Bioactive molecule conjugate, and preparation method and use thereof

Abstract

To provide a bioactive molecule conjugate, and a preparation method and use thereof, particularly a novel bioactive molecule conjugate obtained by improving coupling of a drug and a targeting moiety in an ADC or SMDC, a preparation method thereof, and use thereof in the manufacture of a medicament for the treatment of a disease associated with abnormal cell activity.SOLUTION: The invention provides a compound represented by formula (I) defined by T-[L1-(L2)m1-(L3)m2-(L4)m3-E]-G, or a pharmaceutically acceptable salt thereof. In the formula: T is a fragment of a bioactive molecule, preferably a fragment of a molecule with antitumor bioactivity; L1 and L2 are each an amino acid, a peptide composed of 2-10 amino acids, an oligosaccharide, or the like; L3 is an amino group, 3-8 membered cycloalkylene, or the like; L4 is a specific group; and G is a leaving group for nucleophilic substitutions.SELECTED DRAWING: Figure 1

Description

Field of the Invention

[0001] The present disclosure belongs to the technical field of medical technology, and relates to bioactive molecule conjugates, methods for preparing the same, and, without limitation, uses in the prevention and / or treatment of neoplastic diseases, including uses in the prevention and / or treatment of diseases associated with abnormal cell activity.

Background Art

[0002] Once, chemotherapy was the standard treatment for cancer, but bioactive molecules with high killing effects can accidentally kill normal cells and cause severe side effects. Targeted therapy has become a research topic of interest in the field of oncology due to its targeted nature and antitumor activity. In the transition of the 20th century, great progress has been made in the development of antitumor drugs and tumor-targeted therapies using biopolymer drugs (e.g., therapeutic antibodies or antibody fragments) and targeted small molecule ligands . However, despite their high target specificity, biopolymer drugs have only limited curative effects on solid tumors; in addition, bioactive molecules often lack target specificity despite the high killing effect of the bioactive molecule on cancer cells, damage normal cells inadvertently, and cause severe toxicity and side effects.

[0003] Recent research has found that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). Since the target effect of the antibody and the activity of the bioactive molecule are combined in ADCs, the bioactive molecule has become a "biological missile". ADCs are guided by antibodies to bind to target cells, then internalized by the cells to release the drug, thereby treating related diseases . ​Treat. Due to the specificity and targeting to tumor cell-related targets, the application value of the antibody is not only reflected in the treatment but also serves as an ideal carrier for drug target delivery, reducing the side effects of the drug . Small molecule drug conjugates (SMDCs) are designed based on the same principle as antibody-drug conjugates (A DCs), that is, selectively binding to receptors on the surface of tumor cells through a chemical process and thereby improving the targeting of effector molecules (bioactive molecules) to tumor cells based on the coupling of bioactive molecules with several small molecule ligands that can. The difference between SMDCs and ADCs is that SMDCs use small molecule ligands instead of antibodies , and that there are still no SMDCs available on the market.

[0004] Currently, there are four commercially available ADCs: Mylotarg (gemtuzumab mab-ozogamicin), Adcetris (brentuximab-ved otin, CD30 monoclonal antibody-MMAE), Kadcyla (trastuzumab-emtansine) and Besponsa (inotuzumab-oz ogamicin, CD22 monoclonal antibody-calicheamicin). ADCs generally consist of an antibody , a bioactive molecule and a linker. The bioactive molecule is covalently coupled to the antibody via the linker; the antibody (e.g., a monoclonal antibody) can specifically recognize a specific target on the surface of tumor cells, so it guides the ADC to reach the surface of cancer cells and enables the ADC to penetrate into cancer cells by endocytosis ; then the bioactive molecule is released in cancer cells to achieve the effect of specifically killing cancer cells without damaging normal tissue cells . ​

[0005] Lysine is the most common conjugation site in antibodies, and the ε-amino group of lysine can react with the activated carboxyl group of a linker to form an amide bond. Techniques for site-specific coupling are currently available, i.e., the carboxyl group of the linker is activated and then forms an amide bond with the specific lysine ε-amino group in the antibody to complete the coupling. However, such amide bonds are easily hydrolyzed under the action of enzymes in vivo, resulting in the dissociation of the bioactive molecule and the antibody before reaching the target cell, leading to an increase in toxicity and at the same time the loss of the targeting property of the ADC.

[0006] The thiol groups of antibody cysteines usually exist in the form of disulfide bonds. Opening the disulfide bonds in the antibody can obtain multiple free sulfhydryl groups as coupling sites. One way to couple with the sulfhydryl groups of the antibody is the Michael addition reaction between the free sulfhydryl group of the antibody and maleimide, or two Michael addition reactions between the free sulfhydryl group of the antibody and a specific substrate that forms a sulfur cross-linking bond with a unique structure. However, many literatures report that the ADC obtained by the thiol-Michael addition method will undergo a reverse Michael addition in the systemic circulation, resulting in a toxic reaction. International Publication No. WO 2016 / 142049 discloses a structure containing amatoxin as a bioactive molecule, as well as a bioactive molecule and a linker having the structure of methylsulfonyl-substituted benzobisoxadiazole, but the details of the coupling with the antibody are not specifically described. Summary of the Invention

[0007] ​​​​​​​​​​​​​​​​ The present invention discloses a novel bioactive molecule conjugate obtained by improving the coupling method of a drug and a targeting moiety in an ADC or an SMDC. The conjugate has high stability, extremely high coupling efficiency (90%) and a high DAR (5 - 8). This disclosure is based on the above findings. Through intensive research, in the ADC of the present invention, for example, BT001021 (Example 32), after intravenous administration, the exposure of the bioactive small molecule toxin in the tumor is significantly higher than that in the plasma. On the other hand, it was unexpectedly found that Immu-132 has a plasma exposure significantly higher than the tumor exposure under the same administration route. Therefore, the ADC of the present invention has a better therapeutic window than Immu-132. The inventors also surprisingly found that the ADC of the present invention has higher efficacy than Immu-132 in animal models of gastric cancer, breast cancer and non-small cell lung cancer.

[0008] A first aspect of this disclosure provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. T - [L1 - (L2) m1 - (L3) m2 - (L4) m3 - E] - G Formula (I) [Wherein, T is a fragment of a bioactive molecule, preferably a fragment of a molecule having antitumor bioactivity; L1 is an amino acid, a peptide composed of 2 to 10 amino acids, an oligosaccharide, - (C H2) t1 -, - (CH2CH2O) t1 - (CH2) t2 -,

Chemical formula

Chemical formula

[0009] In some preferred embodiments, L1 is Val, Cit, Phe, Lys, D-Val , a peptide composed of Leu, Gly, Ala, Asn, and 2 to 5 amino acids,

Chemical

[0010] In some preferred embodiments, L1 is Val, Cit, Phe, Lys, D-Val , Leu, Gly, Ala, Asn, Cit-Val, Val-Ala, Lys-Val , Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-L eu-Lys, Gly-Gly-Arg, Ala-Ala-Asn,

Chemical

[0011] In some preferred embodiments, L1 is Lys, Cit, Cit-Val, Val-A la, Lys-Val,

Chemical formula

[0012] In some preferred embodiments, L1 is Lys, Cit, Cit-Val, Val-A la, Lys-Val,

Chemical formula

[0013] In some preferred embodiments, L1 is

Chemical formula

[0014] In some preferred embodiments, L2 is a peptide composed of Val, Cit, Phe, Lys, D-Val , Leu, Gly, Ala, Asn, and 2 to 5 amino acids,

Chemical formula

[0015] In some preferred embodiments, L2 is Val, Cit, Phe, Lys, D-Val , Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Val-Ala , Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac) , D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn,

Chemical formula

[0016] In some preferred embodiments, L2 is

Chemical formula

[0017] In some preferred embodiments, L2 is

Chemical formula

[0018] In some preferred embodiments, L2 is

Chemical formula

[0019] In some preferred embodiments, L3 is the following group which may be substituted with one or more R7: amino, C3-C8 cycloalkylene, C3-C8 aliphatic heterocyclylene, C6-C12 membered bridged heterocyclylene, C6-C12 membered spiroheterocyclylene, C6-C12 membered fused heterocycl ylene, C6-C10 arylene, C5-C12 heteroarylene or C3-C8 cycloalk yl; Selected from Kilen-W; W is oxygen or NR8, and R7 is H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C alkyl, C 1~4 alkyl, C 1~4 alkoxy, C 2~6 alkenyl or C 2~6 alkynyl is independently selected from; preferably, a 3- to 8-membered aliphatic heterocyclylene, a 6- to 12-membered bridged heterocyclylene, a 6- to 12-membered spiroheterocyclylene or a 6- to 12-membered fused heterocyclic ylene has one or more nitrogen atoms; preferably, a 3- to 8-membered aliphatic heterocyclylene, a 6- to 12-membered bridged heterocyclylene, a 6- to 12-membered spiroheterocyclylene or a 6- to 12-membered con densed heterocyclylene has one or more quaternized nitrogen atoms; preferably, a 3- to 8-membered fatty aliphatic heterocyclylene, a 6- to 12-membered bridged heterocyclylene, a 6- to 12-membered spiroheterocyclic ylene or a 6- to 12-membered fused heterocyclylene has one or more nitrogen atoms, and at least one nitrogen atom is substituted with =O; R8 is H (hydrogen), D (deuterium) , C 1~6 alkyl, C 2~6 alkenyl, C 3~6 alkynyl, C 3~6 cycloalkyl yl, C 1~6 alkoxy or cyano C 1~2 alkyl is independently selected from; m2 is 0, 1, 2 or 3.

[0020] In some preferred embodiments, L3 is one or more of the following groups which may be substituted with one or more R7: amino, a 3- to 6-membered aliphatic heterocyclylene or a 5- to 10-membered heteroarylene selected from; R7 is H (hydrogen), D (deuterium), halogen, =O, CF3, CN, ​CH2CN, carboxyl, sulfonic acid group, C 1~4 alkyl, C 1~4 alkoxy, C 2~6 alkenyl or C 2~6 alkynyl, independently selected; preferably, the 3- to 6-membered aliphatic heterocyclylene has one or more nitrogen atoms; preferably, the 3- to 6-membered aliphatic he terocyclylene has one or more quaternized nitrogen atoms; preferably, the 3- to 6-membered aliphatic heterocyclylene has one or more nitrogen atoms, and at least one nitrogen atom is substituted with =O ; m2 is 0, 1 or 2.

[0021] In some preferred embodiments, L3 may be substituted with one or more R7 and is selected from the following groups: amino or 5- to 6-membered heteroarylene; R7 is H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C alkyl, C alkoxy, C 1~4 alkyl, C 1~4 alkoxy, C 2~6 alkenyl or C 2~6 alkynyl, independently selected; m2 is 0 or 1.

[0022] In some preferred embodiments, L3 may be substituted with one or more R7 and is selected from the following groups: amino, N-methylpiperidylene, pyrazolylene or triazolylene; R7 is H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2 CN, carboxyl, sulfonic acid group, C alkyl, C alkoxy, C 1~4 alkyl, C 1~4 alkoxy, C 2~6 alkenyl or C 2~6 alkynyl, independently selected; m2 is 0 or 1. ​

[0023] In some preferred embodiments, L3 is selected from triazolylene; m2 is 0 or 1 and exists.

[0024] In some preferred embodiments, L3 is

Chemical formula

[0025] In some preferred embodiments, L3 is the following group which may be substituted with one or more R7: amino,

Chemical formula

Chemical formula

[0026] In some preferred embodiments, L3 is

Chemical formula

[0027] In some preferred embodiments, L3 is

Chemical formula

[0028] In some preferred embodiments, L4 is

Chemical formula

[0029] In some preferred embodiments, L4 is

Chemical formula

[0030] In some preferred embodiments, L4 is

Chemical formula

[0031] In some preferred embodiments, L4 is

Chemical formula

[0032] In some preferred embodiments, L4 is [Chemical formula] selected from; m3 is 1.

[0033] In some preferred embodiments, L4 is [Chemical formula] selected from; m3 is 1.

[0034] In some preferred embodiments, E is one or more Rs 12 optionally substituted 5 ~10-membered heteroarylene selected from; R 12 is H (hydrogen), D (deuterium) , halogen, CN, nitro, C 1~4 alkyl or halogenated C 1~4 alkyl independently selected.

[0035] In some preferred embodiments, E is one or more Rs 12 optionally substituted with the following groups: selected from pyrimidylene, quinolinylene or pyrrolo[2,3-d]pyrimidylene; R 12 is H (hydrogen), D (deuterium), halogen, CN, nitro, C 1~2 alkyl or halogenated C 1~2 alkyl independently selected.

[0036] In some preferred embodiments, E is one or more Rs 12 optionally substituted pi selected from limidinyl; R 12 is independently selected from H (hydrogen) or D (deuterium) is done.

[0037] In some preferred embodiments, G is halogen, OMs, OTs, OTf, nitro, or is one or more R 13 may be substituted with the following groups: alkylthio, arylth o, heteroarylthio, alkylsulfinyl, arylsulfinyl, heteroaryl sulfinyl, alkylsulfonyl, arylsulfonyl or heteroarylsulf onyl, selected from any one of; R 13 is H (hydrogen), D (deuterium), ha logen, CN, nitro, C 1~6 alkyl, halogenated C 1~6 alkyl, C 1~6 al koxy, 6- to 10-membered aryl or 5- to 12-membered heteroaryl, independently selected.

[0038] In some preferred embodiments, G is F, Cl, Br, I, OMs, OTs, OTf, methylsulfonyl, ethylsulfonyl, p-toluenesulfonyl or naphthalenesulf onyl, selected from.

[0039] In some preferred embodiments, G is F, Cl, Br, OMs, OTs, methylsulf onyl or p-toluenesulfonyl, selected from.

[0040] In some preferred embodiments, G is selected from Cl or methylsulfonyl.

[0041] In some preferred embodiments,

Chemical formula

[0042] In some preferred embodiments, [Chemical formula] is [Chemical formula] wherein m4 is preferably an integer from 0 to 6, and methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0043] In some preferred embodiments, [Chemical formula] is [Chemical formula] wherein m5 is preferably an integer from 0 to 6, and methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0044] In some preferred embodiments, [Chemical formula] is [Chemical formula] wherein m6 is preferably an integer from 0 to 6, and methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0045] In some preferred embodiments, [Chemistry] is [Chemistry] where m7 is an integer from 1 to 5, and methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0046] In some preferred embodiments, [Chemistry] is [Chemistry] where m8 is an integer from 1 to 5, and methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0047] In some preferred embodiments, [Chemistry] is [Chemistry] where m9 is an integer from 1 to 5, and R 13 is hydrogen or C 1~6 alkyl, and methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0048] In some preferred embodiments, [Chemistry] is [Chemistry] and m 10 is an integer from 0 to 6, and Z4 is selected from 5- to 6-membered heteroarylenes; Methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0049] In some preferred embodiments,

Chemical formula

Chemical formula

[0050] In some preferred embodiments,

Chemical formula

Chemical formula

[0051] In some preferred embodiments,

Chemical formula

Chemical formula

[0052] In some preferred embodiments,

Chemical formula

Chemical formula

[0053] In some preferred embodiments,

Chemical formula

Chemical formula

[0054] In some preferred embodiments,

Chemical formula

Chemical formula

[0055] In some preferred embodiments,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0056] In some preferred embodiments, T is a fragment of a bioactive molecule. In some preferred embodiments, the bioactive molecule is a metal complex such as a platinum metal complex (e.g., oxaliplatin) or a gold metal complex; a glycopeptide antibiotic such as bleomycin or pingyangmycin; a DNA topoisomerase inhibitor such as a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, bellotencian or rubitecan) or a topoisomerase II inhibitor (e.g., actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); a drug that interferes with DNA synthesis such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or narabine; a drug that acts on structural proteins such as a tubulin inhibitor, a vinca alkaloid, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel; a tumor cell signaling pathway inhibitor such as a serine / threonine kinase inhibitor, a tyrosine kinase inhibitor, an aspartokinase inhibitor or a histidine kinase inhibitor; a proteasome inhibitor; a histone deacetylase inhibitor; an anti-tumor angiogenesis agent; a cyclin inhibitor; a maytansine derivative; a calicheamicin derivative; an auristatin derivative; a pyrrolobenzodiazepine dimer (PBD) derivative; melphalan; mitomycin C; chlorambucil; and other active substances that inhibit the growth of tumor cells, promote apoptosis or necrosis of tumor cells selected from.

[0057] In some preferred embodiments, the bioactive molecule is [Chemical formula] [wherein, R 14 is selected from acyl or sulfonyl substituted with R 15 , and R 15 is C 1~6 alkyl, halogenated C 1~6 alkyl, 6- to 10-membered aryl or 5- to 12 member heteroaryl; R 16 is H (hydrogen), D (deuterium), C 1~ 6 alkyl, or R 17 substituted C 1~6 alkyl, R 17 is this but is not limited to, selected from aryl or heteroaryl including phenyl and pyridyl selected, m 11 is 0, 1 or 2.] selected from.

[0058] In some preferred embodiments, the bioactive molecule is

Chemical formula

[0059] In some preferred embodiments, the bioactive molecule is [Chemistry] selected from.

[0060] In some preferred embodiments, the bioactive molecule is [Chemistry] selected from.

[0061] In some preferred embodiments, the bioactive molecule is [Chemistry] selected from.

[0062] In some preferred embodiments, the bioactive molecule is [Chemistry] selected from.

[0063] In some preferred embodiments, the bioactive molecule is [Chemistry] selected from.

[0064] In some preferred embodiments, T is [Chemistry] [Chemistry] [Chemistry] selected from.

[0065] In some preferred embodiments, T is

Chem.

Chem.

Chem.

[0066] In some preferred embodiments, T is

Chem.

[0067] In some preferred embodiments, T is

Chem.

[0068] In some preferred embodiments, T is

Chem.

[0069] In some preferred embodiments, the compound represented by formula (I) is

Chem.

Chem.

Chem.

Chem.

[0070] In some preferred embodiments, the compound is [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] selected from

[0071] In a second aspect, the present disclosure provides a conjugate comprising a bioactive molecule, a linker, and a targeting moiety. The targeting moiety is linked to the linker via a reactive group (e.g., a thiol group) to form the conjugate. In some preferred embodiments, the structure of the conjugate is the structure shown in the following formula (II). {T-[L1-(L2)

[0072] -(L3) -E]} {T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A Formula (I I) [wherein, A is a targeting moiety (e.g., a small molecule ligand, a protein, a polypeptide, or a non-protein reagent (e.g., a sugar, RNA, or DNA)); γ is an integer or a decimal number from 1 to 10; preferably, γ is an integer or a decimal number from 5 to 8 (e.g., 5, 6, 7, or 8); the remaining groups are as described in the first aspect of the present disclosure.] ; ; the remaining groups are as described in the first aspect of the present disclosure.]

[0073] In some preferred embodiments, the targets of A are 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, Claudin 18.2. It is selected from BMPR1B, Tyro7, c-Met, ApoE, CD11c, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, 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, CD11b, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b or BCMA.

[0074] In some preferred embodiments, A is a folic acid derivative, a glutamic acid urea derivative, a somato statin derivative, an arylsulfonamide derivative (such as a carbonic anhydrase IX inhibitor), a polyene connecting two aliphatic indoles, a cyanine dye or a small molecule ligand such as IR-783 or its derivative .

[0075] In some preferred embodiments, A is [Chemical formula] selected from.

[0076] In some preferred embodiments, A is an antibody such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof is Fab, Fab’, F(ab’)2, Fd, Fv, dAb, complementarity-determining fragments, single-chain antibodies (e.g., scFv) , non-human antibodies, humanized antibodies, chimeric antibodies, fully humanized antibodies, pro-bodies, bispecific antibodies or multispecific antibodies.

[0077] In some preferred embodiments, A is an anti-Her2 monoclonal antibody such as trastuzumab, pertuzumab; or an anti-Trop-2 monoclonal antibody such as sacituzumab .

[0078] In some preferred embodiments, A is an anti-Trop-2 mo noclonal antibody such as antibody M1, M2 or M3. [Table 1]

[0079] The amino acid designations in each region or domain can follow the definitions of Chothia & Lesk (19 87) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) , Nature 342: 878-883. 1. Heavy and light chain sequences of antibody M1 modified for hydrophobicity Amino acid sequence of the heavy chain variable region of M1: (121 aa) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVK QAPGQGLKWMGWINTDSGEPTYTDDFKGRFAFSLDTSVST AYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVT VSS (SEQ ID NO: 11) Amino acid sequence of the light chain variable region of M1: (107 aa) DIQLTQSPSSLSASVGDRVSITCKASQDVSSAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSL QPEDFAVYYCQQHYSTPLTFGAGTKVEIK (SEQ ID NO: 12) 2. Hydrophobically modified heavy and light chain sequences of antibody M2 Amino acid sequence of the heavy chain variable region of M2: (121 aa) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVK QAPGQGLKWMGWINTDSGEPTYTDDFKGRFAFSLDTSVST AYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVT VSS (SEQ ID NO: 13) Amino acid sequence of the light chain variable region of M2: (107 aa) DIQLTQSPSSLSASVGDRVSITCKASQDVSSAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSL QPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 14) 3. Hydrophobically modified heavy and light chain sequences of antibody M3 Amino acid sequence of the heavy chain variable region of M3: (121 aa) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVK QAPGQGLKWMGWINTDSGEPTYTDDFKGRFAFSLDTSVST AYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVT VSS (SEQ ID NO: 15) Amino acid sequence of the light chain variable region of M3: (107 aa) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSL QPEDFAVYYCQQHYSTPLTFGAGTKVEIK (SEQ ID NO: 16) Sequences of the light chain constant regions of M1, M2, and M3: (107 aa) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9) Sequences of the heavy chain constant regions of M1, M2, and M3: (330 aa) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 10)

[0080] The C-terminal Lys of the heavy chain is easily deleted, but such deletions do not affect biological activity See Dick, L.W. et al., Biotechnol. Bioeng., 100: 1132 - 1143. The above monoclonal antibodies M1, M2, M3 in which Lys is deleted at the C-terminus of the heavy chain, and their sequences or fragments all correspond to the M1, M2, M3 monoclonal antibodies of the present invention.

[0081] In some preferred embodiments, A is an RGD peptide that recognizes a cell surface integrin receptor ; a growth factor that recognizes a cell surface growth factor receptor such as EGF, PDGF or VEGF ; or a peptide that can recognize a functional cell surface plasminogen activator, bombesin, bradykinin, somatostatin or a prostate specific membrane antigen receptor, selected from .

[0082] In some preferred embodiments, A is selected from CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, or DR5 ligand.

[0083] In some preferred embodiments, the conjugate is selected from the following.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0084] In some preferred embodiments, the conjugate is selected from the following. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [wherein, γ is an integer or a decimal number from 1 to 10, and mAb is an anti-Trop-2 monoclonal antibody or an anti-Her2 monoclonal antibody; preferably, the anti-Trop-2 monoclonal body or an anti-Her2 monoclonal antibody; preferably, the anti-Trop-2 monoclonal The Ru antibody is selected from trastuzumab, and the anti-Her2 monoclonal antibody is selected from trastuzumab or pertuzumab; preferably, γ is an integer or a decimal from 5 to 8 (for example, 5, 6, 7, or 8).

[0085] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Wherein, A1 is trastuzumab, and γ is an integer or a decimal from 1 to 10; preferably, γ is an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 8, or 7.5 to 8.]

[0086] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Chemical formula] [Wherein, A1 is trastuzumab, and γ is an integer or a decimal from 1 to 10; preferably, γ is an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 8, or 7.5 to 8.]

[0087] ​In some preferred embodiments, the conjugate is as follows.

Chemical formula

[0088] In some preferred embodiments, the conjugate is as follows.

Chemical formula

[0089] In some preferred embodiments, the conjugate is as follows.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0090] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Chemical formula] [Wherein, A2 is trastuzumab, and γ is an integer or a decimal between 1 and 10; preferably , γ is an integer or a decimal between 5 and 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6. It is an integer or a decimal between 5 and 7.5, 6.5 and 8, 7 and 8, or 7.5 and 8.

[0091] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Wherein, A2 is trastuzumab, and γ is an integer or a decimal between 1 and 10; preferably , γ is an integer or a decimal between 5 and 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6. It is an integer or a decimal between 5 and 7.5, 6.5 and 8, 7 and 8, or 7.5 and 8.

[0092] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Wherein, A3 is pertuzumab, and γ is an integer or a decimal from 1 to 10; preferably, γ is an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 ~7.5, 6.5 to 8, 7 to 8 or 7.5 to 8.]

[0093] In some preferred embodiments, the conjugate is as follows.

Chemical formula

Chemical formula

[0094] In some preferred embodiments, the conjugate is as follows.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0095] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Wherein, A4 is antibody M1, and γ is an integer or a decimal number between 1 and 10; preferably, γ is , an integer or a decimal number between 5 and 8, such as between 6 and 7, between 6 and 7.5, between 6 and 8, between 6.5 and 7, between 6.5 and 7 .5, an integer or a decimal number between 6.5 and 8, between 7 and 8, or between 7.5 and 8.

[0096] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Wherein, A5 is antibody M2, and γ is an integer or a decimal number between 1 and 10; preferably, γ is , an integer or a decimal number between 5 and 8, such as between 6 and 7, between 6 and 7.5, between 6 and 8, between 6.5 and 7, between 6.5 and 7 .5, an integer or a decimal number between 6.5 and 8, between 7 and 8, or between 7.5 and 8.

[0097] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Wherein, A5 is antibody M2, and γ is an integer or a decimal number between 1 and 10; preferably, γ is an integer or decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, an integer or decimal number from 6.5 to 8, 7 to 8 or 7.5 to 8.]

[0098] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [wherein A6 is antibody M3 and γ is an integer or decimal number from 1 to 10; preferably, γ is an integer or decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, an integer or decimal number from 6.5 to 8, 7 to 8 or 7.5 to 8.]

[0099] In some preferred embodiments, the conjugate is as follows. [Chemical formula] [wherein A6 is antibody M3 and γ is an integer or decimal number from 1 to 10; preferably, γ is an integer or decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, an integer or decimal number from 6.5 to 8, 7 to 8 or 7.5 to 8.]

[0100] In another aspect, the present disclosure is a method for preparing the conjugate of the second aspect, comprising coupling the linker of the compound of formula (I) with the active group of the targeting moiety Provide a method comprising P.

[0101] In some preferred embodiments, the method comprises reducing the disulfide bond of the targeting moiety to open it with a reducing agent (e.g., TCEP) to obtain a sulfhydryl group.

[0102] In some preferred embodiments, the method comprises forming a C-S bond between the linker of the compound of formula (I) and the sulfhydryl group of the targeting moiety.

[0103] In some preferred embodiments, the targeting moiety is an anti-Her2 monoclonal antibody (e.g., trastuzumab, pertuzumab) or an anti-Trop-2 monoclonal antibody (e.g., sacituzumab, M1, M2 or M3), or an active fragment or variant thereof.

[0104] In some preferred embodiments, the molar ratio of the targeting moiety to the compound of formula (I) is 1:(1 - 20); preferably, the coupling is carried out in water and / or an organic solvent ; preferably, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide , N-methylpyrrolidone, nitrile (e.g., acetonitrile), alcohol (e.g., methanol , ethanol) or any combination thereof.

[0105] In some preferred embodiments, the method further comprises purifying the coupling product; preferably, the coupling product is purified by chromatography (e.g., one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography).

[0106] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the first aspect of the present disclosure or a pharmaceutically acceptable salt thereof or a conjugate of the second aspect, and one or more pharmaceutical additives. In another aspect, the present disclosure provides the use of a compound of the first aspect of the present disclosure or a pharmaceutically acceptable salt thereof or a conjugate of the second aspect in the manufacture of a medicament for treating a disease associated with abnormal cell activity (such as cancer).

[0107] In some preferred embodiments, the cancer is esophageal cancer (e.g., esophageal adenocarcinoma, esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin lymphoma, central nervous system tumor (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer

[0108] such as solid tumors or non-solid tumors.

[0109] In another aspect, the present disclosure provides the use of a compound of the first aspect of the present disclosure or a pharmaceutically acceptable salt thereof or a conjugate of the second aspect or a pharmaceutical composition in the treatment of a disease associated with abnormal cell activity (such as cancer).

[0110] In another aspect, the present disclosure provides a method for treating a disease associated with abnormal cell activity (such as cancer), the method comprising administering to an individual in need thereof an effective amount of a compound of the first aspect of the present disclosure or a pharmaceutically acceptable salt thereof or a conjugate of the second aspect or a pharmaceutical composition. ​

[0111] Unless otherwise specified, all scientific and technical terms used in this disclosure have the meanings that are generally understood by those skilled in the art. Furthermore, all cell culture, molecular genetics, nucleic acid chemistry, and immunology experimental procedures used in this specification are routine steps widely used in the corresponding fields. In addition, definitions and explanations of related terms are provided below for a better understanding of this disclosure. For a better understanding of this disclosure, definitions and explanations of related terms are provided below. For a better understanding of this disclosure, definitions and explanations of related terms are provided below.

