Bioactive substance conjugate and preparation method and use thereof

Abstract

The present disclosure relates to bioactive conjugates, preparation methods and uses thereof. Specifically, the present disclosure relates to a class of novel bioactive molecule conjugates obtained by improving the conjugation mode between a drug and a targeting portion in an ADC or SMDC, as well as preparation methods and uses thereof in the preparation of drugs for treating diseases associated with abnormal cell activity.

Description

[0001] This application is a divisional application of the application with Chinese national application number 201880069543.5, the date of entry into the Chinese national phase on April 24, 2020, and the invention name being “Biologically active conjugates, preparation methods and uses thereof”. Technical Field

[0002] The present disclosure belongs to the field of medical technology, and relates to bioactive substance conjugates, and preparation methods thereof and uses thereof in preventing and / or treating diseases associated with abnormal cell activities, including but not limited to preventing and / or treating tumor diseases. Background Art

[0003] Chemotherapy was once the standard treatment for cancer, but highly lethal bioactive molecules can kill normal cells by mistake, causing serious side effects. Targeted anti-tumor drugs have become a hot topic in today's tumor research field because they have both targeting and anti-tumor activity. Since the 20th century, breakthrough progress has been made in the development of anti-tumor drugs and tumor targeted therapy using biomacromolecular drugs (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, although biomacromolecular drugs have strong targeting, their therapeutic effect on solid tumors is limited; and although bioactive molecules have a high killing effect on cancer cells, they often lack targeting and often accidentally damage normal cells, causing serious toxic side effects.

[0004] In recent years, studies have found that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting effect of antibodies with the activity of bioactive molecules, becoming a "biological missile". Antibodies guide ADCs to bind to target cells, which are then internalized by the cells, release drugs, and treat diseases. Since antibodies are specific and targeted to tumor cell-related targets, their application value is not only reflected in treatment, but also becomes an ideal carrier for targeted drug delivery, reducing the side effects of drugs. The design principles of small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs) are the same, that is, bioactive molecules are coupled to some small molecule ligands that can selectively bind to tumor cell surface receptors by chemical methods, thereby improving the targeting of effector molecules to tumor cells. The difference between SMDC and ADC is that SMDC uses small molecule ligands instead of antibodies. At present, no SMDC is on the market.

[0005] Currently, there are four ADCs on the market: Mylotarg (Gemtuzumab Ozogamicin), Adcetris (Brentuximab Vedotin, CD30 monoclonal antibody-MMAE), Kadcyla (Trastuzumab Emtansine, Trastuzumab-Maytansine alkaloid) and Besponsa (Inotuzumab ozogamicin, CD22 monoclonal antibody-calicheamicin). Generally, ADC drugs are composed of antibodies, bioactive molecules and linkers. The bioactive molecules are covalently coupled to the antibodies through linkers; antibodies (such as monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide ADCs to the surface of cancer cells, and allow ADCs to enter cancer cells through endocytosis; then the bioactive molecules are released inside the cancer cells, achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

[0006] Lysine is the most common linking site in antibodies, and its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond. Currently, there is a technology that can achieve site-specific coupling, that is, the carboxyl group of the linker is activated with an activating group, and then an amide bond is formed with the specific lysine ε-amino group in the antibody to complete the coupling. However, this type of amide bond is easily hydrolyzed under the action of enzymes in the body, causing the bioactive molecule and the antibody to fall off before reaching the target cell, which not only loses the targeting ability of the ADC, but also increases the toxicity.

[0007] Usually, the sulfur groups of antibody cysteine ​​exist in the form of disulfide bonds. Opening the disulfide bonds in the antibody can provide multiple free thiol groups as coupling sites. One method of coupling with the antibody thiol group is that the free thiol group on the antibody undergoes a Michael addition reaction with maleimide, or a specific substrate and the free thiol group on the antibody undergo two Michael addition reactions to form a uniquely structured sulfur bridge. However, there are many literature reports that the ADC obtained by the thiol Michael addition method will undergo reverse Michael addition in the systemic circulation, resulting in toxic reactions. In patent WO2016142049, a bioactive molecule and a linker structure containing a benzoxadiazole structure substituted with a mesyl group are disclosed, but there is no specific description of the coupling content with the antibody. Summary of the invention

[0008] The present invention has discovered a novel class of bioactive conjugates, which are obtained by improving the conjugation mode of the drug and the targeting part in the ADC or SMDC drug. The conjugate has high stability, extremely high coupling efficiency (90%) and high drug loading (DAR value is 5-8), and the present disclosure is completed based on the above findings. Through in-depth research, we surprisingly found that the ADC of the present invention, such as BT001021 (Example 32), after intravenous administration, the exposure of biologically active small molecule toxins in tumor tissue is significantly higher than the exposure in plasma tissue. However, under the same administration route, the plasma exposure of Immu-132ADC is significantly higher than the exposure in tumor tissue, so the ADC of the present invention has a better therapeutic window than Immu-132. At the same time, we also surprisingly found that the ADC of the present invention has a better therapeutic effect than Immu-132 in animal models of gastric cancer, breast cancer and non-small cell lung cancer tumors.

[0009] The first aspect of the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0010] T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]-G

[0011] Formula (I)

[0012] Wherein, T is a biologically active molecular fragment, preferably a molecular fragment having anti-tumor biological activity;

[0013] L1 is selected from amino acids, peptides consisting of 2-10 amino acids, oligosaccharides, -(CH2) t1 -、-(CH2CH2O) t1 -(CH2) t2 -、

[0014] wherein each of R, R', R1 and R2 is independently H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, cyano, C 1-6 Alkyl, halogenated C 1-6 Alkyl (e.g. -CF3), cyano substituted C 1-6 Alkyl (e.g. -CH2CN), C 1-6 Alkoxy, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-6Cycloalkyl, 6-10 membered aryl or 5-12 membered heteroaryl, each Z1 is independently an amino acid or a peptide composed of 2-10 amino acids, each t1 and t2 is independently 0, 1, 2, 3, 4, 5 or 6, each x1 and x2 is independently 0, 1, 2, 3, 4, 5 or 6, each x3 is independently 0, 1, 2, 3 or 4, and the 1 position of L1 is connected to T;

[0015] L2 is selected from amino acids, peptides consisting of 2-10 amino acids, oligosaccharides, -(CH2) t1 -、-(CH2CH2O) t1 -(CH2) t2 -、 Wherein, R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CN, C 1-6 Alkyl, halogenated C 1-6 Alkyl, cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 Alkenyl, C 2-10 Alkynyl and C 3-6 Cycloalkyl, or R3, R4 or R5, R6 or R3, R5 together with the carbon atoms to which they are attached form a 3-8 membered ring, t1 and t2 are each independently 0, 1, 2, 3, 4, 5 or 6, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the 1 position of L2 is connected to L1;

[0016] L3 is selected from the following groups optionally substituted by one or more R7: amino, 3-8 membered cycloalkyl, 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spiro heterocyclic group, 6-12 membered fused heterocyclic group, 6-10 membered aryl (e.g., phenyl or naphthyl), 5-12 membered heteroaryl and 3-8 membered cycloalkyl-W-; wherein W is oxygen or NR8, and R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CN, carboxyl, sulfonic acid, C 1-6 Alkyl, halogenated C 1-6 Alkyl, cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 Alkenyl and C 2-10 Alkynyl, R8 is independently selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, C 3-6 Cycloalkyl, C 1-6 Alkoxy and cyano C 1-2 An alkyl group, and the 1 position of L3 is connected to L2;

[0017] L4 is selected from Among them, Z5 is preferably selected from C2-6 Olefins, C 2-6 alkyne, amide, sulfone, sulfoxide, 6-10 membered aryl, 5-6 membered heteroaryl; Z2 is selected from C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-8 Cycloalkylene, 6-10 membered aryl and 5-14 membered heteroaryl; R9 is selected from H (hydrogen), C 1-6 Alkyl; Z3 is absent or selected from C 1-6 Alkylene, halogenated C 1-6 Alkylene and alkoxy substituted C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6; and the 2 position of L4 is connected to E;

[0018] E is selected from optionally one or more R 12 The following groups substituted: 6-10 membered aryl, 5-14 membered heteroaryl; wherein R 12 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl and halogenated C 1-6 alkyl;

[0019] G is a leaving group of a nucleophilic substitution reaction, such as a halogen, a sulfonyl group, a sulfonate group, a nitro group, etc.;

[0020] m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0021] In some preferred embodiments, L1 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide consisting of 2-5 amino acids, wherein each of R, R', R1 and R2 is independently H (hydrogen), D (deuterium), C 1-6 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl or C 3-6 Cycloalkyl, Z1 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x1 is 0, 1, 2 or 3, and x3 is 0, 1, 2, 3 or 4.

[0022] In some preferred embodiments, L1 is selected from 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-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Wherein, R, R' and R1 are each independently H (hydrogen), D (deuterium), C 1-6 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl or C 3-6 Cycloalkyl, Z1 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x1 and x3 are each independently 0, 1, 2 or 3.

[0023] In some preferred embodiments, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, Wherein, R, R' and R1 are each independently H (hydrogen), D (deuterium) or C 1-4 Alkyl, Z1 is Cit, Lys, Cit-Val, Cit-Ala, Val-Ala or Lys-Val, and x1 and x3 are each independently 0, 1 or 2.

[0024] In some preferred embodiments, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val,

[0025] In some preferred embodiments, L1 is selected from

[0026] In some preferred embodiments, L2 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide consisting of 2-5 amino acids, Wherein, R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CF3, CN, CH2CN, C 1-4Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl and C 3-6 Cycloalkyl, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and the 1 position of L2 is connected to L1;

[0027] m1 is 0, 1, 2, or 3.

[0028] In some preferred embodiments, L2 is selected from 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, Wherein, R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CF3, CN, CH2CN, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl and C 3-6 Cycloalkyl, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and the 1 position of L2 is connected to L1;

[0029] m1 is 0, 1, or 2.

[0030] In some preferred embodiments, L2 is selected from Wherein, R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium) and C 1-4 Alkyl, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and the 1 position of L2 is connected to L1;

[0031] m1 is 1.

[0032] In some preferred embodiments, L2 is selected from

[0033] In some preferred embodiments, L2 is selected from

[0034] In some preferred embodiments, L3 is selected from the following groups optionally substituted by one or more R7: amino, 3-8 membered cycloalkyl, 3-8 membered alicyclic, 6-12 membered bridged heterocyclic, 6-12 membered spiro heterocyclic, 6-12 membered fused heterocyclic, 6-10 membered aryl, 5-12 membered heteroaryl and 3-8 membered cycloalkyl-W-; wherein W is oxygen or NR8, and R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl and C 2-6 alkynyl; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spiro heterocyclic group or 6-12 membered fused heterocyclic group contains one or more nitrogen atoms; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spiro heterocyclic group or 6-12 membered fused heterocyclic group contains one or more quaternized nitrogen atoms; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spiro heterocyclic group or 6-12 membered fused heterocyclic group contains one or more nitrogen atoms, wherein at least one nitrogen atom is substituted by =O; R8 is independently selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, C 2-6 Alkenyl, C 3-6 Alkynyl, C 3-6 Cycloalkyl, C 1-6 Alkoxy and cyano C 1-2 alkyl;

[0035] m2 is 0, 1, 2 or 3.

