. The bioactive conjugate of claim 13 or 14, wherein

16. A composition comprising one or more bioactive conjugates according to any one of claims 13 to 15, Preferably, the DAR of the composition is about 1 to 10, preferably about 1 to 5, more 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 kit 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 an optionally selected package insert.

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 for use in the manufacture of a pharmaceutical 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 the prevention or treatment of tumors or autoimmune diseases.

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

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