[0112] In this disclosure, pharmaceutical additives refer to excipients and additives used in pharmacy and formulation, and are substances that have been evaluated as reasonable in terms of safety. They are included in pharmaceutical preparations in addition to the active ingredients. In addition to being used as excipients, carriers, and stability promoters, pharmaceutical additives also have important functions such as solubilization and sustained release, and are important components that can affect the quality, safety, and efficacy of drugs. Pharmaceutical additives can be divided into natural substances, semi-synthetic substances, and fully synthetic substances according to their sources; they can be divided into solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating substances, fragrances, anti-adhesion agents, antioxidants, chelating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, encapsulating agents, wetting agents, absorbents, diluents, aggregating agents and resolubilizing agents, film-forming aids, and release retardants according to their actions and uses; they can be divided into oral administration, injection, mucosal, transdermal or topical administration, nasal or oral inhalation, and ophthalmic administration according to the administration route. The same pharmaceutical additive can be used for pharmaceutical preparations with different administration routes and has different effects and uses. In addition to being used as excipients, carriers, and stability promoters, pharmaceutical additives also have important functions such as solubilization and sustained release, and are important components that can affect the quality, safety, and efficacy of drugs. Pharmaceutical additives can be divided into natural substances, semi-synthetic substances, and fully synthetic substances according to their sources; they can be divided into solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating substances, fragrances, anti-adhesion agents, antioxidants, chelating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, encapsulating agents, wetting agents, absorbents, diluents, aggregating agents and resolubilizing agents, film-forming aids, and release retardants according to their actions and uses; they can be divided into oral administration, injection, mucosal, transdermal or topical administration, nasal or oral inhalation, and ophthalmic administration according to the administration route. The same pharmaceutical additive can be used for pharmaceutical preparations with different administration routes and has different effects and uses. In addition to being used as excipients, carriers, and stability promoters, pharmaceutical additives also have important functions such as solubilization and sustained release, and are important components that can affect the quality, safety, and efficacy of drugs. Pharmaceutical additives can be divided into natural substances, semi-synthetic substances, and fully synthetic substances according to their sources; they can be divided into solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating substances, fragrances, anti-adhesion agents, antioxidants, chelating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, encapsulating agents, wetting agents, absorbents, diluents, aggregating agents and resolubilizing agents, film-forming aids, and release retardants according to their actions and uses; they can be divided into oral administration, injection, mucosal, transdermal or topical administration, nasal or oral inhalation, and ophthalmic administration according to the administration route. The same pharmaceutical additive can be used for pharmaceutical preparations with different administration routes and has different effects and uses. In addition to being used as excipients, carriers, and stability promoters, pharmaceutical additives also have important functions such as solubilization and sustained release, and are important components that can affect the quality, safety, and efficacy of drugs. they can be divided into solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating substances, fragrances, anti-adhesion agents, antioxidants, chelating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, encapsulating agents, wetting agents, absorbents, diluents, aggregating agents and resolubilizing agents, film-forming aids, and release retardants according to their actions and uses; they can be divided into oral administration, injection, mucosal, transdermal or topical administration, nasal or oral inhalation, and ophthalmic administration according to the administration route. The same pharmaceutical additive can be used for pharmaceutical preparations with different administration routes and has different effects and uses. The same pharmaceutical additive can be used for pharmaceutical preparations with different administration routes and has different effects and uses. The same pharmaceutical additive can be used for pharmaceutical preparations with different administration routes and has different effects and uses.

[0113] Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate. Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate. Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate. Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate. Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate. Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate. Depending on the administration route, the pharmaceutical composition can be formulated into various suitable dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermal agents, aerosols, powders and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate.

[0114] The pharmaceutical composition can be administered in the form of an injection, including injection solutions, sterile powders for injection, and concentrated injection solutions. Acceptable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an injection, including injection solutions, sterile powders for injection, and concentrated injection solutions. Acceptable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an injection, including injection solutions, sterile powders for injection, and concentrated injection solutions. Acceptable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an injection, including injection solutions, sterile powders for injection, and concentrated injection solutions. Acceptable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspending media.

[0115] In the present disclosure, the term "individual" includes human individuals or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., the diseases described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, and reptiles), non-human primates, domestic animals, and / or household animals (e.g., sheep, dogs, cats, cows, and pigs). In the present disclosure, the term "individual" includes human individuals or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., the diseases described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, and reptiles), non-human primates, domestic animals, and / or household animals (e.g., sheep, dogs, cats, cows, and pigs). In the present disclosure, the term "individual" includes human individuals or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., the diseases described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, and reptiles), non-human primates, domestic animals, and / or household animals (e.g., sheep, dogs, cats, cows, and pigs). In the present disclosure, the term "individual" includes human individuals or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., the diseases described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, and reptiles), non-human primates, domestic animals, and / or household animals (e.g., sheep, dogs, cats, cows, and pigs). In the present disclosure, the term "individual" includes human individuals or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., the diseases described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, and reptiles), non-human primates, domestic animals, and / or household animals (e.g., sheep, dogs, cats, cows, and pigs). In the present disclosure, the term "individual" includes human individuals or non-human animals. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., the diseases described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, and reptiles), non-human primates, domestic animals, and / or household animals (e.g., sheep, dogs, cats, cows, and pigs).

[0116] In the present disclosure, the term "effective amount" means one or more symptoms of the treated disease after administration. Refers to the amount of the compound that is reduced to some extent.

[0117] In the present disclosure, the term "conjugate" refers to a substance obtained by linking a bioactive molecule to a targeting moiety. In some embodiments of the present disclosure, the bioactive molecule is linked to the targeting moiety via a linker. The linker is cleaved in a specific environment (e.g., intracellular low pH environment) or under a specific action (e.g., the action of lysosomal proteases), thereby dissociating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly linked to the targeting moiety by a covalent bond that is cleaved in a specific environment or under a specific action, thereby dissociating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the bioactive molecule is linked to the targeting moiety via a linker. The linker is cleaved in a specific environment (e.g., intracellular low pH environment) or under a specific action (e.g., the action of lysosomal proteases), thereby dissociating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly linked to the targeting moiety by a covalent bond that is cleaved in a specific environment or under a specific action, thereby dissociating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the bioactive molecule is linked to the targeting moiety via a linker. The linker is cleaved in a specific environment (e.g., intracellular low pH environment) or under a specific action (e.g., the action of lysosomal proteases), thereby dissociating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly linked to the targeting moiety by a covalent bond that is cleaved in a specific environment or under a specific action, thereby dissociating the bioactive molecule from the targeting moiety. In a specific environment (e.g., intracellular low pH environment) or under a specific action (e.g., the action of lysosomal proteases), the linker is cleaved, thereby dissociating the bioactive molecule from the targeting moiety. In a specific environment (e.g., intracellular low pH environment) or under a specific action (e.g., the action of lysosomal proteases), the linker is cleaved, thereby dissociating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly linked to the targeting moiety by a covalent bond that is cleaved in a specific environment or under a specific action, thereby dissociating the bioactive molecule from the targeting moiety. In a specific environment or under a specific action, the covalent bond is cleaved, thereby dissociating the bioactive molecule from the targeting moiety. In a specific environment or under a specific action, the covalent bond is cleaved, thereby dissociating the bioactive molecule from the targeting moiety.

[0118] In the present disclosure, the terms "bioactive substance" and "bioactive molecule" refer to substances that inhibit or block cell functions and / or bring about cell death or destruction. In some embodiments of the present disclosure, the bioactive substance or bioactive molecule in the conjugate is a molecule having antitumor bioactivity. For example: radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 or radioisotopes of Lu; metal complexes such as platinum metal complexes, gold metal complexes or oxaliplatin; glycopeptide antibiotics such as bleomycin or pingyangmycin; topoisomerase I inhibitors such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, velotencian or rubitecan, or topoisomerase II inhibitors such as DNA topoisomerase inhibitors such as actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide; drugs that interfere with DNA synthesis such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or nelarabine; drugs that act on structural proteins such as tubulin inhibitors, vincblastine alkaloids, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel; tumor cell signaling pathway inhibitors such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartokinase inhibitors or histidine kinase inhibitors; also, proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine (PBD) derivatives; melphalan; mitomycin C; chlorambucil; and other active substances that inhibit the growth of tumor cells, promote apoptosis or necrosis of tumor cells; enzymes such as nucleolytic enzymes and fragments thereof; antibiotics; toxins such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, including fragments and / or variants thereof; growth inhibitors; and drug molecules. The term "toxin" refers to substances that have a harmful effect on cell growth or proliferation.

[0119] In the present disclosure, the term "small molecule" refers to a small molecule drug having biological activity.

[0120] In the present disclosure, the term "linker" refers to a fragment that links a bioactive molecule to a targeting moiety.

[0121] In the present disclosure, the term "targeting moiety" refers to a moiety of a conjugate that can specifically bind to a target (or a part of a target) on the cell surface. The conjugate can be delivered to a specific cell population by the interaction between the targeting moiety and the target.

[0122] In the present disclosure, when the targeting moiety of a conjugate is an antibody, the conjugate can be referred to as a "drug - antibody conjugate". In the present disclosure, "drug - antibody conjugate" and "immunoconjugate" are interchangeable.

[0123] In the present disclosure, the term "antibody", in its broadest sense, is construed to include full - monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two full antibodies, provided that the antibody has the required biological activity. In the present disclosure, the terms "antibody" and "immunoglobulin" are interchangeable.

[0124] In the present disclosure, the term "monoclonal antibody" refers to an antibody from a substantially homogeneous group of antibodies. That is, except for a few possible natural mutations, the antibodies that make up the group are identical. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, while in contrast, polyclonal antibodies have various specificities for various determinants (epitopes). ​​​​​​​​​contains the antibody. In addition to specificity, monoclonal antibodies have the advantage of not being contaminated by other antibodies during synthesis. Here, the modifying phrase "monoclonal" indicates that the antibody is characterized by being derived from a substantially homogeneous group of antibodies and should not be construed as being prepared by a special method.

[0125] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies. That is, if the antibody has the required biological activity, part of the heavy chain and / or light chain is the same or of the same kind as an antibody of a certain type, class or subclass, and the rest is of another type , of the same or of the same kind as an antibody of another class or subclass (see, for example, U.S. Patent No. 4, 816,567; and Morrison et al., 1984, PNAS, 81:6851-6 855). Chimeric antibodies available in the present disclosure include primatized antibodies containing variable region antigen-binding sequences derived from non-human primates (e.g., old world monkeys or orangutans) and human constant region sequences.

[0126] The term "antibody fragment" refers to a part of an antibody, preferably the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fd, Fv, dAb and complementary determining fragments, diabodies, linear antibodies and single-chain antibody molecules.

[0127] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) via a linker arm. This conjugate maintains the activity of each antibody and thus has bifunctional and bispecific properties.

[0128] The term "multispecific antibody" includes, for example, a trispecific antibody that is an antibody having three different antigen-binding specificities, and a tetra specific antibody that is an antibody having four different antigen-binding specificities.

[0129] The term "full antibody" refers to an antibody containing an antigen-binding variable region, a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. The full antibody is preferably a full antibody having one or more effector functions.

[0130] The term "probody" refers to a modified antibody that can specifically bind to its target and can be coupled to a masked group, where the masked group here refers to a cleavage constant (cleavage const ant) related to the binding ability of the antibody or antibody fragment to the target that is at least 100-fold or 1000-fold or 10000-fold higher than the cleavage constant related to the binding ability of the antibody or antibody fragment not coupled to the masked group to the target.

[0131] In the present disclosure, a "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimal non-human immunoglobulin sequence. Most humanized antibodies have residues in the hypervariable regions of human recipient immunoglobulins replaced with residues in the hypervariable regions (donor antibodies) of non-human ( e.g., mouse, rat, rabbit, or non-human primate) that have the required specificity, affinity, and function. In some embodiments, the framework regions of human immunoglobulins ​​​​​​​​​​​(FR) The residues in it are also replaced with non-human residues. Furthermore, the humanized antibody may also contain residues that are not present in the recipient antibody or donor antibody. Such modifications are made to further optimize antibody performance. Humanized antibodies generally contain at least one variable region, typically two variable regions, where all or almost all of the hypervariable loops correspond to non-human immunoglobulins, but all or almost all of the FRs are of human immunoglobulin sequences. The humanized antibody may contain at least a portion of the immunoglobulin constant region (Fc, usually a human immunoglobulin Fc). For details, see, for example, Jones et al., 1986, Nature, 321:522-525; Reich mann et al., 1988, Nature, 332:323-329; and Presta, 1 992, Curr Op Struct Bwl 2:593-596. mann et al., 1988, Nature, 332:323-329; and Presta, 1 992, Curr Op Struct Bwl 2:593-596.

[0132] Intact antibodies can be classified into different "groups" according to the amino acid sequence of the heavy chain constant region. The main five groups are IgA, IgD, IgE, IgG, and IgM, some of which are also further classified into different " subclasses" (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The different groups of the heavy chain constant region of the antibody are called α, β, ε, γ, and μ, respectively. The subunit structure of immunoglobulins and the various classes of 3 D arrangements are well known in the art.

[0133] In the present disclosure, amino acid substitutions in antibodies are often replaced with L-amino acids, but the embodiments are not limited thereto. In some embodiments, the antibody peptide chain is one or multiple ., but the embodiments are not limited thereto. In some embodiments, the antibody peptide chain is one or multiple The peptides containing D-amino acids may be administered intravenously through the oral cavity, intestinal tract or blood. In plasma, it is more stable and less degradable than peptides containing only L-amino acids. There is a saying.

[0134] The monoclonal antibodies used in this disclosure can be generated by a number of methods. For example, the monoclonal antibodies used in this disclosure are isolated from a number of species (mouse, hamster, etc. Hybridoma methods (e.g., Kohler et al., J. Immunol. 1999, 113:1311-1323, including rat and human cells) (see, e.g., W. et al., 1975, Nature 256:495), or recombinantly. DNA techniques (see, e.g., U.S. Pat. No. 4,816,567). or can be isolated from a phage antibody library (e.g., Cla ckson et al., 1991, Nature 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:58 1-597). Monoclonal antibodies that can be used in the present disclosure include: Anti-Her2 monoclonal antibodies, such as, but not limited to, trastuzumab and pertuzumab monoclonal antibody, or sacituzumab (i.e., isactuzumab or Anti-Trop-2 monoclonal antibodies, such as hRS7 antibody, M1, M2 or M3 can be.

[0135] In some preferred embodiments, the targets of A are 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 dothelin 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, I L20Rα, 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, Claudin 18.2, BMPR1B, Tyro7, c-Met, ApoE, CD11c, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINE1, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CX selected from CL11, 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, CD11b, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b or BCMA.

[0136] In some embodiments of the present disclosure, the target of targeting moiety A is a cell surface integrin an RGD peptide that recognizes a receptor; a growth factor that recognizes a cell surface growth factor receptor such as EGF, PDGF or VEGF; and a functional cell surface plasminogen activator, bombe ninogen activator receptor, urokinase plasminogen activator receptor, or tissue plasminogen activator receptor. A peptide capable of recognizing syn, bradykinin, somatostatin, or prostate-specific membrane antigen receptor is selected from.

[0137] In some embodiments of the present disclosure, the target of targeting moiety A is CD40 ligand, C D30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, and DR5 ligand.

[0138] In some embodiments of the present disclosure, targeting moiety A is an anti-Her2 monoclonal antibody such as trastuzumab or pertuzumab; or an anti-Trop-2 monoclonal antibody such as sacituzumab, M1, M2, or M3. is.

[0139] In some embodiments of the present disclosure, the targeting moiety is trastuzumab or pertuzumab . Trastuzumab is an anti-Her2 monoclonal antibody, and its amino acid sequence is known to those skilled in the art. For an overview of the sequence, see, for example, CN103319599 . .

[0140] In some embodiments of the present disclosure, the terminal Lys of the heavy chain of the targeting moiety can be easily deleted, but such deletion does not affect biological activity. See Dick, L.W. et al., Bio technol. Bioeng., 100:1132-1143. For example , the targeting moiety is an anti-Trop-2 monoclonal antibody such as sacituzumab with the terminal Lys of the heavy chain deleted, M1, M2, or M3. For example, the targeting moiety is an anti-Her2 monoclonal antibody such as trastuzumab or pertuzumab with the terminal Lys of the heavy chain deleted . is. is. is.

[0141] For exemplary heavy and light chain sequences of trastuzumab, see SEQ ID NO: 17 and SEQ ID NO: 18 See. In the present disclosure, the heavy and light chain sequences of trastuzumab that are referenced or related are described using the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively. For exemplary heavy and light chain sequences of pertuzumab, see SEQ ID NO: 16 and SEQ ID NO: 15 of US Patent No. 7,560,111

[0142] SEQ ID NO: 17 (heavy chain sequence) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQ APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG(K)

[0143] SEQ ID NO: 18 (light chain sequence) ​​​DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQK PGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQ PEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0144] In some embodiments of the present disclosure, the targeting moiety anti-Trop-2 antibody is RS7 (i.e., sacituzumab of the present disclosure) described in U.S. Pat. No. 7,517,964; and U.S. Pat. hRS7 (i.e., the sacs of the present disclosure) described in Patent Application Publication No. 2012 / 0237518 The anti-Trop-2 antibodies available in the present disclosure were screened to identify The carrier design, construction and antibody display disclosed in Japanese Patent No. 103476941A Alternatively, the antibody may be obtained by constructing a library of antibodies that are similar to those of Sorrento T Screening of G-MAB® library from Herapeutics, Inc. It can also be obtained by searching.

[0145] The heavy and light chain amino acid sequences of the monoclonal antibody sacituzumab are See, for example, SEQ ID NO:19 and SEQ ID NO:20.

[0146] SEQ ID NO:19 (heavy chain sequence) QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQ APGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTA YLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG(K)

[0147] The terminal K (or lys) of the heavy chain is easily deleted, but such a deletion does not affect biological activity. See Dick, L.W. et al., Biotechnol. Bioeng., 10 0:1132~1143. Please refer to.

[0148] SEQ ID NO: 20 (light chain sequence) DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQK PGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQ PEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0149] In the present disclosure, ErbB2 and Her2 / neu are interchangeable, and both represent the native sequence of the human Her2 protein (Genebank CAS number X03363, see, for example, Se mba et al., 1985, PNAS, 82:6497-6501; and Yamamoto et al., 1986, Nature, 319:230-234) and its functional derivatives such as amino acid sequence var iants. ErbB2 represents the gene encoding human Her2, and neu represents the gene encoding rat p185neu. In some embodiments , the compounds or conjugates of the present disclosure can inhibit or kill cells expressing the ErbB2 receptor, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, kidney cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells or liver cancer cells.

[0150] In the present disclosure, Trop-2 or TROP2 refers to human trophoblast cell surface antigen 2, also known as TACSTD2, M1S1, GA733 -1, EGP-1, which is a cell surface receptor expressed in many human tumors (e.g., breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer and cervix ical cancer). In some embodiments, the compounds or conjugates of the present disclosure can inhibit or kill cells expressing the TROP2 receptor, such as breast cancer cells, colorectal cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, prostate cancer cells or cervical cancer cells.

[0151] As used herein, the [Chemical] When the targeting moiety is an antibody, it shows a specific linking pattern of sulfhydryl groups and linkers in the antibody. It shows the specific linking pattern of the sulfhydryl groups and the linker in the antibody when the targeting moiety is an antibody.

[0152] As used herein, the term "C 1~6 alkyl" refers to a straight-chain or branched alkyl containing 1 to 6 carbon atoms, including, for example, "C 1~4 alkyl" and "C alkyl". Specific examples include, but are not limited to, methyl, ethyl, n-prop 1~3 yl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n -pentyl, iso-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpent yl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethyl butyl and 1,2-dimethylpropyl. It includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl and 1,2-dimethylpropyl. It includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl and 1,2-dimethylpropyl.

[0153] As used herein, the term "C 2~6 alkenyl" refers to a straight-chain, branched or cyclic alkenyl containing at least one double bond and 2 to 6 carbon atoms, including, for example, "C 2~4 alkenyl". "C alkenyl" examples include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1, 2~6 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-penta enyl, 2,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2,4-hexadienyl and 1,3,5-hexatriene. 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-penta Dienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl , 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclo Includes hexenyl and 1,4-cyclohexadienyl.

[0154] As used herein, "C 2~6 The term alkynyl includes, for example, "C 2~4 Alkynyl: A straight chain containing at least one triple bond and 2 to 6 carbon atoms. or branched alkynyl. 2~6 Examples of "alkynyl" include, but are not limited to, , ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl -2-pentynyl, 2-hexynyl, 3-hexynyl and 5-methyl-2-hexynyl Included.

[0155] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine. Contains elements.

[0156] As used herein, "3- to 8-membered cycloalkyl" or "C 3~8 Cycloalkyl " The term "cycloalkyl" includes, for example, "3- to 6-membered cycloalkyl," "4- to 6-membered cycloalkyl," 3 to 8 carbon atoms including "5 to 7-membered cycloalkyl" or "5 to 6-membered cycloalkyl" Specific examples include, but are not limited to, cycloalkyl, cycloalkyl groups containing aryl groups. Cyclopropanyl, cyclobutyl alkyl, pentanyl, cyclohexyl, cycloheptyl and and cyclooctadecyl.

[0157] As used herein, "C 1~6 The term "alkoxy" refers to 1~6 Alkyl- Refers to a group having the structure of O—, where C 1~6 Alkyl is as defined previously. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isoprop oxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-but oxy, pentyloxy, and hexyloxy.

[0158] As used herein, the term “3- to 8-membered aliphatic heterocyclyl” refers to a cyclic group containing 3 to 8 ring-forming atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom . Optionally, the ring-forming atoms in the cyclic structure (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) may be substituted with oxygen. “3- to 8-membered aliphatic heterocyclyl” includes, for example, but is not limited to, oxiranyl, oxo cyclobutyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl , tetrahydropyranyl, and homopiperazinyl, including “3- to 8-membered nitrogen-containing aliphatic heterocy clyl”, “3- to 8-membered oxygen-containing aliphatic heterocyclyl”, “3- to 6-membered aliphatic heterocyclyl” , “3- to 6-membered oxygen-containing aliphatic heterocyclyl”, “4- to 7-membered aliphatic heterocyclyl” , “4- to 6-membered aliphatic heterocyclyl”, “5- to 7-membered aliphatic heterocyclyl”, “5- to 6-membered aliphatic heterocyclyl”, and “5- to 6-membered nitrogen-containing aliphatic heterocyclyl”. As used herein, the term “6- to 12-membered spirocyclyl” refers to a cyclic structure formed by two or more cyclic structures containing 6 to 12

[0159] ring-forming carbon atoms and sharing one carbon atom. Optionally, the carbon atoms in the cyclic structure may be substituted with oxygen. formed by two or more cyclic structures sharing one carbon atom. Optionally, the carbon atoms in the cyclic structure may be substituted with oxygen. "6- to 12-membered spirocyclic group" includes, for example, "6- to 11-membered spirocyclic group", "6- to 1 0-membered spirocyclic group", "7- to 10-membered spirocyclic group", "7- to 9-membered spirocyclic group", "7- to 8-membered spirocyclic group", "9- to 10-membered spirocyclic group", and "3- to 10-membered spiro cyclic group". Specific examples include, but are not limited to,

Chem.

[0160] As used herein, the term "6- to 12-membered bridged cyclic group" refers to a cyclic structure formed by two or more cyclic structures containing 6 to 12 ring-forming carbon atoms and sharing two adjacent carbon atoms. Optionally, the carbon atoms in the cyclic structure may be substituted with oxygen. "6- to 12-membered bridged cyclic group" includes, for example, "6- to 11-membered bridged cyclic group", "5- to 10 membered bridged cyclic group", "7- to 10-membered bridged cyclic group", "7- to 9-membered bridged cyclic group", "7- to 8 membered bridged cyclic group", "9- to 10-membered bridged cyclic group", and "3- to 10-membered bridged cyclic group". Specific examples include, but are not limited to, 。 membered bridged cyclic group", "7- to 10-membered bridged cyclic group", "7- to 9-membered bridged cyclic group", "7- to 8 membered bridged cyclic group", "9- to 10-membered bridged cyclic group", and "3- to 10-membered bridged cyclic group" are included . Specific examples include, but are not limited to,

Chem.

[0161] As used herein, the term "6- to 12-membered fused cyclic group" includes "6- to 11-membered fused cyclic group", "6- to 10-membered fused cyclic group", "6- to 8-membered fused cyclic group", "10- to 12 membered fused cyclic group", "7- to 10-membered fused cyclic group", and refers to a cyclic structure formed by two or more cyclic structures containing 6 to 12 ring-forming carbon atoms and sharing two adjacent atoms. ​refers to. Examples of "6- to 12-membered fused heterocyclyl" include, but are not limited to, [Chem.] are included.

[0162] As used herein, the term "6- to 12-membered spiroheterocyclyl" refers to a cyclic structure formed by two or more cyclic structures that contain 6 to 1 2 ring-forming carbon atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom and share one ring-forming atom. Optionally, the ring-forming atoms (e.g., carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure may be substituted with oxygen. "6- to 12-membered spirohetero cycllyl" includes, for example, "6- to 11-membered spiroheterocyclyl", "5- to 10-membered spiro heterocyclyl", "7- to 11-membered spiroheterocyclyl", "7- to 10-membered spirohetero cyclyl", "7- to 9-membered spiroheterocyclyl", "7- to 8-membered spiroheterocyclyl", " 9- to 10-membered spiroheterocyclyl" and "3- to 10-membered spiroheterocyclyl". Specific examples include, but are not limited to, . [Chem.] are included.

[0163] As used herein, the term "6- to 12-membered bridged heterocyclyl" refers to a cyclic structure formed by two or more cyclic structures that contain 6 to 12 ring-forming atoms, at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom and share two non-adjacent ring-forming atoms. Optionally, the ring-forming atoms (e.g., carbon atoms) in the cyclic structure... The (atom, nitrogen atom or sulfur atom) may be substituted with oxygen. "6- to 12-membered bridged hetero "Cyclic" includes, for example, "6- to 11-membered bridged heterocyclic", "6- to 9-membered bridged heterocyclic "Cyclic", "6- to 10-membered bridged heterocyclic", "7- to 10-membered bridged heterocyclic", " "7- to 9-membered bridged heterocyclic", "7- to 8-membered bridged heterocyclic", "8-membered bridged heterocyclic "Cyclic", "9- to 10-membered bridged heterocyclic" and "3- to 10-membered bridged heterocyclic" are included. Specific examples include, but are not limited to,

Chemical formula

[0164] As used herein, the term "6- to 12-membered fused heterocyclic" refers to a cyclic structure containing 6 to 12 ring-forming atoms (at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom), formed by two or more cyclic structures sharing two adjacent atoms. Optionally, the ring-forming atoms (e.g., carbon atoms in the cyclic structure, nitrogen atoms or sulfur atoms) may be substituted with oxygen. "6- to 12-membered fused heterocyclic "Cyclic" includes, for example, "6- to 11-membered fused heterocyclic", "5- to 10-membered fused heterocyclic "Cyclic", "7- to 10-membered fused heterocyclic", "3- to 10-membered fused heterocyclic", "3 "To 10-membered nitrogen-containing fused heterocyclic", "9- to 10-membered fused heterocyclic", "9- to 1 "0-membered nitrogen-containing fused heterocyclic" and "6- to 12-membered oxygen-containing fused heterocyclic" are included . Specific examples include, but are not limited to, tetrahydroimidazo[4,5-c pyridyl, 3,4-dihydroquinazolinyl, 1,2-dihydroquinoxalinyl, benzo pyridyl, etc. [d][1,3]Dioxolyl, 1,3-dihydroisobenzofuranyl, 4H-1,3- Benzoxazinyl, 4,6-dihydro-1H-furo[3,4-d]imidazolyl, 3a ,4,6,6a-tetrahydro-1H-furo[3,4-d]imidazolyl, 4,6-dih dro-1H-thieno[3,4-d]imidazolyl, 4,6-dihydro-1H-pyrrolo[3 ,4-d]imidazolyl, benzimidazolidinyl, octahydro-benzo[d]imid azolyl, decahydroquinolyl, hexahydrothienoimidazolyl, hexahydrofuroimid azolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, octahyd rocyclopenta[c]pyrrolyl, dihydroindolyl, dihydroisoindolyl, be nzoxazolidinyl, benzothiazolidinyl, 1,2,3,4-tetrahydroisoqui nolyl, 1,2,3,4-tetrahydroquinolinyl and 4H-1,3-benzoxazi nyl are included.

[0165] As used herein, the term "aryl" refers to monocyclic or polycyclic hydrocarbo nyls having aromaticity, such as 6- to 20-membered aryl, 6- to 10- membered aryl, and 5- to 8-membered aryl. Specific examples include, but are not limited to, phenyl, naphthyl, anthra cenyl, and phenanthryl. "6- to 20-membered aryl" refers to aryl containing 6 to 20 ring-forming atoms.

[0166] As used herein, the term "heteroaryl" refers to a cyclic group having aromaticity in which at least one ring-forming atom is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring-forming atoms in the cyclic structure (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) The (substituent) may be replaced with oxygen. Specific examples include, but are not limited to, furyl , thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-tri zolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-ox adiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyrid yl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxacyclohexadien yl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxa dinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-ox azinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-tri azinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-dia zacycloheptatrienyl, and azacyclooctatetraenyl, etc., 5- to 10-membered heteroaryl, 5- to 10-membered nitrogen-containing heteroaryl, 6- to 10-membered oxygen-containing heteroaryl, 6- to 8-membered nitrogen-containing heteroaryl, and 5- to 8-membered oxygen-containing heteroaryl are included.

[0167] Advantageous Effects of the Invention The present disclosure provides certain novel bioactive molecule conjugates by improving the coupling method of the drug and the targeting moiety in an ADC or SMDC. In some embodiments of the present disclosure, the bioactive molecule conjugate is obtained by nucleophilic substitution of the heteroaryl ring on the ADC linker by the free sulfhydryl in the antibody. The conjugate obtained by this coupling has the following technical effects: At least one of the following can be achieved: (1) High stability; (2) High DAR, and in some embodiments, the DAR value of the conjugate can reach 5 - 8. ; (3) Extremely high coupling efficiency, and in some embodiments, the coupling efficiency can reach 90%. ; (4) The conjugate obtained by coupling can effectively improve the stability of the circulating drug and reduce the unexpected dissociation of the drug in non - target cells. ; (5) The conjugate can also increase the effective release of the bioactive molecule in cells, achieving the purpose of reducing toxicity and increasing efficacy. ; (6) The conjugate has good tumor tissue targeting; and (7) The conjugate has high efficacy against animal models of tumors.

[0168] In addition, the coupling method described in the present disclosure has a wide range of applications and can be widely used when coupling bioactive molecules with antibodies or targeted small - molecule ligands.

Brief Description of the Drawings

[0169]

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Figure 49A

Figure 49B

[0170] The present disclosure is further illustrated in combination with specific embodiments, but the present disclosure is not limited to those embodiments. Those skilled in the art should understand that various changes or improvements can be made in accordance with the teachings of the present disclosure without departing from the basic ideas and scope of the present disclosure.