[0036] In some preferred embodiments, L3 is selected from the following groups optionally substituted by one or more R7: amino, 3-6 membered alicyclic group and 5-10 membered heteroaryl; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl and C 2-6 Alkynyl; preferably, the 3-6 membered alicyclic group contains one or more nitrogen atoms; preferably, the 3-6 membered alicyclic group contains one or more quaternized nitrogen atoms; preferably, the 3-6 membered alicyclic group contains one or more nitrogen atoms, wherein at least one nitrogen atom is substituted by =O;

[0037] m2 is 0, 1 or 2.

[0038] In some preferred embodiments, L3 is selected from the following groups optionally substituted by one or more R7: amino or 5-6 membered heteroaryl; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl and C 2-6 Alkynyl; m2 is 0 or 1.

[0039] In some preferred embodiments, L3 is selected from the following groups optionally substituted by one or more R7: amino, N-methylpiperidine, pyrazole and triazole; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl and C 2-6 Alkynyl; m2 is 0 or 1.

[0040] In some preferred embodiments, L3 is selected from triazole; m2 is 0 or 1.

[0041] In some preferred embodiments, L3 is selected from m2 is 0 or 1; preferably, the 1 position of L3 is connected to L2.

[0042] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7: amino,

[0043]

[0044] R7 is independently selected from H (hydrogen), D (deuterium), =O, CN, CH2CN, methyl, CF3;

[0045] W is NR8, R8 is selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, C 2-6 Alkenyl, C 3-6 Alkynyl and C 3-6 Cycloalkyl.

[0046] In some preferred embodiments, L3 is selected from

[0047]

[0048] Among them, R q Each independently selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 3-6 Alkynyl and C 3-8 Cycloalkyl; β1 is 0, 1 or 2; β2 is 1, 2 or 3.

[0049] In some preferred embodiments, L3 is selected from

[0050] In some preferred embodiments, L4 is selected from Wherein Z4 is a 6-10 membered aryl group or a 5-6 membered heteroaryl group; R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl; Z3 is absent or selected from C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are connected form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and the 2 position of L4 is connected to E;

[0051] m3 is selected from 0, 1, 2 or 3.

[0052] In some preferred embodiments, L4 is selected from Among them, Z4 is a benzene ring, R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl; Z3 is absent or selected from C 1-6 Alkylene, or R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and the 2 position of L4 is connected to E;

[0053] m3 is selected from 0, 1, 2 or 3.

[0054] In some preferred embodiments, L4 is selected from Wherein Z4 is a 5-6 membered heteroaryl group; R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylidene, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl; Z3 is absent or selected from C 1-6Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are connected form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and the 2 position of L4 is connected to E;

[0055] m3 is selected from 0, 1, 2 or 3.

[0056] In some preferred embodiments, L4 is selected from

[0057] m3 is 1.

[0058] In some preferred embodiments, L4 is selected from

[0059] m3 is 1.

[0060] In some preferred embodiments, L4 is selected from

[0061] m3 is 1.

[0062] In some preferred embodiments, E is selected from the group consisting of 12 substituted 5-10 membered heteroaryl; wherein R 12 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and halogenated C 1-4 alkyl.

[0063] In some preferred embodiments, E is selected from the group consisting of 12 Substituted from the following groups: pyrimidine, quinazoline and pyrrolo[2,3-d]pyrimidine; wherein R 12 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl and halogenated C 1-2 alkyl.

[0064] In some preferred embodiments, E is selected from the group consisting of 12 Substituted pyrimidine; wherein R 12 Independently selected from H (hydrogen) and D (deuterium).

[0065] In some preferred embodiments, G is selected from halogen, OMs, Ots, Otf, nitro, and optionally substituted by one or more R 13 The following groups substituted: alkyl sulfide, aryl sulfide, heteroaryl sulfide, alkyl sulfoxide, aryl sulfoxide, heteroaryl sulfoxide, alkyl sulfonyl, aryl sulfonyl, heteroaryl sulfonyl; wherein R 13 are independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, 6-10 membered aryl and 5-12 membered heteroaryl.

[0066] In some preferred embodiments, G is selected from F, Cl, Br, I, Oms, Ots, Otf, methanesulfonyl, ethanesulfonyl, p-toluenesulfonyl, and naphthylsulfonyl.

[0067] In some preferred embodiments, G is selected from F, Cl, Br, Oms, Ots, methanesulfonyl, and p-toluenesulfonyl.

[0068] In some preferred embodiments, G is selected from Cl and mesyl.

[0069] In some preferred embodiments, In the above, G is preferably methanesulfonyl, E is preferably pyrimidine, and m3 is 1.

[0070] In some preferred embodiments, for Among them, m4 is preferably an integer of 0-6, and the methylsulfonyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0071] In some preferred embodiments, for Among them, m5 is preferably an integer of 0-6, and the methylsulfonyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0072] In some preferred embodiments, for Among them, m6 is preferably an integer from 0 to 6, and the methylsulfonyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0073] In some preferred embodiments, for Wherein, m7 is selected from an integer of 1-5, and the methylsulfonyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0074] In some preferred embodiments, for wherein m8 is selected from an integer of 1-5, and the methylsulfonyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0075] In some preferred embodiments, for Wherein, m9 is selected from an integer of 1-5, R 13 Selected from hydrogen, C 1-6 Alkyl and methanesulfonyl are substituents of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0076] In some preferred embodiments, for Where m 10 is an integer selected from 0-6, Z4 is selected from 5-6 membered heteroaryl; and the methylsulfonyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0077] In some preferred embodiments, for Z4 is selected from pyridine, pyrimidine, pyrazole, thiazole, oxazole and triazole, and the mesyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring. More preferably, m 10 An integer selected from 0-6.

[0078] In some preferred embodiments, for Z4 is selected from pyridine, pyrimidine, pyrazole and triazole. More preferably, m 10 An integer selected from 0-6.

[0079] In some preferred embodiments, for Z4 is selected from oxazole and thiazole, and the mesyl group is a substituent of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring. More preferably, m 10 An integer selected from 0-6.

[0080] In some preferred embodiments, for Where m 10 is an integer selected from 0 to 6, Z4 is selected from a 6-10 membered aromatic group; and the mesyl group is a substituent of a carbon atom adjacent to the nitrogen atom. More preferably, m 10 An integer selected from 0-6.

[0081] In some preferred embodiments, for Where m 10 An integer selected from 0-6, Z4 is a benzene ring.

[0082] In some preferred embodiments, for

[0083] In some preferred embodiments, in formula (I) Selected from the following structural fragments:

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] In some preferred embodiments, T is a bioactive molecule fragment. In some preferred embodiments, the bioactive molecule is selected from metal complexes, such as metal platinum complexes (such as oxaliplatin), metal gold complexes; glycopeptide antibiotics, such as bleomycin or bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan or rubitecan), topoisomerase II inhibitors (such as actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, Gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or nelarabine; drugs acting on structural proteins, such as microtubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cycle protein inhibitors; maytansine derivatives; calicheamicin derivatives; auristatin derivatives; pyrrolobenzodiazepine dimers (PBD) derivatives; melphalan; mitomycin C; chlorambucil; and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0090] In some preferred embodiments, the bioactive molecule is selected from Where R 14 Selected from R 15 Substituted acyl or sulfonyl, R 15 Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, 6-10 membered aryl and 5-12 membered heteroaryl; R 16 Selected from H (hydrogen), D (deuterium), C 1-6 The alkyl group, R 17 Substituted C 1-6 The alkyl group, R 17 Selected from aryl, heteroaryl, including but not limited to phenyl, pyridyl, m 11 Select from 0, 1, 2.

[0091] In some preferred embodiments, the bioactive molecule is selected from Where R 14 Selected from R 15 Substituted acyl or sulfonyl, R 15 Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, 6-10 membered aryl and 5-12 membered heteroaryl; R 16 Selected from H (hydrogen), D (deuterium), C 1-6 The alkyl group, R 17 Substituted C 1-6 The alkyl group, R 17 Selected from aryl, heteroaryl, including but not limited to phenyl, pyridyl, m 11 Select from 0, 1, 2.

[0092] In some preferred embodiments, the bioactive molecule is selected from

[0093]

[0094] In some preferred embodiments, the bioactive molecule is selected from

[0095] In some preferred embodiments, the bioactive molecule is selected from

[0096] In some preferred embodiments, the bioactive molecule is selected from

[0097]

[0098] In some preferred embodiments, the biologically active molecule is selected from

[0099]

[0100] In some preferred embodiments, T is selected from

[0101]

[0102]

[0103] In some preferred embodiments, T is selected from

[0104]

[0105] In some preferred embodiments, T is selected from

[0106]

[0107] In some preferred embodiments, T is selected from

[0108]

[0109] In some preferred embodiments, T is selected from

[0110]

[0111] In some preferred embodiments, the compound represented by formula (I) is selected from

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] In some preferred embodiments, the compound is selected from

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] In a second aspect, the present disclosure provides a conjugate comprising a bioactive molecule, a linker and a targeting moiety, wherein the targeting moiety is connected to the linker via its active group (eg, thiol) to form a conjugate.

[0131] In some preferred embodiments, the conjugate structure is as shown in formula (II):

[0132] {T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A

[0133] Formula (II)

[0134] Wherein, A is a targeting moiety (e.g., a small molecule ligand, a protein, a polypeptide, a non-protein agent (e.g., a sugar, RNA, or DNA)); γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8 (e.g., 5, 6, 7, or 8);

[0135] The remaining groups are as described in the first aspect of the present disclosure.

[0136] In some preferred embodiments, the target of A is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, 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, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, I L-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, AKR1C 2. CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and BCMA. .

[0137] In some preferred embodiments, A is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aromatic sulfonamide derivative (such as a carbonic anhydrase IX inhibitor), a polyene connecting two aliphatic indoles, a cyanine dye, or IR-783 or its derivatives.

[0138] In some preferred embodiments, A is selected from

[0139]

[0140]

[0141] 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 includes Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity determining region fragment, a single-chain antibody (e.g., scFv), a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody, a probody, a bispecific antibody or a multispecific antibody.

[0142] In some preferred embodiments, A is an anti-Her 2 monoclonal antibody, such as Trastuzumab, Pertuzumab, or an anti-Trop-2 monoclonal antibody, such as Sacituzumab.

[0143] In some preferred embodiments, A is an anti-Trop-2 monoclonal antibody, such as M1, M2, and M3.

[0144]

[0145]

[0146] The distribution of amino acids in various regions or domains can follow the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0147] 1. Hydrophobic modification of the heavy and light chain sequences of antibody M1

[0148] Amino acid sequence of the M1 heavy chain variable region: (121aa)

[0149] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID No.:11)

[0150] Amino acid sequence of the M1 light chain variable region: (107aa)

[0151] DIQLTQSPSSSLSASVGDRVSITCKASQDVSSAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSTPLTFGAGTKVEIK(SEQ ID No.:12)

[0152] 2. Hydrophobicity-modified heavy and light chain sequences of antibody M2

[0153] Amino acid sequence of the M2 heavy chain variable region: (121aa)

[0154] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID No.:13)

[0155] Amino acid sequence of the M2 light chain variable region: (107aa)

[0156] DIQLTQSPSSSLSASVGDRVSITCKASQDVSSAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK(SEQ ID No.:14)

[0157] 3. Hydrophobicity-modified heavy and light chain sequences of antibody M3

[0158] Amino acid sequence of the M3 heavy chain variable region: (121aa)

[0159] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID No.:15)

[0160] Amino acid sequence of the M3 light chain variable region: (107aa)

[0161] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSTPLTFGAGTKVEIK(SEQ ID No.:16)

[0162] M1, M2, M3 light chain constant region sequence: (107aa)

[0163] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID No.:9)

[0164] M1, M2, M3 heavy chain constant region sequence: (330aa)

[0165] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.:10)

[0166] The last Lys of the heavy chain is easily deleted, but this deletion does not affect the biological activity, see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143. The above-mentioned M1, M2, M3 monoclonal antibodies and sequences or fragments thereof with the last Lys of the heavy chain deleted belong to the M1, M2, M3 monoclonal antibodies of the present invention.

[0167] In some preferred embodiments, A is selected from RGD peptides that recognize cell surface integrin receptors; growth factors such as EGF, PDGF or VEGF that recognize cell surface growth factor receptors; and peptides that can recognize functional cell surface plasminogen activator, bombesin, bradykinin, somatostatin or prostate specific membrane antigen receptors.

[0168] In some preferred embodiments, A is selected from the group consisting of CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand and DR5 ligand.

[0169] In some preferred embodiments, the conjugate is selected from:

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Wherein, γ is selected from an integer or decimal between 1-10, mAb is an anti-Trop-2 monoclonal antibody or an anti-Her 2 monoclonal antibody; preferably, the anti-Trop-2 monoclonal antibody is selected from Sacituzumab, M1, M2 and M3 antibodies, and the anti-Her 2 monoclonal antibody is selected from Trastuzumab and Pertuzumab; preferably, γ is selected from an integer or decimal between 5-8 (e.g., 5, 6, 7 or 8).

[0180] In some preferred embodiments, the conjugate is selected from:

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Wherein, γ is selected from an integer or decimal between 1-10, mAb is an anti-Trop-2 monoclonal antibody or an anti-Her 2 monoclonal antibody; preferably, the anti-Trop-2 monoclonal antibody is selected from Sacituzumab, and the anti-Her 2 monoclonal antibody is selected from Trastuzumab and Pertuzumab; preferably, γ is selected from an integer or decimal between 5-8 (e.g., 5, 6, 7 or 8).

[0189] In some preferred embodiments, the conjugate is:

[0190]

[0191]

[0192]

[0193]

[0194] Wherein, A1 is Sacituzumab antibody, γ is selected from an integer or decimal between 1-10; preferably γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0195] In some preferred embodiments, the conjugate is:

[0196]

[0197]

[0198] Wherein, A1 is Sacituzumab antibody, γ is selected from an integer or decimal between 1-10; preferably γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0199] In some preferred embodiments, the conjugate is:

[0200]

[0201] Wherein, A1 is Sacituzumab antibody, γ is selected from an integer or decimal between 1-10; preferably γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0202] In some preferred embodiments, the conjugate is:

[0203]

[0204] Wherein, A1 is a fragment of the Sacituzumab antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0205] In some preferred embodiments, the conjugate is:

[0206]

[0207]

[0208]

[0209]

[0210] Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0211] In some preferred embodiments, the conjugate is:

[0212]

[0213]

[0214] Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0215] In some preferred embodiments, the conjugate is:

[0216]

[0217] Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0218] In some preferred embodiments, the conjugate is:

[0219]

[0220]

[0221]

[0222]

[0223] Wherein, A3 is pertuzumab, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0224] In some preferred embodiments, the conjugate is:

[0225]

[0226]

[0227] Wherein, A3 is pertuzumab, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0228] In some preferred embodiments, the conjugate is:

[0229]

[0230]

[0231]

[0232]

[0233] Wherein, A4 is M1 antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0234] In some preferred embodiments, the conjugate is:

[0235]

[0236] Wherein, A4 is M1 antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0237] In some preferred embodiments, the conjugate is:

[0238]

[0239]

[0240]

[0241]

[0242] Wherein, A5 is M2 antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0243] In some preferred embodiments, the conjugate is:

[0244]

[0245] Wherein, A5 is M2 antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0246] In some preferred embodiments, the conjugate is:

[0247]

[0248]

[0249]

[0250] Wherein, A6 is M3 antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0251] In some preferred embodiments, the conjugate is:

[0252]

[0253] Wherein, A6 is M3 antibody, and γ is selected from an integer or decimal between 1-10; preferably, γ is selected from an integer or decimal between 5-8, for example, an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0254] In another aspect, the present disclosure provides a method for preparing the conjugate described in the second aspect, which comprises the step of conjugating a linker of the compound of formula (I) to an active group of a targeting moiety.

[0255] In some preferred embodiments, the method comprises opening the disulfide bonds of the targeting moiety using a reducing agent (eg, TCEP) to yield a sulfhydryl group.

[0256] In some preferred embodiments, the method comprises the step of forming a CS bond between a linker of the compound of formula (I) and a thiol group of the targeting moiety.

[0257] In some preferred embodiments, the targeting moiety is an anti-Her 2 monoclonal antibody (eg, Trastuzumab, Pertuzumab) or an anti-Trop-2 monoclonal antibody (eg, Sacituzumab, M1, M2 or M3), or an active fragment or variant thereof.

[0258] In some preferred embodiments, the ratio of the amount of the targeting moiety to the amount of 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, nitriles (such as acetonitrile), alcohols (such as methanol, ethanol) and any combination thereof.

[0259] In some preferred embodiments, the method further comprises the step of purifying the coupling product; preferably, the coupling product is purified by a chromatography method (eg, one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography).

[0260] In another aspect, the present disclosure provides a pharmaceutical composition comprising the compound described in the first aspect of the present disclosure or a pharmaceutically acceptable salt thereof, or the conjugate described in the second aspect, and one or more pharmaceutical excipients.

[0261] In another aspect, the present disclosure provides use of the compound or pharmaceutically acceptable salt thereof of the first aspect or the conjugate of the second aspect in the preparation of a drug for treating a disease associated with abnormal cell activity (eg, cancer disease).

[0262] In some preferred embodiments, the cancer disease is a solid tumor or a non-solid tumor, for example, selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and 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's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

[0263] In another aspect, the present disclosure provides use of the compound or pharmaceutically acceptable salt thereof according to the first aspect or the conjugate or pharmaceutical composition according to the second aspect for treating diseases associated with abnormal cell activity (eg, cancer diseases).

[0264] On the other hand, the present disclosure provides a method for treating a disease associated with abnormal cell activity (e.g., a cancer disease), comprising administering to an individual in need thereof an effective dose of the compound or a pharmaceutically acceptable salt thereof provided in the first aspect of the present disclosure or the conjugate or pharmaceutical composition provided in the second aspect.

[0265] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0266] In this disclosure, the pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations in addition to active ingredients. In addition to excipients, carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc. According to their administration routes, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and ocular administration, etc. The same pharmaceutical excipients can be used in drug preparations with different administration routes and have different functions and uses.

[0267] The pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or a pharmaceutically acceptable salt or conjugate thereof, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

[0268] The pharmaceutical composition can be used in the form of injection, including injection, sterile powder for injection and concentrated solution for injection. Among them, the carriers and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.

[0269] In the present disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals (referred to as patients) suffering from diseases (e.g., 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, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0270] As used herein, the term "effective dose" refers to that amount of a compound which, when administered, will relieve to some extent one or more of the symptoms of the condition being treated.

[0271] In the present disclosure, the term "conjugate" refers to a substance obtained by connecting a biologically active molecule to a targeting portion. In some embodiments of the present disclosure, the biologically active molecule is connected to the targeting portion via a connector. The connector can be broken in a specific environment (e.g., an intracellular low pH environment) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the biologically active molecule from the targeting portion. In some embodiments of the present disclosure, the connector comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the biologically active molecule is directly connected to the targeting portion via a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the biologically active molecule from the targeting portion.

[0272] In the present disclosure, the terms "bioactive substance" and "bioactive molecule" refer to substances that inhibit or prevent cell function and / or cause cell death or destruction. In some embodiments of the present disclosure, the bioactive substance or bioactive molecule in the conjugate is a molecule with anti-tumor biological activity. For example: radioactive isotopes, such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212and radioactive isotopes of Lu; metal complexes, such as metal platinum complexes, metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin and bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, etc.; drugs that act on structural proteins, such as tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel The invention also includes inhibitors of tumor signaling pathways, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors, etc.; proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cell cycle protein inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell growth, promote tumor cell apoptosis and necrosis; enzymes and their fragments, such as nuclear lytic enzymes; antibiotics; toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; growth inhibitors; drug modules. The term "toxin" refers to a substance that can have a harmful effect on the growth or proliferation of cells.

[0273] In the present disclosure, the term "small molecule" refers to a small molecule drug with biological activity.

[0274] In the present disclosure, the term "linker" refers to a fragment that connects a biologically active molecule to a targeting moiety.

[0275] In the present disclosure, the term "targeting moiety" refers to a portion of the conjugate that can specifically bind to a target (or a portion of a target) on the cell surface. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.

[0276] In the present disclosure, when the targeting moiety in the conjugate is an antibody, the conjugate may be referred to as a “drug-antibody conjugate.” In the present disclosure, “drug-antibody conjugate” and “immunoconjugate” may be used interchangeably.

[0277] In the present disclosure, the term "antibody" is interpreted in its broadest sense, including complete monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, as long as they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" can be used interchangeably.

[0278] In the present disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the antibodies constituting the population are identical except for a small number of naturally occurring mutations that may be present. Monoclonal antibodies have a high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, contain different antibodies for different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" herein indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by a particular method.

[0279] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies, that is, a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class or subclass of antibody, and the remaining portion is identical or homologous to another type, class or subclass of antibody, as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies, which contain variable region antigen binding sequences from non-human primates (e.g., ancient monkeys, orangutans, etc.) and human constant region sequences.

[0280] The term "antibody fragment" refers to a portion of an antibody, preferably an antigen binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single-chain antibody molecules.

[0281] 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) through a coupling arm, which retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0282] The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0283] The term "complete antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2 and CH3). The constant region can be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. A complete antibody is preferably a complete antibody with one or more effector functions.

[0284] The term "probody" is a modified antibody, including an antibody or an antibody fragment, that can specifically bind to its target and can be coupled to a masking group, wherein the masking group refers to a cleavage constant for the binding ability of the antibody or antibody fragment to its target that is at least 100 times, 1000 times, or 10,000 times greater than the cleavage constant for the binding ability of the antibody or antibody fragment to its target without the coupled masking group.

[0285] In the present disclosure, the "humanized" form of non-human (e.g., mouse) antibodies refers to chimeric antibodies containing a minimum amount of non-human immunoglobulin sequences. Most humanized antibodies are hypervariable region residues of human recipient immunoglobulins that are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibodies) with desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of human immunoglobulins are also replaced with non-human residues. Moreover, humanized antibodies may also include residues that are not present in the recipient antibody or the donor antibody. These modifications are to further optimize the performance of the antibody. Humanized antibodies generally include at least one, usually two variable regions, in which all or nearly all hypervariable loops correspond to those of non-human immunoglobulins, while FRs are completely or almost completely sequences of human immunoglobulins. Humanized antibodies may also include at least a portion of an immunoglobulin constant region (Fc, usually human immunoglobulin Fc). For details, see, e.g., Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Bwl 2:593-596.

[0286] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different classes of antibodies are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.

[0287] In the present disclosure, although in most cases the amino acid substitution in the antibody is substituted with L-amino acids, it is not limited thereto. In some embodiments, one or more D-amino acids may be included in the antibody peptide chain. Peptides containing D-amino acids are more stable and less prone to degradation in the oral cavity, intestinal tract or plasma than peptides containing only L-amino acids.