[0171] The abbreviations in the present invention have the following meanings:

Table 2

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

[0173] Nuclear magnetic resonance ( 1 1H NMR) was determined by using a Bruker 400 MHz NMR spectrometer. Deuterated methanol (CD3OD), deuterated chloroform ( CDCl3), or deuterated dimethyl sulfoxide (DMSO-D6) was used as the solvent for the determination, and tetramethylsilane (TMS) was used as the internal standard substance. CDCl3), or deuterated dimethyl sulfoxide (DMSO-D6) was the solvent for the determination, and tetramethylsilane (TMS) was the internal standard substance.

[0174] The abbreviations of the nuclear magnetic resonance (NMR) spectra used in the examples are shown below. s: singlet, d: doublet, t: triplet, q: quartet, dd: doublet of doublets, qd: quartet doublet, ddd: doublet of doublet of doublets, ddt: doublet of doublet of triplets, dddd: doublet of doublet of doublet of doublets Heavy line, m: multiple line, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. The δ value was expressed in ppm. Hydrogenated dimethyl sulfoxide. The δ value was expressed in ppm.

[0175] The mass spectrum (MS) was determined using an Agilent (ESI) mass spectrometer (model: Agilent 6120B).

[0176] Preparative liquid chromatography: Method A: Chromatography column: Daisogel C18 10μm 100×25 0mm Mobile phase A: water; Mobile phase B: acetonitrile [Table 3]

[0177] Method B: Chromatography column: Daisogel C18 10μm 50×250 mm Mobile phase A: water; Mobile phase B: acetonitrile [Table 4]

[0178] Method C: Chromatography column: Daisogel C18 10μm 50×250 mm Mobile phase A: water containing 0.1% trifluoroacetic acid; Mobile phase B: acetonitrile [Table 5]

[0179] Method D: Chromatography column: Waters SunFire C18 5μm 1 9×250mm Mobile phase A: Acetonitrile; Mobile phase B: Water containing 0.05% formic acid Time: 0 minutes to 16 minutes; Mobile phase A: 10% to 90%; Flow rate: 28 mL / min

[0180] I. Synthesis of bioactive molecules Example 1: (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R, 2R)-3-((1-((4-Aminobenzyl)amino)-1-oxo-3-phenylprop yl)amino)-1-methoxy-2-methyl-3-propionyl)pyrrolid in-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-(( S)-2-(Dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyl amide (T001) synthesis

Chemical formula

[0181] Step 1: Synthesis of tert-butyl (4-((2-((2R,3R)-3-((S)-1 -((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3 -methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5methyl lheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamide )-3-phenylpropionamide)methyl)phenyl)carbamate At room temperature, 1-hydroxybenzotriazole (2.0 mg, 14.74 μmol) was dissolved in N, N-dimethylformamide (4 mL), cooled to 0 °C, and then tert-butyl 4-methylaminobenzylcarbamate (4.0 mg, 16.1 μmol), N,N-di isopropylethylamine (8.5 mg, 66.8 μmol), ((2R,3R)-3- ((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethyl amino)-3-methylbutanoyl)amino)-N,3-dimethylbutanoyl)amino)-3-meth oxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylprop ionyl)-L-phenylalanine (10.0 mg, 13.5 μmol, commercially available) was added sequentially. After stirring for 5 minutes, 1H-benzotriazol-1-yl-oxytripyr rolidinophosphonium hexafluorophosphate (10.0 mg, 20.1 μmol) was added thereto and stirred at 0 °C for 1 hour. The reaction of the starting materials was monitored by high performance liquid chromatography-mass spectrometry. After the starting materials were consumed, the reaction solution was purified by preparative liquid chromatography (Method D) to obtain the title compound (9.0 mg of white solid). ESI + .

[0182] Step 2: (2S)-N-((3R,4S,5S)-1-((2S)-2-((1 R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenyl propane-2-yl)amino)-1-methoxy-2-methyl-3-propionyl)pyr rolidin-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2- ((S)-2-(dimethylamino)-3-methylbutanoyl)amino)-N,3-dimethylbut anamide synthesis At room temperature, tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3 R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methyl butanoyl)amino)-N,3-dimethylbutanoyl)-3-methoxy-5-methylheptanoy ((S)-Pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-3-phenyl Propaneamide)methyl)phenyl)carbamate (9.0 mg, 0.02 mmol) was dissolved in 1,4-dioxane (0.5 mL), cooled to 0 °C, and then a solution of hydrogen chloride in dioxane (1 mL, 4.0 M) was added, and the mixture was reacted at room temperature for 3 hours with stirring. The reaction of the starting material was monitored by high performance liquid chromatography-mass spectrometry. After the starting material was consumed, the solvent was evaporated under reduced pressure, and the + crude product was purified by preparative liquid chromatography (Method

[0183] C) to obtain the trifluoroacetate salt of the title compound (5.0 mg of a white solid). ESI -MS (m / z): 850.5 [M+H] Example 2: (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R )-3-((S)-1-((4-aminobenzyl)amino)1-oxo-3-phenylpro pyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolid

Chemical formula

[0184] Step 1: Synthesis of tert-butyl (S)-(4-((2-(((((9H-fluoren- 9-yl)methoxy)carbonyl)amino)-3-phenylpropionamide)methyl) phenyl)lcarbamate At 0 °C, 4-aminobenzylamine (222 mg, 1.0 mmol) and N-methylmo L-holin (306 mg, 1.5 mmol) was placed in N,N-dimethylformamide (5 mL). to a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) -3-phenylpropionic acid (387 mg, 1.0 mmol), and then 1 -hydroxybenzotriazole (203 mg, 1.5 mmol) and 1-(3-dimethyl laminopropyl)-3-ethylcarbodiimide hydrochloride (288 mg, 1.5 m mol) were successively added. The resulting mixture was reacted at 0 °C overnight. The reaction solution was poured into water ( 50 mL), and a white solid precipitated. The solid was filtered, and the filter cake was washed with water (2 0 mL × 3). The solid was purified by silica gel column chromatography to obtain the title compound (380 mg of white solid). ESI-MS (m / z): 592.3 [M +H] + .

[0185] Step 2: Synthesis of tert-butyl (S)-(4-((2-amino-3-phenylprop onamide)methyl)phenyl)carbamate Lithium hydroxide monohydrate (21 mg, 0.51 mmol) was dissolved in water (1 mL), and t ert-butyl (S)-(4-((2-((((9H-fluoren-9-yl)methoxy ))carbonyl)amino)-3-phenylpropionamide)methyl)phenyl)carbama te (102 mg, 0.17 mmol) in tetrahydrofuran (2 mL) was added . The resulting mixture was reacted at room temperature for 2 hours. Water (20 mL) was added to the reaction solution, and acetic acid ethyl (30 mL × 4) was used for extraction. The organic phases were combined, washed with saturated saline (30 mL × 2) , and dried over anhydrous sodium sulfate. Then, the desiccant was removed by filtration, and the solvent Evaporate under reduced pressure and purify the residue by preparative liquid chromatography (Method D) to obtain the title compound (65 mg of white solid). ESI-MS (m / z): 370.2 [M + H] + .

[0186] Step 3: Synthesis of (4-((S)-2-((2R,3R)-3-((S)-1-((3R ,4S,5S)-4-((S)-3-((methylamino)-3-methylbutyrylamino) -N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrol idine-2-yl)-3-methoxy-2-methylpropionamide)-3-phenylprop onamide)methyl)phenyl)carbamate At 0 °C, tert-butyl (S)-(4-((2-amino-3-phenylpropionamido )methyl)phenyl)carbamate (15 mg, 0.04 mmol) and N-methyl morpholine (12 mg, 0.12 mmol) were added to a solution of (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2- in N,N-dimethylformamide (2 mL) ((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbut yrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl-3- methoxy-2-methylpropionic acid (24 mg, 0.04 mmol), and then 1-hydroxybenzotriazole (8 mg, 0.06 mmol) and 1-(3-dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (12 mg, 0.0 6 mmol) were successively added. The resulting mixture was reacted at 0 °C overnight. The reaction solution was purified by preparative liquid chromatography (Method D) to obtain the title compound (24 m of white solid g) was obtained. ESI-MS (m / z): 950.6 [M+H] + .

[0187] Step 4: (S)-N-((3R,4S,5S)-1-((S)-2-((1R, 2R)-3-((S)-1-((4-Aminobenzyl)amino)-1-oxo-3-phen ylpropyl)amino)-1-methoxy-2-methyl-3-oxopropyl)pi rolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)- 2-((S)-2-(Dimethylamino)-3-methylbutyrylamino)-N,3-dimethyl lbutylamide synthesis Trifluoroacetic acid (0.5 mL) was added to a solution of (4-((S) -2-((2R,3R)-3-((S)-1-(3R,4S,5S)-4-((S)-3 -(Methylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino) -3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2 -methylpropanamide)-3-phenylpropionamide)methyl)phenyl)carb amate (14.0 mg, 0.015 mmol). The resulting mixture was reacted at room temperature for 1 hour. Then, the solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography y (Method C) to obtain the trifluoroacetate salt of the title compound (4.2 mg, white solid). ESI-MS (m / z): 850.6 [M+H] + .

[0188] The following molecules can be synthesized by a similar synthetic method.

Table 6

[0189] Example 3: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione- 3,4,12,14-tetrahydro-1H-pyrano[3’,4’,6,7]indolo[1, 2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide

Chemical formula

[0190] Vortecan hydrochloride (1.0 g, 2.13 mmol) and triethylamine (0.65 g, 0.9 mL) were dissolved in dichloromethane (50 mL) at room temperature, and acetic anhydride (0.22 g, 2 .13 mmol) was slowly added dropwise. The resulting mixture was reacted at room temperature for 1 hour . The organic phase was washed with water (10 mL × 2) and dried over anhydrous sodium sulfate. Insoluble substances were removed by filtration, the solvent was evaporated, and the residue was purified by silica gel column chromatography (di chloromethane / methanol = 50 / 1) to obtain the title compound (1 g). ESI-MS (m / z): 476.2 [M+H] + .

[0191] Example 4: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione -3,4,12,14-tetrahydro-1H-pyrano[3’,4’,6,7]indolizidino [1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide

Chemical formula

[0192] Methylsulfonyl chloride (462 mg, 12.77 mmol, purity: about 70%) was added to dichloro ​A solution of verotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in romethane (40 mL) was added dropwise. The resulting mixture was reacted at room temperature for 2 hours. Suction filtration was performed, and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound (2.2 g). and the resulting mixture was reacted at room temperature for 2 hours. Suction filtration was performed, and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound (2.2 g). Suction filtration was carried out, and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound (2.2 g). to give the title compound (2.2 g).

[0193] The structure characteristic data are as follows. 1 H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 8.4 Hz, 1 H), 8.20 (dd, J = 8.4, 1.2 Hz, 1H), 7.93 - 7.84 (m, 1 H), 7.79 (t, J = 7.6 Hz, 1H), 7.35 (s, 1H), 6.56 (s, 1H), 5.44 (d, J = 9.2 Hz, 4H), 3.98 (p, J = 6.7 Hz, 1H ), 3.50 (t, J = 8.0 Hz, 2H), 3.42 - 3.35 (m, 2H), 3.0 0 (s, 3H), 1.93 - 1.82 (m, 2H), 1.15 (d, J = 6.7 Hz, 6 H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 512.2 [M + H] + . [α] D 20 is +28.19° (c = 0.101 g / 100 mL, CH3C N).

[0194] The remaining bioactive molecules for which the synthesis method is not described are either commercially available or can be prepared by the methods disclosed in the prior art. The remaining bioactive molecules for which the synthesis method is not described are either commercially available or can be prepared by the methods disclosed in the prior art.

[0195] II. Synthesis of Compounds Containing Bioactive Molecules and Linkers Example 5: (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3- d]Pyrimidin-7-yl)butyrylamide)-3-methylbutyrylamide)-N-(4 -(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4 -((S)-2-((S)-2-(Dimethylamino)-3-methylbutyrylamino)-N ,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine -2-yl)-3-methoxy-2-methylpropionamide)-3-phenylpropion amide)methyl)phenyl)-5-ureidovaleramide synthesis

Chemical Structure

[0196] Step 1: Synthesis of tert-butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyr imidin-7-yl)butyrate (Compound 1-2) At room temperature, Compound 1-1 (500 mg, 3.27 mmol) was dissolved in N,N-dimethylform amide (10 mL), and sodium hydride (130 mg, 3.27 mmol) was slowly added thereto in portions. The resulting mixture was stirred at room temperature for 10 minutes, and then tert-butyl 4-bromo butyrate (725 mg, 3.27 mmol) was added dropwise. Subsequently, the reaction was carried out at room temperature for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution and extracted with ethyl acetate (50 mL × 3). Then, the organic phases were combined, washed with saturated saline (50 mL × 3), dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the title compound (500 mg). ESI-MS (m / z): 296.1 [M + H + .

[0197] ​​​​Step 2: Synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (Compound 1-3) ) At room temperature, Compound 1-2 (500 mg, 1.69 mmol) was dissolved in dichloromethane (6 mL) , trifluoroacetic acid (3 mL) was added, and the mixture was reacted at room temperature for 4 hours. Then, the solvent was evaporated under reduced pressure to obtain the title compound (400 mg). ESI-MS (m / z): 240.1 [M+H] + .

[0198] Step 3: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)- 1-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino )-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxo butyl-2-yl)-carbamate (Compound 1-5) At room temperature, 4-(N-Boc-aminomethyl)-aniline (6.0 g, 27 mmol), Compound 1-4 (3.35 g, 6.75 mmol), and 2-ethoxyl-1-ethoxycarbonyl-1,2-dihydroquinoline (3.34 g, 13.5 mmol) were dissolved in a mixed solvent of dichloromethane (140 mL) and methanol (70 mL). Then, the temperature was raised to 45 °C and the mixture was reacted at that temperature for 8.0 hours. After cooling to room temperature, a large amount of solid precipitated, and it was subjected to suction filtration to obtain the title compound (3.65 g). ESI-MS (m / z): 70 1.4 [M+H] . + .

[0199] Step 4: (9H-fluoren-9-yl)-methyl-((S)-1-(((S) -1-((4-(aminomethyl)phenyl))amino)-1-oxo-5-5-ureido (Pentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate -Synthesis of (Compound 1-6) At room temperature, trifluoroacetic acid (15 mL) was added to Compound 1-5 (3.0 g, 4.29 mmol) and stirred at room temperature for 1.0 hour. Then, the solvent was evaporated under reduced pressure to obtain a yellow oil . The addition of anhydrous diethyl ether (20 mL) resulted in the precipitation of a large amount of solid. After vigorously stirring for 0. 5 hours, suction filtration was carried out to obtain the trifluoroacetate salt of the title compound (3. 06 g). ESI-MS (m / z): 601.3 [M+H] + .

[0200] Step 5: (9H-Fluoren-9-yl)-methyl-((S)-1-(((S) -1-((4-(((R)-2-((tert-butyloxycarbonyl)amino)-3-phen ylpropionamide)methyl)phenyl)amino-1-oxo-5-ureidopentyl -2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Comp ound 1-7) synthesis At room temperature, Boc-D-phenylalanine (1.1 g, 4.2 mmol) and the trifluoroacetate salt of Compound 1-6 (3.0 g, 4.2 mmol) were dissolved in N,N-dimethylformamide ( 40 mL), cooled to 0 °C, and then 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (1.2 g, 6.3 mmol), 1-hydroxybe nzotriazole (0.9 g, 6.3 mmol) and N-methylmorpholine (1.7 g, 16.8 mmol) were added successively. The reaction system was stirred at that temperature for 1.0 hour. Next ly, the reaction solution was added dropwise to ice water (400 mL) and vigorously stirred for 0.5 hour, resulting in a large amount of The solid precipitated. Suction filtration was carried out to obtain the title compound (3.3 g). ESI-MS (m / z): 848.4 [M+H] + .

[0201] Step 6: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S) -1-((4-(((R)-2-amino-3-phenylpropionamide)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl -1-oxobutyl-2-yl)-carbamate (Compound 1-8) At room temperature, Compound 1-7 (3.0 g, 3.3 mmol) was dissolved in trifluoroacetic acid (30 mL) and stirred at room temperature for 1.0 h. The solvent was evaporated under reduced pressure to give a yellow oil. Diethyl ether (100 mL) was added and stirred vigorously for 0.5 h, then a large amount of solid precipitated. Suction filtration was carried out to obtain the trifluoroacetate salt of the title compound (2.1 g). ESI-MS (m / z): 748.4 [M+H] . + .

[0202] Step 7: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S) -1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S ,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamide )-N,3-dimethylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl )-3-methoxy-2-methylpropionamide)-3-phenylpropionamide) methyl)phenyl)amino)-1-oxo-5-ureidopenta-2-yl)amino)- 3-methyl-1-oxobutane-2-yl carbamate (Compound 1-9) (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)- 2-((S)-(dimethylamino)-3-butyrylamino)-N,3-dimethylbutyryl amino)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy- 2-methylpropionic acid (1.3 g, 2.17 mmol) and trifluoro acetic acid salt of compound 1-8 (1.8 g, 2.17 mmol) were dissolved in N,N-dimethylformamide (20 mL) and cooled to 0 °C, then 1-hydroxybenzotriazole (440 mg, 3. 26 mmol) and N-methylmorpholine (658 mg, 6.51 mmol) were added successively and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro chloride (624 mg, 1.38 mmol) was added. The reaction solution was stirred at 0 °C for 5 hours and purified by preparative liquid chromatography (Method D) to obtain the title compound (1.8 g) ESI-MS (m / z): 1329.2 [M+H] + .

[0203] Step 8: Synthesis of (S)-2-((S)-2-amino-3-butyrylamino)-N-(4 -(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4 -((S)-2-((S)-2-(dimethylamino)-3-butyrylamino)-N,3- dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl) -3-methoxy-2-methylpropionamide)-3-phenylpropionamide)methyl phenyl)-5-ureidovaleramide (Compound 1-10) At room temperature, compound 1-9 (500 mg, 0.38 mmol) was dissolved in N,N-dimethylform Dissolved in MeOH (5 mL), piperidine (324 mg, 3.8 mmol) was added, and the mixture was stirred at room temperature for 3 h. Then purification was performed by preparative liquid chromatography (Method D) to obtain the title compound (350 mg). ESI-MS (m / z): 1107.2 [M+H] + .

[0204] Step 9: Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2, 3-d]pyrimidin-7-yl)butyrylamido)-3-methylbutyrylamido)-N- (4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S) -4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino) -N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrol idin-2-yl)-3-methoxy-2-methylpropionamide)-3-phenylprop onamide)methyl)phenyl)-5-ureidovaleramide (Compound TL001) At room temperature, Compound 1-10 (60 mg, 0.054 mmol) and 4-(4-chloro-7H -pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (26 mg, 0.066 mmol) were dissolved in N,N-dimethylformamide (3 mL), cooled to 0 °C, and N,N-diisop ropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazole- 1-oxytripyrrolidinophosphonium hexafluorophosphate (281 mg, 0. 54 mmol) were added successively. The reaction mixture was stirred at room temperature for 3 h. Then purification was performed by preparative liquid chromatography (Method D) to obtain the title compound (30 mg). ESI-M S (m / z): 664.5 [M / 2+H] . + .

[0205] Example 6: (S)-N-(4-(((S)-2-((2R,3R)-3-((S) -1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino) -3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-meth thylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionam ido)-3-phenylpropionamide)methyl)phenyl)-2-((S)-3-methyl -2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7 -yl)-butyrylamido)-butyrylamido)-5-ureidovaleramide [Chemical formula]

[0206] Step 1: Synthesis of 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)butyric acid (Compound 2-2) At room temperature, 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (300 mg, 1.25 mmol) was dissolved in methanol (8 mL), and sodium methan ethanethiol (351 mg, 5.02 mmol) was added all at once, then heated to 50 °C, and reacted overnight. Purification was carried out by preparative liquid chromatography (Method D) to obtain the title compound (1 20 mg). ESI-MS (m / z): 252.1 [M+H] + .

[0207] Step 2: (S)-N-(4-(((S)-2-((2R,3R)-3-((S) -1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino) -3-Methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5 -Methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropion Amide)-3-phenylpropionamide)methyl)phenyl)-2-((S)-3-me thyl-2-(4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine- 7-yl)-butyrylamide)-butyrylamide)-5-ureidovaleramide (Compound 2-3) synthesis 4-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)butyric acid was 4-( 4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)butyric acid was replaced except that, the same operations as described in Step 9 of Example 5 were carried out. Purification was performed using preparative liquid chromatography (Method D) to obtain the title compound (20 mg). ESI-MS (m / z): 670.5 [M / 2 + H] + .

[0208] Step 3: (S)-N-(4-(((S)-2-((2R,3R)-3-((S) -1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino) -3-Methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5 -Methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropion Amide)-3-phenylpropionamide)methyl)phenyl)-2-((S)-3-me thyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidine -7-yl)-butyrylamide)-butyrylamide)-5-ureidovaleramide (Compound TL002) synthesis At room temperature, compound 2-3 (20 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL) and m-chloroperoxybenzoic acid (4.0 mg, 0.022 mmol) was added . The resulting mixture was reacted at room temperature for 2 hours. Purification was carried out by preparative liquid chromatography (method D) to obtain the title compound (5.0 mg). ESI-MS (m / z): 686.5 [M / 2 + H] + .

[0209] Example 7: N-((S)-1-(((S)-1-((4-(((S)-2-((2R, 3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2 -(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino )-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy- 2-methylpropionamido-3-phenylpropionamide)methyl)phenyl)amino no)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutane -2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexyne amide

Chemical Structure

[0210] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexy noate (Compound 3-2) At room temperature, methyl 5-hexynoate (500 mg, 3.97 mmol) and 5-bromo -2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 ml), and then triethylamine (3 ml), copper(I) iodide (75 mg, 0.4 mmol) and bis(tri ​Phenylphosphine)palladium(II) dichloride (279 mg, 0.4 mmol) was added continuously. The resulting mixture was heated to 95 °C under nitrogen protection and reacted for 6 hours with stirring , quenched with water, and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated physiological saline (20 mL × 2), and dried over anhydrous sodium sulfate. The drying agent was removed by filtration , and the solvent was evaporated under reduced pressure. Purification was carried out by preparative liquid chromatography (Method D) to obtain the title compound (300 mg). ESI-MS (m / z): 251.3 [M+H + .

[0211] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid ( Compound 3-3) At room temperature, Compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixed solution of tetrahydrofuran and water ( 4 mL / 4 mL), lithium hydroxide monohydrate (235 mg, 5.6 m mol) was added, and the reaction was carried out at room temperature for 4 hours with stirring. Then, it was diluted with water and extracted with ethyl acetate ( 20 ml × 2). The aqueous phase was adjusted to pH = 3 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL × 3). Then, the organic phases were combined, washed with saturated physiological saline (20 mL × 2), dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the title compound (120 mg).

[0212] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexy noic acid (Compound 3-4) At room temperature, Compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL) , m-chloroperoxybenzoic acid (22 mg, 0.127 mmol) was added,​ The reaction was allowed to proceed overnight at room temperature with stirring. Purification was carried out by preparative liquid chromatography (Method D) to give the title compound (20 mg). ESI-MS (m / z): 269.1 [M+H] + .

[0213] Step 4: N-((S)-1-(((S)-1-((4-(((S)-2-((2 R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S) -2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyryla mino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy -2-methylpropionamide)-3-phenylpropionamide)methyl)phenyl )amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxy butan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-he xynamide (Compound TL003) 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid was replaced with 6-( 2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid, and the procedure described in Step 9 of Example 5 was carried out in the same manner. Purification was carried out by preparative liquid chro matography (Method D) to give the title compound (14 m). ESI-MS (m / z): 679.0 [M / 2+H] (m / z): 679.0 [M / 2+H] + .

[0214] Example 8: (S)-4-Ethyl-11-(2-(N-isopropylmethylsulfona mido)-ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyra no[3’,4’,6,7]-indolizino[1,2-b]-quinolin-4-yl (4-( (S)-42-(2-(Methylsulfonyl)pyrimidin-5-yl)-4,8,37-tri oxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27, 30,33-nonoxy-3,9,36-azatetracosan-41-amide)benzyl) carbonate

Chemical formula

[0215] Step 1: Synthesis of Methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino )-1-oxo-5-ureidopentan-2-yl)-(9H-fluorenyl)carbamate (Compound 19-2) At room temperature, Fmoc-L-citrulline (5.0 g, 12.58 mmol), p-aminoben zyl alcohol (6.20 g, 50.32 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (6.22 g, 25.16 mmol) were dissolved in dichloro methane (100 mL), heated to 45 °C, and reacted for 6 hours. The reaction solution was concentrated under reduced pressure and stirred with anhydrous diethyl ether (100 mL) to obtain the title compound (6.0 g). ESI-MS (m / z): 503.3 [M+H] +

[0216] Step 2: Synthesis of (S)-2-Amino-N-(4-(hydroxymethyl)phenyl)-5 -ureidovaleramide (Compound 19-3) At room temperature, Compound 19-2 (1.0 g, 1.99 mmol) was dissolved in N,N-dimethylformamide (8 mL), and piperidine (339 mg, 3.98 mmol) was added dropwise at room temperature for 30 minutes to react. Then dichloromethane (10 mL) was added, followed by 10 minutes ​​​​It was stirred. The reaction solution was concentrated under reduced pressure and purified by flash column chromatography to obtain the title compound (400 mg). ESI-MS (m / z): 281.2 [M + H] + .

[0217] Step 3: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18 ,21,24,27,30-nonyloxa-6-azatriacetamide)-N-(4-( hydroxymethyl)phenyl)-5-ureidovaleramide (Compound 19-4) Compound 19-3 (150 mg, 0.54 mmol) and 32-azido-5-oxo-3 ,9,12,15,18,21,24,27,30-nonoxy-6-azatricycloundecane acid (296 mg, 0.54 mmol) were dissolved in dichloromethane (10 mL), cooled to 0 °C, and then 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinone phosphine (145 mg, 0.58 mmol) was added. The resulting mixture was brought to room temperature and reacted overnight. The reaction solution was concentrated under reduced pressure and purified by flash column chromatography to obtain the title compound (200 mg). ESI-MS (m / z): 817.5 [M + H] + .

[0218] Step 4: 4-((S)-35-azido-4,8-dioxo-2-(3-ureido propyl)-6,12,15,18,21,24,27,30,33-nonoxy-3, 9-azatetracosan)benzyl ((S)-4-ethyl-11-(2-(N-isopropyl methylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’,6,7]indolizino[1,2-b]quinoline- ​ Synthesis of (S)-N-(2-(4-Ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4’,6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (Compound 19-5) At room temperature, (S)-N-(2-(4-Ethyl-4-hydroxy-3,14-dioxo-3,4 ,12,14-tetrahydro-1H-pyrano[3’,4’,6,7]indolizino[1, 2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (2 00 mg, 0.39 mmol) was dissolved in dichloromethane (10 mL), cooled to 0 °C, and a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL) was added, followed by slow dropwise addition of a solution of triphosgene (1 16 mg, 0.39 mmol) in dichloromethane (1.0 mL). The resulting mixture was stirred at 0 °C for 1 hour. A solution of Compound 19-4 (15 9 mg, 0.18 mmol) in dichloromethane (2.0 mL) was added to the reaction solution, and the reaction was carried out at room temperature for 1 hour. Purification was performed by preparative high-performance liquid chromatography (Method D) to obtain the title compound (160 mg) . ESI-MS (m / z): 678.0 [M / 2 + H] + .

[0219] Step 5: Synthesis of 4-((S)-35-Amino-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonoxo-3, 9-azatetracosan)benzyl ((S)-4-Ethyl-11-(2-(N-isopropyl methylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetra hydro-1H-pyrano[3’,4’,6,7]indolizino[1,2-b]quinolin- 4-yl)carbonate (Compound 19-6) At room temperature, Compound 19-5 (80 mg, 0.059 mmol) was dissolved in tetrahydrofuran (1. 0 mL) Dissolved in 0 ml), cooled to 0 °C, and then 4-dimethyl A solution of (573 mg, 4.69 mmol) of thylaminopyridine was added, and platinum dioxide (1 5 mg, 0.059 mmol) was added in one batch under nitrogen protection, and then the air was replaced with hydrogen And replaced three times, and reacted at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product This was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (40 mg). ESI-MS (m / z): 665.0 [M / 2 + H] + .

[0220] Step 6: (S)-4-Ethyl-11-(2-(N-isopropylmethylsulfone Amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pi rano[3’,4’,6,7]indolizino[1,2-b]quinolin-4-yl(4-(( (S)-42-(2-(Methylsulfonyl)pyrimidin-5-yl)-4,8,37-tri Oxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,3 0,33-nonoxy-3,9,36-azatetracosan-41-amide)benzyl)car bonate (Compound TL019) synthesis Compound 19-6 (30 mg, 0.016 mmol) and 6-(2-methylsulfonylpyr imidine-5-yl)-5-hexynoic acid (6.4 mg, 0.024 mmol) were dissolved in N,N- dimethylformamide (1 mL), cooled to 0 °C, and then benzotriazole- 1-yl-oxytripyrrolidinyl hexafluorophosphate (16.5 mg, 0.0 32 mmol), N,N-diisopropylethylamine (6.2 mg, 0.047 mmo (l) was added continuously. The resulting mixture was reacted at room temperature for 2 hours. Purification was carried out by preparative high performance liquid chromatography (Method D) to obtain the title compound (10 mg). ESI-MS (m / z): 790.0 [M / 2 + H] (m / z): 790.0 [M / 2 + H] + .

[0221] Example 9: (S)-4-Ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3’,4’,6,7]indolizino[1,2-b]quinolin-4-yl-(4-((S )-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5 -yl))-5-hexynamido)butylamido-5-ureidovalerylamido)benzyl l) carbonate l) carbonate

Chemical formula

[0222] Step 1: Methyl ((S)-1-(((S)-1-((4-(hydroxymethyl) phenyl)amino)-1-oxo-5-ureidovaleryl amido-2-yl)amino)) -3-methyl-butyl amido-2-yl)-(9H-fluorenyl)carbamate A compound of formula 19-1 was replaced with a compound of formula 28-1, and the procedure described in Step 1 of Example 8 was carried out to obtain the title compound (310 mg). ESI-MS( m / z): 602.3 [M + H] m / z): 602.3 [M + H] + .