[0288] The monoclonal antibodies used in the present disclosure can be produced by many methods. For example, the monoclonal antibodies used in the present disclosure can be obtained by hybridoma methods using many species (including cells of mice, hamsters, rats and humans) (see, for example, Kohler et al., 1975, Nature, 256: 495), or prepared by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352: 624-628; and Marks et al., 1991, Journal of Molecular Biology, 222: 581-597). The monoclonal antibodies that can be used in the present disclosure include, but are not limited to, anti-Her 2 monoclonal antibodies, such as trastuzumab, pertuzumab, or anti-Trop-2 monoclonal antibodies, such as sacituzumab (i.e., isactuzumab or hRS7 antibody), M1, M2 or M3.

[0289] In some embodiments of the present disclosure, the target of the targeting portion A is selected from the group consisting of epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, I L-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, AKR1C 2. CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and BCMA. .

[0290] In some embodiments of the present disclosure, the target of the targeting portion A is selected from: RGD peptides that recognize cell surface integrin receptors; growth factors such as EGF, PDGF, VEGF that recognize cell surface growth factor receptors; and peptides that can recognize functional cell surface plasminogen activator, bombesin, bradykinin, somatostatin, and prostate-specific membrane antigen receptors.

[0291] In some embodiments of the present disclosure, the target of the targeting moiety A is selected from the group consisting of: CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand and DR5 ligand.

[0292] In some embodiments of the present disclosure, the targeting moiety A is an anti-Her 2 monoclonal antibody, such as Trastuzumab and Pertuzumab; or the targeting moiety is an anti-Trop-2 monoclonal antibody, such as Sacituzumab, M1, M2 or M3.

[0293] In some embodiments of the present disclosure, the targeting moiety is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her 2, and its amino acid sequence is known to those skilled in the art, and its schematic sequence can be found in, for example, CN103319599.

[0294] In some embodiments of the present disclosure, the last Lys of the heavy chain of the targeting moiety is easily deleted without affecting the biological activity, see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143. For example, the targeting moiety is an anti-Trop-2 monoclonal antibody, such as Sacituzumab, M1, M2 or M3 heavy chain last Lys deleted, for example, the targeting moiety is an anti-Her 2 monoclonal antibody, such as trastuzumab, Pertuzumab heavy chain last Lys deleted.

[0295] Exemplary heavy chain sequences and light chain sequences of trastuzumab can be found, for example, in SEQ ID No.: 17 and SEQ ID No.: 18. In the present disclosure, when the heavy chain sequence and light chain sequence of trastuzumab are mentioned or referred to, they are described using the sequences shown in SEQ ID No.: 17 and SEQ ID No.: 18, respectively. Exemplary heavy chain sequences and light chain sequences of pertuzumab can be found in SEQ ID No.: 16 and SEQ ID No.: 15 of US7560111.

[0296] SEQ ID No.: 17 (heavy chain sequence)

[0297] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)

[0298] SEQ ID No.: 18 (light chain sequence)

[0299] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0300] In some embodiments of the present disclosure, the targeted anti-Trop-2 antibody is RS7 described in U.S. Patent No. 7,517,964 (i.e., Sacituzumab of the present disclosure); and hRS7 described in US2012 / 0237518 (i.e., Sacituzumab of the present disclosure). The anti-Trop-2 antibody that can be used in the present disclosure can also be obtained by screening the vector design, construction, and antibody library for constructing display antibodies disclosed in CN103476941A, or can be obtained by screening the antibody library of Sorrento Therapeutics, Inc. The library was screened.

[0301] The heavy chain sequence and light chain amino acid sequence of sacituzumab monoclonal antibody can be found in, for example, SEQ ID No.: 19 and SEQ ID No.: 20, respectively.

[0302] SEQ ID No.: 19 (heavy chain sequence)

[0303] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQ GSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)

[0304] The last K (or lys) of the heavy chain is easily deleted, but this deletion does not affect the biological activity, see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143.

[0305] SEQ ID No.: 20 (light chain sequence)

[0306] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0307] In the present disclosure, ErbB2 and Her2 / neu are used interchangeably, both of which represent the native sequence human Her2 protein (Genebank accession number: X03363, see, e.g., Semba 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 variants. 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 ErbB2 receptors, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, renal cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells or liver cancer cells.

[0308] In the present disclosure, Trop-2 or TROP2 refers to human trophoblast cell-surface antigens 2, also known as TACSTD2, M1S1, GA733-1, EGP-1, which is a cell surface receptor expressed by many human tumors (such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer) cells. In some embodiments, the compounds or conjugates of the present disclosure can inhibit or kill cells expressing TROP2 receptors, such as breast cancer cells, colorectal cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, prostate cancer cells or cervical cancer cells.

[0309] In this context, the conjugates of the present invention are When the targeting moiety is an antibody, it indicates the specific connection method between the thiol group in the antibody and the linker.

[0310] In this article, the term “C 1-6 "Alkyl" means a straight or branched chain alkyl group containing 1 to 6 carbon atoms, including, for example, "C 1-4 Alkyl", "C 1-3 Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 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, 1,2-dimethylpropyl, and the like.

[0311] In this article, the term “C 2-6 "Alkenyl" refers to a linear, branched or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, including, for example, "C 2-4 Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, and the like.

[0312] In this article, the term “C 2-6 "Alkynyl" refers to a straight or branched chain alkynyl group containing at least one triple bond and having 2 to 6 carbon atoms, including, for example, "C 2-4 Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0313] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0314] As used herein, the term "3-8 membered cycloalkyl" or "C 3-8 "Cycloalkyl" refers to a saturated cyclic alkyl group containing 3-8 carbon atoms, including, for example, "3-6 membered cycloalkyl", "4-6 membered cycloalkyl", "5-7 membered cycloalkyl" or "5-6 membered cycloalkyl", etc. Specific examples include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, etc.

[0315] In this article, the term “C 1-6 "Alkoxy" refers to a C 1-6 Alkyl-O- structure group, where C 1-6 Alkyl is as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0316] In this article, the term "3-8 membered alicyclic group" refers to a cyclic group containing 3-8 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxo-substituted. "3-8 membered alicyclic group" includes, for example, "3-8 membered nitrogen-containing alicyclic group", "3-8 membered oxygen-containing alicyclic group", "3-6 membered alicyclic group", "3-6 membered oxygen-containing alicyclic group", "4-7 membered alicyclic group", "4-6 membered alicyclic group", "5-7 membered alicyclic group", "5-6 membered alicyclic group", "5-6 membered nitrogen-containing alicyclic group", including but not limited to oxirane, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.

[0317] In this article, the term "6-12 membered spirocyclyl" refers to a cyclic structure containing 6-12 ring carbon atoms formed by two or more cyclic structures sharing one carbon atom. Optionally, the carbon atoms in the cyclic structure can be oxo-substituted. "6-12 membered spirocyclyl" includes, for example, "6-11 membered spirocyclyl", "6-10 membered spirocyclyl", "7-10 membered spirocyclyl", "7-9 membered spirocyclyl", "7-8 membered spirocyclyl", "9-10 membered spirocyclyl", "3-10 membered spirocyclyl", etc. Specific examples include but are not limited to:

[0318] wait.

[0319] In this article, the term "6-12 membered bridged ring group" refers to a ring structure containing 6-12 ring carbon atoms formed by two or more ring structures sharing two non-adjacent carbon atoms. Optionally, the carbon atoms in the ring structure can be oxoed. "6-12 membered bridged ring group" includes, for example, "6-11 membered bridged ring group", "5-10 membered bridged ring group", "7-10 membered bridged ring group", "7-9 membered bridged ring group", "7-8 membered bridged ring group", "9-10 membered bridged ring group", "3-10 membered bridged ring group" and the like. Specific examples include but are not limited to:

[0320] wait.

[0321] In this article, the term "6-12 membered fused cyclic group" refers to a cyclic structure containing 6-12 ring carbon atoms formed by two or more cyclic structures sharing two adjacent atoms, including "6-11 membered fused cyclic group", "6-10 membered fused cyclic group", "6-8 membered fused cyclic group", "10-12 membered fused cyclic group", "7-10 membered fused cyclic group". Its examples include but are not limited to:

[0322] wait.

[0323] In this article, the term "6-12 yuan spiro heterocyclic group" refers to a cyclic structure formed by two or more cyclic structures sharing a ring atom with each other, containing 6-12 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxoed. "6-12 yuan spiro heterocyclic group" includes, for example, "6-11 yuan spiro heterocyclic group", "5-10 yuan spiro heterocyclic group", "7-11 yuan spiro heterocyclic group", "7-10 yuan spiro heterocyclic group", "7-9 yuan spiro heterocyclic group", "7-8 yuan spiro heterocyclic group", "9-10 yuan spiro heterocyclic group", "3-10 yuan spiro heterocyclic group" etc. Specific examples include but are not limited to:

[0324] wait.

[0325] In this article, the term "6-12 membered bridged heterocyclic group" refers to a ring structure containing 6-12 ring 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 ring structures sharing two non-adjacent ring atoms. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the ring structure can be oxo-substituted. "6-12 membered bridged heterocyclic group" includes, for example, "6-11 membered bridged heterocyclic group", "6-9 membered bridged heterocyclic group", "6-10 membered bridged heterocyclic group", "7-10 membered bridged heterocyclic group", "7-9 membered bridged heterocyclic group", "7-8 membered bridged heterocyclic group", "8 membered bridged heterocyclic group", "9-10 membered bridged heterocyclic group", "3-10 membered bridged heterocyclic group" and the like. Specific examples include but are not limited to:

[0326] wait.

[0327] In this article, the term "6-12 membered fused heterocyclic group" refers to a cyclic structure containing 6-12 ring atoms (wherein at least one ring atom 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 atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxoed. "6-12 membered fused heterocyclic group" includes, for example, "6-11 membered fused heterocyclic group", "5-10 membered fused heterocyclic group", "7-10 membered fused heterocyclic group", "3-10 membered fused heterocyclic group", "3-10 membered nitrogen-containing fused heterocyclic group", "9-10 membered fused heterocyclic group", "9-10 membered nitrogen-containing fused heterocyclic group", "6-12 membered oxygen-containing fused heterocyclic group" and the like. Specific examples include, but are not limited to, tetrahydroimidazo[4,5-c]pyridinyl, 3,4-dihydroquinazolinyl, 1,2-dihydroquinoxalinyl, benzo[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-dihydro-1H-thieno[3,4-d]imidazolyl, 4,6-dihydro-1H-pyrrolo[3,4-d]imidazolyl, benzimidazolidinyl, octahydro-benzo[d]imidazolyl, decahydroquinolinyl, hexahydrothienoimidazolyl, hexahydrofuranoimidazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, octahydrocyclopenta[c]pyrrolyl, dihydroindolyl, dihydroisoindolyl, benzoxazolidinyl, benzothiazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl, 4H-1,3-benzoxazinyl, and the like.

[0328] As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity, such as a 6-20-membered aryl, a 6-10-membered aryl, a 5-8-membered aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, etc. The "6-20-membered aryl" refers to an aryl group containing 6-20 ring atoms.

[0329] In this article, the term "heteroaryl" refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl oxazolyl, pyridinyl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0330] Advantageous Effects of the Invention

[0331] The present disclosure improves the coupling method of the drug and the targeting part in ADC or SMDC to obtain a novel class of bioactive molecule conjugates. In some embodiments of the present disclosure, the bioactive molecule conjugate is obtained by a nucleophilic substitution reaction between the heteroaromatic ring on the ADC drug linker and the free thiol group in the antibody molecule. The conjugate obtained by the above coupling method can achieve at least one of the following technical effects:

[0332] (1) High stability;

[0333] (2) having a high drug loading capacity. In some embodiments, the DAR value of the conjugate can reach 5-8;

[0334] (3) having extremely high coupling efficiency. In some embodiments, the coupling efficiency can reach 90%;

[0335] (4) The conjugate obtained by the above conjugation method can effectively improve the stability of drug molecules in the circulation and reduce the shedding of non-target drugs in non-target cells;

[0336] (5) The conjugate can also increase the effective release of bioactive molecules in cells, thereby achieving the purpose of increasing efficacy and reducing toxicity;

[0337] (6) The conjugate has good tumor tissue targeting ability; and

[0338] (7) The conjugate has a good therapeutic effect on tumor animal models.

[0339] In addition, the coupling method disclosed in the present invention has a wide range of applications and can be widely used for coupling biologically active molecules with antibodies or targeted small molecule ligands. BRIEF DESCRIPTION OF THE DRAWINGS

[0340] Figure 1 This is the TIC diagram (total ion current diagram) of BT001002.

[0341] Figure 2 This is the deconvolution image of BT001002 coupled light chain.

[0342] Figure 3 This is the deconvoluted image of BT001002 coupled heavy chain.

[0343] Figure 4 This is the TIC diagram (total ion current diagram) of BT001004.

[0344] Figure 5 This is the deconvoluted image of BT001004 coupled light chain.

[0345] Figure 6 This is the deconvoluted image of BT001004 coupled heavy chain.

[0346] Figure 7 This is the SEC chromatogram of BT001002.

[0347] Figure 8 This is the molecular weight marker SEC chromatogram of BT001002.

[0348] Fig. 9 This is the SEC chromatogram of BT001004.

[0349] Fig.10 This is the deconvoluted image of BT001012 coupled light chain.

[0350] Fig.11 This is the deconvoluted image of BT001012 coupled heavy chain.

[0351] Fig.12 This is the deconvoluted image of BT001013 coupled light chain.

[0352] Fig.13 This is the deconvoluted image of BT001013 coupled heavy chain.

[0353] Fig.14 This is the deconvolution image of BT001018 coupled light chain.

[0354] Fig.15 This is the deconvolution image of BT001018 coupled heavy chain.

[0355] Fig.16 This is the deconvoluted image of BT001021 coupled light chain.

[0356] Fig.17 This is the deconvoluted image of BT001021 coupled heavy chain.

[0357] Fig.18 This is the deconvoluted image of BT001023 coupled light chain.

[0358] Fig.19 This is the deconvoluted image of BT001023 coupled heavy chain.

[0359] Fig. 20 This is the deconvoluted image of BT001040 coupled light chain.

[0360] Fig.21 This is the deconvoluted image of BT001040 coupled heavy chain.

[0361] Fig. 22 This is the deconvoluted image of BT001041 coupled light chain.

[0362] Fig.23 This is the deconvoluted image of BT001041 coupled heavy chain.

[0363] Fig.24 This is the deconvoluted image of BT001042 coupled light chain.

[0364] Fig.25 This is the deconvoluted image of BT001042 coupled heavy chain.

[0365] Fig.26 This is the deconvoluted image of BT001043 coupled light chain.

[0366] Fig. 27 This is the deconvoluted image of BT001043 coupled heavy chain.

[0367] Fig.28 This is the deconvoluted image of BT001044 coupled light chain.

[0368] Fig.29 This is the deconvoluted image of BT001044 coupled heavy chain.

[0369] Fig.30 This is the deconvoluted image of BT001046 coupled light chain.

[0370] Fig.31 This is the deconvoluted image of BT001046 coupled heavy chain.

[0371] Fig.32 This is the deconvoluted image of BT001047 coupled light chain.

[0372] Fig.33 This is the deconvoluted image of BT001047 coupled heavy chain.

[0373] Fig.34 This is the SEC chromatogram of BT001012.

[0374] Fig.35 The SEC chromatogram was obtained for BT001013.

[0375] Fig.36 SEC chromatogram was obtained for BT001018.

[0376] Fig.37 This is the SEC chromatogram of BT001021.

[0377] Fig.38 This is the SEC chromatogram of BT001023.

[0378] Fig.39 This is the SEC chromatogram of BT001042.

[0379] Fig.40 This is the SEC chromatogram of BT001043.

[0380] Fig.41 This is the SEC chromatogram of BT001044.

[0381] Fig.42 This is the SEC chromatogram of BT001046.

[0382] Fig.43 This is the SEC chromatogram of BT001047.

[0383] Fig.44 Growth changes of tumor volume in each group of mice in the NCI-N87 human gastric cancer model.

[0384] Fig.45 Changes in body weight of mice in each group in the NCI-N87 human gastric cancer model.

[0385] Fig.46 Growth changes of tumor volume in each group of mice in the HCC1806 human breast cancer model.

[0386] Fig.47A Growth changes of tumor volume in each group of mice in the HCC827 human non-small cell lung cancer transplant tumor model.

[0387] Fig.47B Changes in body weight of mice in each group in the HCC827 human non-small cell lung cancer transplant tumor model.

[0388] Fig.48AGrowth changes of tumor volume in each group of mice in the NCI-N87 human gastric cancer transplant model.

[0389] Fig.48B Changes in body weight of mice in each group in the NCI-N87 human gastric cancer transplant tumor model.

[0390] Fig.49A .The growth changes of tumor volume in each group of mice in the MDA-MB-231 human breast cancer tumor-bearing mouse model.

[0391] Fig.49B .Changes in body weight of each group of mice in the MDA-MB-231 human breast cancer tumor-bearing mouse model. DETAILED DESCRIPTION

[0392] The present disclosure is further described below by describing specific implementations, but this is not intended to limit the present disclosure. Those skilled in the art may make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure.

[0393] The abbreviations in the present invention have the following meanings:

[0394]

[0395] Preparation plan

[0396] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0397] Nuclear magnetic resonance (NMR) 1 H NMR) was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the measuring solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or hexadeuterated dimethyl sulfoxide (DMSO-d6); the internal standard substance was tetramethylsilane (TMS).

[0398] The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the Examples are shown below.

[0399] s: singlet, d: doublet, t: triplet, q: quartet, dd: doubledoublet, qd: quartet doublet, ddd: double doubledoublet, ddt: doubledouble triplet, dddd: double doubledoubledoublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0400] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0401] The preparative liquid chromatography method is as follows:

[0402] Method A:

[0403] Chromatographic column: Daisogel C18 10μm 100x250mm

[0404] Mobile phase A: water; mobile phase B: acetonitrile

[0405]

[0406] Method B:

[0407] Chromatographic column: Daisogel C18 10μm 50x250mm

[0408] Mobile phase A: water; mobile phase B: acetonitrile

[0409]

[0410] Method C:

[0411] Chromatographic column: Daisogel C18 10μm 50x250mm

[0412] Mobile phase A: water containing 0.1% trifluoroacetic acid; Mobile phase B: acetonitrile

[0413]

[0414] Method D: Column: Waters SunFire C18 5μm 19x250mm

[0415] Mobile phase A: acetonitrile; Mobile phase B: water containing 0.05% formic acid

[0416] Time: 0min-16min; Mobile phase A: 10%-90%; Flow rate: 28mL / min

[0417] 1. Synthesis of Bioactive Molecules

[0418] Example 1 Synthesis of (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-propionyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide (T001)

[0419]

[0420] Step 1: Synthesis of tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)carbamate

[0421] At room temperature, 1-hydroxybenzotriazole (2.0 mg, 14.74 μmol) was dissolved in N,N-dimethylformamide (4 mL), cooled to 0°C, and (4-methylaminobenzyl)-carbamic acid tert-butyl ester (4.0 mg, 16.1 μmol), N,N-diisopropylethylamine (8.5 mg, 66.8 μmol), ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-( (S)-2-(dimethylamino)-3-methylbutyrylamide)-N,3-dimethylbutyrylamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (10.0 mg, 13.5 μmol, commercially available), stirred for 5 min, 1H-benzotriazol-1-yloxytripyrrolidyl hexafluorophosphate (10.0 mg, 20.1 μmol) was added, and the reaction was stirred at 0°C for 1 h after the addition. The reaction of the raw materials was monitored by HPLC-MS chromatography, and the reaction solution was purified by preparative liquid chromatography (method D) to obtain the title compound as a white solid 9.0 mg. ESI-MS (m / z): 950.5 [M+H] + .

[0422] Step 2: Synthesis of (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-propionyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0423] At room temperature, tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanoyl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-3-phenylpropanamido)methyl)phenyl)carbamate (9.0 mg, 0.02 mmol) was dissolved in 1,4-dioxane (0.5 mL), the temperature was cooled to 0°C, hydrochloric acid-dioxane solution (1 mL, 4.0 M) was added, and the reaction was stirred at room temperature for 3 h. The reaction of the raw material was monitored by HPLC-MS / MS, the solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography (method C) to obtain the trifluoroacetate salt of the title compound as a white solid (5.0 mg). ESI-MS (m / z): 850.5 [M+H] + .

[0424] Example 2 Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino)1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide (T011)

[0425]

[0426] Step 1: Synthesis of tert-butyl (S)-(4-((2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido)methyl)phenyl)carbamate

[0427] At 0°C, 4-aminobenzylamine (222 mg, 1.0 mmol) and N-methylmorpholine (306 mg, 1.5 mmol) were added to a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionic acid (387 mg, 1.0 mmol) in N,N-dimethylformamide (5 mL), and 1-hydroxybenzotriazole (203 mg, 1.5 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (288 mg, 1.5 mmol) were added in sequence. The reaction was allowed to proceed overnight at 0°C. The reaction solution was poured into water (50 mL), and a white solid precipitated. The mixture was filtered, and the filter cake was washed with water (20 mL×3). The solid was purified by silica gel column chromatography to obtain the title compound as a white solid (380 mg). ESI-MS (m / z): 592.3 [M+H] + .

[0428] Step 2: Synthesis of tert-butyl (S)-(4-((2-amino-3-phenylpropionamido)methyl)phenyl)carbamate

[0429] Lithium hydroxide monohydrate (21 mg, 0.51 mmol) was dissolved in water (1 mL) and added to a solution of tert-butyl (S)-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido)methyl)phenyl)carbamate (102 mg, 0.17 mmol) in tetrahydrofuran (2 mL) and reacted at room temperature for 2 h. Water (20 mL) was added to the reaction solution, and extracted with ethyl acetate (30 mL×4). The organic phases were combined, washed with saturated brine (30 mL×2), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the solvent was evaporated under reduced pressure, and the residue was purified by preparative liquid phase (method D) to obtain the title compound as a white solid (65 mg). ESI-MS (m / z): 370.2 [M+H] + .

[0430] 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)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)carbamate

[0431] At 0°C, (S)-tert-butyl (4-((2-amino-3-phenylpropionamido)methyl)phenyl)carbamate (15 mg, 0.04 mmol) and N-methylmorpholine (12 mg, 0.12 mmol) were added to (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl- 1-Hydroxybenzotriazole (8 mg, 0.06 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12 mg, 0.06 mmol) were added to a solution of 3-methoxy-2-methylpropionic acid (24 mg, 0.04 mmol) in N,N-dimethylformamide (2 mL) in sequence. The reaction was allowed to proceed overnight at 0°C. The reaction solution was purified by preparative liquid chromatography (method D) to obtain the title compound as a white solid (24 mg). ESI-MS (m / z): 950.6 [M+H] + .