[0223] Step 2: Synthesis of (S)-2-((S)-2-amino-3-methylbutylamido)-N- (4-(hydroxymethyl)phenyl)-5-ureidovaleramide (Compound 28-2) of (S)-2-((S)-2-amino-3-methylbutylamido)-N- The same operations as described in Step 2 of Example 8 were carried out except that Compound 19-2 was replaced with Compound 28-2 to obtain the title compound (150 mg). ESI-MS( m / z): 380.3 [M+H] + .

[0224] Step 3: Synthesis of N-((S)-1-(((S)-1-((4-Hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl- 1-oxobutan-2-yl)-6-(2-(Methylsulfonyl)pyrimidin-5-yl )-5-hexynamide (Compound 28-4) At room temperature, benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate (313 mg, 0.6 mmol) and N,N-diisopropylethylamine (19 4 mg, 1.50 mmol) were added to a solution of 6-(2 -Methylsulfonylpyrimidin-5-yl)-5-hexynoic acid (135 mg, 0.5 mmol) and (2S)-2-(((2S)-2-amino-3-methyl-butyryl)amino) -N-(4-(Hydroxymethyl)phenyl)-5-ureido-valeramide (190 mg, 0.5 mmol) in N,N-dimethylformamide (10 mL), and the mixture was reacted at room temperature for 3 hours with stirring. The reaction solution was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (78 mg). ESI-MS (m / z): 630.3 [M+H] + .

[0225] Step 4: (S)-4-Ethyl-11-(2-(N-Isopropylmethanesulfon amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pi ​​​​​​​Rano[3’,4’,6,7]indolizino[1,2-b]quinolin-4-yl-(4-( (S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidine -5-yl))-5-hexynamide)butylamide-5-ureidovalerylamide)be nzyl)carbonate (Compound TL028) synthesis Except for replacing Compound 19-4 with Compound 28-4, in Step 4 of Example 8 the same operations as described were carried out to obtain the title compound (1.76 mg). ESI-MS (m / z): 1167.4 [M+H] + .

[0226] Example 10: (S)-4-Ethyl-11-(2-(N-isopropylmethylsulfona mide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyra no[3’,4’,6,7]indolizino[1,2-b]quinolin-4-yl-(4-(( (2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)py rimidine-5-yl)-5-hexynamide)methyl)-1H-1,2,3-triazoe le-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15 ,18,21,24,27,30,33,36-nonoxo-3,6,12-triazot riacontylamide)benzyl)carbonate

Chemical Structure

[0227] Step 1: (S)-2-((S)-35-azido-2-isopropyl-4,8-di oxo-6,12,15,18,21,24,27,30,33-nonoxo-3,9- Azatetracosyl)-N-(4-(hydroxymethyl)phenyl)-5-ureidoval Synthesis of amide (Compound 29-1) Except for replacing Compound 19-3 with Compound 28-3, in Step 3 of Example 8 The same operations as described were carried out to obtain the title compound (180 mg). ESI-MS( m / z): 916.5 [M+H] + .

[0228] Step 2: 4-((2S,5S)-38-azido-5-isopropyl-4,7,1 1-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27, 30,33,36-nonoxy-3,6,12-triazatriacontylamide)benz yl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)eth yl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’, 4’,6,7]indolizino[1,2-b]quinolin-4-yl) carbonate (Compound 29-2) synthesis Except for replacing Compound 19-4 with Compound 29-1, in Step 4 of Example 8 The same operations as described were carried out to obtain the title compound (30 mg). ESI-MS (m / z): 727.5 [M / 2+H] + .

[0229] Step 3: (S)-4-ethyl-11-(2-(N-isopropylmethylsulfon amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pi rano[3’,4’,6,7]indolizino[1,2-b]quinolin-4-yl-(4-( (2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl) (pyrimidin-5-yl)-5-hexynamide)methyl)-1H-1,2,3-triazolo -l-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,1 5,18,21,24,27,30,33,36-nonoxy-3,6,12-triazolo triacontylamide)benzyl)carbonate (Compound TL029) Synthesis At room temperature, Compound 29-2 (20 mg, 0.014 mmol) and 6-(2-(methyls ulfonyl)pyrimidin-5-yl)-N-(2-propyn-1-yl)-5-hexynamide (4.3 mg, 0.014 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water ( 1 mL / 0.25 mL), then copper(II) bromide (3.95 mg, 0.027 mmol ) was added and the reaction was carried out with stirring for 1 hour. Purification was performed by preparative high performance liquid chromatography (Method D ) to obtain the title compound (15 mg). ESI-MS (m / z): 880.0 [M / 2 + H] + .

[0230] Example 11: (S)-4-Ethyl-11-(2-(N-isopropylmethanesulfona mide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyr ano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-((2 (S,5S)-5-isopropyl-45-(2-(methylsulfonyl)pyrimidin-5-yl )-4,7,11,40-tetraoxo-2-(3-ureidopropyl)-9,15, 18,21,24,27,30,33,36-nonoxy-3,6,12,39-tetra azapentatetracontane-44-carbamoyl)benzyl)carbonate

Chemical Structure

[0231] Step 1: Synthesis of (S)-2-((S)-35-amino-2-isopropyl-4,8-di oxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9- diazapentatriacontamide)-N-(4-(hydroxymethyl)phenyl)-5- ureidovaleramide At 20 °C, compound 29-1 (400 mg, 0.44 mmol) was dissolved in methanol and tetrahydro furan (2.0 mL:4.0 mL). After complete dissolution, platinum dioxide (40 m g) was added in one portion under nitrogen protection, and then the mixed solution was flushed with hydrogen three times. Hydrogenation was carried out at 20 °C for 2 hours. The reaction solution was filtered. The filter cake was washed with methanol. The filtrate was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (200 mg). ESI-MS (m / z): 890.4 [M+H + .

[0232] Step 2: Synthesis of N-((6S,9S)-1-amino-6-((4-(hydroxymethyl phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo- 13,19,22,25,28,31,34,37,40-nonoxy-2,7,10, 16-tetraazadotetracont-42-yl)-6-(2-(methylsulfonyl)pyr imidin-5-yl)hex-5-ynylamide At 20 °C, compound 22-1 (250 mg, 0.28 mmol) was dissolved in N,N-dimethylform amide (1.0 mL), and then HATU (160 mg, 0.42 mmol) and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were successively added ​​​Added, followed by stirring overnight at room temperature. Purification was carried out by preparative high performance liquid chromatography (Method D) to obtain the title compound (250 mg).

[0233] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfon amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-py rano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-(( (2S,5S)-5-isopropyl-45-(2-(methylsulfonyl)pyrimidin-5- yl)-4,7,11,40-tetraoxo-2-(3-ureidopropyl)-9,15 ,18,21,24,27,30,33,36-nonaoxy-3,6,12,39-tetra aza pentatetracontane-44-carbamoyl)benzyl)carbonate (Compound T L022) At 20 °C, (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3, 4,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1 ,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide ( 70 mg, 0.14 mmol) was dissolved in dichloromethane (4.0 mL), cooled to 0 °C , then a solution of p-dimethylaminopyridine (200 mg , 1.64 mmol) in dichloromethane (1.0 ml) was added, and triphosgene (40.6 mg, 0.14 mmol) in dichloromethane (1.0 ml) was slowly added dropwise. The resulting mixture was reacted at 0 °C for 1 hour with stirring. Nitrogen was bubbled through the unreacted triphosgene, and a solution of compound 22-2 (139 mg, 0.12 mmol) in dichloromethane (2.0 mL) was added to the reaction ​It was added to the solution and reacted at 0 °C for 1 hour with stirring. Purification was carried out by preparative high performance liquid chromatography —(Method D) to obtain the title compound (1.5 mg). ESI-MS (m / z): 83 9.5 [M / 2 + H] + 。

[0234] Example 12: 4-((S)-2-(4-Aminobutyl)-42-(2-(methylsulf onyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,2 1,24,27,30,33-nonaoxa-3,9,36-triazatetratetracontanyl -41-alkynamide)benzyl-((S)-4-ethyl-11-(2-(N-isop ropylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetra hydro-1H-pyrone[3’,4’:6,7]indolizino[1,2-b]quinolin-4 -yl)carbonate

Chemical formula

[0235] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulf onamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pi rone[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-(( S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-4 2-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo- 6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-t riazatetratetracontanyl-41-alkynamide)benzyl carbonate 6-(-2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-yn-1-ol at room temperature The acid (12 mg, 0.045 mmol) was dissolved in dichloromethane (2 mL), and then 2 -(7-Azabenzotriazole)-N,N,N’,N’-tetramethylurea hexafluor ophosphate (21.2 mg, 0.056 mmol) and N,N-diisopropyle thylamine (8.6 mg, 0.067 mmol) were added, and the mixture was stirred for 10 minutes. Compound 24 -1 (35 mg, 0.022 mmol) was added, and the reaction was carried out with stirring for 1 hour. Purification was performed by preparative high-performance liquid chromatography (Method B) to obtain the title compound (20 mg). ESI -MS (m / z): 1821.8 [M+H] + .

[0236] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-42-(2-(methylsulf onyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18, 21,24,27,30,33-nonaoxa-3,9,36-triazadotetratetracont yl-41-alkynamide)benzyl-((S)-4-ethyl-11-(2-(N-iso propylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetra hydro-1H-pyrrolo[3’,4’:6,7]indolizino[1,2-b]quinolin- 4-yl)carbonate (Compound TL024) At room temperature, compound 24-2 (20 mg, 0.011 mmol) was dissolved in acetonitrile (1 mL ), and a solution of trifluoroacetic acid (0.5 m l) in acetonitrile (0.5 ml) was added dropwise over 20 minutes, and the mixture was stirred. Purification was performed by preparative high-performance liquid chromatography (Method C) to obtain the trifluoroacetate salt (12 mg) of the title compound. ESI-MS (m / z) : 1549.6 [M+H] + 。

[0237] Example 13: (S)-4-Ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrido[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl-4-((2 (S,5S)-5-isopropyl-2-methyl-38-(4-((6-(methylsulfonyl)pyrimidin-5-yl)yl)hex-5-enamide)methyl)-1H-1, 2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21 ,24,27,30,33,36-nonaoxo-3,6,12-triazatriacontaoic amide)benzyl)carbonate synthesis

Chemical Structure

[0238] Step 1: Preparation of (S)-(9H-fluoren-9-yl)-methyl(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)carbamate At room temperature, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.31 g, 5.30 mmol) and p-aminobenzyl alcohol (593 mg, 4.82 mmol) were added to a solution of compound 30-1 (1.5 g, 4.82 mmol) in dichloromethane (35 mL), and the mixture was reacted with stirring for 3 hours. Purification was performed by silica gel column chromatography to obtain the title compound (1.8 g). ESI-MS (m / z): 417.2 M+H] +

[0239] ​​​​​​​ Step 2: Preparation of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)pro pionamide At room temperature, ethylenediamine (5 mL) was added to a solution of compound 30- 2 (1.8 g, 4.32 mmol) in dichloromethane (20 mL), and the mixture was reacted for 2 hours. Purification was carried out by silica gel column chromatography to obtain the title compound (820 mg). ESI-MS( m / z): 195.1 [M+H]+

[0240] Step 3: Preparation of (9H-fluoren-9-yl)-methyl ((S)-1-(((S)- 1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl )amino)-3-methyl-1-oxobutan-2-yl)-carbamate At room temperature, (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)- 3-methyl-butyric acid (875 mg, 2.58 mmol), O-benzotriazol-1-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (1.45 g, 3.83 mmol), N, N-diisopropylethylamine (1.00 g, 7.74 mmol) and 1-hydroxy benzotriazole (525 mg, 3.89 mmol) were successively added to a solution of compound 30-3 (503 mg, 2.58 mmol) in dichloromethane (2 mL), and the mixture was reacted with stirring for 4 hours. Purification was carried out by silica gel column chromatography to obtain the title compound (1.1 g). ESI-MS (m / z): 516.2 [M+H]+ g) was obtained. ESI-MS (m / z): 516.2 [M+H]+

[0241] Step 4: Preparation of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl )phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide At room temperature, ethylenediamine (2 mL) was added to a solution of compound 30-4 (1.1 g, 2.13 mmol) in dichloromethane (8 mL), and the mixture was reacted with stirring for 1 hour. Purification was carried out by silica gel column chromatography to obtain the title compound (610 mg). ESI-M S (m / z): 294.2 [M+H] +

[0242] Step 5: Preparation of (S)-2-(32-azido-5-oxo-3,9,12,15,18 ,21,24,27,30-nonaoxy-6-diazapentatriacontamide)-N- ((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan -2-yl)-3-methylbutylamide At room temperature, O-benzotriazolyl-tetramethyluronium hexafluorophosphate (160 mg, 0.42 mmol), 1-hydroxybenzotriazole (57 mg, 0.42 mmol), N,N-diisopropylethylamine (109 mg, 0.84 mm ol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27, 30-nonaoxy-6-azatricyclodecane-1-carboxylic acid (156 mg, 0.28 mmol ) were added to a solution of compound 30-5 (84 mg, 0.28 mmol) in dichloromethane (3 mL), and the mixture was reacted with stirring for 4 hours. Purification was carried out by silica gel column chromatography to obtain the title compound (163 mg). ESI-MS (m / z): 830.4 [M+H] +

[0243] Step 6: 4-((2S,5S)-38azido-5-isopropyl-2-methyl- 4,7,11-trioxo-9,15,18,21,24,27,30,33,36-non ​​(S)-4-ethyl-3,6,12-triazatriacontamino)benzyl -11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-diamino Xo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]India Preparation of lysino[1,2-b]quinolin-4-yl) carbonate Under nitrogen protection, triphosgene (16 mg, 0. A solution of 0.05 mmol) of 4-dimethylaminopyridine in dichloromethane (0.7 mL) (65 mg, 0.53 mmol) and (S)-N-(2-(4-ethyl-4-hydroxy -3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4' :6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropyl Pyrmethansulfonamide (45 mg, 0.09 mmol) was added dropwise to the mixture, and 0 The reaction was allowed to proceed at 5° C. for 1 hour. Compound 30-6 (73 ml) in dichloromethane (1 mL) was then added. g, 0.09 mmol) was added dropwise to the reaction solution and reacted at 0° C. for 1 hour. The title compound (33 mg) was obtained by silica gel column chromatography. -MS(m / z): 1367.6 [M + H] +

[0244] Step 7: (S)-4-Ethyl-11-(2-(N-isopropyl methyl sulfone) Amido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyridin lano[3',4':6,7 indolizino[1,2-b]quinolin-4-yl-4-((2 S,5S)-5-isopropyl-2-methyl-38-(4-((6-(2-(methylsulfonyl) pyrimidin-5-yl)yl)hex-5-ynamido)methyl)-1H-1, (2,3-Triazol-1-yl)-4,7,11-trioxo-9,15,18,21 ,24,27,30,33,36-nonoxa-3,6,12-triazatriacontao zanamide)benzyl carbonate (Compound TL030) Preparation At room temperature, copper bromide (5 mg, 0.04 mmol) and Compound 30-7 (20 mg, 15 u mol) were added dropwise to a solution of 6- (2-(Methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl l)-hexa-5-ynylamide (9 mg, 0.007 mmol) in water and N,N-dimethylformamide (0.2 ml:0.8 ml), and the reaction was carried out with stirring for 4 hours. Purification was performed by preparative high performance liquid chromatography (Method D) to obtain the title compound (4.15 mg). ESI-MS (m / z): 1672.7 [M+H] +

[0245] Example 14: 4-((S)-2-(4-Aminobutyl)-35-(4-((6-(2- (Methylsulfonyl)pyrimidin-5-yl)hexa-5-ynamide)methyl)-1H -1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18, 21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamide )benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonam do)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate [Chemical Structure]

[0246] Step 1: 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(pro Synthesis of (p-2-yn-1-yl)hex-5-ynamide At 25°C, prop-2-ynyl-1-amine (189 mg, 3.4 mmol) and compound Compound 3-4 (800 mg, 2.83 mmol) was dissolved in dichloromethane (10 mL) and then N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-( 7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) was added continuously while stirring. The reaction was allowed to proceed for 2 hours under stirring. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash silica gel column chromatography. The title compound was purified by chromatography (ethyl acetate / petroleum ether=3 / 1). The product (700 mg) was obtained. ESI-MS (m / z): 306.1 [M+H] + .

[0247] Step 2: 4-((S)-35-azido-2-(4-(((4-methoxyphenyl )Benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21 ,24,27,30,33-Non-azo-3,9-diazapentatriacanthoamino)benzene Di((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl) ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2, 3-b]-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline- Synthesis of 4-yl)carbonates Under nitrogen protection, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane at 25 °C. (10 mL), cooled to 0° C., and then dissolved in 4-dimethylformamide (3 mL) in dichloromethane (10 mL). A solution of l-aminopyridine (478 mg, 3.91 mmol) was added, followed by a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) dropwise added slowly, and the reaction was carried out at 0 °C for 20 minutes with stirring. Nitrogen was bubbled through the reaction solution for 20 minutes, and then a solution of (S)-2-(32-azido-5-oxo-3, 9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)benz hydryl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added, and the reaction was carried out at 0 °C for 1 hour with stirring. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Method A) to obtain the title compound (500 mg). ESI-MS (m / z): 1597.5 [M+H] + .

[0248] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfon amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyr rano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-(( (S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)- 35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexa-5- ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo -6,12,15,18,21,24,27,30,33-nonaoxy-3,9-dia zapentatriacontamide)benzyl)carbonate At room temperature, compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and​​​​​ Dissolved in water (2.0 mL: 0.5 mL), and subsequently, copper(II) bromide (11 mg, 0.08 mmol ) was added, and the reaction was carried out with stirring for 1 hour. Purification was performed by preparative high-performance liquid chromatography (Method B ) to obtain the title compound (30 mg). ESI-MS (m / z): 815.9 [(M - 273) / 2 + H] + .

[0249] Step 4: Synthesis of 4 - ((S)-2-(4 - aminobutyl)-35-(4 - ((6-(2 -(methylsulfonyl)pyrimidin - 5 - yl)hexa - 5 - inamide)methyl)-1 H - 1,2,3 - triazol - 1 - yl)-4,8 - dioxo - 6,12,15,18 ,21,24,27,30,33 - nonaoxa - 3,9 - diazapentatriacontamide d)benzyl((S)-4 - ethyl - 11-(2-(N - isopropylmethylsulfonam ide)ethyl)-3,14 - dioxo - 3,4,12,14 - tetrahydro - 1H - pyr ano[3’,4’:6,7]indolizino[1,2 - b]quinolin - 4 - yl)carbonate (Compound TL033) Compound 33 - 2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) , and trifluoroacetic acid (0.2 mL) was added to the reaction solution, and the reaction was carried out at room temperature for 30 minutes . Purification was performed by preparative high-performance liquid chromatography (Method C) to obtain the trifluoro acetate (20.0 mg) of the title compound. The identification of the title compound is as follows 1 1H NMR (400 MHz, DMSO - d6) δ 10.18 (s, 1H), 9.10 (s, 2H), 8.38 (t, J = 5.56 Hz, 1H), 8.32 (d, J = 8.40 Hz, 1H), 8.22 - 8.20 (m, 2H), 8.09 (t, J = 5.68 Hz, 1 H), 7.91 - 7.87 (m, 2H), 7.82 - 7.78 (m, 1H), 7.69( brs, 3H), 7.61 (d, J = 8.56 Hz, 2H), 7.32 (d, J = 8.5 6 Hz, 2H), 7.06 (s, 1H), 5.56 (d, J = 16.96 Hz, 1H), 5.51 (d, J = 16.96 Hz, 1H), 5.47 (d, J = 19.28 Hz, 1H ), 5.42 (d, J = 19.28 Hz, 1H), 5.14 (d, J = 12.20 Hz, 1H), 5.07 (d, J = 12.16 Hz, 1H), 4.48 (t, J = 5.24 Hz , 2H), 4.46 - 4.43 (m, 1H), 4.29 (d, J = 5.60 Hz, 2H) , 4.08 - 3.95 (m, 5H), 3.79 (t, J = 5.28 Hz, 2H), 3.5 1 - 3.43 (m, 32H), 3.40 (s, 3H), 3.39 - 3.35 (m, 2H) , 3.30 - 3.26 (m, 2H), 3.00 (s, 3H), 2.82 - 2.74 (m, 2H), 2.56 (t, J = 7.08 Hz, 2H), 2.29 (t, J = 7.36 Hz, 2H), 2.23 - 2.13 (m, 2H), 1.82 (p, J = 7.24 Hz, 2H), 1.78 - 1.63 (m, 2H), 1.61 - 1.49 (m, 2H), 1.42 - 1.2 7 (m, 2H), 1.15 (d, J = 6.80 Hz, 3H), 1.13 (d, J = 6.7 6 Hz, 3H), 0.90 (t, J = 7.32 Hz, 3H). ESI - MS (m / z): 816.0 [M / 2 + H] + 。[α] D 20 is - 19.55° (c = 1.000 g / 10 0 mL, CH3CN).

[0250] Example 15: 4 - ((S) - 2 - (4 - aminobutyl) - 35 - (4 - ((6 - (2 - ((Methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H -1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18, 21,24,27,30,33-nonoxy-3,9-diazapentatriacontamide )benzyl((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4, 12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1,2 -b]quinoline-4-carbonate

Chemical formula

[0251] Step 1: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl )diphenylmethyl)amino)butyl)-4,8-dioxo6,12,15,18,21 ,24,27-nonoxy-((S)-9-((tert-butyldimethylsilyl)oxy )-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro- 1,2,3,4-tetrahydroquinoline-1H-pyrano[3’,4’:6,7]indoli dino[1,2-b]quinolin-4-yl)carbonate At room temperature, compound 34-1 (100 mg, 0.2 mmol) was dissolved in anhydrous dichloromethane (2 ml ) under nitrogen protection, then cooled to 0 °C, and subsequently anhydrous dichloromethane (0.5 ml) containing a solution of 4-dimethylaminopyridine (144 mg, 1.18 mmol) was added, and then a solution of triphosgene (41 mg, 0.14 mmol ) in anhydrous dichloromethane (0.5 ml) was slowly added dropwise. The resulting mixture was stirred at 0 °C for 1 hour . Then, (S)-2-(32-azido-5-oxo Kiso-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatri acetamide)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyp enyl)benzhydryl)amino)acetamide (160 mg, 0.15 μmol) of the sol ution was added to the reaction solution and reacted at room temperature for 1 hour. Purification was carried out by preparative high performance liquid chromatography - (Method B) to obtain the title compound (60 mg). ESI-MS (m / z): 159 2.7 [M+H] + .

[0252] Step 2: (S)-9-(tert-butyldimethylsilyl)oxy)-4,11 -diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano 3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl-4-((S)- 2-(4-(((6-2-(methylsulfonyl)pyrimidin-5-yl)-35-(4- ((6-2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-inamide)me thyl)-1H-1,2,3-triazol-1-yl)-dioxo6,12,15,18 ,21,24,27,30,33-nonoxo-3,9-diazapentatriacontamide d) carbonate synthesis At room temperature, compound 34-2 (40 mg, 0.03 mmol) and 6-(2-(methylsulf onyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hexa-5-inamide (11.50 mg, 0.04 mmol) were dissolved in dimethyl sulfoxide and water (0. 5 ml:0.1 ml), and copper bromide (9.01 mg, 0.06 mmol) was added . The resulting mixture was reacted with stirring for 1 hour. Purification was carried out by preparative high performance liquid chromatography ([[]] Method B) was performed to obtain the title compound (20 mg). ESI-MS (m / z): 1897. 5[M+H].

[0253] Step 3: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2 -(methylsulfonyl)pyrimidin-5-yl)hexa-5-ynamide)methyl)-1 H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18 ,21,24,27,30,33-nonaoxy-3,9-diazapentatriacontamide d)benzyl((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4 ,12,14-tetrahydro-1H-pyrano[3’,4’:6,7]indolizino[1, 2-b]quinoline-4-carbonate (Compound TL034) synthesis At room temperature, compound 34-3 (30 mg, 0.018 mmol) was dissolved in acetonitrile and water ( 0.4 mL:0.1 mL), then a mixed solution of trifluoroacetic acid and acetonitrile (0 .5 mL:0.5 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. Purification was carried out by preparative high speed liquid chromatography (Method C) to obtain the trifluoroacetate salt (12 m g) of the title compound. ESI-MS (m / z): 1511.5 [M+H] + .

[0254] Example 16: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2- (methylsulfonyl)pyrimidin-5-yl)hexa-5-ynamide)methyl)-1H -1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18, 21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamide ((S)-4-Ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate Synthesis of

Chemical Structure

[0255] Step 1: ((S)-35-Azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21, 24,27,30,33-nonoxo-3,9-diazapentatriacontamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamide)ethyl)- 3,14-dioxo-3,4,12,14-tetrahydro-1,2,3,6-triazasichroheptane-1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin- 4-yl)carbonate synthesis Except for replacing compound 34-1 with compound 35-1, the same operations as described in Step 1 of Example 15 were carried out to obtain the title compound (60 mg). ESI-MS( m / z): 1561.5 [M+H] . +

[0256] Step 2: (S)-4-Ethyl-11-(2-(N-isopropylacetamide)ethyl)-3,14-dioxo 3,4,12,14-tetrahydro-1H-pyrano[3’ ,4’:6,7]indolizino[1,2-b]quinolin-4-yl)-4-((S)-2 -(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-( ​​​​4-((6-2-(Methylsulfonyl)pyrimidin-5-yl)hexa-5-ynamide )(methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,1 2,15,18,21,24,27,30,33-nonaxy-3,9-diazapentatriacontamide]carbonate synthesis Except for replacing compound 34-2 with compound 35-2, step 2 of Example 15 Adopt a synthesis method similar to the synthesis method described in step 2 to obtain the title compound (20 mg). ESI -MS (m / z): 1866.5 [M+H].

[0257] Step 3: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2 -(Methylsulfonyl)pyrimidin-5-yl)hexa-5-ynamide)methyl)-1 H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18 ,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamide d)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamide)eth yl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’ ,4’:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL035) synthesis Except for replacing compound 34-3 with compound 35-3, step 3 of Example 15 Adopt a synthesis method similar to the synthesis method described in step 3 to obtain the trifluoroacetate salt of the title compound (4. 9 mg). ESI-MS (m / z): 1594.5 [M+H] + .

[0258] Example 17: 4-((S,Z)-2-(4-aminobutyl)-42-(2-(methyls ​(Rufonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18 ,21,24,27,30,33-nonaoxa-3,9,36-triazatetracont yl-41-alkeneamide)benzyl-((S)-4-ethyl-11-(2-(N-iso propylacetamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro -1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-4- yl)carbonate synthesis

Chemical formula

[0259] Step 1: Synthesis of (Z)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enoic acid At 20 °C, compound 3-4 (200 mg, 0.67 mmol) was dissolved in methanol (8.0 mL ), Lindlar catalyst (20 mg) was added under nitrogen protection, and then the solution was subjected to hydrogen replacement three times. Hydrogenation was carried out at 20 °C for 3 hours. After filtration, the filtrate was passed through a dehydrator to obtain the title compound (150 mg). ESI-MS (m / z): 271.1 [M+H] + .

[0260] Step 2: (S)-4-Ethyl-11-(2-(N-isopropylacetamide) ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3 ’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S,Z) -2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-42- (2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6, ​12,15,18,21,24,27,30,33-Nonaoxa-3,9,36-triaza Synthesis of ((Z)-2-(4-Aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadotetracos-41-enamide)benzyl Carbonate At room temperature, compound 45-2 (8 mg, 0.030 mmol) was dissolved in dichloromethane (2 mL) and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyl uronium hexafluorophosphate (14.9 mg, 0.039 mmol) and N,N-diisopropylethylamine (8.8 mg, 0.068 mmol) were added. The reaction solution was stirred at room temperature for 10 minutes, then compound 48-1 (30 mg, 0.020 mm ol) was added and the reaction was carried out at room temperature for 1 hour with stirring. Purification was performed by preparative high performance liquid chromatography (Method B) to obtain the title compound (30 mg). ESI-MS (m / z): 17 87.8 [M+H] + .

[0261] Step 3: Synthesis of 4-((S,Z)-2-(4-Aminobutyl)-42-(2-(methyl sulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,1 8,21,24,27,30,33-Nonaoxa-3,9,36-triazadotetrac hyl-41-enamide)benzyl-((S)-4-ethyl-11-(2-(N- isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tet rahydro-1H-pyrrolo[3',4':6,7]indolizino[1,2-b]quinolin- 4-yl) Carbonate (Compound TL045) At room temperature, compound 45-3 (30 mg, 0.017 mmol) was dissolved in acetonitrile (1 ml ) and a solution of trifluoroacetic acid (0.5 ml) in acetonitrile (0.5 ml) was added was added dropwise. The reaction solution was stirred at room temperature for 20 minutes. Purification was carried out by preparative high performance liquid chromatography using Method C to obtain the trifluoroacetate salt (9 mg) of the title compound. ESI- MS (m / z): 1515.6 [M+H] + .

[0262] Example 18: 4-((S)-2-(4-Aminobutyl)-42-(2-(methylsulfonyl pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,2 1,24,27,30,33-nonaoxa-3,9,36-triazatetratetracontanyl -41-alkynamide)benzyl-((S)-4-ethyl-11-(2-(N-isop ropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro -1H-pyrrolo[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl carbonate [Chemical Structure]

[0263] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido) ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrrolo[3 ’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2 -(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-42-(2 -(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12 ,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazado tetratetracontanyl-41-alkynamide)benzyl carbonate Step 1 of Example 12, except that compound 24-1 was replaced with compound 48-1 A synthesis method similar to the synthesis method described in was adopted to obtain the title compound (15 mg). ESI -MS (m / z): 1785.8 [M+H] + .