[0432] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide

[0433] 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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)carbamate (14.0 mg, 0.015 mmol) in dichloromethane (1.5 mL) and reacted at room temperature for 1 h. The solvent was evaporated under reduced pressure and the residue was purified by preparative liquid chromatography (method C) to obtain the trifluoroacetate salt of the title compound as a white solid (4.2 mg). ESI-MS (m / z): 850.6 [M+H] + .

[0434] The following molecules can be synthesized using a similar synthetic approach:

[0435]

[0436] 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]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide

[0437]

[0438] At room temperature, belothione hydrochloride (1.0 g, 2.13 mmol) and triethylamine (0.65 g, 0.9 mL) were dissolved in dichloromethane (50 mL), and acetic anhydride (0.22 g, 2.13 mmol) was slowly added dropwise. The reaction was continued at room temperature for 1 h. The organic phase was washed with water (10 mL × 2) and dried over anhydrous sodium sulfate. The insoluble matter was filtered off, the solvent was evaporated, and the title compound was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain 1 g of the title compound. ESI-MS (m / z): 476.2 [M+H] + .

[0439] 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]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide

[0440]

[0441] Methanesulfonyl chloride (462 mg, 12.77 mmol, purity about 70%) was added dropwise to a solution of belotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in dichloromethane (40 mL), and reacted for 2 h at room temperature. The mixture was filtered and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound, 2.2 g.

[0442] The structural characterization data are as follows:

[0443] 1H NMR (400MHz, DMSO-d6) δ8.32 (d, J=8.4Hz, 1H), 8.20 (dd, J=8.4, 1.2Hz, 1H), 7.93-7. 84(m,1H),7.79(t,J=7.6Hz,1H),7.35(s,1H),6.56(s,1H),5.44(d,J=9.2Hz,4H),3 .98(p,J=6.7Hz,1H),3.50(t,J=8.0Hz,2H),3.42-3.35(m,2H),3.00(s,3H),1.93-1 .82(m,2H),1.15(d,J=6.7Hz,6H),0.88(t,J=7.3Hz,3H).ESI-MS(m / z):512.2[M+H] + .[α] D 20 +28.19°(c=0.101g / 100mL,CH3CN).

[0444] The remaining biologically active molecules whose synthesis methods are not described are commercially available or prepared by methods disclosed in the prior art.

[0445] 2. Synthesis of compounds containing cell-bioactive molecules and linkers

[0446] Example 5 Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyramido)-3-methylbutyramido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide

[0447]

[0448] Step 1: Synthesis of tert-butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyrate (Compound 1-2)

[0449] At room temperature, compound 1-1 (500 mg, 3.27 mmol) was dissolved in N,N-dimethylformamide (10 mL), sodium hydride (130 mg, 3.27 mmol) was slowly added in batches, and stirred at room temperature for 10 min. 4-bromobutyric acid tert-butyl ester (725 mg, 3.27 mmol) was added dropwise, and the reaction was carried out at room temperature for 2 h. The mixture was quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, the desiccant was filtered out, and the solvent was evaporated under reduced pressure to obtain the title compound, 500 mg. ESI-MS (m / z): 296.1 [M+H] + .

[0450] Step 2: Synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (Compound 1-3)

[0451] At room temperature, compound 1-2 (500 mg, 1.69 mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (3 mL) was added. After addition, the mixture was reacted at room temperature for 4 h, and the solvent was evaporated under reduced pressure to obtain the title compound, 400 mg. ESI-MS (m / z): 240.1 [M+H] + .

[0452] Step 3: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((tert-butyloxycarbonyl)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentanyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-5)

[0453] At room temperature, 4-(N-Boc-aminomethyl)-aniline (6.0 g, 27 mmol), compound 1-4 (3.35 g, 6.75 mmol), 2-ethoxy-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), and the temperature was raised to 45°C and kept for 8.0 h. After cooling to room temperature, a large amount of solid precipitated, which was filtered to obtain the title compound, 3.65 g. ESI-MS (m / z): 701.4 [M+H] + .

[0454] Step 4: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-6)

[0455] 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 h. The solvent was evaporated under reduced pressure to obtain a yellow oil. Anhydrous ether (20 mL) was added to precipitate a large amount of solid. The mixture was stirred vigorously for 0.5 h and filtered to obtain the trifluoroacetate salt of the title compound, 3.06 g. ESI-MS (m / z): 601.3 [M+H] + .

[0456] Step 5: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-((tert-butyloxycarbonyl)amino)-3-phenylpropionamido)methyl)phenyl)amino-1-oxo-5-ureidopentanyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-7)

[0457] At room temperature, Boc-D-phenylalanine (1.1 g, 4.2 mmol) and trifluoroacetate 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-hydroxybenzotriazole (0.9 g, 6.3 mmol), and N-methylmorpholine (1.7 g, 16.8 mmol) were added in sequence. The reaction system was stirred for 1.0 h. The reaction solution was dripped into ice water (400 mL) drop by drop and stirred vigorously for 0.5 h. A large amount of solid precipitated and was filtered to obtain the title compound, 3.3 g. ESI-MS (m / z): 848.4 [M+H] + .

[0458] Step 6: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-amino-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentanyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-8)

[0459] Compound 1-7 (3.0 g, 3.3 mmol) was dissolved in trifluoroacetic acid (30 mL) at room temperature and stirred at room temperature for 1.0 h. The solvent was evaporated under reduced pressure to obtain a yellow oil, anhydrous ether (100 mL) was added, and the mixture was stirred vigorously for 0.5 h. A large amount of solid precipitated, which was filtered to obtain the trifluoroacetate salt of the title compound, 2.1 g. ESI-MS (m / z): 748.4 [M+H] + .

[0460] 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-butyryl)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionyl)-3-phenylpropionyl)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (Compound 1-9)

[0461] At room temperature, (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-(dimethylamino)-3-butyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropanoic acid (1.3 g, 2.17 mmol) and trifluoroacetate of compound 1-8 (1.8 g, 2.17 mmol) were dissolved in N,N-dimethylformamide (20 mL), cooled to 0°C, 1-hydroxybenzotriazole (440 mg, 3.26 mmol) and N-methylmorpholine (658 mg, 6.51 mmol) were added in sequence, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (624 mg, 1.38 mmol) was added at last. After the addition was completed, the reaction solution was stirred at 0°C for 5 h. Purification by preparative liquid chromatography (method D) gave the title compound, 1.8 g. ESI-MS (m / z): 1329.2 [M+H] + .

[0462] Step 8: Synthesis of (S)-2-((S)-2-amino-3-butyrylamide)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamide)-N,3-dimethylbutyrylamide)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide (Compound 1-10)

[0463] At room temperature, compound 1-9 (500 mg, 0.38 mmol) was dissolved in N,N-dimethylformamide (5 mL), piperidine (324 mg, 3.8 mmol) was added, and stirred at room temperature for 3 h. Purification by preparative liquid chromatography (method D) gave the title compound, 350 mg. ESI-MS (m / z): 1107.2 [M+H] + .

[0464] Step 9: Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanamido)-3-methylbutanamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidopentanamide (Compound TL001)

[0465] 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-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazole-1-oxytripyrrolidine hexafluorophosphate (281 mg, 0.54 mmol) were added in sequence. After addition, the reaction system was stirred at room temperature for 3 h. Purification by preparative liquid chromatography (method D) gave the title compound, 30 mg. ESI-MS (m / z): 664.5 [M / 2+H] + .

[0466] Example 6 (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butyramido)-butyramido)-5-ureidopentanamide

[0467]

[0468] Step 1: Synthesis of 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (Compound 2-2)

[0469] At room temperature, 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid (300 mg, 1.25 mmol) was dissolved in methanol (8 mL), sodium thiomethoxide (351 mg, 5.02 mmol) was added at once, and the temperature was raised to 50°C for overnight reaction. Purification by preparative liquid chromatography (method D) gave the title compound, 120 mg. ESI-MS (m / z): 252.1 [M+H] + .

[0470] Step 2: Synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butyramido)-butyramido)-5-ureidopentanamide (Compound 2-3)

[0471] A similar procedure as described in step nine of Example 5 was used, with 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid replacing 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid, and purification by preparative liquid chromatography (method D) was performed to obtain the title compound, 20 mg. ESI-MS (m / z): 670.5 [M / 2+H] + .

[0472] Step 3: Synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butanamido)-butanamido)-5-ureidopentanamide (Compound TL002)

[0473] At room temperature, compound 2-3 (20 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL), and m-chloroperbenzoic acid (4.0 mg, 0.022 mmol) was added. After addition, the mixture was reacted at room temperature for 2 h. Purification by preparative liquid chromatography (method D) gave the title compound, 5.0 mg. ESI-MS (m / z): 686.5 [M / 2+H]+ .

[0474] 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-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0475]

[0476] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (Compound 3-2)

[0477] At room temperature, 5-hexynoic acid methyl ester (500 mg, 3.97 mmol) and 5-bromo-2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 mL), triethylamine (3 mL), cuprous iodide (75 mg, 0.4 mmol) and ditriphenylphosphine palladium dichloride (279 mg, 0.4 mmol) were added in sequence, the temperature was raised to 95°C under nitrogen protection, stirred and reacted for 6 h, water was added to quench, ethyl acetate was extracted (20 mL×3), the organic phases were combined, washed with saturated brine (20 mL×2), dried over anhydrous sodium sulfate, the desiccant was filtered off, the solvent was evaporated under reduced pressure, and purified by preparative liquid chromatography (method D) to obtain the title compound, 300 mg. ESI-MS (m / z): 251.3 [M+H] + .

[0478] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-3)

[0479] 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 mmol) was added, and the reaction was stirred at room temperature for 4 h, diluted with water, extracted with ethyl acetate (20 mL×2), the aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate (20 mL×3), the organic phases were combined, washed with saturated brine (20 mL×2), dried over anhydrous sodium sulfate, the desiccant was filtered out, and the solvent was evaporated under reduced pressure to obtain 120 mg of the title compound.

[0480] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-4)

[0481] At room temperature, compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL), and m-chloroperbenzoic acid (22 mg, 0.127 mmol) was added. After the addition was completed, the mixture was stirred at room temperature overnight and purified by preparative liquid chromatography (method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 269.1 [M+H] + .

[0482] Step 4: Synthesis of 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-methylpropanoylamino)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (Compound TL003)

[0483] A similar procedure as described in step nine of Example 5 was used, with 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid replacing 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid, and purification by preparative liquid chromatography (method D) was performed to obtain the title compound, 14 mg. ESI-MS (m / z): 679.0 [M / 2+H] + .

[0484] Example 8 (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(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonyloxy-3,9,36-azatetracosane-41-amide)benzyl)carbonate

[0485]

[0486] Step 1: Synthesis of (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)-(9H-fluorenyl)-carbamic acid methyl ester (Compound 19-2)

[0487] At room temperature, Fmoc-L-citrulline (5.0 g, 12.58 mmol), p-aminobenzyl alcohol (6.20 g, 50.32 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (6.22 g, 25.16 mmol) were dissolved in dichloromethane (100 mL), and the temperature was raised to 45 ° C for 6 h. The reaction solution was concentrated under reduced pressure and slurried with anhydrous ether (100 mL) to obtain the title compound, 6.0 g. ESI-MS (m / z): 503.3 [M+H] + .

[0488] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 19-3)

[0489] 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. After addition, the mixture was reacted at room temperature for 30 min, and dichloromethane (10 mL) was added and stirred for 10 min. The reaction solution was concentrated under reduced pressure and purified by rapid silica gel column to obtain the title compound, 400 mg. ESI-MS (m / z): 281.2 [M+H] + .

[0490] Step 3: Synthesis of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azatriacetamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 19-4)

[0491] Compound 19-3 (150 mg, 0.54 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatricycloundecanoic acid (296 mg, 0.54 mmol) were dissolved in dichloromethane (10 mL), cooled to 0°C, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (145 mg, 0.58 mmol) was added, and the mixture was moved to room temperature for overnight reaction. The reaction solution was concentrated under reduced pressure and purified by rapid silica gel column to obtain the title compound, 200 mg. ESI-MS (m / z): 817.5 [M+H] + .

[0492] Step 4: Synthesis of 4-((S)-35-azido-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-azacosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound 19-5)

[0493] At room temperature, (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 (200 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, and then a solution of triphosgene (116 mg, 0.39 mmol) in dichloromethane (1.0 mL) was slowly added dropwise. After addition, the mixture was stirred at 0°C for 1 h. A solution of compound 19-4 (159 mg, 0.18 mmol) in dichloromethane (2.0 mL) was added to the reaction solution. After addition, the mixture was reacted at room temperature for 1 h. Purification by preparative HPLC (method D) gave the title compound, 160 mg. ESI-MS (m / z): 678.0 [M / 2+H] + .

[0494] Step 5: Synthesis of 4-((S)-35-amino-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-azacosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound 19-6)

[0495] Compound 19-5 (80 mg, 0.059 mmol) was dissolved in tetrahydrofuran (1.0 mL) at room temperature, cooled to 0°C, and a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL) was added. Platinum dioxide (15 mg, 0.059 mmol) was added at once under nitrogen protection. After addition, the air was replaced by hydrogen three times, and the reaction was carried out at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product, which 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]+ .

[0496] Step 6: Synthesis of (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(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonyloxy-3,9,36-azatetracosane-41-amide)benzyl)carbonate (Compound TL019)

[0497] Compound 19-6 (30 mg, 0.016 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-5-hexynoic acid (6.4 mg, 0.024 mmol) were dissolved in N,N-dimethylformamide (1 mL), cooled to 0°C, and benzotriazole-1-yl-oxytripyrrolidino hexafluorophosphate (16.5 mg, 0.032 mmol) and N,N-diisopropylethylamine (6.2 mg, 0.047 mmol) were added in sequence. After addition, the mixture was reacted at room temperature for 2 h. The title compound was purified by preparative high performance liquid chromatography (method D), 10 mg. ESI-MS (m / z): 790.0 [M / 2+H] + .

[0498] Example 9 (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)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)butyramido)-5-ureidopentanamido)benzyl)carbonate

[0499]

[0500] Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentanamido-2-yl)amino)-3-methyl-butyramido-2-yl)-(9H-fluorenyl)methyl-carbamate

[0501] Using a similar operation as in step 1 of Example 8, compound 28-1 was substituted for compound 19-1 to obtain the title compound, 310 mg. ESI-MS (m / z): 602.3 [M+H] + .

[0502] Step 2: Synthesis of (S)-2-((S)-2-amino-3-methylbutyramide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 28-2)

[0503] Using a similar operation as in step 2 of Example 8, compound 28-2 was substituted for compound 19-2 to obtain the title compound, 150 mg. ESI-MS (m / z): 380.3 [M+H] + .

[0504] Step 3: Synthesis of N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (Compound 28-4)

[0505] Benzotriazole-1-yl-oxytripyrrolidino hexafluorophosphate (313 mg, 0.6 mmol) and N,N-diisopropylethylamine (194 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-pentanamide (190 mg, 0.5 mmol) in N,N-dimethylformamide (10 mL) at room temperature, and the reaction was stirred at room temperature for 3 h. The title compound was purified by preparative HPLC (method D), 78 mg. ESI-MS (m / z): 630.3 [M+H] + .

[0506] Step 4: Synthesis of (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)butyramido)-5-ureidopentanamido)benzyl)carbonate (Compound TL028)

[0507] Using a similar operation as in step 4 of Example 8, compound 28-4 was substituted for compound 19-4 to obtain the title compound, 1.76 mg. ESI-MS (m / z): 1167.4 [M+H] + .

[0508] Example 10 (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-(4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatricarboxamido)benzyl)carbonate

[0509]

[0510] Step 1: Synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-azatetracosane)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 29-1)

[0511] Using a similar operation as in step 3 of Example 8, compound 28-3 was substituted for compound 19-3 to obtain the title compound, 180 mg. ESI-MS (m / z): 916.5 [M+H] + .

[0512] Step 2: Synthesis of 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonyloxy-3,6,12-triazatricarboxamido)benzyl((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 (Compound 29-2)

[0513] Using a similar operation as in step 4 of Example 8, compound 29-1 was substituted for compound 19-4 to obtain the title compound, 30 mg. ESI-MS (m / z): 727.5 [M / 2+H] + .

[0514] Step 3: Synthesis of (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-(4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonyloxy-3,6,12-triazatricarboxamido)benzyl)carbonate (Compound TL029)

[0515] At room temperature, compound 29-2 (20 mg, 0.014 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(2-propynyl-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), and cuprous bromide (3.95 mg, 0.027 mmol) was added and stirred for 1 h. The title compound was purified by preparative high performance liquid chromatography (method D), 15 mg. ESI-MS (m / z): 880.0 [M / 2+H] + .

[0516] Example 11 (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[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-nonyloxy-3,6,12,39-tetraazapentadecane-44-carbamoyl)benzyl)carbonate

[0517]

[0518] Step 1: Synthesis of (S)-2-((S)-3,5-amino-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

[0519] At 20°C, compound 29-1 (400 mg, 0.44 mmol) was dissolved in methanol and tetrahydrofuran (2.0 mL: 4.0 mL). After complete dissolution, platinum dioxide (40 mg) was added at once under nitrogen protection, and hydrogen was replaced three times. The reaction was hydrogenated at 20°C for 2 hours. Filter, wash the filter cake with methanol, concentrate the filtrate under reduced pressure, and purify the residue by preparative high performance liquid chromatography (method D) to obtain the title compound, 200 mg. ESI-MS (m / z): 890.4 [M+H] + .

[0520] 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-nonyloxy-2,7,10,16-tetraazatetradecane-42-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0521] At 20°C, compound 22-1 (250 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (1.0 mL), and HATU (160 mg, 0.42 mmol) and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were added in sequence. After the addition was completed, the mixture was stirred at room temperature overnight. The title compound was purified by preparative HPLC (method D), 250 mg.

[0522] Step 3: Synthesis of (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(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-nonyloxy-3,6,12,39-tetraazapentadecane-44-carbamoyl)benzyl)carbonate (Compound TL022)

[0523] 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, a solution of p-dimethylaminopyridine (200 mg, 1.64 mmol) in dichloromethane (1.0 mL) was added, and then a solution of triphosgene (40.6 mg, 0.14 mmol) in dichloromethane (1.0 mL) was slowly added dropwise. After the addition was completed, the reaction was stirred at 0°C for 1 h. The phosgene that did not participate in the reaction was blown away with nitrogen, and a solution of compound 22-2 (139 mg, 0.12 mmol) in dichloromethane (2.0 mL) was added to the reaction solution, and the mixture was stirred at 0°C for 1 h. The title compound was purified by preparative HPLC (method D), 1.5 mg. ESI-MS (m / z): 839.5 [M / 2+H] + .

[0524] Example 12 4-((S)-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-triazatetradecanoyl-41-ynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0525]

[0526] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[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-triazatetradecanoyl-41-ynamido)benzyl carbonate

[0527] At room temperature, 6-(-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (12 mg, 0.045 mmol) was dissolved in dichloromethane (2 mL), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.2 mg, 0.056 mmol) and N,N-diisopropylethylamine (8.6 mg, 0.067 mmol) were added, stirred for 10 min, compound 24-1 (35 mg, 0.022 mmol) was added, and the reaction was stirred for 1 h. Purification by preparative high performance liquid chromatography (method B) gave the title compound, 20 mg. ESI-MS (m / z): 1821.8 [M+H] + .

[0528] Step 2: Synthesis of 4-((S)-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-triazatetradecanoyl-41-ynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound TL024)

[0529] 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 mL) in acetonitrile (0.5 mL) was added dropwise and stirred for 20 min. The title compound was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 12 mg. ESI-MS (m / z): 1549.6 [M+H] + .

[0530] Example 13 Synthesis of (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-4-((2S,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-nonyloxy-3,6,12-triazatriacontamido)benzyl carbonate

[0531]

[0532] Step 1: Preparation of (S)-(9H-fluoren-9-yl)-methyl(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate

[0533] 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 stirred for 3 h. Purification by silica gel column chromatography gave the title compound, 1.8 g. ESI-MS (m / z): 417.2 [M+H] +

[0534] Step 2: Preparation of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)propionamide

[0535] 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 reacted for 2 h. Purification by silica gel column chromatography gave the title compound, 820 mg. ESI-MS (m / z): 195.1 [M+H]+

[0536] 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

[0537] At room temperature, (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyric acid (875 mg, 2.58 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (1.45 g, 3.83 mmol), N,N-diisopropylethylamine (1.00 g, 7.74 mmol) and 1-hydroxybenzotriazole (525 mg, 3.89 mmol) were added to a solution of compound 30-3 (503 mg, 2.58 mmol) in dichloromethane (2 mL) in sequence and stirred for 4 h. Purification by silica gel column chromatography gave the title compound, 1.1 g. ESI-MS (m / z): 516.2 [M+H]+

[0538] Step 4: Preparation of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0539] 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). The mixture was stirred for 1 h. The mixture was purified by silica gel column chromatography to obtain the title compound, 610 mg. ESI-MS (m / z): 294.2 [M+H] +

[0540] Step 5: Preparation of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-diazapentatriacontamide)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutanamide

[0541] At room temperature, O-benzotriazole-tetramethyluronium hexafluorophosphate (160 mg, 0.42 mmol), 1-hydroxybenzotriazole (57 mg, 0.42 mmol), N,N-diisopropylethylamine (109 mg, 0.84 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatricyclodecane-1-acid (156 mg, 0.28 mmol) were added to dichloromethane (3 mL) of compound 30-5 (84 mg, 0.28 mmol) and stirred for 4 h. Purification by silica gel column chromatography gave the title compound, 163 mg. ESI-MS (m / z): 830.4 [M+H] +

[0542] Step 6: Preparation of 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonyloxy-3,6,12-triazatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0543] Under nitrogen protection, a solution of triphosgene (16 mg, 0.05 mmol) in dichloromethane (0.3 mL) was added dropwise to a mixed solution of 4-dimethylaminopyridine (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-isopropylmethanesulfonamide (45 mg, 0.09 mmol) in dichloromethane (0.7 mL) at 0°C, and the mixture was reacted for 1 h at 0°C. Compound 30-6 (73 mg, 0.09 mmol) in dichloromethane (1 mL) was added dropwise to the reaction solution, and the mixture was reacted for 1 h at 0°C. Purification by silica gel column chromatography gave the title compound, 33 mg. ESI-MS (m / z): 1367.6 [M+H] +

[0544] Step 7: Preparation of (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-4-((2S,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-nonyloxy-3,6,12-triazatriacontamido)benzyl carbonate (Compound TL030)

[0545] Under room temperature, cuprous bromide (5 mg, 0.04 mmol) and compound 30-7 (20 mg, 15 umol) were added dropwise to a mixed solution of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (9 mg, 0.007 mmol) in water and N,N-dimethylformamide (0.2 mL: 0.8 mL), and the mixture was stirred for 4 h. The title compound was purified by preparative HPLC (method D) to obtain 4.15 mg. ESI-MS (m / z): 1672.7 [M+H] +

[0546] Example 14 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((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

[0547]

[0548] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide

[0549] At 25°C, prop-2-yn-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL), and N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were added in sequence, and the reaction was stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by rapid silica gel column (ethyl acetate / petroleum ether=3 / 1) to obtain the title compound, 700 mg. ESI-MS (m / z): 306.1 [M+H] + .