[0264] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-42-(2-(methylsulf onyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18, 21,24,27,30,33-nonaoxa-3,9,36-triazadotetracos yl-41-alkynamide)benzyl-((S)-4-ethyl-11-(2-(N-iso propylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro -1H-pyrone[3’,4’:6,7]indolizino[1,2-b]quinolin-4- yl)carbonate (compound TL048) Step 2 of Example 12, except that compound 24-2 was replaced with compound 48-2 A synthesis method similar to the synthesis method described in was adopted to obtain the trifluoroacetate salt of the title compound (11 .35 mg). ESI-MS (m / z): 1513.7 [M+H] + .

[0265] Example 19: 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2- ((methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxamido)methyl yl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,1 5,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacont amine)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethyls (Ruvonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1 H-pyrone[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl)car Ruvonate

Chemical Structure

[0266] Step 1: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)thiazole-4-car Ruvonic acid Compound 49-1 (100 mg, 0.40 mmol), 2-bromo-4-thiazolecar boxylic acid (99.01 mg, 0.48 mmol), potassium carbonate (137.03 mg, 0. 99 mmol) and [1,1’-bis(diphenylphosphino)ferrocenyl]palladi um dichloride (29.02 mg, 0.04 mmol) were dissolved in N,N-dimethylformamide ( 4 mL) and water (1 ml) under nitrogen protection, and the reaction system was heated to 100 °C and stirred for 4 hours Then, the reaction solution was cooled to room temperature and dropped into water. After filtration, the filtrate was collected and extracted with ethyl acetate ( 10 mL×3). The aqueous phase was collected, adjusted to pH = 3 with dilute hydrochloric acid, and the solid was precipitated and filtered. The filter cake was collected to obtain the title compound (70 mg). ESI-M S (m / z): 254.0 [M+H] + .

[0267] Step 2: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazole- 4-carboxylic acid Compound 49-2 (73 mg, 0.29 mmol) was dissolved in dichloromethane (15 mL) and m-chloroperoxybenzoic acid (175.53 mg, 0.87 mmol, 85%) was It was added. The reaction system was stirred at room temperature overnight. The solvent was concentrated under reduced pressure. Purification was performed by preparative high performance liquid chromatography (Method D) to obtain the title compound (20 mg). ESI-MS( m / z): 286.0 [M+H] + .

[0268] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(pro par-2-yn-1-yl)thiazole-4-carboxamide Compound 49-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL) , and O-(7-benzotriazol)-N,N,N,N-tetramethyluronium hexafluor luorophosphate (39.98 mg, 0.11 mmol) was added. The resulting reaction system was cooled to 0 °C, and then N,N-diisopropylethylamine (22.65 mg, 0.1 8 mmol) and propargylamine (4.63 mg, 0.09 mmol) were added to the reaction system . The reaction solution was stirred at room temperature for 3 hours. Purification was performed by preparative high performance liquid chromatography (meth od D) to obtain the title compound (10 mg). ESI-MS (m / z): 323.0 [M+H] + .

[0269] Step 4: (S)-4-Ethyl-11-(2-(N-isopropylmethanesulfon amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pi rolo[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl-(4-( (S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)- 35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazole- 4-carboxamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8 -Dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9 -Diaza pentatriacontamino)benzyl)carbonate synthesis At room temperature, compound 33-1 (30 mg, 0.02 mmol) and compound 49-4 (9.0 8 mg, 0.03 mmol) were dissolved in dimethyl sulfoxide and water (2 mL / 0.5 mL), copper bromide (5.39 mg, 0.04 mmol) was added, and the mixture was reacted with stirring for 2 hours. After filtration, the filtrate was purified by preparative high performance liquid chromatography (Method B) to obtain the title compound (20 mg). ESI-MS (m / z): 1647.3 [M+H-273] + .

[0270] Step 5: 4-((S)-2-(4-Aminobutyl)-35-(4-((2-(2 -((Methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxamide)meth yl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12, 15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatri contamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethyl sulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrone[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl) carbonate synthesis At room temperature, compound 49-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (0.2 mL) was added dropwise. The resulting reaction solution was stirred at room temperature for 20 minutes. Then the reaction solution was concentrated. The residue was purified by preparative high performance liquid chromatography (Method C) to obtain the trifluoroacetate salt (8 mg) of the title compound. ESI -MS (m / z): 1647.9 [M+H] + 。

[0271] Example 20: 4-((S)-2-(4-Aminobutyric acid)-35-(4-((2-(2-( Methylsulfonyl)pyrimidin-5-yl)-oxazole-4-formylamino)methyl yl)-1H-1,2,3-triazole-1-yl)-4,8-dioxo-6,12,1 5,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacont amide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethyl sulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl) Carbonate

Chemical formula

[0272] Step 1: Synthesis of ethyl 2-(2-(methylthio)pyrimidin-5-yl)oxazole -4-carboxylate At 25 °C, ethyl 2-bromooxazole-4-carboxylate (100 mg, 0. 45 mmol) and compound 49-1 (126 mg, 0.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane and water (4 mL / 2 mL), then potassium carbonate (125 mg, 0.9 mmol) and [1,1’-bis(diphenylphosphino)ferrocene]palladium dichloride (33 mg, 0.05 mmol) were successively added under N2 protection, and the mixture was heated to 9 0 °C and reacted for 3 hours. The reaction solution was filtered through diatomaceous earth. The filtrate was washed with water( It was diluted with (50 mL), and extracted with ethyl acetate (30 mL × 3). The organic phases were combined and dried. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product. This was purified by preparative thin-layer chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain the title compound (40 mg). ESI-MS (m / z): 266.1 [M+H] + .

[0273] Step 2: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)oxazole-4- carboxylic acid At 25 °C, compound 50-1 (50 mg, 0.19 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (4 mL / 2 mL). After complete dissolution, lithium hydroxide monohydrate (40 mg, 0.94 mmol) was added thereto, and the mixture was reacted at 25 °C for 1 hour. The reaction solution was diluted with water ( 15 mL), and extracted with ethyl acetate (20 mL × 2). The aqueous phase was adjusted to pH = 2-3 with 1N dilute hydrochloric acid, and then extracted with a mixed solvent of dichloromethane / methanol (v:v = 10:1) (20 mL × 3). The organic phases were combined, washed with saturated saline (30 mL × 1) , and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated to obtain the title compound (40 mg). This was used as it was in the subsequent reaction without purification. ES I-MS (m / z): 238.1 [M+H] + .

[0274] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole -4-carboxylic acid At 25 °C, compound 50-2 (40 mg, 0.17 mmol) was dissolved in dichloromethane (6 mL) . After complete dissolution, m-chloroperoxybenzoic acid (29 mg, 0.17 mmol ) was added thereto, and the reaction was carried out at 25 °C for 14 hours with stirring. The reaction solution was concentrated, and the residue was separated and purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (20 mg) . ESI-MS (m / z): 269.9 [M+H] + .

[0275] Step 4: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propargyl-2-yl)-oxazole-4-carboxamide At 25 °C, compound 50-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (4 mL ), then O-(7-azabenzotriazol-1-yl)-N,N,N',N '-tetramethyluronium hexafluorophosphate (42 mg, 0.11 mmol ) and N,N-diisopropylethylamine (19 mg, 0.15 mmol) were successively added, and the mixture was stirred for 5 minutes. Subsequently, propargylamine (5.0 mg, 0.09 mmol) was added, and then the resulting mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (5.0 m g). ESI-MS (m / z): 306.9 [M+H] . + .

[0276] Step 5: (S)-4-Ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrido[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 4-((S )-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-3 5-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole- )-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-3 5-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole- 4-(Formylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8 -dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3, 9-diazapentatriacontamide)benzyl)carbonate synthesis At 25 °C, compound 50-4 (6.0 mg, 0.02 mmol) and compound 33-1 (3 0 mg, 0.02 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (2 mL / 0.5 mL), and copper bromide (5.0 mg, 0.04 mmol) was added in one batch. The resulting mixture was reacted at room temperature for 2 hours. The reaction solution was filtered and purified by preparative high-performance liquid chromatography (Method B) to obtain the title compound (25 mg). ESI-MS( m / z): 1631.3[(M - 273 + H] + 。

[0277] Step 6: 4-((S)-2-(4-Aminobutyric acid)-35-(4-((2-(2- (Methylsulfonyl)pyrimidin-5-yl)oxazole-4-formylamino)methyl yl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,1 5,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacont amide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethyl sulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl) carbonate synthesis At 25 °C, compound 50-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2.0 m L). After completion of dissolution, trifluoroacetic acid (0.2 mL) was added to the reaction mixture , reacted at 25 °C for 10 minutes. The reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography y (Method C) to obtain the trifluoroacetate salt (3.0 mg) of the title compound was obtained. ESI-MS (m / z): 816.5 [M / 2 + H] + .

[0278] Example 21: N-((1-((6S,9S)-1-Amino-6-((4-(((S)- 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2 -((S)-2-(Dimethylamino)-3-methylbutanamide)-N,3-dimethylbut anamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3- methoxy-2-methylpropanamide)-3-phenylpropanamide)methyl)phenyl yl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxa-13,1 9,22,25,28,31,34,37,40-nonaoxa-2,7,10,16-tetra azaanthracen-42-yl)-1H-1,2,3-triazol-4-yl)meth yl)-6-(2-(Methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

Chemical Structure

[0279] Step 1: 9-Fluorenylmethyl ((S)-1-(((S)-1-((4-(( (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-(( S)-2-((S)-2-(Dimethylamino)-3-butyrylamido)-N,3-dimethyl rubutyrylamido)-3-methoxy-5-methylheptanoyl)pyro-2-yl)-3- (Methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)f enyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl -1-oxobutan-2-yl)carbamate synthesis At room temperature, compound 51-1 (100 mg, 0.17 mmol) and 9-fluorenylmethyl ((S)-1-(((S)-1-((4-(((S)-2-amino-3-phenylpropa namido)methyl)phenyl)amino)-1-oxo-5-pentylureido-2-yl )amino)-3-methyl-1-oxobutan-2-yl)carbamate trifluoroacet ate (144 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (2 mL) and cooled to 0 °C, then 1-hydroxybenzotriazole (34 mg, 0.25 mmol), N-methylmorpholine (51 mg, 0.51 mmol) and 1-(3-dime thylaminopropyl)-3-ethylcarbodiimide hydrochloride (48 mg, 0.25 mmol) were added successively. After the addition, the reaction solution was stirred at 0 °C for 5 hours. The reaction solution was poured into water (20 mL) to precipitate a white solid, which was then suction filtered. The filter cake was washed and dried to obtain the title compound (200 mg). ESI-MS (m / z): 13 29.2 [M + H] + .

[0280] Step 2: (S)-2-((S)-2-Amino-3-butyrylamino)-N-(4 -(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4 -((S)-2-((S)-2-(dimethylamino)-3-butyrylamino)-N,3- dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl) -3-Methoxy-2-methylpropionamide)-3-phenylpropionamide)methyl Synthesis of phenyl)-5-ureidovaleramide At room temperature, compound 51-2 (200 mg, 0.12 mmol) was dissolved in N,N-dimethylform amide (5 mL), and piperidine (0.5 mL) was added. The reaction solution was stirred at room temperature for 2 hours, and then purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (65 mg). ESI-MS (m / z): 1107.2 [M+H] + .

[0281] Step 3: (S)-2-((S)-35-Azido-2-isopropyl-4,8-di oxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9- diazapentatriacontamino)-N-(4-(((S)-2-((2R,3R)-3 -((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethyl amino)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy -5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpro panamide)-3-phenylpropanamide)methyl)phenyl)-5-ureidovaler amide synthesis 32-Azido-5-oxo-3,9,12,15,18,21,24,27,30-nona oxa-6-azatricarboxylic acid (33.1 mg, 0.06 mmol) was dissolved in N,N-dime thylformamide (5 mL), and then O-(7-azabenzotriazole)-N ,N,N,N-tetramethyluronium hexafluorophosphate (38 mg, 0.1 0 mmol) and N,N-diisopropylethylamine (26 mg, 0.20 mmol) was added. The reaction solution was stirred at room temperature for 10 minutes, then cooled to 0 °C, and Compound 51-3 (55 mg, 0.05 mmol) was added. The reaction solution was stirred at room temperature for 2 hours and purified by preparative high-speed liquid chromatography (Method D) to obtain the title compound (56 mg). ESI-MS (m / z): 821.8 [M / 2 + H] + .

[0282] Step 4: N-((1-((6S,9S)-1-Amino-6-((4-(((S) -2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)- 2-((S)-2-(Dimethylamino)-3-methylbutanamide)-N,3-dimethyl butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3 -methoxy-2-methylpropanamide)-3-phenylpropanamide)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13, 19,22,25,28,31,34,37,40-nonaoxa-2,7,10,16- tetraazaanthracen-42-yl)-1H-1,2,3-triazol-4-yl) methyl)-6-(2-(Methylsulfonyl)pyrimidin-5-yl)-5-hexynamide synthesis At room temperature, Compound 51-4 (56 mg, 0.04 mmol) and 6-(2-(methylsulf onyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-5-hexynamide (16 mg, 0.05 mmol) were dissolved in a mixed solution of dimethyl sulfoxide and water (2 m L / 0.5 mL), and copper(II) bromide (10 mg, 68.17 μmol) was added. The resulting mixture was stirred for 2 hours and then filtered. The filtrate was purified by preparative high-performance liquid chromatography (Method D). Purification by Method D gave the title compound (50 mg). ESI-MS (m / z): 9 74.3 [M / 2 + H] + .

[0283] Example 22: 4 - ((2S,5S)-5 - Isopropyl - 38 - (4 - ((6 - (2 - (Methylsulfonyl)pyrimidin - 5 - yl)-5 - hexynamide)methyl)-1H - 1,2,3 - triazol - 1 - yl)-4,7,11 - trioxo - 2 - (3 - ureido propyl)-9,15,18,21,24,27,30,33,36 - nonaoxa - 3 ,6,12 - triazatetracontanyl)benzyl - ((S)-1 - (((S)-1 - ( ((3R,4S,5S)-1 - ((S)-2 - ((1R,2R)-3 - (((1S,2R )-1 - hydroxy - 1 - phenylpropan - 2 - yl)amino)-1 - methoxy - 2 - methyl - 3 - oxypropyl)pyrrolidin - 1 - yl)-3 - methoxy - 5 - methyl - 1 - oxyheptan - 4 - yl)(methyl)amino)-3 - methyl - 1 - oxobutan - 2 - yl)(methyl)carbamate [Chemical Structure]

[0284] Step 1: 4 - ((2S,5S)-38 - azido - 5 - isopropyl - 4,7,1 1 - trioxo - 2 - (3 - ureidopropyl)-9,15,18,21,24,27, 30,33,36 - nonaoxa - 3,6,12 - triazatetracontanyl)benzyl - ((S)-1 - (((S)-1 - (((3R,4S,5S)-1 - ((S)-2 - ((1 R,2R)-3 - (((1S,2R)-1 - hydroxy - 1 - phenylpropan - 2 - yl ((2S,5S)-5-Isopropyl-38-(4-((6-(2-((methylsulfonyl)pyrimidin-5-yl)-5-hexynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatetracont-3-yl)benzyl-((S)-1-(((S)-1-((((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate )-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)- (3-methyl-1-oxobutan-2-yl)(methyl)carbamate At room temperature, compound 53-1 (100 mg, 0.09 mmol) was dissolved in N,N-dimethylform amide (3 mL), and then 1-hydroxybenzotriazole (13 mg, 0. 09 mmol), N,N-diisopropylethylamine (36 mg, 0.28 mmol) and compound 52-1 (67 mg, 0.09 mol) were added. The reaction solution was stirred at room temperature for 16 hours and then purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (120 mg). ESI-MS (m / z): 830.1 [M / 2+H] + .

[0285] Step 2: Synthesis of 4-((2S,5S)-5-isopropyl-38-(4-((6-(2 -(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide)methyl)-1H -1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ure idopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa- 3,6,12-triazatetracont-3-yl)benzyl-((S)-1-(((S)-1- (((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2 R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2 -methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl- 1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan- 2-yl)(methyl)carbamate At room temperature, compound 52-2 (22 mg, 0.07 mmol) was dissolved in a mixed solution of dimethyl sulfoxide and water (3 mL / 0.3 mL), and then copper bromide (18 mg, 0.13 m mol) was added, and the mixture was stirred for 1 hour. The reaction solution was filtered. The filtrate was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (92 mg). ESI-MS( m / z): 982.8 [M / 2 + H] + .

[0286] Example 23: Synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S, 5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(( (4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methyl sulfonyl)pyrimidin-5-yl)hex-5-ylcarbamoyl)methyl)-1H-1 ,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21, 24,27,30-nonoxy-6-azatriacontamino)butyrylamino)-5- ureidovaleryl)amino)benzyl)oxy)carbonyl)amino)butyrylamino)- 3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methyl propionyl)-L-phenylalanine

Chemical Structure

[0287] Step 1: 4-((2S,5S)-38-azido-5-isopropyl-4,7,1 1-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27, 30,33,36-nonoxy-3,6,12-triazaoctatriacontamino)be Synthesis of Nzyl-(4-nitrophenyl)-carbonate At 25 °C, compound 29-1 (500 mg, 0.55 mmol) was dissolved in N,N-dimethylform amide (10 mL), N,N-diisopropylethylamine (141 mg, 1 .09 mmol) was added, and then a solution of di(p-nitrobenz yl) carbonate (332 mg, 1.09 mmol) in dichloromethane (1 mL) was added dropwise. After the addition, the mixture was reacted at 25 °C for 3 hours with stirring. The reaction solution was purified by reverse column (C18) chromato graphy (acetonitrile / water = 1:2) to obtain the title compound (400 mg). ESI-MS (m / z): 1081.9 [M+H] + .

[0288] Step 2: (S)-2-((2R,3R)-3-((2S)-1-((3R,4S ,5S)-4-((S)-2-((S)-2-((((4-((S)-2-((S)-2 -(32-azido-5-oxo-3,9,12,15,18,21,24,27,30- nonaoxy-6-azatriacontamide)-3-methylbutyrylamino)-5-ureido dipentanoylamino)benzyl)oxy)carbonyl)(methyl)amino)-3-methyl lbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylhe ptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phe nylalanine synthesis At 25 °C, compound 53-1 (60 mg, 0.06 mmol) and ((2R)-3-((2 S)-1-((3R,5S)-4-((S)-N,3-dimethyl-2-((S)-3-me thyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-me ​((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl((4-((S)-2-((S)-3-methyl-2-(3-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-carbamoyl)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontanamino)butyrylamino)-5-ureidovaleryl)amino)benzyl)oxy)carbonyl)amino)butyryl)amino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L- (S)-2-((2R,3R)-3-((2S)-1-((3R,4S ,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl( ((4-((S)-2-((S)-3-methyl-2-(3-(2-(methyl sulfonyl)pyrimidin-5-yl)hexan-5-carbamoyl)methyl)-1H- 1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21 + .

[0289] Step 3: (S)-2-((2R,3R)-3-((2S)-1-((3R,4S ,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl( ((4-((S)-2-((S)-3-methyl-2-(3-(2-(methyl sulfonyl)pyrimidin-5-yl)hexan-5-carbamoyl)methyl)-1H- 1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21 ,24,27,30-nonaoxa-6-azatriacontanamino)butyrylamino)-5 -ureidovaleryl)amino)benzyl)oxy)carbonyl)amino)butyrylamino) -3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methyl propyl)-L-phenylalanine synthesis At 25 °C, 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2 -yn-1-yl)-oxazole-4-formamide (9 mg, 0.03 mmol) and compound 53-2 (50 mg, 0.03 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (1 mL / 0.25 mL). After complete dissolution, copper(II) bromide (11 mg, 0.08 mmol) was added. (mmol) was added. After the addition, the mixture was stirred for 1 hour under N2 protection. Then, filtration was carried out and the filtrate was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound ( 25 mg). ESI-MS (m / z): 989.9 [M / 2+H] + .

[0290] Example 24: 4-((S)-2-(4-Aminobutyl)-35-(4-((6-(2- (Methylsulfonyl)pyrimidin-5-yl)-5-hexynamide)methyl)-1H- 1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,2 1,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino) Benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)- 2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropa ne-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidine -1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl) amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-ox obutan-2-yl)(methyl)carbamate

Chemical Structure

[0291] Step 1: Synthesis of (S)-4-(35-Azido-2-(4-(((4-Methoxyphenyl )benzhydryl)amino)butyryl)-4,8-dioxo-6,12,15,18,2 1,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino) Benzyl-(4-nitrophenyl)-carbonate At room temperature, 1 g (0.95 mmol) of Compound 54-1 was dissolved in dichloromethane (20 mL). Then N,N-diisopropylethylamine (488 mg, 3.77 mmol) was added, followed by dropwise addition of a solution of di-(p-nitrophenyl)-carbonate (860 mg, 2.83 mmol) in dichloromethane (10 mL). The resulting reaction solution was stirred at room temperature for 6 hours and purified by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to obtain the title compound (900 mg). ESI-MS (m / z): 953.0 [M+H-273] . + .

[0292] Step 2: Synthesis of benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate, 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamine) At room temperature, 1-hydroxybenzotriazole (33 mg, 0.25 mmol) and N,N-diisopropylethylamine (48 mg, 0.37 mmol) were added to Compound 54-2 (2 mL). ​​​​​​​​​​​), followed by the addition of compound 52-1 (88 mg, 0.12 mmol). The mixture was stirred at room temperature for 16 hours and then purified by preparative high performance liquid chromatography (Method B). The title compound was purified to give 150 mg. ESI-MS (m / z): 1803.6 M+H] + .

[0293] Step 3: 4-((S)-2-(4-(((4-Methoxyphenyl)benzhydrylidene 35-(4-((6-(2-(methylsulfonyl)pyrimidine) -5-yl)-5-hexyneamido)methyl)-1H-1,2,3-triazole-1- 4,8-dioxo-6,12,15,18,21,24,27,30,33-yl Naoxa-3,9-diazapentatriacanthoamino)benzyl ((S)-1-(((S )-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((( 1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-meth 2-Methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5- Methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxo (butan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)ca Synthesis of rubamate Compound 54-3 (100 mg, 0.06 mmol) and 6-(2-(methylphenyl)- (sulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-5-hexyl The amide (26 mg, 0.08 mmol) was dissolved in dimethyl sulfoxide (2 mL) and water (0 Then copper bromide (16 mg, 0.11 mmol) was added and the mixture was stirred for 2 h. It was stirred. Then, filtration was performed, and the filtrate was purified by preparative high performance liquid chromatography (Method B) to obtain the title compound (70 mg). ESI-MS (m / z): 1936.6 [M+H-273] [M+H-273] + .

[0294] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2 -(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide)methyl)-1H -1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18, 21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamino )benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S) -2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpro pan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolid in-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl )amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxo butan-2-yl)(methyl)carbamate At room temperature, compound 54-4 (70 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (0.2 mL) was added dropwise. The resulting reaction solution was stirred at room temperature for 20 minutes, then concentrated, and the residue was purified by preparative high performance liquid chromatography (Method C) to obtain the trifluoroacetate salt of the title compound (55 mg). ESI-MS (m / z): 918.8 [M / 2+H] [M / 2+H] + .

[0295] Example 25: 4-((S)-2-(4-Aminobutyl)-35-(4-((4-(2- (Methylsulfonyl)pyrimidin-5-yl)benzamide)methyl)-1H-1,2, 3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24 ,27,30,33-nonoxy-3,9-diazapentatriacontamide)benzyl ((S)-4-Ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl )-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’,4 ’:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate synthesis

Chemical formula

[0296] Step 1: Synthesis of methyl 4-(2-(methylthio)pyrimidin-5-yl)benzoate Synthesis At 25 °C, compound 49-1 (252 mg, 1.0 mmol), water (3 mL), Pd(dp pf)Cl2 (40 mg, 0.05 mmol) and potassium carbonate (277 mg, 2.0 m mol) were successively added to a solution of methyl p-bromobenzoate (215 mg, 1.0 mmol) in 1,4-dioxane (5 mL), and the mixture was stirred at 80 °C for 4 hours. The reaction solution was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried, and then the insoluble substances were removed by filtration , and the residue was purified by silica gel column chromatography to obtain the title compound (220 mg). ESI-MS (m / z): 261.0 [M+H] . + .

[0297] Step 2: Synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic acid At 25 °C, lithium hydroxide monohydrate (322 mg, 7.68 mmol) and water (3 ml ) were each added to a solution of compound 55-1 (500 mg, 1.92 mmol) in tetrahydrofuran (3 ml), and the mixture was stirred for 4 hours. The reaction solution was adjusted to pH = 3-4 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL × 3). The combined organic phases were dried. The insoluble substances were removed by filtration, and the residue was purified by preparative high-performance liquid chromatography (Method D) to give the title compound (430 mg). ESI-MS (m / z): 246.9 [M+H] + .

[0298] Step 3: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid At 25 °C, m-chloroperoxybenzoic acid (420 mg, 2.44 mmol) was added to a solution of compound 55-2 (200 mg, 0.81 mmol) in dichloro methane (5 ml), and the mixture was stirred for 5 hours and then purified by silica gel column chromatography to give the title compound (180 mg). ESI-MS (m / z): 279.0 [M+H] . + .

[0299] Step 4: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop -2-yn-1-yl)benzamide At 25 °C, benzotriazol-N,N,N’,N’-tetramethyluronium hexa fluorophosphate (100 mg, 0.26 mmol) was added to a solution of compound 55-3 (50 mg, 0.18 mmol) in dichloromethane (10 mL) , and the mixture was stirred for 30 minutes. Then, propynylamine (10 mg, 0.2 mmol) and N,N-diisopropyle were added, and the mixture was stirred for another 30 minutes. Then, N,N-diisopropyle ​Chiramine (70 mg, 0.5 mmol) was added to the reaction solution and reacted for 2.5 hours under stirring. The reaction solution was purified by silica gel column chromatography to obtain the title compound (2 0 mg). ESI-MS (m / z): 316.0 [M+H] + .

[0300] Step 5: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfon amide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pi rano[3’,4’:6,7]indolizino[1,2-b]quinolin-4-yl(4-(( (S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl) -35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzam ido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6, 12,15,18,21,24,27,30,33-nonoxo-3,9-diazapenta triacontamide)benzyl)carbonate Under N2 protection, at 25 °C, copper iodide (10 mg, 0.05 mmol) and water (2 mL) were successively added to a dimethyl sulfoxide solution (2 mL) of compound 55-4 (16 mg, 0.05 mmol) and compound 33-1 (80 mg, 0.0 5 mmol), and the mixture was reacted for 1 hour under stirring. Purification (Method B) was performed to obtain the title compound (79 mg). ESI-MS (m / z): 1641.5 [M-273+H] + .

[0301] Step 6: 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2 -(methylsulfonyl)pyrimidin-5-yl)benzamide)methyl)-1H-1,2 ,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,2 4,27,30,33-nonoxy-3,9-diazapentatriacontamide)benz yl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)eth yl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3’, 4’:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate synthesis At 25 °C, compound 55-5 (55 mg, 0.029 mmol) was added to trifluoroacetic acid (0.5 mL) in a mixed solvent of water / acetonitrile (0.1 mL / 0.5 mL), and the mixture was reacted with stirring for 15 minutes. The reaction solution was purified by preparative high-performance liquid chromatography (Method C) to obtain the trifluoroacetate salt (42 mg) of the title compound. ESI-MS (m / z ): 821.0 [M / 2 + H] + .

[0302] Example 26: N-((1-((6S,9S)-1-amino-6-((4-((S)-3 -azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-( (S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3 -dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl )-3-methoxy-2-methylpropionamide)propyl)phenyl)carbamoyl )-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25, 28,31,34,37,40-nonoxy-2,7,10,16-tetraazadotetra cont-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-( 2-(methylsulfonyl)pyrimidin-5-yl)hexa-5-ynylamide​

Chemical

[0303] Step 1: Synthesis of 32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azadotriacontanoic acid At 20 °C, compound 56-1 (750 mg, 1.28 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (496 mg, 1.54 mmol) were dissolved in dimethyl sulfoxide (10 mL), and copper(II) bromide (465 mg, 3.21 mmol) was added in one batch. After the addition, the mixture was reacted with stirring for 12 hours. The reaction solution was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method D) to obtain the title compound (500 mg). ESI-MS (m / z): 860.4 [M+H] -5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6- azadotriacontanoic acid At 20 °C, compound 56-1 (750 mg, 1.28 mmol) and 6-(2-(methyl sulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5 -ynamide (496 mg, 1.54 mmol) were dissolved in dimethyl sulfoxide (10 mL) and copper(II) bromide (465 mg, 3.21 mmol) was added in one batch. After the addition , the mixture was reacted with stirring for 12 hours. The reaction solution was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method D) to obtain the title compound (500 mg). ESI-M S (m / z): 860.4 [M+H] + .

[0304] Step 2: (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1 -((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R ,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbut yrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylhept yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamide)propyl) ((S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(4-aminophenyl)-3-azidopropyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamine(185mg,0.24mmol)wasdissolvedinN,N-dimethylformamide(5mL),thenHATU(137mg,0.36mmol)wasaddedandstirredfor5minutes,followedbytheadditionof(S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanoicacid(131mg,0.26mmol).Themixturewasstirredatroomtemperaturefor30minutes.Thereactionsolutionwasusedasitisforfurtherreaction.ESI-MS(m / z):626.0[M / 2+H] )-3-methyl-1-oxobutan-2-yl)carbamate synthesis At 25 °C, (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R )-3-(((S)-1-(4-aminophenyl)-3-azidopropyl-2-yl)a mino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3 -methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimeth ylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamine(185 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then , HATU (137 mg, 0.36 mmol) was added and stirred for 5 minutes, followed by (S)- 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)ami no)-3-methylbutyrylamino)-5-ureidopentanoic acid (131 mg, 0.26 m mol) was added. The mixture was stirred at room temperature for 30 minutes. The reaction solution was used as it was for further reaction. ESI-MS (m / z): 626.0 [M / 2+H] ESI-MS (m / z): 626.0 [M / 2+H] + .

[0305] Step 3: Synthesis of (S)-2-((S)-2-amino-3-methylbutyrylamino)-N -(4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R, 4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbut ylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl )pyrrolidin-2-yl)-3-methoxy-2-methylpropionamide)propyl)ph enyl)-5-ureidovaleramide At 25 °C, diethylamine (0.5 mL) was added to the reaction solution obtained in Step 2, and the mixture was stirred for 30 minutes after addition to cause a reaction. The reaction solution was purified by preparative high performance liquid chromatography (Method D ) to obtain the title compound (70 mg). ESI-MS (m / z): 515. 0 [M / 2 + H] + .

[0306] Step 4: Synthesis of N-((1-((6S,9S)-1-amino-6-((4-((S)- 3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4- ((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N, 3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2- yl)-3-methoxy-2-methylpropionamide)propyl)phenyl)carbamoyl yl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25 ,28,31,34,37,40-nonaoxo-2,7,10,16-tetraazadotetra laconta-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6- (2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide At 25 °C, (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-( 4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S ,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryl amino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)py rrolidin-2-yl)-3-methoxy-2-methylpropionamide)propyl)phenyl ​L)-5-Ureidovaleryl amide (95 mg, 0.092 mmol) and 32-(4-( (6-(2-(Methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)meth yl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,1 5,18,21,24,27,30-nonaoxy-6-azadotriacontanoic acid (79 m g, 0.092 mmol) was dissolved in N,N-dimethylformamide (4 mL), and HAT U (70 mg, 0.184 mmol) was added in one batch. The mixture was stirred at room temperature for 1 hour and purified by preparative high performance liquid chromatography (Method D) to obtain the title compound (30 mg). ESI-MS (m / z): 935.8 [M / 2 + H] + .

[0307] III. Coupling of Compounds Containing Bioactive Molecules and Linkers with Antibodies Example 27: Preparation of BT001002 0.3 mL of the antibody sacituzumab (anti-Trop-2, 33.5 mg / mL) was diluted with 0.25 ml of a solution containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate (pH 7. 6), and 0.45 ml of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added thereto and mixed uniformly. The mixture was adjusted to pH = 7.4 with 1 M K2HP O4 solution, then 10 mM TCEP (tris(2-carboxy ethyl)phosphine) solution was added, mixed uniformly, and left at room temperature for 30 minutes. To this solution system, TL003 dissolved in dimethyl sulfoxide was added in an amount of 15 equivalents, mixed uniformly, and left at room temperature for 2 hours. After the addition, 6.1 μl of 100 mM cysteine was added to terminate the reaction and the mixture was left at room temperature for 2 hours. After the addition, 6.1 μl of 100 mM cysteine was added to terminate the reaction and the mixture was left at room temperature for 2 hours. After the addition, 6.1 μl of 100 mM cysteine was added to terminate the reaction and the mixture was left at room temperature for 2 hours. After the addition, 6.1 μl of 100 mM cysteine was added to terminate the reaction The reaction was stopped. Finally, the buffer was replaced with a 20 mM PB buffer solution at pH 6.44 using a G-25 gel column to obtain the coupling product of TL003 and trastuzumab, which was named BT001002.

Chemical formula

[0308] Example 28: Preparation of BT001004 0.285 mL of trastuzumab (anti-Trop-2, 17.6 mg / mL) was diluted with 0.095 mL of diluent (a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate, pH 7.6). Then, the diluted solution was adjusted to pH 7.4 with 1 M Na2HPO4 solution, 10 mM TCEP solution was added, mixed uniformly, and left at room temperature for 30 minutes. To this solution system, TL019 dissolved in dimethyl sulfoxide was added in a 9-fold equivalent amount, mixed uniformly, and left at room temperature for 2 hours. Finally, the buffer was replaced with a PBS buffer solution at pH 6.5 using a G-25 gel column to obtain the coupling product of TL019 and trastuzumab, which was named BT001004.

Chemical formula

[0309] Example 29: Preparation of BT001012 A method similar to that described in Example 27 was employed to obtain the coupling product of TL024 and trastuzumab, except that TL003 was replaced with the trifluoroacetate salt of TL024, which was named BT001012.

Chemical formula

[0310] Example 30: Preparation of BT001013 A method similar to that described in Example 27 was employed, except that TL003 was replaced with the trifluoroacetate of TL048, to generate a coupling product of TL048 and trastuzumab. This product was obtained and named BT001013.

Chemical formula

[0311] Example 31: Preparation of BT001018 A method similar to that described in Example 27 was employed, except that TL003 was replaced with TL030, to obtain a coupling product of TL030 and trastuzumab. This product was named BT001018.

Chemical formula

[0312] Example 32: Preparation of BT001021 0.3 mL of trastuzumab (anti-Trop-2, 33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6), and then 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added and mixed uniformly. The mixture was adjusted to pH = 7.4 with 1 M Na2HPO4 solution, then 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and mixed uniformly, and the mixture was left at room temperature for 30 minutes. To this solution system, 10 equivalents of the trifluoroacetate of TL033 dissolved in dimethyl sulfoxide was added and mixed uniformly, and the mixture was left at room temperature for 2 hours. Then, 6.1 of 100 mM cysteine was added and mixed uniformly, and the mixture was left at room temperature for 1 hour. This solution system was purified by size exclusion chromatography to obtain a product, which was named BT001021. was added and mixed uniformly, and the mixture was left at room temperature for 1 hour.​​ μl was added to stop the reaction. Finally, the buffer was replaced with a PBS buffer solution of p H6.5 to obtain a coupling product of TL033 and trastuzumab, which was named BT001021.

Chemical formula

[0313] Example 33: Preparation of BT001022 A method similar to the method described in Example 27 was adopted, except that TL003 was replaced with the trifluoroacetate salt of TL034, to obtain a coupling product of TL034 and trastuzumab, which was named BT001022.

Chemical formula

[0314] Example 34: Preparation of BT001023 A method similar to the method described in Example 27 was adopted, except that TL003 was replaced with the trifluoroacetate salt of TL035, to obtain a coupling product of TL035 and trastuzumab, which was named BT001023.

Chemical formula

[0315] Example 35: Preparation of BT001032 A method similar to the method described in Example 27 was adopted, except that TL003 was replaced with the trifluoroacetate salt of TL045, to obtain a coupling product of TL045 and trastuzumab, which was named BT001032.

Chemical formula

[0316] Example 36: Preparation of BT001033 TL003 was replaced with the trifluoroacetate of TL033, and trastuzumab was replaced with antibody M1. Except for this replacement, a method similar to the method described in Example 27 was adopted to obtain a coupling product of TL033 and antibody M1, and this was named BT001033.

Chemical formula

[0317] Example 37: Preparation of BT001034 TL003 was replaced with the trifluoroacetate of TL033, and trastuzumab was replaced with antibody M2. Except for this replacement, a method similar to the method described in Example 27 was adopted to obtain a coupling product of TL033 and antibody M2, and this was named BT001034.

Chemical formula

[0318] Example 38: Preparation of BT001035 0.3 mL of antibody M3 (anti-Trop-2, 33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate (pH 7.6), and then 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added and mixed uniformly. The mixture was adjusted to pH = 7.4 with 1 M Na2HPO4 solution, then a 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and mixed uniformly, and left at room temperature for 30 minutes. To the solution system, the trifluoroacetate of TL033 dissolved in dimethyl sulfoxide was added in an amount of 10 equivalents. ​​​​​​​​​​, were uniformly mixed. The resulting mixture was left at room temperature for 2 hours. Then, 6.1 μl of 100 mM cyste ine was added to stop the reaction. Finally, the buffer was replaced with a PBS buffer solution at pH 6.5 by a G-25 gel column to obtain a coupling product of TL033 and antibody M3, which was named BT001035.

Chemical Structure

[0319] Example 39: Preparation of BT001036 A method similar to the method described in Example 27 was employed, except that TL003 was replaced with the trifluoroacetate salt of TL033 and trastuzumab was replaced with trastuzumab to obtain a coupling product of TL033 and trastuzumab, which was named BT001036.

Chemical Structure

[0320] Example 40: Preparation of BT001040 A method similar to the method described in Example 27 was employed, except that TL003 was replaced with the trifluoroacetate salt of TL049, to obtain a coupling product of TL049 and trastuzumab which was named BT001040.

Chemical Structure

[0321] Example 41: Preparation of BT001041 A method similar to the method described in Example 27 was employed, except that TL003 was replaced with the trifluoroacetate salt of TL050, to obtain a coupling product of TL050 and trastuzumab which was named BT001041. ​​​​A substance was obtained and named BT001041.

Chemical formula

[0322] Example 42: Preparation of BT001042 A method similar to the method described in Example 27 was adopted, except that TL003 was replaced with TL051, to obtain a coupling product of TL051 and trastuzumab, and this was named BT0 01042.

Chemical formula

[0323] Example 43: Preparation of BT001043 A method similar to the method described in Example 27 was adopted, except that TL003 was replaced with TL052, to obtain a coupling product of TL052 and trastuzumab, and this was named BT0 01043.

Chemical formula

[0324] Example 44: Preparation of BT001044 A method similar to the method described in Example 27 was adopted, except that TL003 was replaced with TL053, to obtain a coupling product of TL053 and trastuzumab, and this was named BT0 01044.

Chemical formula

[0325] Example 45: Preparation of BT001045 A method similar to that of Example 27 was adopted, except that TL003 was replaced with the trifluoroacetate of TL054. ​​​Adopt a method similar to the method described in [reference], and generate the coupling of TL054 with satuzumab to obtain a product, which was named BT001045.

Chemical formula

[0326] Example 46: Preparation of BT001046 Except that TL003 was replaced with the trifluoroacetate salt of TL055, Example 27 Adopt a method similar to the method described in [reference], and generate the coupling of TL055 with satuzumab to obtain a product, which was named BT001046.

Chemical formula

[0327] Example 47: Preparation of BT001047 Except that TL003 was replaced with TL056, a method similar to the method described in Example 27 was adopted to obtain the coupling product of TL056 and satuzumab, and this was named BT001047.

Chemical formula

[0328] Example 48: Determination of the molecular weight of BT001002 by LC-MS The molecular weight of BT001002 obtained by coupling was analyzed by LC-MS . LC conditions: Liquid chromatography column: ACQUITU UPLC (registered trademark) Prot ein BEH C4 1.7μm, 2.1mm×100mm; Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2 % H2O / 98% ACN; Flow rate: 0.25 mL / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Sample size: 1 μg;

Table 7

[0329] The theoretical and measured molecular weights of BT001002

Table 8

[0330] In the table, mAb represents monoclonal antibody; LC represents the light chain of the antibody; HC represents the heavy chain of the antibody; DAR1 represents a conjugate containing one light chain / heavy chain of an antibody and one bioactive molecule; DAR2 represents a conjugate containing one light chain / heavy chain of an antibody and two bioactive molecules; DAR3 represents a conjugate containing one light chain / heavy chain of an antibody and three bioactive molecules; DAR4 represents a conjugate containing one light chain / heavy chain of an antibody and four bioactive molecules; Glycoform represents the structure of glycans composed of two heavy chains; G0F represents fucosylation lacking galactosylation. The following mAb, LC, HC, DAR1, DAR2, DAR3, DAR4, and G0F in this specification are as described above.

[0331] As can be seen from Figures 1 - 3, the molecular weights of both the light and heavy chains of the antibody are both TL003 and KA ​​​​​​​​​After being looped, it changes, where the light chain is coupled to one bioactive molecule, and the heavy chain is coupled to three bioactive molecules. Therefore, it can be inferred that the DAR of the antibody against the bioactive molecule was 8.

[0332] Example 49: Determination of the molecular weight of BT001004 by LC-MS The molecular weight of BT001004 obtained by coupling was analyzed by LC-MS. LC conditions: Liquid chromatography column: ACQUITU UPLC (registered trademark) Prot ein BEH C18 1.7μm, 2.1mm×100mm; Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2 % H2O / 98% ACN; Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Sample size : 1 μg;

Table 9

[0333] The theoretical and measured molecular weights of BT001004

Table 10

[0334] ​​As can be seen from FIGS. 4 to 6, in BT001004, the light chain of the antibody was coupled to 0 to 1 bioactive molecule (LC and DAR1 accounted for 14% and 86% respectively), and the heavy chain was coupled to 1 to 3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 13%, 19% and 68% respectively). Therefore, the DAR of the antibody to the bioactive molecule can be calculated to be 7.0.

[0335] Example 50: Determination of the molecular weight of BT001012 by LC-MS The same method as described in Example 48 was adopted, and the results are shown in FIGS. 10 and 11.

[0336] The theoretical and measured molecular weights (calculated from the main glycoform G0F) of the light and heavy chains of BT001012 obtained by coupling TL024 and the antibody are shown in the following table .

Table 11

[0337] As can be seen from FIGS. 10 and 11, in BT001012, the light chain of the antibody was coupled to 0 to 1 toxin (LC and DAR1 accounted for 12.9% and 87.1% respectively ), and the heavy chain was coupled to 1 to 3 toxins (DAR1, DAR2 and DAR3 accounted for 3.4%, 10.8% and 75.8% respectively). Therefore, the DAR of the antibody to the toxin can be calculated to be 7.0.

[0338] Example 51: Determination of the molecular weight of BT001013 by LC-MS The same method as described in Example 48 was adopted, and the results are shown in FIGS. 12 and 13.

[0339] ​The light chain of BT001013 obtained by coupling TL048 and an antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the table below as follows.

Table 12

[0340] As can be seen from Figures 12 and 13, in BT001013, the light chain of the antibody was coupled with 0 to 1 toxin (LC and DAR1 accounted for 6.8% and 93.2% respectively) , and the heavy chain was coupled with 1 to 4 toxins (DAR1, DAR2, DAR3 and DAR4 accounted for 12.8%, 12.8%, 64.9% and 9.5% respectively). Therefore, the DAR of the antibody against the toxin can be calculated as 7.3 .

[0341] Example 52: Determination of the molecular weight of BT001018 by LC-MS The same method as described in Example 48 was adopted, and the results are shown in Figures 14 and 15

[0342] The light chain of BT001018 obtained by coupling TL030 and an antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the table below as follows.

Table 13

[0343] As can be seen from Figures 14 and 15, in BT001018, the light chain of the antibody was coupled with 0 to 1 toxin (LC and DAR1 accounted for 55.3% and 44.7% respectively) , and the heavy chain was coupled with 1 to 3 toxins (DAR1, DAR2, DAR3 and D AR3) ​​AR4 accounted for 19.6%, 23.3% and 49.6% respectively). Therefore, the DAR of the antibody against the toxin can be calculated as 5.2.

[0344] Example 53: Determination of the molecular weight of BT001021 by LC-MS The molecular weight of the coupled BT001021 was analyzed by LC-MS. LC conditions: Liquid chromatography column: ACQUITU UPLC (registered trademark) Prot ein BEH C4 1.7μm, 2.1mm × 100mm; Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2 % H2O / 98% ACN; Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Sample sa mples: 1 μg; [Table 14] MS conditions: Mass spectrometer model: Triple TOF 5600+; GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP 300; CE10 m / z600 - 5000; The results are shown in Figures 16 and 17.

[0345] The light chain and the theoretical and measured molecular weights of the heavy chain of BT001021 obtained by coupling TL033 and the antibody (calculated based on the main glycoform G0F) are shown in the following table below. [Table 15]

[0346] As can be seen from Figures 16 and 17, in BT001021, the light chain of the antibody has 0 to 1 toxin coupled to the moiety (LC and DAR1 accounted for 4.5% and 95.5% respectively) , the heavy chain was coupled to 1 to 3 toxins (DAR1, DAR2, DAR3 and DA R4 accounted for 15.3%, 17.6% and 67.1% respectively). Therefore, the DAR of the antibody against the toxin can be calculated as 6.9.

[0347] Example 54: Determination of the molecular weight of BT001023 by LC-MS The same method as described in Example 48 was adopted, and the results are shown in Figures 18 and 19.

[0348] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light chain and heavy chain of BT001023 obtained by coupling TL035 and the antibody are shown in the table below as follows.

Table 16

[0349] As can be seen from Figures 18 and 19, in BT001023, the light chain of the antibody was coupled to 0 to 1 toxin (LC and DAR1 accounted for 15% and 85% respectively), and the heavy chain was coupled to 0 to 3 toxins (HC, DAR1, DAR2 and DAR3 accounted for 6.7%, 16.7%, 12.7% and 63.9% respectively). Therefore, the DAR of the antibody against the toxin can be calculated as 6.4.

[0350] Example 55: Determination of the molecular weight of BT001040 by LC-MS The molecular weight of BT001040 obtained by coupling was analyzed by LC-MS . Liquid chromatography column: Thermo MabPacTM RP 4μm , 3.0 mm × 100 mm Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2 % H2O / 98% ACN Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Sample sa mple: 1 μg

Table 17

[0351] The light chain of BT001040 obtained by coupling TL049 and the antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the table below as follows.

Table 18

[0352] As can be seen from Figures 20 and 21, in BT001040, the light chain of the antibody is coupled to 0 to 1 bioactive molecule (LC and DAR1 account for 4.9% and 95.1% respectively) ), and the heavy chain is coupled to 1 to 4 bioactive molecules (DAR1, DAR2, D AR3 and DAR4 account for 16.5%, 14.3%, 52.6% and 16.6% respectively) ). Therefore, the DAR of the antibody to the bioactive molecule can be calculated as 7.3.

[0353] Example 56: Determination of the molecular weight of BT001041 by LC-MS The same method as described in Example 55 was adopted, and the results are shown in Figures 22 and 23.

[0354] ​ The light chain of BT001041 obtained by coupling TL050 and an antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the table below as follows.

Table 19

[0355] As can be seen from Figures 22 and 23, in BT001041, the light chain of the antibody is coupled with 0 to 1 biologically active molecule (LC and DAR1 accounted for 10.5% and 89.5% respectively), and the heavy chain is coupled with 1 to 4 biologically active molecules (DAR1, DAR2 , DAR3 and DAR4 accounted for 21.3%, 14.8%, 57.9% and 6.0% respectively). Therefore, the DAR of the antibody to the biologically active molecule can be calculated as 6.8.

[0356] Example 57: Determination of the molecular weight of BT001042 by LC-MS The same method as described in Example 55 was adopted, and the results are shown in Figures 24 and 25.

[0357] The light chain of BT001042 obtained by coupling TL051 and an antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the table below as follows.

Table 20

[0358] As can be seen from Figures 24 and 25, in BT001042, the light chain of the antibody is coupled with 0 to 1 biologically active molecule (LC and DAR1 accounted for 14.9% and 85.1% respectively), and the heavy chain is coupled with 1 to 3 biologically active molecules (DAR1, DAR2 and DAR3 accounted for 19.7%, 9.4% and 70.9% respectively). Therefore, the DAR of the antibody against the bioactive molecule can be calculated as 6.7.

[0359] Example 58: Determination of the molecular weight of BT001043 by LC-MS The same method as described in Example 55 was adopted, and the results are shown in Figures 26 and 27.

[0360] The light chain of BT001043 obtained by coupling TL052 and the antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the following table as follows. [Table 21]

[0361] As can be seen from Figures 26 and 27, in BT001043, the light chain of the antibody was coupled with 0 to 1 bioactive molecule (LC and DAR1 accounted for 9.1% and 90.9% respectively), and the heavy chain was coupled with 1 to 3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 20.1%, 11.4% and 68.4% respectively). Therefore, the DAR of the antibody against the bioactive molecule can be calculated as 6.8.

[0362] Example 59: Determination of the molecular weight of BT001044 by LC-MS The same method as described in Example 55 was adopted, and the results are shown in Figures 28 and 29.

[0363] The light chain of BT001044 obtained by coupling TL053 and the antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the following table as follows. [Table 22]

[0364] As can be seen from Figures 28 and 29, in BT001044, the light chain of the antibody is 0 to 1 nucleotide. The LC and DAR1 were coupled to bioactive molecules at 23.0% and 77.0%, respectively. The heavy chains were coupled to one to three biologically active molecules (DAR1, DAR2 and DAR3 accounted for 19.4%, 11.4%, and 69.3%, respectively). , the DAR of the antibody to the bioactive molecule can be calculated to be 6.5.

[0365] Example 60: Determination of the molecular weight of BT001046 by LC-MS A method similar to that described in Example 55 was employed, and the results are shown in Figures 30 and 31.

[0366] Light chain of BT001046 obtained by coupling TL055 and antibody The theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the table below. did. [Table 23]

[0367] As can be seen from Figures 30 and 31, in BT001046, the light chain of the antibody is 0 to 1 nucleotide. The LC and DAR1 were coupled to bioactive molecules at 33.8% and 66.2%, respectively. The heavy chains were coupled to 0 to 3 bioactive molecules (DAR0, DAR1 DAR2 and DAR3 accounted for 21.9%, 6.1%, 9.6% and 62.3%, respectively. Therefore, the DAR of the antibody against the bioactive molecule can be calculated to be 5.6.

[0368] Example 61: Determination of the molecular weight of BT001047 by LC-MS The same method as described in Example 55 was adopted, and the results are shown in Figures 32 and 33.

[0369] The light chain of BT001047 obtained by coupling TL056 and the antibody and the theoretical and measured molecular weights of the heavy chain (calculated based on the main glycoform G0F) are shown in the following table below.

Table 24

[0370] As can be seen from Figures 32 and 33, in BT001047, the light chain of the antibody is coupled with 0 to 1 bioactive molecule (LC and DAR1 accounted for 13.7% and 86.3% respectively), and the heavy chain is coupled with 1 to 3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 22.2%, 13.5% and 64.3% respectively). Therefore , the DAR of the antibody to the bioactive molecule can be calculated to be 6.6.

[0371] Example 62: Size exclusion chromatography analysis The coupling reaction was monitored by SEC-HPLC, and the conjugate was tested by SEC as follows. Chromatography conditions: Liquid chromatography column: TOSOH TSKgel SuperSW m Ab, 4μm, 7.8mm × 300mm; Mobile phase: 100 mmol / L Na2HPO4, 100 mmol / L NaCl, 5 % isopropanol, pH 7.0; Flow rate: 0.5 ml / min; Detection wavelength: 280 nm; Column temperature: room temperature; Sample chamber temperature : 8°C; Sample size: 30 μg; Isocratic operation: 30 minutes.

[0372] The SEC chromatogram of BT001002 obtained by coupling TL003 with the antibody and the molecular weight marker SEC chromatogram are shown in FIGS. 7 and 8, respectively. According to the molecular weight marker, the molecular weight of the main peak of the coupling product is about 150 kD. That is, in BT001002 obtained by coupling TL003 with the antibody, the light chain and the heavy chain are not dissociated, and it is confirmed that the antibody still maintains its complete structure.

[0373] The SEC chromatogram of BT001004 obtained by coupling TL019 with the antibody is shown in FIG. 9. According to the retention time and peak area ratio in SEC, the molecular weight of the main coupling product is about 150 kD. That is, it is confirmed that BT001004 obtained by coupling TL019 with the antibody still maintains the complete structure of the antibody.

[0374] The SEC chromatogram of BT001012 obtained by coupling TL024 with the antibody is shown in FIG. 34. According to the retention time and peak area ratio in SEC, the molecular weight of the main coupling product is about 150 kD. That is, it is confirmed that BT001012 obtained by coupling TL024 with the antibody still maintains the complete structure of the antibody.

[0375] The SEC chromatogram of BT001013 obtained by coupling TL048 with the antibody is shown in FIG. 35. According to the retention time and peak area ratio in SEC, the molecular weight of the main coupling ​​​​​The molecular weight of the ring product is approximately 150 kD, that is, the coupling of TL048 and the antibody The obtained BT001013 by the ring still maintains the complete structure of the antibody, which is confirmed.

[0376] The SEC chromatogram of BT001018 obtained by the coupling of TL030 and the antibody is shown in Figure 36. According to the retention time and peak area ratio in SEC, the main coupling The molecular weight of the ring product is approximately 150 kD, that is, the coupling of TL030 and the antibody The obtained BT001018 by the ring still maintains the complete structure of the antibody, which is confirmed.

[0377] The SEC chromatogram of BT001021 obtained by the coupling of TL033 and the antibody is shown in Figure 37. According to the retention time and peak area ratio in SEC, the main coupling The molecular weight of the ring product is approximately 150 kD, that is, the coupling of TL033 and the antibody The obtained BT001021 by the ring still maintains the complete structure of the antibody, which is confirmed.

[0378] The SEC chromatogram of BT001023 obtained by the coupling of TL035 and the antibody is shown in Figure 38. According to the retention time and peak area ratio in SEC, the main coupling The molecular weight of the ring product is approximately 150 kD, that is, the coupling of TL035 and the antibody The obtained BT001023 by the ring still maintains the complete structure of the antibody, which is confirmed.

[0379] The SEC chromatogram of BT001042 obtained by the coupling of TL051 and the antibody The togram is shown in Figure 39. According to the retention time and peak area ratio in SEC, the main cut The molecular weight of the pring product is about 150 kD, that is, BT001042 obtained by the coupling of TL051 and the antibody is still confirmed to maintain the complete structure of the antibody.

[0380] The SEC chromatogram of BT001043 obtained by the coupling of TL052 and the antibody is shown in Figure 40. According to the retention time and peak area ratio in SEC, the main cut The molecular weight of the pring product is about 150 kD, that is, BT001043 obtained by the coupling of TL052 and the antibody is still confirmed to maintain the complete structure of the antibody.

[0381] The SEC chromatogram of BT001044 obtained by the coupling of TL053 and the antibody is shown in Figure 41. According to the retention time and peak area ratio in SEC, the main cut The molecular weight of the pring product is about 150 kD, that is, BT001044 obtained by the coupling of TL053 and the antibody is still confirmed to maintain the complete structure of the antibody.

[0382] The SEC chromatogram of BT001046 obtained by the coupling of TL055 and the antibody is shown in Figure 42. According to the retention time and peak area ratio in SEC, the main cut The molecular weight of the pring product is about 150 kD, that is, BT001046 obtained by the coupling of TL055 and the antibody is still confirmed to maintain the complete structure of the antibody.

[0383] ​​​​The SEC chromatogram of BT001047 obtained by coupling TL056 with an antibody is shown in Figure 43. According to the retention time and peak area ratio in SEC, the molecular weight of the main coupling product is about 150 kD, that is, it is confirmed that BT001047 obtained by coupling TL056 with an antibody still maintains the complete structure of the antibody .

[0384] Example 63: Test on the inhibitory effect of bioactive molecules and antibody-drug conjugates on cell activity in vitro First, tumor cells MDA-MB-468 (Trop-2 positive cell line) and HCC180 6 (Trop-2 positive cell line) were cultured. The bioactive molecules and ADC molecules disclosed in the present disclosure were co-cultured with the tumor cells, and then CCK8 reagent (Dojindo Molecular Technologies, Inc., Cat: CK04, Lot: JJ744) was added. The activity of dehydrogenase in mitochondria was tested by the readings from a microplate reader (manufacturer: Molecular Devices, model: SpectraMax M2) (the detection wavelength was 450 nm), and the inhibitory effect of ADC on cell proliferation was evaluated. The sources of the tumor cells are shown in Table 1 .

Table 25

[0385] In vitro cell activity test: Bioactive molecules or ADCs were diluted in the corresponding test medium (containing 2% FBS ). Tumor cells were trypsinized by the conventional method, collected, counted, and then resuspended in the corresponding test medium (containing 2% FBS ). ​​It was turbid. The diluted bioactive molecule or ADC was added to a 96-well plate, and then the cells were added. 20 μL of CCK8 reagent was added to each well and reacted for 4 hours, and the readings (the detection wavelength was 450 nm) were taken from a microplate reader. The experimental conditions and test results are shown in Tables 2 and 3.

[0386]

Table 26

[0387] The test results showed that all of the bioactive molecules had a killing effect on tumor cells. .

[0388]

Table 27

[0389] The test results showed that the ADC molecules obtained by the novel coupling method had a killing effect on tumor cells. This indicates that the ADC formed by the novel coupling method has a killing effect on tumor cells and that the novel coupling method is feasible in the synthesis of ADC molecules. .

[0390] Example 64 : Pharmacodynamic tests of antibody-drug conjugates and bioactive molecules in vivo Test drug Drug name, source and preparation method: BT001021, the liquid aliquot was stored at -20 °C at a concentration of 5.44 mg / ml and diluted to the dose with physiological saline before use to obtain the test solution; Immu-132 (prepared according to Example 2 of International Publication No. WO2015 / 012904A2, also described as IMMU-132), the liquid aliquot was stored at -20 °C at 1 3. Store at a concentration of 158 mg / ml and dilute to the required dose with saline before use to obtain the test solution. Got; Solid powder of T-030 was diluted with 100% DMSO (Sigma) at a concentration of 5.2 mg / mL. The solution was prepared and stored in liquid aliquots at -20°C and diluted to the desired dose with saline prior to use. Dilution was performed to obtain test solutions; SN-38 (also referred to as SN38) solid powder was dissolved in 100% DMSO (Sigma). A solution of 3.23 mg / ml was prepared using 100% ethanol and stored at -20°C in liquid aliquots. The test solutions were obtained by pre-dilution with saline to the dose. NOTE: Toxin was prepared and administered at an equimolar ratio to the ADC sample.

[0391] The structures of T-030, SN-38 and Immu-132 are as follows: [ka]

[0392] Experimental animals and cell lines Balb / c-nu mouse (Beijing Vital River Labora tory Animal Technology Co., Ltd., Production License Number: SCXK(Beijing)2016-0011); gastric cancer cell line NCI-N87(AT CC), breast cancer cell line HCC1806 (COBIOER Nanjing).

[0393] Experimental grouping and evaluation method 100~200mm 3 Tumor-bearing mice (6 mice / group) with tumor volumes of The number of groups was determined according to the number of samples. The administration volume was 10 ml / kg. The administration route was intravenous injection into the tail vein. The mice were administered the drug twice a week, and the tumor diameter was measured with a caliper. The tumor volume was calculated based on the following formula: V = 0.5a × b 2 [where a and b represent the major axis and minor axis of the tumor, respectively. The death of the animals was observed and recorded daily.

[0394] The tumor growth inhibition rate TGI (%) was calculated from the following formula to evaluate the tumor inhibitory effect of the antibody-drug conjugate. TGI (%) = [1 - (V Tend - V Tstart ) / (V Cend - V Csta rt )] × 100% [where V Tend : the average tumor volume at the end of the experiment in the treatment group V Tstart : the average tumor volume at the start of administration in the treatment group V Cend : the average tumor volume at the end of the experiment in the control group V Cstart : the average tumor volume at the start of administration in the control group]

[0395] In the following Experimental Examples 1 and 2, the inhibition of the antibody conjugate BT001021 on tumor growth in tumor-bearing mice constructed by subcutaneous xenografts of human tumor cells was evaluated. Specifically in Experimental Examples 1 and 2, the tumor-bearing mouse model was constructed by subcutaneous xenografts of the human gastric cancer cell line NCI-N87 or the human triple-negative breast cancer cell line HCC1806. After the tumor volume reached approximately 100 mm the mice were randomly grouped and BT001021 was intravenously administered twice a week for a total of 6 times. The changes in tumor volume and body weight of the animals were measured twice a week to evaluate the efficacy (tumor inhibitory effect) of the antibody-drug conjugate on tumor-bearing mice. 3

[0396] Experimental Example 1 ​​​. Inhibition of NCI-N87 by Antibody-Drug Conjugates and Bioactive Molecules Experimental Method: NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c-nu mice to establish a gastric cancer model. When the average tumor volume reached approximately 90 mm 3 the mice were randomly grouped into a physiological saline group, a BT001021 (3 mg / kg, IV, BIW×3W) group, a positive drug Immu -132 (3 mg / kg, IV, BIW×3W) group, a T030 group, and an SN38 group, and then the corresponding drugs were intravenously injected into the tail vein twice a week for a total of six times. After administration, the tumor volume and body weight of the mice were regularly observed and measured. The specific results are shown in Tables 4, Figures 4

[0397] Conclusion: In the experimental example, a subcutaneous xenograft model of human gastric cancer was established using the human gastric cancer cell line NCI-N87, and the efficacy of BT00102 1 in the NCI-N87 human gastric cancer-bearing mouse model was evaluated.

[0398] The experimental results showed that BT001021 (3 mg / kg, IV, BIW×3W) significantly inhibited the tumor growth of the xenograft model mice of NCI-N8 7 gastric cancer, and tumor regression could occur at the end of administration, indicating that the anti-tumor activity was superior to that of the positive control Immu-132. During the observation period, neither animal death nor significant animal weight loss occurred in all treatment groups, indicating that

[0399] BT001021 had no significant toxicity.

Table 28

[0400] Experimental Example 2 . Inhibition of HCC1806 by Antibody-Drug Conjugates Experimental Method: HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC1806 cells in the exponential growth phase were collected, resuspended in PBS at an appropriate concentration, and subcutaneously inoculated into female Balb / c-nu mice to establish a breast cancer model . When the average tumor volume reached approximately 130 mm 3 , the mice were randomly grouped into a physiological saline group, a BT001021 (10 mg / kg, IV, BIW×3W) group, and a positive drug Imm mu-132 (10 mg / kg, IV, BIW×3W) group, and then the corresponding drugs were intravenously injected into the tail vein twice a week for a total of 5 times. After administration, the tumor volume of the mice was regularly observed and measured. The specific results are shown in Table 5 and Figure 46.

[0401] Conclusion: In the experimental example, a subcutaneous xenograft model of human breast cancer was established using the human breast cancer cell line HCC1806, and the efficacy of BT00102 1 in the HCC1806 human breast cancer-bearing mouse model was evaluated.

[0402] The experimental results showed that BT001021 (10 mg / kg, IV, BIW×3W) could significantly inhibit the tumor growth of xenograft model mice with HCC18 06 breast cancer, and its antitumor activity was superior to that of the positive control Immu-132.

[0403]

Table 29

[0404] According to Tables 4 and 5 and Figures 44 to 46, the antibody drug BT001021 of the present invention is NCI- BT001021 was able to significantly inhibit tumor growth in the N87 mouse model. It was significantly superior to Immu-132 at the dose level, with no significant weight loss or significant drug toxicity. In the HCC1806 mouse model, the dose was adjusted due to the high tumor aggressiveness. When increased to 10 mg / kg, Immu-132 showed no significant inhibitory activity. However, BT001021 was able to significantly inhibit tumor growth. 1 showed high efficacy and excellent safety.

[0405] In the subcutaneous xenograft models of Experimental Examples 1 and 2, the antitumor activity of BT001021 was The antitumor activity was significantly superior to that of Immu-132 at the same dose, which was consistent with that of BT00. 1021 has the potential to treat solid tumors and is clinically seen in more patients than Immu-132 These results suggest that the use of GABA in the treatment of chronic conditions may be expected to be beneficial for patients with chronic conditions.

[0406] Experimental Example 3. Inhibition of HCC827 by antibody-drug conjugates Using Experimental Example 3, we investigated the effect of subcutaneous xenograft human tumor cells of HCC827 non-small cell lung cancer. BT001021 and BT001035 on the proliferation of tumor-bearing mice models established using Specifically, the study evaluated the inhibitory effect of human non-small cell lung cancer (NSCLC) on tumor-bearing mice. The tumor volume was approximately 100 mm. 3 To After the mice were grown, they were randomly assigned to groups and administered BT001021 and BT001035 twice weekly. It was administered intravenously a total of 6 times. Subsequently, changes in tumor volume and body weight of the animals were measured every two weeks to calculate the efficacy (tumor inhibitory effect) of BT001021 and BT001035 on tumor-bearing mice. The HCC827 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 °C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended at an appropriate concentration in PBS, and subcutaneously inoculated into female Balb / c-nu mice to construct a xenograft model of lung cancer. When the average tumor volume reached approximately 80 mm

[0407] Experimental method: The HCC827 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 °C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended at an appropriate concentration in PBS, and subcutaneously inoculated into female Balb / c-nu mice to construct a xenograft model of lung cancer. The HCC827 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 °C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended at an appropriate concentration in PBS, and subcutaneously inoculated into female Balb / c-nu mice to construct a xenograft model of lung cancer. The HCC827 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 °C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended at an appropriate concentration in PBS, and subcutaneously inoculated into female Balb / c-nu mice to construct a xenograft model of lung cancer. The HCC827 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 °C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended at an appropriate concentration in PBS, and subcutaneously inoculated into female Balb / c-nu mice to construct a xenograft model of lung cancer. 3 When the average tumor volume reached approximately 80 mm The mice were randomly grouped into a physiological saline group, a positive drug Immu-132 (10 mg / kg, IV, BIW×3W) group, a BT001021 (10 mg / kg, IV, BIW×3W) group, and a BT001035 ( 10 mg / kg, IV, BIW×3W) group. Subsequently, the corresponding drugs were injected into the tail vein twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The results are shown in Table 6, Figure 47A, and Figure 47B. 10 mg / kg, IV, BIW×3W) group. Subsequently, the corresponding drugs were injected into the tail vein twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The results are shown in Table 6, Figure 47A, and Figure 47B. 10 mg / kg, IV, BIW×3W) group. Subsequently, the corresponding drugs were injected into the tail vein twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The results are shown in Table 6, Figure 47A, and Figure 47B. 10 mg / kg, IV, BIW×3W) group. Subsequently, the corresponding drugs were injected into the tail vein twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The results are shown in Table 6, Figure 47A, and Figure 47B.

[0408] Conclusion: The experimental results showed that BT001021 and BT001035 significantly inhibited the tumor growth of xenograft model mice with HCC827 non-small cell lung cancer, and tumor regression could occur at the end of administration. The anti-tumor activity was superior to that of the positive control Immu-132 group. During the observation period, neither animal death nor significant animal weight loss occurred in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated. The experimental results showed that BT001021 and BT001035 significantly inhibited the tumor growth of xenograft model mice with HCC827 non-small cell lung cancer, and tumor regression could occur at the end of administration. The anti-tumor activity was superior to that of the positive control Immu-132 group. During the observation period, neither animal death nor significant animal weight loss occurred in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated. The experimental results showed that BT001021 and BT001035 significantly inhibited the tumor growth of xenograft model mice with HCC827 non-small cell lung cancer, and tumor regression could occur at the end of administration. The anti-tumor activity was superior to that of the positive control Immu-132 group. During the observation period, neither animal death nor significant animal weight loss occurred in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated. The experimental results showed that BT001021 and BT001035 significantly inhibited the tumor growth of xenograft model mice with HCC827 non-small cell lung cancer, and tumor regression could occur at the end of administration. The anti-tumor activity was superior to that of the positive control Immu-132 group. During the observation period, neither animal death nor significant animal weight loss occurred in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated. The experimental results showed that BT001021 and BT001035 significantly inhibited the tumor growth of xenograft model mice with HCC827 non-small cell lung cancer, and tumor regression could occur at the end of administration. The anti-tumor activity was superior to that of the positive control Immu-132 group. During the observation period, neither animal death nor significant animal weight loss occurred in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated. The experimental results showed that BT001021 and BT001035 significantly inhibited the tumor growth of xenograft model mice with HCC827 non-small cell lung cancer, and tumor regression could occur at the end of administration. The anti-tumor activity was superior to that of the positive control Immu-132 group. During the observation period, neither animal death nor significant animal weight loss occurred in any of the treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated.

[0409]

Table 30

[0410] According to Table 6, Figure 47A, and Figure 47B, both BT001021 and BT001035 showed significant inhibitory activity against tumor growth during the evaluation period, but they were significantly superior to the inhibitory activity of Immu-132 at the same dose. During the treatment, no significant weight loss or significant drug toxicity was observed in all groups. The results indicated that both BT001021 and BT00 1035 had excellent antitumor activity. In the subcutaneous xenograft model, the antitumor activities of both BT001021 and BT001035 were significantly superior to the antitumor activity of Immu-132 at the same dose, suggesting that both BT001

[0411] 021 and BT001035 have the potential to treat solid tumors and may be beneficial to more patients clinically than Immu-132.

[0412] Experimental Example 4 . Inhibition of NCI-N87 by antibody-drug conjugate Using Experimental Example 4, the inhibition of the antibody-drug conjugate BT001036 on tumor growth of tumor-bearing mice constructed by subcutaneous xenografts of human tumor cells was evaluated. Specifically, in the experiment, a tumor-bearing mouse model was constructed by subcutaneous xenograft of the human gastric cancer cell line NCI-N87. After the tumor volume reached approximately 140 mm 3 3 3 001036 was intravenously administered twice a week for a total of 6 times. The changes in tumor volume and animal body weight were measured twice a week to evaluate the efficacy of the antibody-drug conjugate on tumor-bearing mice (tumor inhibition The harmful effects were evaluated.

[0413] Experimental method: NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected and resuspended in PBS at an appropriate concentration, and then subcutaneously inoculated into female Balb / c-nu mice to establish a xenograft model of gastric cancer . When the average tumor volume reached approximately 140 mm 3 , the mice were randomly grouped according to tumor size into a physiological saline group, a BT001036 (1.5 mg / kg, IV, BIW×3W) group, and a B T001036 (3 mg / kg, IV, BIW×3W) group. Subsequently, the corresponding drugs were intravenously injected into the tail vein twice a week for a total of six times. After administration, the tumor volume and body weight of the mice were regularly observed and measured. The specific results are shown in Table 7, Figures 48A and 48B .

[0414]

Table 31

[0415] Conclusion: In the experimental example, a subcutaneous xenograft model of human gastric cancer was established using a subcutaneous xenograft of the human gastric cancer cell line NCI-N87, and the efficacy of B T001036 in the NCI-N87 human gastric cancer-bearing mouse model was evaluated.

[0416] The experimental results showed that both the high and low doses of BT001036 (1.5 mg / kg, 3 mg / kg ) had excellent antitumor activity, significantly inhibited the tumor growth in the xenograft model mice of NCI-N87 gastric cancer, and tumor regression could occur at the end of administration. During the observation period, all ​​​In all treatment groups, animal death and significant animal weight loss did not occur, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated.

[0417] Experimental Example 5 . Inhibition of MDA-MB-231 by antibody-drug conjugate Using Experimental Example 5, the inhibitory effect of BT001021 on the growth of a tumor-bearing mouse model constructed with subcutaneous xenograft human tumor cells of MDA-MB-231 breast cancer was evaluated. Specifically, in the experiment, a tumor-bearing mouse model was constructed with a subcutaneous xenograft of the human breast cancer cell line MDA-MB-231. After the tumor volume reached approximately 130 mm 3, the mice were randomly 3 grouped, and BT001021 was intravenously administered 2 times a week for a total of 6 times. Subsequently, the changes in tumor volume and animal body weight were measured, and the efficacy (tumor inhibitory effect) of BT001021 on the tumor-bearing mice was calculated.

[0418] Experimental method: NCI-MDA-MB-231 cells were cultured in RPMI1640 culture medium containing 10% fetal bovine serum at 37 °C and 5% CO2. MDA-MB-231 cells in the exponential growth phase were collected, resuspended in PBS at an appropriate concentration, and subcutaneously inoculated into female Balb / c-nu mice to construct a xenograft model of breast cancer. When the average tumor volume reached approximately 130 mm3, the mice were randomly grouped into a physiological saline group and a BT001021 (3 mg / kg) group according to tumor size, and then the corresponding drugs were injected into the tail vein 2 times a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were regularly observed and measured. The results are shown in Table 8, Figure 49A, and Figure 49B.

[0419] Conclusion: The results showed that BT001021 inhibited the tumors of the mice in the xenograft model of MDA-MB-231 breast cancer. It was shown that tumor growth was significantly inhibited and tumor regression could occur at the end of administration. During the observation period, in all treatment groups, neither animal death nor significant animal weight loss occurred, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated.

[0420]

Table 32

[0421] In the subcutaneous xenograft model, BT001021 had significant antitumor activity. During the observation period, in all treatment groups, neither animal death nor significant animal weight loss occurred, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all the drugs evaluated.

[0422] Example 65: In vivo Pharmacokinetics Study of Antibody-Drug Conjugates and Bioactive Molecules Using Experimental Example 6, the in vivo pharmacokinetics of antibody-drug conjugates and bioactive molecules were evaluated. Specifically, in the experiment, a tumor-bearing mouse model was constructed by subcutaneous xenograft of human gastric cancer cell line NCI-N87 into Balb / c-nu mice. After the tumor volume reached 100 ~200 mm 3 , the mice were randomly grouped and a single dose of BT001021 and T-030 was administered intravenously. The concentrations of T-030 in tumor tissue and serum were determined to evaluate the in vivo pharmacokinetics of antibody conjugate BT001021 and bioactive molecule T-030 in tumor-bearing mice.

[0423] Test Drug Drug Name and Preparation Method: ​​​BT001021, the liquid aliquot was stored at -20 °C at a concentration of 20 mg / ml and diluted with physiological saline to the desired dose before use to obtain a test solution; T-030 was prepared to 1 mg / ml with dimethyl sulfoxide and diluted with physiological saline to the desired dose to obtain a test solution. Experimental animals and cell lines: Balb / c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: S CXK(Beijing)2016 - 0011); gastric cancer cell line NCI-N87 (ATCC ). Experimental groups and evaluation methods: Tumor-bearing mice (4 mice / group) with a tumor volume of 100 - 200 mm 3 were randomly grouped (the number of groups was determined according to the number of samples), and the administration route was a single intravenous injection into the tail . .

[0424] Experimental Example 6. In vivo pharmacokinetic study of BT001021 and T-030 in tumor-bearing mice Experimental method: NCI-N87 cells were cultured at 37°C and 5% CO2 in RPMI 1640 culture medium containing 10% heat-inactivated fetal bovine serum. NCI-N87 cells in the exponential growth phase were collected, resuspended at an appropriate concentration in PBS, and subcutaneously inoculated into Balb / c-nu mice to establish a xenograft model of gastric cancer. When the average tumor volume reached approximately 100 - 200 mm3, the mice were randomly grouped according to tumor size into a physiological saline group, a T-030 (0.23 mg / kg, IV, single dose) group, and a BT001021 (10 mg / kg, IV, single dose) group, and then the corresponding drugs were injected into the tail vein. In the T-030 group, serum and tumor tissues were collected 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, and 72 hours after administration (T-030 was not detected in serum and tumor tissues 72 hours after administration, so serum and tumor tissues were not collected 168 hours after administration). In the BT001021 group, serum and tumor tissues were collected 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 72 hours, and 168 hours after administration, and the concentrations of T-030 in serum and tumor were tested by LC-MS / MS. The specific results are shown in Table 9. The administered dose of T-030 (0.23 mg / kg) was changed to an equimolar dose (10 mg / kg) of BT001021.

[0425]

Table 33

[0426] Conclusion: The AUC of the drug in the tumor and serum of the BT001021 (10 mg / kg) administration group last was 850.1 h×ng / ml and 174.97 h×ng / ml, respectively, while the AUC of the drug in the tumor and serum of the T -030 administration group last was 3.85 h×ng / ml and 5.58 h×ng / ml, respectively. The comparison showed that the exposure of T-0 30 in the BT001021 administration group was significantly increased compared with the exposure in the T-030 administration group. In addition Furthermore, the exposure of the bioactive molecule T-030 in the tumors of the BT001021 administration group was significantly higher than that in the serum. However, the exposure of the bioactive molecules in the serum and tumors of the T-030 administration group was essentially the same, indicating that the antibody-drug conjugate (BT001021) had high tumor tissue targeting properties.

[0427] The C of the bioactive molecule T-030 in the tumors and serum of the BT001021 (10 mg / kg) administration group was 7.82 ng / ml and 11.7 ng / ml, respectively, while the C of the bioactive molecule T-030 in the tumors and serum of the T- 030 administration group was max 1.2 0 ng / ml and 1.81 ng / ml, respectively, indicating that the antibody-drug conjugate (BT0 max 01021) had higher concentrations of the bioactive molecule (T-030) in the tumor tissue and serum.

[0428] The T of the bioactive molecule T-030 in the tumors of the BT001021 (10 mg / kg) administration group was 93.14 hours, while the T of the bioactive molecule T -030 in the tumors of the T-030 administration group was 1 / 2 2.55 hours, indicating that the antibody-drug conjugate (BT0 1 / 2 01021) had a longer half-life in the tumor tissue.

[0429] In conclusion, BT001021 had significant tumor tissue targeting properties and good pharmacokinetic characteristics compared to the corresponding bioactive molecule (T-030).

[0430] Experimental Example 7. Pharmacokinetic study of the antibody-drug conjugates BT001021 and Immu-132 in vivo. In the experiment, a tumor-bearing mouse model was constructed by subcutaneous xenografts of human gastric cancer cell line NCI -N87 in Balb / c-nu mice. When the tumor volume reached 100-200 mm 3 , the mice were randomly grouped, and a single dose of BT001021 and Immu-132 was intravenously administered. The concentrations of the bioactive molecules T-030 and SN-38 corresponding to BT001021 and the concentration of Immu-132 in tumor tissues and sera were measured respectively to evaluate the pharmacokinetics of antibody conjugates BT001021 and Immu-132 in tumor-bearing mice in vivo.

[0431] Test drug Drug name and preparation method: BT001021, liquid aliquots were stored at -20 °C at a concentration of 20 mg / ml and diluted to the desired dose with physiological saline before use to obtain a test solution; Immu-132 was diluted to the desired dose with physiological saline to obtain a test solution. Experimental animals and cell lines: Balb / c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: S CXK(Beijing)2016-0011); gastric cancer cell line NCI-N87 (ATCC ). Experimental groups and evaluation methods: Tumor-bearing mice (4 mice / group) with a tumor volume of 100-200 mm 3 were randomly grouped (the number of groups was determined according to the number of samples), and the administration route was a single intravenous injection into the tail vein

[0432] Experimental method: ​​​​​​NCI-N87 cells were cultured in RPMI 1640 culture medium containing 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS at an appropriate concentration, and subcutaneously inoculated into Balb / c-nu mice to establish a xenograft model of gastric cancer. When the average tumor volume reached approximately 100 - 200 mm3, the mice were randomly grouped according to tumor size into the BT001021 (5 mg / kg, IV, single dose) group and the Immu-132 (5 mg / kg, IV, single dose) group, and then the corresponding drugs were injected into the tail vein. Serum and tumor tissues were collected 2 hours, 24 hours, 48 hours, and 72 hours after administration, respectively, and the concentrations of T-030 or SN-38 in serum and tumors were tested by LC-MS / MS.

[0433]

Table 34

[0434] Conclusion: The AUC of small toxin molecules in tumors and serum of the BT001021 administration group last was 427.2 h×ng / ml and 115.3 h×ng / ml, respectively, while the AUC of small toxin molecules in tumors and serum of the Immu-1 32 administration group last was 116.8 h×ng / ml and 422.7 h×ng / ml, respectively. The C max of small toxin molecules in tumors of the BT001021 administration group was 6.8 ng / ml, while the C max of small toxin molecules in tumors of the Immu-132 administration group was 2.8 ng / ml. The results indicated that BT00 1021 had better tumor tissue targeting, better pharmacokinetic

[0435] Although specific modes for carrying out the present invention have been described in detail, various changes Modifications and substitutions can be made in accordance with all published teachings, and it should be understood by those skilled in the art that such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended patent claims and any equivalents thereof. ​

Claims

1. A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. T - [L １ - (L ２ ) ｍ１ - (L ３ ) ｍ２ - (L ４ ) ｍ３ - E] - G formula (I) [In the formula, T is a fragment of a bioactive molecule, preferably a fragment of a molecule having antitumor bioactivity; L １ is an amino acid, a peptide composed of 2 to 10 amino acids, an oligosaccharide, -(C H ２ ) ｔ１ -, -(CH ２ CH ２ O) ｔ１ -(CH ２ ) ｔ２ -, 【Chemical 1】 selected from; R, R', R １ and R ２ each of which is independently H (hydrogen), D (deuterium), ha Halogen, carboxylic acid group, sulfonic acid group, cyano, C １～６ alkyl, halogenated C １～６ C substituted with alkyl and cyano １～６ alkyl (e.g., -CH ２ CN), C １～６ Alkoxy, C ２～１０ Alkenyl, C ２～１０ Alkynyl, C ３～６ Cycloalkyl, 6-10 membered aryl or 5-12 membered heteroaryl, and each Z １ is independently an amino acid or is a peptide composed of 2 to 10 amino acids, and t １ and t ２ each of which is independent is 0, 1, 2, 3, 4, 5 or 6, and x １ and x ２ each independently is 0, 1, 2, is 3, 4, 5 or 6, and each x ３ is independently 0, 1, 2, 3 or 4, and L １ is L １ of 1 is bonded to T at the position; L ２ is an amino acid, a peptide composed of 2 to 10 amino acids, an oligosaccharide, -(C H ２ ) ｔ１ -, -(CH ２ CH ２ O) ｔ１ -(CH ２ ) ｔ２ -, 【Chemical Formula 2】 is selected from; R ３ , R ４ , R ５ and R ６ each of which is H (hydrogen), D (deuterium) mu), halogen, carboxylic acid group, sulfonic acid group, CN, C １～６ alkyl, halogenated C １～６ C substituted with alkyl and cyano １～６ alkyl, C １～６ alkoxy, C ２ ～１０ Alkenyl, C ２～１０ Alkynyl or C ３～６ Independently selected from cycloalkyl is formed, or R ３ / R ４ 、R ５ / R ６ or R ３ / R ５ is bonded to the carbon atom to which it is attached Together form a 3- to 8-membered ring, and t １ and t ２ each of which is independently 0, 1, 2, 3, 4 , 5 or 6, and y １ and y ２ each of which is independently 0, 1, 2, 3, 4, 5, 6, 7, is 8, 9 or 10, and L ２ is L ２ is in the 1st place of L １ and is bound to L; L ３ is the following group which may be substituted with one or more R ７ : amino, 3- to 8-membered cyclo alkylene, 3- to 8-membered aliphatic heterocyclylene, 6- to 12-membered bridged heterocyclylene, 6- 12-membered spiroheterocyclylene, 6- to 12-membered fused heterocyclylene, 6- to 10-membered arylene selected from 5- to 12-membered heteroarylene or 3- to 8-membered cycloalkylene-W-; W is oxygen or NR ８ wherein R ７ is H (hydrogen), D (deuterium), halogen, =O , CN, carboxyl, sulfonic acid group, C １～６ alkyl, halogenated C １～６ alkyl , C substituted with cyano １～６ alkyl, C １～６ alkoxy, C ２～１０ alkeni Ru or C ２～１０ independently selected from alkynyl, and R ８ is H (hydrogen), D (deuterium) Umu), C １～６ Alkyl, C ３～６ Cycloalkyl, C １～６ Alkoxy or cyano C １～２ Independently selected from alkyl, L ３ is L ３ bonded to L at the 1-position of L ２ ; L ４ is 【Chemical Formula 3】 selected from, and Z ５ is preferably C ２～６ alkenyl, C ２～６ alkynyl, amide a group, sulfuryl, sulfinyl, 6- to 10-membered arylene or 5- to 6-membered heteroarylene are selected; Z ２ is C １～６ alkylene, C ２～１０ alkenylene, C ２～１０ alkyl Nylene, C ３～８ Cycloalkylene, 6- to 10-membered arylene or 5- to 14-membered heteroary selected from REN; R ９ is selected from H (hydrogen) or C １～６ selected from alkyl; Z ３ is present Or C １～６ alkylene, halogenated C １～６ alkylene, or substituted with alkoxy Replaced C １～６ is selected from alkylene; or R ９ and Z ３ are attached thereto together with the nitrogen atom forms a 4- to 8-membered heterocyclyl; α is independently 0, 1, 2 , 3, 4, 5 or 6; L ４ is L ４ is attached to E at the 2-position of L; E is the following group which may be substituted with one or more Rs １２ : pyrimidylene, quinolylyl which may be substituted with is selected from pyrrolo[2,3-d]pyrimidylene; R １２ is H (hydrogen), D (di Ytterbium), halogen, CN, nitro, C １～６ alkyl or halogenated C １～６ A selected independently from alkyl; G is a leaving group for nucleophilic substitution; m １ 、 m ２ and m ３ each of which is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or is 10. ]

2. L １ wherein Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, A sn, a peptide composed of 2 to 5 amino acids, 【Chemical Formula 4】 selected from; R, R', R １ and R ２ each of which is independently H (hydrogen), D (deuterium) lithium), C １～６ alkyl, C ２～１０ alkenyl, C ２～１０ alkynyl or C ３～ ６ is cycloalkyl, and Z １ is Val, Cit, Phe, Lys, D-Val, Le u, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Va l-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Ly s(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala -Asn, where x １ is 0, 1, 2 or 3, and x ３ is 0, 1, 2, 3 or 4 The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. L １ is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, A sn, Cit-Val, Val-Ala, Lys-Val, Val-Lys(Ac), P he-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly -Arg, Ala-Ala-Asn, [Chemical Formula 5] selected from; each of R, R' and R １ is independently H (hydrogen), D (deuterium) ), C １～６ alkyl, C ２～１０ alkenyl, C ２～１０ alkynyl or C ３～６ cyclo is lower alkyl, and Z １ is Val, Cit, Phe, Lys, D-Val, Leu, G ly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-A la, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(A c), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-As is n, and x １ and x ３ each of which is independently 0, 1, 2 or 3, according to claim 1 or 2 The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

4. L １ wherein L is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, 【Chemical Formula 6】 selected from; each of R, R' and R １ is independently H (hydrogen), D (deuterium) ) or C １～４ is alkyl, and Z １ is Cit, Lys, Cit-Val, Cit-A is la, Val-Ala or Lys-Val, and x １ and x ３ each of which is independently 0, 1 or 2, the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

5. L １ wherein L is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, 【Chemical Formula 7】 selected from, the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. L １ is 【Chemical 8】 selected from, the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. L ２ is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, A sn, a peptide composed of 2 to 5 amino acids, 【Chemical Formula 9】 selected from; R ３ , R ４ , R ５ and R ６ each of which is H (hydrogen), D (deuterium) mu), halogen, carboxylic acid group, sulfonic acid group, CF ３ , CN, CH ２ CN, C １～４ a Lukil, C １～４ Alkoxy, C ２～６ Alkenyl, C ２～６ Alkynyl or C ３～６ Shi Independently selected from chloroalkyl, y １ and y ２ each of which is independently 0, 1, 2, 3, 4 is 5, 6, 7 or 8, and L ２ where L ２ is in the 1st place of L １ is coupled to; m １ where m is 0, 1, 2, or 3 The compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

8. L ２ wherein, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, A sn, Val-Cit, Cit-Val, Val-Ala, Lys-Val, Val-L ys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys , Gly-Gly-Arg, Ala-Ala-Asn, 【Chemical 10】 selected from; R ３ , R ４ , R ５ and R ６ each of which is H (hydrogen), D (deuterium) mu), halogen, carboxylic acid group, sulfonic acid group, CF ３ , CN, CH ２ CN, C １～４ a Lukil, C １～４ Alkoxy, C ２～６ Alkenyl, C ２～６ Alkynyl or C ３～６ Shi Independently selected from chloroalkyl, y １ and y ２ each of which is independently 0, 1, 2, 3, 4 is 5, 6, 7 or 8, and L ２ is L ２ is at the 1st position of L １ is coupled to; m １ is 0, 1 or 2, The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. L ２ is 【Chemical 11】 selected from; R ３ , R ４ , R ５ and R ６ each of which is H (hydrogen), D (deuterium) (mu) or C １～４ independently selected from alkyl, y １ and y ２ each of which is independently 0, 1 is 2, 3, 4, 5, 6, 7 or 8, and L ２ is L ２ is in the 1st place of L １ is coupled to; m １ is 1, The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

10. L ２ is 【Chemical 12】 Selected from, the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof.

11. L ２ is 【Chemical 13】 Selected from, the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof.

12. L ３ is optionally substituted with one or more R ７ and represents the following group: amino, 3- to 8-membered cyclo Selected from alkylene, 3- to 8-membered aliphatic heterocyclylene, 6- to 12-membered bridged heterocyclylene, 6- to 12-membered spiroheterocyclylene, 6- to 12-membered fused heterocyclylene, 6- to 10-membered arylene , 5- to 12-membered heteroarylene or 3- to 8-membered cycloalkylene-W-; W is oxygen or NR ８ where R ７ is H (hydrogen), D (deuterium), halogen, =O , CF ３ , CN, CH ２ CN, carboxyl, sulfonic acid group, C １～４ alkyl, C １～ ４ alkoxy, C ２～６ alkenyl or C ２～６ independently selected from alkynyl; preferably is, or said 3- to 8-membered aliphatic heterocyclylene, 6- to 12-membered bridged heterocyclylene, 6- to 1 2-membered spiroheterocyclylene or 6- to 12-membered fused heterocyclylene has one or more nitrogen atoms; preferably, said 3- to 8-membered aliphatic heterocyclylene, 6- to 12-membered bridged hetero cyclylene, 6- to 12-membered spiroheterocyclylene or 6- to 12-membered fused heterocyclylene has one or more quaternized nitrogen atoms; preferably, said 3- to 8-membered aliphatic heterocycli lene, 6- to 12-membered bridged heterocyclylene, 6- to 12-membered spiroheterocyclylene or 6- to 1 2-membered fused heterocyclylene has one or more nitrogen atoms, and at least one nitrogen atom is replaced by =O; R ８ is H (hydrogen), D (deuterium), C １～６ alkyl Ru, C ２～６ Alkenyl, C ３～６ Alkynyl, C ３～６ Cycloalkyl, C １～６ Al Coxy or cyano C １～２ independently selected from alkyl; m ２ where m is 0, 1, 2, or 3 The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof.

13. L ３ is optionally substituted with one or more R ７ and is the following group: amino, 3- to 6-membered aliphatic Selected from heterocyclylene or 5- to 10-membered heteroarylene; R ７ is H (hydrogen), D (deuterium), halogen, =O, CF ３ , CN, CH ２ CN, carboxyl, s Rufonic acid group, C １～４ alkyl, C １～４ alkoxy, C ２～６ alkenyl or C ２～６ Independently selected from alkynyl; preferably, said 3- to 6-membered aliphatic heterocyclylene has 1 or more nitrogen atoms; preferably, said 3- to 6-membered aliphatic heterocyclylene has 1 or more quaternized nitrogen atoms; preferably, said 3- to 6-membered aliphatic heterocyclylene has one or more nitrogen atoms, and at least one nitrogen atom is substituted with =O; m ２ is 0, 1 or 2, The compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

14. L ３ is optionally substituted with one or more R ７ and is selected from 5- to 6-membered heteroarylenes which may be substituted is selected; R ７ is H (hydrogen), D (deuterium), halogen, =O, CF ３ , CN, CH ２ CN, carboxyl, sulfonic acid group, C １～４ alkyl, C １～４ alkoxy, C ２～６ selected independently from alkenyl or C ２～６ alkynyl; m ２ where m is 1 The compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof.

15. L ３ is the following group which may be substituted with one or more Rs ７ : amino, N-methylpiperidine Selected from rylen, pyrazolylen or triazolylen; R ７ is H (hydrogen), D (di Ytterium), halogen, =O, CF ３ , CN, CH ２ CN, carboxyl, sulfone Acid group, C １～４ Alkyl, C １～４ Alkoxy, C ２～６ Alkenyl or C ２～６ Alk Independently selected from nil; m ２ is 0 or 1, The compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

16. L ３ is selected from triazolylene; m ２ where m is 0 or 1 The compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof.

17. L ４ is 【Chemical 14】 selected from, Z ４ is a 6- to 10-membered arylene or a 5- to 6-membered heteroarylene; R １ ０ is H (hydrogen) or C １～６ is selected from alkyl; Z ２ is C １～６ alkylene, C ２ ～１０ alkenylene, C ２～１０ alkynylene or C ３～８ selected from cycloalkylene ; R ９ is H (hydrogen) or C １～６ selected from alkyl; Z ３ is absent or C １ ～６ is selected from alkylene; or R ９ and Z ３ are, with the nitrogen atom to which they are attached Together they form a 4- to 8-membered heterocyclylene; α is independently 0, 1, 2, 3, 4, 5 or is 6, and L ４ is L ４ is bonded to E at the 2-position of L; L ４ is L ４ is bonded to E at the 2-position of L ; m ３ where m is 0, 1, 2 or 3, The compound according to any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof.

18. L ４ is 【Chemical 15】 selected from, and Z ４ is a benzene ring, and R １０ is H (hydrogen) or C １～６ from alkyl is selected; Z ２ is C １～６ alkylene, C ２～１０ alkenylene, C ２～１０ alkynyl Lene or C ３～８ selected from cycloalkylene; R ９ is H (hydrogen) or C １～６ alkyl selected from Ru; Z ３ is absent or C １～６ selected from alkylene, or R ９ and Z ３ together with the nitrogen atom to which it is attached form a 4- to 8-membered heterocyclylene wherein; α is independently 0, 1, 2, 3, 4, 5 or 6, and L ４ is L ４ is bonded to E at the 2-position of and; L ４ where L ４ is coupled to E at the 2-position of L; m ３ is 0, 1, 2 or 3, The compound according to claim 17 or a pharmaceutically acceptable salt thereof.

19. L ４ is 【Chemical 16】 selected from, Z ４ is a 5- or 6-membered heteroarylene; R １０ is H (hydrogen) or C １～ ６ selected from alkyl; Z ２ is C １～６ alkylene, C ２～１０ alkenylene, C ２ ～１０ Selected from alkynylene or C ３～８ Selected from cycloalkylene; R ９ is H (hydrogen) or C １～６ is selected from alkyl; Z ３ is absent or is selected from C １～６ alkylene or R ９ and Z ３ forms, together with the nitrogen atom to which it is attached, a 4- to 8-membered heterocycle forming a relen; α is independently 0, 1, 2, 3, 4, 5 or 6; L ４ is L ４ of Bonded to E at the 2-position; m ３ where m is 0, 1, 2, or 3 The compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof.

20. L ４ is 【Chemical 17】 selected from; m ３ The compound according to any one of claims 1 to 19, wherein m Pharmaceutically acceptable salts.

21. L ４ is 【Chemical Formula 18】 selected from; m ３ The compound according to any one of claims 1 to 20, or its, wherein m is 1 Pharmaceutically acceptable salts.

22. L ４ is 【Chemical Formula 19】 selected from; m ３ The compound according to any one of claims 1 to 21, wherein m is 1, or a Pharmaceutically acceptable salts.

23. E is selected from pyrimidilene which may be substituted by one or more Rs １２ ; R １ ２ independently selected from H (hydrogen) or D (deuterium), any one of claims 1 to 22 The compound according to any one of the preceding claims or a pharmaceutically acceptable salt thereof.

24. G is halogen, OMs, OTs, OTf, nitro or one or more Rs １３ substituted with The following group which may be substituted: alkylthio, arylthio, heteroarylthio, alkyl sulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylsulfonyl selected from nil, arylsulfonyl or heteroarylsulfonyl; R １３ is H ( hydrogen), D (deuterium), halogen, CN, nitro, C １～６ alkyl, halogen C compound １～６ alkyl, C １～６ alkoxy, 6- to 10-membered aryl or 5- to 12-membered heteroaryl selected independently from arylsulfonyl, the compound according to any one of claims 1 to 23 or its pharmaceutically acceptable salt.

25. G is selected from F, Cl, Br, I, OMs, OTs, OTf, methylsulfonyl, ethylsulfonyl sulfonyl, p-toluenesulfonyl or naphthalenesulfonyl, the compound according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof.

26. G is selected from F, Cl, Br, OMs, OTs, methylsulfonyl or p-toluenesulfonyl selected from the group consisting of the compound according to any one of claims 1 to 25 or a pharmaceutically acceptable salt thereof.

27. G is selected from Cl or methylsulfonyl, the compound according to any one of claims 1 to 26 or a pharmaceutically acceptable salt thereof.

28. 【Fig. 20】 wherein G is preferably methylsulfonyl and E is preferably pyrimidylene , m ３ wherein m is 1, the compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salts.

29. 【Fig. 21】 is 【Chemical 22】 and m ４ is preferably an integer of 0 to 6, and methylsulfonyl is in the pyrimidine ring a substituent on the carbon atom adjacent to the nitrogen atom, the compound according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof.

30. 【Fig. 23】 is 【Chemical 24】 and m ５ is preferably an integer of 0 to 6, and methylsulfonyl is in the pyrimidine ring a substituent on the carbon atom adjacent to the nitrogen atom, the compound according to any one of claims 1 to 29 or a pharmaceutically acceptable salt thereof.

31. 【Fig. 25】 is 【Chemical 26】 and m ６ is preferably an integer of 0 to 6, and methylsulfonyl is in the pyrimidine ring a substituent on the carbon atom adjacent to the nitrogen atom, the compound according to any one of claims 1 to 30 or a pharmaceutically acceptable salt thereof.

32. 【Fig. 27】 is 【Chemical Formula 28】 and m ７ is an integer of 1 to 5, and methylsulfonyl is attached to the nitrogen atom in the pyrimidine ring The compound according to any one of claims 1 to 31, which is a substituent on adjacent carbon atoms, or its pharmaceutically acceptable salt.

33. 【Fig. 29】 is 【Chemical Formula 30】 and m ８ is an integer from 1 to 5, and methylsulfonyl is attached to the nitrogen atom in the pyrimidine ring The compound according to any one of claims 1 to 32, which is a substituent on adjacent carbon atoms, or its pharmaceutically acceptable salt.

34. 【Fig. 31】 is 【Chemical 32】 and m ９ is an integer from 1 to 5, and R １３ is hydrogen or C １～６ is selected from alkyl, Methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring, The compound according to any one of claims 1 to 33 or its pharmaceutically acceptable salt.

35. 【Fig. 33】 is 【Chemical 34】 and m １０ is an integer from 0 to 6, and Z ４ is selected from 5- to 6-membered heteroarylenes; Methylsulfonyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring, The compound according to any one of claims 1 to 34 or its pharmaceutically acceptable salt.

36. 【Fig. 35】 is 【Chemical Formula 36】 and Z ４ is pyridylene, pyrimidylene, pyrazolylene, thiazolylene, oxazoli Selected from ren or triazolylene; methylsulfonyl is the nitrogen atom in the pyrimidine ring The compound according to any one of claims 1 to 35 which is a substituent on the adjacent carbon atom or its pharmaceutically acceptable salt.

37. 【Fig. 37】 is 【Chemical Formula 38】 and Z ４ is selected from oxazolylene or thiazolylene, and methylsulfonyl is Is a substituent on the carbon atom adjacent to the nitrogen atom in the limidine ring, any one of claims 1 to 36 The compound according to one item or its pharmaceutically acceptable salt.

38. 【Fig. 39】 is 【Chemical Formula 40】 The compound according to any one of claims 1 to 37 or its pharmaceutically acceptable salt.

39. - [L １ - (L ２ ) ｍ１ - (L ３ ) ｍ２ - (L ４ ) ｍ３ - E] - G is the following fragment: 【Chemical Formula 41-1】 【Chemical Formula 41-2】 【Chemical Formula 41-3】 【Chemical Formula 41-4】 The compound according to claim 1 or its pharmaceutically acceptable salt selected from

40. T is a fragment of a bioactive molecule, and the bioactive molecule is a metal complex such as a platinum metal complex (for example, oxali Platin) or a gold metal complex; a glycopeptide antibiotic such as bleomycin or pingyangmycin; DNA topoisomerase inhibitors such as topoisomerase I inhibitors (for example, camptothecin, hydro Xycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, verotencian or rubitecan) or topoisomerase II inhibitors (for example, actin Nomycin D, adriamycin, doxorubicin, duocarmycin, daunorubicin, Mitoxantrone, podophyllotoxin or etoposide) and other DNA topoisomer Inhibitors; drugs that interfere with DNA synthesis such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, me Lukaptoprine, pentostatin, fludarabine, cladribine or nelarabine; tubulin inhibitors, vincblastine alkaloids, vincristine Tin, vincblastine, paclitaxel, docetaxel or cabazitaxel and other drugs that act on structural proteins; serine / threonine kinase inhibitors, tyrosine kinase inhibitors or A drug that acts on a structural protein such as paclitaxel, docetaxel or cabazitaxel; a serine / threonine kinase inhibitor, a tyrosine kinase inhibitor or , a tumor cell signal transduction pathway inhibitor such as an aspartokinase inhibitor or a histidine kinase inhibitor; a proteasome inhibitor; a histone deacetylase inhibitor; a tumor angiogenesis inhibitor; a cyclin inhibitor; a maytansine derivative; a calicheamicin derivative; an auristatin derivative; a pyrrolobenzodiazepine dimer (PBD) derivative; melphalan; mitomycin C; chlorambucil; or another active substance that inhibits the growth of tumor cells and promotes apoptosis or necrosis of tumor cells, the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 39.

41. The bioactive molecule is , but not limited to, selected from aryl or heteroaryl containing phenyl and pyridyl , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 40.

42. The bioactive molecule is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 41. 【Chemical Formula 42】 [Wherein, R １４ is selected from acyl or sulfonyl substituted with R １５ , and R １５ is C １～６ alkyl, halogenated C １～６ alkyl, 6- to 10-membered aryl or 5- to 12 selected from member heteroaryl; R １６ is H (hydrogen), D (deuterium), C １～ ６ alkyl, or R １７ substituted C １～６ selected from alkyl, and R １７ is this

43. is selected, and m １１ is 0, 1, or 2.] The bioactive molecule is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 42.

44. 【Chemical 43】 [wherein, R １４ is selected from acyl or sulfonyl substituted by R １５ , and R １５ is C １～６ alkyl, halogenated C １～６ alkyl, 6- to 10-membered aryl or 5- to 12 selected from member heteroaryl; R １６ is H (hydrogen), D (deuterium), C １～ ６ Alkyl, R １７ C substituted with １～６ selected from alkyl, and R １７ is aryl selected from aryl or heteroaryl, and m １１ is 0, 1, or 2.] The bioactive molecule is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 43.

45. 【Chemical Formula 44】 The bioactive molecule is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 44.

46. 【Chemical 45】 The bioactive molecule is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 45.

47. 【Chemical 46】 The bioactive molecule is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 46.

48. 【Chemical 47】 T is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 47.

49. 【Chemical 48】 T is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 48.

50. 【Chemical Formula 49-1】 【Chemical Formula 49-2】 【Chemical Formula 49-3】 T is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 49.

51. 【Chemical Formula 50-1】 【Chemical Formula 50-2】 T is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 50.

52. 【Chemical Formula 51】 T is , selected from , the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 51.

53. 【Chemical 52】 ​ ​ ​ ​ 【Chemical 53】 ​ ​ ​ The compound is 【Chemical 54-1】 【Chemical 54-2】 【Chemical Formula 54-3】 【Chemical 54-4】 [Chemical 54-5] 【Chemical 54-6】 【Chemical Formula 54-7】 【Chemical Formula 54-8】 【Chemical 54-9】 【Chemical 54-10】 selected from the group consisting of the compounds according to any one of claims 1 to 52 or a pharmaceutically acceptable salt thereof.

54. The compound is 【Chemical Formula 55-1】 【Chemical Formula 55-2】 【Chemical 55-3】 【Chemical Formula 55-4】 【Chemical Formula 55-5】 【Chemical Formula 55-6】 【Chemical Formula 55-7】 【Chemical Formula 55-8】 selected from the group consisting of the compounds according to any one of claims 1 to 53 or a pharmaceutically acceptable salt thereof.

55. A conjugate comprising a bioactive molecule, a linker, and a targeting moiety, wherein the targeting moiety is linked to the linker via a reactive group (e.g., a thiol group) to form a conjugate .

56. The conjugate according to claim 55, having a structure represented by the following formula (II). {T - [L １ - (L ２ ) ｍ１ - (L ３ ) ｍ２ - (L ４ ) ｍ３ - E]} γ - A formula (I I) [Wherein, A is a targeting moiety (e.g., a small molecule ligand, a protein, a polypeptide, or a non- protein reagent (e.g., a sugar, RNA, or DNA)); γ is an integer or a decimal number from 1 to 10; preferably, γ is an integer or a decimal number from 5 to 8 (e.g., 5, 6, 7, or 8) and the remaining groups are as described in any one of claims 1 to 54.] ；

57. The target of A is epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFR VIII, Mesothelin, Folate eceptor1, Mucin 1 , CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Ti ssue factor, Mucin16, Endothelinreceptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CC D79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-1 6, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD 2, DR5, E16, STEAP1, 0772P, MPF, Nap i3b, Sema 5 b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIP TO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevi can, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22 , CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH 1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FG ​ FR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, E pCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SL ITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR 1B, E16, STEAP1, TyrO7, 0772P, MPF, Nap13b, Sem a5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, C RIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Br evican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD7 9a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IR TA2, c-Met, ApoE, CD1 lc, CD40, CD45 (PTPRC), C D49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS 4A7, PIK3AP1, PIK3CD, CCR5, IFNγ, IL10RA1, IL- 6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NO G, SERPINE1, STAT1, TGFBR1, CTSS, PGF, VEGFA, C 1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP 3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KIS S1R, STC2, DIT4, PFKFB3, PGK1, PDK1, AKR1C1, A KR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT3 3A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, T CF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, T NFRSF21, UPK1B, TNFRSF9, MMP16, MFI2, IGF-1R, R NF43, NaPi2b, BCMA or TENB2 selected from, claim 55 or 56 the conjugate according to.

58. A is a folic acid derivative, a glutamic acid urea derivative, a somatostatin derivative, an arylsulfon amide derivative (for example, a carbonic anhydrase IX inhibitor), a polyene connecting two aliphatic indoles, a small molecule ligand such as a cyanine dye or IR-783 or a derivative thereof The conjugate according to any one of claims 55 to 57.

59. A is 【Chemical Formula 56】 The conjugate according to any one of claims 55 to 58, selected from

60. A is an antibody such as a monoclonal antibody or an antigen-binding fragment thereof, and the monoclonal The antibody or its antigen-binding fragment is Fab, Fab', F(ab') ２ , Fd, Fv, dA b, complementarity-determining fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully humanized antibody, probody, bispecific antibody or multispecific antibody, the conjugate according to any one of claims 55 to 59.

61. A is an anti-Her2 monoclonal antibody or an anti-Trop-2 monoclonal antibody, preferably Preferably, the anti-Trop-2 monoclonal antibody is selected from the antibodies of sacituzumab, M1, M2 or M 3; preferably, the anti-Her2 monoclonal antibody is selected from trastuzumab b or pertuzumab; The heavy chain of the sacituzumab has the amino acid sequence set forth in SEQ ID NO: 19; the light chain has the amino acid sequence set forth in SEQ ID NO: 20; The heavy chain variable region of antibody M1 has the amino acid sequence set forth in SEQ ID NO: 11; the light chain variable region has The amino acid sequence set forth in SEQ ID NO: 12; The heavy chain variable region of antibody M2 has the amino acid sequence set forth in SEQ ID NO: 13; the light chain variable region has The amino acid sequence set forth in SEQ ID NO: 14; The heavy chain variable region of antibody M3 has the amino acid sequence set forth in SEQ ID NO: 15; the light chain variable region has The amino acid sequence set forth in SEQ ID NO: 16; The heavy chain constant regions of antibodies M1, M2 and M3 have the amino acid sequence set forth in SEQ ID NO: 10; The light chain constant region has the amino acid sequence set forth in SEQ ID NO: 9, The conjugate according to any one of claims 55 to 60.

62. A is an anti-Her2 monoclonal antibody or an anti-Trop-2 monoclonal antibody, preferably Preferably, the anti-Trop-2 monoclonal antibody is selected from sacituzumab, and the anti- Her2 monoclonal antibody is selected from trastuzumab or pertuzumab, the conjugate according to any one of claims 55 to 61.

63. A is an RGD peptide that recognizes a cell surface integrin receptor; a growth factor that recognizes a cell surface growth factor receptor such as EGF, PDGF or VEGF; or a functional cell surface pro Plasminogen activator, bombesin, bradykinin, somatostatin or prostate A conjugate according to any one of claims 55 to 62, selected from peptides capable of recognizing a specific membrane antigen receptor. The conjugate according to any one of claims 55 to 62, selected from peptides capable of recognizing a specific membrane antigen receptor. **Claim 64** A conjugate according to any one of claims 55 to 63, wherein A is selected from CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, or DR5 ligand. The conjugate according to any one of claims 55 to 63, wherein A is selected from CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand,

65. 【Fig. 57-1】 【Chemical Formula 57-2】 【Chemical Formula 57-3】 【Chemical 57-4】 【Chemical Formula 57-5】 【Chemical Formula 57-6】 【Chemical Formula 57-7】 【Chemical Formula 57-8】 【Chemical Formula 57-9】 ErbB ligand, Her2 ligand, TACSTD2 ligand, or DR5 ligand. [wherein γ is an integer or decimal from 1 to 10, and mAb is an anti-Trop-2 monoclonal antibody or an anti-Her2 monoclonal antibody; preferably, the anti-Trop-2 monoclonal antibody is selected from sacituzumab, M1, M2, or M3, and the anti-Her2 monoclonal antibody is selected from trastuzumab or pertuzumab; preferably, γ is an integer or decimal from 5 to 8 (e.g., 5, 6, 7, or 8).]

66. 【Fig. 58-1】 【Chemical Formula 58-2】 【Chemical Formula 58-3】 【Chemical Formula 58-4】 【Chemical Formula 58-5】 【Chemical Formula 58-6】 【Chemical Formula 58-7】 A conjugate according to any one of claims 55 to 64, selected from [wherein γ is an integer or decimal from 1 to 10, and mAb is an anti-Trop-2 monoclonal antibody or an anti-Her2 monoclonal antibody; preferably, the anti-Trop-2 monoclonal antibody is selected from sacituzumab, and the anti-Her2 monoclonal antibody is selected from trastuzumab or pertuzumab; preferably, γ is an integer or decimal from 5 to 8 (e.g., 5, 6, 7, or 8).] A conjugate according to any one of claims 55 to 65, selected from

67. 【Fig. 59-1】 【Chemical Formula 59-2】 【Chemical Formula 59-3】 【Chemical Formula 59-4】 [wherein A1 is sacituzumab, and γ is an integer or decimal from 1 to 10; preferably, γ is an integer or decimal from 5 to 8.] A conjugate according to any one of claims 55 to 66, selected from

68. 【Fig. 60-1】 【Chemical Formula 60-2】 [wherein A1 is sacituzumab, and γ is an integer or decimal from 1 to 10; preferably, γ is an integer or decimal from 5 to 8, e.g., an integer or decimal from 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 8, or 7.5 to 8.] A conjugate according to any one of claims 55 to 67, selected from

69. 【Fig. 61】 [wherein A1 is sacituzumab, and γ is an integer or decimal from 1 to 10; preferably, γ is an integer or decimal from 5 to 8, e.g., an integer or decimal from 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 8, or 7.5 to 8.] A conjugate according to any one of claims 55 to 68, selected from

70. 【Fig. 62-1】 【Chemical Formula 62-2】 【Chemical Formula 62-3】 [Chemical Formula 62-4] [wherein, A2 is trastuzumab, and γ is an integer or a decimal number from 1 to 10; preferably , γ is an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6. 5 to 7.5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. The conjugate according to any one of claims 55 to 69, selected from

71. 【Chemical Formula 63-1】 【Chemical Formula 63-2】 [wherein, A2 is trastuzumab, and γ is an integer or a decimal number from 1 to 10; preferably , γ is an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6. 5 to 7.5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. The conjugate according to any one of claims 55 to 70, selected from

72. 【Fig. 64】 [wherein, A2 is trastuzumab, and γ is an integer or a decimal number from 1 to 10; preferably , γ is an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6. 5 to 7.5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. The conjugate according to any one of claims 55 to 71, selected from

73. 【Fig. 65-1】 【Chemical Formula 65-2】 【Chemical Formula 65-3】 【Chemical 65-4】 [wherein, A3 is pertuzumab, and γ is an integer or a decimal number from 1 to 10; preferably, γ is an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. The conjugate according to any one of claims 55 to 72, selected from

74. 【Fig. 66-1】 【Chemical Formula 66-2】 [wherein, A3 is pertuzumab, and γ is an integer or a decimal number from 1 to 10; preferably, γ is an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7.5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. The conjugate according to any one of claims 55 to 73, selected from

75. 【Fig. 67-1】 【Chemical Formula 67-2】 【Chemical Formula 67-3】 [Chemical Formula 67-4] [wherein, A4 is antibody M1, and γ is an integer or a decimal number from 1 to 10; preferably, γ is , an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. The conjugate according to any one of claims 55 to 74, selected from

76. 【Fig. 68】 [wherein, A4 is antibody M1, and γ is an integer or a decimal number from 1 to 10; preferably, γ is , an integer or a decimal number from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, 6.5 to 8, 7 to 8 or 7.5 to 8.]. A conjugate according to any one of claims 55 to 75, selected from

77. 【FIG. 69-1】 【Chemical Formula 69-2】 【Chemical Formula 69-3】 【Chemical 69-4】 [wherein, A5 is antibody M2, and γ is an integer or a decimal from 1 to 10; preferably, γ is , an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, 6.5 to 8, 7 to 8 or 7.5 to 8 of an integer or a decimal.] A conjugate according to any one of claims 55 to 76, selected from

78. 【Fig. 70】 [wherein, A5 is antibody M2, and γ is an integer or a decimal from 1 to 10; preferably, γ is , an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, 6.5 to 8, 7 to 8 or 7.5 to 8 of an integer or a decimal.] A conjugate according to any one of claims 55 to 77, selected from

79. 【Fig. 71-1】 【Chemical Formula 71-2】 【Chemical Formula 71-3】 【Chemical Formula 71-4】 [wherein, A6 is antibody M3, and γ is an integer or a decimal from 1 to 10; preferably, γ is , an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, 6.5 to 8, 7 to 8 or 7.5 to 8 of an integer or a decimal.] A conjugate according to any one of claims 55 to 78, selected from

80. 【Fig. 72】 [wherein, A6 is antibody M3, and γ is an integer or a decimal from 1 to 10; preferably, γ is , an integer or a decimal from 5 to 8, such as 6 to 7, 6 to 7.5, 6 to 8, 6.5 to 7, 6.5 to 7 .5, 6.5 to 8, 7 to 8 or 7.5 to 8 of an integer or a decimal.] A conjugate according to any one of claims 55 to 79, selected from

81. A method for preparing a conjugate according to any one of claims 55 to 80, which comprises a step of coupling a linker of a compound of formula (I) with an active group of the targeting moiety.

82. The method according to claim 81, which comprises a step of coupling a linker of a compound of formula (I) with an active group of the targeting moiety to form a C -S bond.

83. The targeting moiety of the conjugate is an anti-Her2 monoclonal antibody or an anti-Trop-2 monoclonal antibody or an active fragment or variant thereof; preferably , the anti-Trop-2 monoclonal antibody is selected from the antibodies of sacituzumab, M1, M2 or M3, and the anti-Her2 monoclonal antibody is selected from trastuzumab or pertuzumab. The method according to claim 81 or 82.

84. ​ The targeting moiety of the conjugate is an anti-Her2 monoclonal antibody or an anti-Trop-2 monoclonal antibody or an active fragment or variant thereof; preferably, the anti-Trop-2 monoclonal antibody is selected from sacituzumab, and the anti-Her2 monoclonal antibody is selected from trastuzumab or pertuzumab, according to any one of claims 81 to 83.

85. The molar ratio of the targeting moiety of the conjugate to the compound of formula (I) is 1 :(1 to 20); preferably, the coupling is carried out in water and / or an organic solvent; preferably, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitrile (e.g., acetonitrile), alcohol (e.g., methanol, ethanol) or any combination thereof, according to any one of claims 81 to 84.

86. The method further includes a step of purifying the coupling product; preferably, the coupling product is purified by chromatography; preferably, the chromatography includes one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography, according to any one of claims 81 to 85.

87. A pharmaceutical composition comprising the compound according to any one of claims 1 to 54 or a pharmaceutically acceptable salt thereof or the conjugate according to any one of claims 55 to 80, and one or more pharmaceutical additives.

88. Use of the compound according to any one of claims 1 to 54 or a pharmaceutically acceptable salt thereof or the conjugate according to any one of claims 55 to 80 or the pharmaceutical composition according to claim 87 in the manufacture of a medicament for treating a disease (e.g., cancer) associated with abnormal cell activity.

89. Use of the compound according to any one of claims 1 to 54 or a pharmaceutically acceptable salt thereof or the conjugate according to any one of claims 55 to 80 or the pharmaceutical composition according to claim 87 for treating a disease (e.g., cancer) associated with abnormal cell activity.

90. The cancer is esophageal cancer (e.g., esophageal adenocarcinoma, esophageal squamous cell carcinoma), brain tumor, lung cancer ( ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ For example, the use according to claim 88 or 89, selected from solid tumors or non-solid tumors such as small cell lung cancer, non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin lymphoma, central nervous system tumors (for example, glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer. cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin lymphoma, central nervous system tumors (for example, glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer. For example, glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer and other solid tumors or non-solid tumors tumors, the use according to claim 88 or 89.

91. A method for treating a disease (such as cancer) associated with abnormal cell activity, comprising administering to an individual in need thereof an effective amount of a compound according to any one of claims 1 to 54 or a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 55 to 80 or a pharmaceutical composition according to claim 87.

91. A method for treating a disease (such as cancer) associated with abnormal cell activity, comprising administering to an individual in need thereof an effective amount of a compound according to any one of claims 1 to 54 or a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 55 to 80 or a pharmaceutical composition according to claim 87. A method for treating a disease (such as cancer) associated with abnormal cell activity, comprising administering to an individual in need thereof an effective amount of a compound according to any one of claims 1 to 54 or a pharmaceutically acceptable salt thereof or a conjugate according to any one of claims 55 to 80 or a pharmaceutical composition according to claim 87. a step of administering the drug to the individual in need thereof.

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