[0550] Step 2: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0551] Under nitrogen protection at 25°C, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, and then a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the addition was completed, the mixture was stirred at 0°C for 20 min, and the reaction solution was purged with nitrogen for 20 min. Add 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)benzhydryl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) and stir at 0°C for 1 h. 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] + .

[0552] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)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)hexane-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)benzyl)carbonate

[0553] At room temperature, compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (11 mg, 0.08 mmol) was added. The mixture was stirred for 1 h. The title compound was purified by preparative HPLC (method B) to obtain 30 mg of the title compound. ESI-MS (m / z): 815.9 [(M-273) / 2+H] + .

[0554] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontanamido)benzyl((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 (Compound TL033)

[0555] 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. The reaction was allowed to react at room temperature for 30 min. The title compound was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 20.0 mg. Its structural characterization is as follows:

[0556] 11H 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, 1H), 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 = 8.56 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), 3.51 - 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 - 1.27 (m, 2H), 1.15 (d, J = 6.80 Hz, 3H), 1.13 (d, J = 6.76 Hz, 3H), 0.90 (t, J = 7.32 Hz, 3H). ESI-MS (m / z): 816.0 [M / 2 + H] + 。[α] D 20 was -19.55° (c = 1.000 g / 100 mL, CH3CN).

[0557] 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-nonyloxy-3,9-diazapentatriacontamido)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

[0558]

[0559] Step 1: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27-nonyloxy-((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]indolizino[1,2-b]quinolin-4-yl)carbonate

[0560] Compound 34-1 (100 mg, 0.2 mmol) was dissolved in dry dichloromethane (2 mL) at room temperature under nitrogen protection, and the temperature was lowered to 0°C. A solution of 4-dimethylaminopyridine (144 mg, 1.18 mmol) in dry dichloromethane (0.5 mL) was added, and then a solution of triphosgene (41 mg, 0.14 mmol) in dry dichloromethane (0.5 mL) was slowly added dropwise. After the addition was completed, the reaction was stirred at 0°C for 1 h. To the reaction solution was added 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)benzhydryl)amino)acetamide (160 mg, 0.15 μmol) in dry dichloromethane (0.5 mL). After addition, the mixture was reacted at room temperature for 1 h. The title compound was purified by preparative HPLC (method B), 60 mg. ESI-MS (m / z): 1592.7 [M+H] + .

[0561] Step 2: Synthesis of (S)-9-(tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine[1,2-b]quinolin-4-yl-4-((S)-2-(4-(((6-2-(methylsulfonyl)pyrimidin-5-yl)-35-(4-((6-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatricarboxamido)carbonate

[0562] At room temperature, compound 34-2 (40 mg, 0.03 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (11.50 mg, 0.04 mmol) were dissolved in dimethyl sulfoxide and water (0.5 mL: 0.1 mL), and cuprous bromide (9.01 mg, 0.06 mmol) was added and stirred for 1 h. Purification by preparative HPLC (method B) gave the title compound, 20 mg. ESI-MS (m / z): 1897.5 [M+H].

[0563] Step 3: Synthesis of 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-nonyloxy-3,9-diazapentatriacontamido)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)

[0564] Compound 34-3 (30 mg, 0.018 mmol) was dissolved in acetonitrile and water (0.4 mL: 0.1 mL) at room temperature, and a mixed solution of trifluoroacetic acid and acetonitrile (0.5 mL: 0.5 mL) was added dropwise, and stirred at room temperature for 2 h. The title compound was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 12 mg. ESI-MS (m / z): 1511.5 [M+H] + .

[0565] Example 16 Synthesis of 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-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0566]

[0567] Step 1: Synthesis of ((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontanoyl)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1,2,3,6-triazacycloheptane-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0568] A synthetic method similar to that in step 1 of Example 15 was used, with compound 35-1 replacing compound 34-1, to obtain the title compound, 60 mg. ESI-MS (m / z): 1561.5 [M+H] + .

[0569] Step 2: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetylamino)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)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontriacontamido]carbonate

[0570] A similar synthesis method as in step 2 of Example 15 was used, with compound 35-2 replacing compound 34-2, to obtain the title compound, 20 mg. ESI-MS (m / z): 1866.5 [M+H].

[0571] Step 3: Synthesis of 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-nonaoxa-3,9-diazapentacontanamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound TL035)

[0572] A synthetic method similar to that in step 3 of Example 15 was used, with compound 35-3 replacing compound 34-3, to obtain the trifluoroacetate salt of the title compound, 4.9 mg. ESI-MS (m / z): 1594.5 [M+H] + .

[0573] Example 17 Synthesis of 4-((S,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-triazatetradecanoyl-41-enamide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0574]

[0575] Step 1: Synthesis of (Z)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enoic acid

[0576] At 20°C, compound 3-4 (200 mg, 0.67 mmol) was dissolved in methanol (8.0 mL), and Lindela catalyst (20 mg) was added under nitrogen protection. The hydrogen was replaced three times, and the hydrogenation reaction was carried out at 20°C for 3 h. Filter and spin-dry the filtrate to obtain the title compound, 150 mg. ESI-MS (m / z): 271.1 [M+H] + .

[0577] Step 2: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[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-triazatetradecanoyl-41-enamide)benzyl carbonate

[0578] At room temperature, compound 45-2 (8 mg, 0.030 mmol) was dissolved in dichloromethane (2 mL), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium 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 min, compound 48-1 (30 mg, 0.020 mmol) was added, and the reaction solution was stirred at room temperature for 1 h. The title compound was purified by preparative high performance liquid chromatography (method B), 30 mg. ESI-MS (m / z): 1787.8 [M+H] + .

[0579] Step 3: Synthesis of 4-((S,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-triazatetradecanoyl-41-enamide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate (Compound TL045)

[0580] Compound 45-3 (30 mg, 0.017 mmol) was dissolved in acetonitrile (1 ml) at room temperature, and a solution of trifluoroacetic acid (0.5 ml) in acetonitrile (0.5 ml) was added dropwise. The reaction solution was stirred at room temperature for 20 min. The title compound was purified by preparative HPLC (method C) to obtain trifluoroacetate salt, 9 mg. ESI-MS (m / z): 1515.6 [M+H] + .

[0581] Example 18 4-((S)-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-triazatetradecanoyl-41-ynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0582]

[0583] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[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-triazatetradecanoyl-41-ynamido)benzyl carbonate

[0584] A similar synthesis method as in step 1 of Example 12 was used, with compound 48-1 replacing compound 24-1. The title compound was obtained, 15 mg. ESI-MS (m / z): 1785.8 [M+H] + .

[0585] Step 2: Synthesis of 4-((S)-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-triazatetradecanoyl-41-ynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound TL048)

[0586] A similar synthesis method as in step 2 of Example 12 was used, with compound 48-2 replacing compound 24-2. The trifluoroacetate salt of the title compound was obtained, 11.35 mg. ESI-MS (m / z): 1513.7 [M+H] + .

[0587] Example 19 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontriadecylamino)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0588]

[0589] Step 1: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)thiazole-4-carboxylic acid

[0590] Compound 49-1 (100 mg, 0.40 mmol), 2-bromo-4-thiazolecarboxylic acid (99.01 mg, 0.48 mmol), potassium carbonate (137.03 mg, 0.99 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (29.02 mg, 0.04 mmol) were added to 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. The reaction solution was cooled to room temperature, dripped into water, filtered, and the filtrate was collected. The ethyl acetate was extracted (10 mL×3), and the aqueous phase was collected. The pH was adjusted to 3 with dilute hydrochloric acid, and the solid was precipitated. The filter cake was collected to obtain the title compound, 70 mg. ESI-MS (m / z): 254.0 [M+H] + .

[0591] Step 2: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxylic acid

[0592] Compound 49-2 (73 mg, 0.29 mmol) was dissolved in dichloromethane (15 mL), and m-chloroperbenzoic acid (175.53 mg, 0.87 mmol, 85%) was added. The reaction system was stirred at room temperature overnight. The solvent was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 286.0 [M+H] + .

[0593] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)thiazole-4-carboxamide

[0594] Compound 49-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL), and O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (39.98 mg, 0.11 mmol) was added. The reaction system was cooled to 0°C, and N,N-diisopropylethylamine (22.65 mg, 0.18 mmol) and propargylamine (4.63 mg, 0.09 mmol) were added. The reaction solution was stirred at room temperature for 3 h. Purification by preparative HPLC (method D) gave the title compound, 10 mg. ESI-MS (m / z): 323.0 [M+H] + .

[0595] Step 4: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[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-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontriacontylamino)benzyl)carbonate

[0596] At room temperature, compound 33-1 (30 mg, 0.02 mmol) and compound 49-4 (9.08 mg, 0.03 mmol) were dissolved in dimethyl sulfoxide and water (2 mL / 0.5 mL), and cuprous bromide (5.39 mg, 0.04 mmol) was added and stirred for 2 h. Filtered, the filtrate was purified by preparative HPLC (method B) to obtain the title compound, 20 mg. ESI-MS (m / z): 1647.3 [M+H-273] + .

[0597] Step 5: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazatriacontriadecylamino)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0598] Compound 49-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2 mL) at room temperature, and trifluoroacetic acid (0.2 mL) was added dropwise. The reaction solution was stirred at room temperature for 20 min. The reaction solution was concentrated, and the residue was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 8 mg. ESI-MS (m / z): 1647.9 [M+H] + .

[0599] Example 20 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)-oxazole-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)benzyl-((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

[0600]

[0601] Step 1: Synthesis of ethyl 2-(2-(methylthio)pyrimidin-5-yl)oxazole-4-carboxylate

[0602] 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), potassium carbonate (125 mg, 0.9 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (33 mg, 0.05 mmol) were added in sequence, and the temperature was raised to 90°C for reaction for 3 h under N2 protection. The reaction solution was filtered through diatomaceous earth, the filtrate was diluted with water (50 mL), extracted with ethyl acetate (30 mL×3), and the organic phases were combined and dried. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the crude product 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] + .

[0603] Step 2: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)-oxazole-4-carboxylic acid

[0604] 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. After addition, the mixture was reacted at 25°C for 1 h. The reaction solution was diluted with water (15 mL), extracted with ethyl acetate (20 mL×2), the aqueous phase was adjusted to pH=2-3 with 1N dilute hydrochloric acid, extracted with a mixed solvent of dichloromethane / methanol (v:v=10:1) (20 mL×3), the organic phases were combined, washed with saturated brine (30 mL×1), and dried over anhydrous sodium sulfate. The desiccant was filtered off and the filtrate was concentrated to obtain the title compound, 40 mg. It was used directly in the next step without purification. ESI-MS (m / z): 238.1 [M+H] + .

[0605] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole-4-carboxylic acid

[0606] At 25°C, compound 50-2 (40 mg, 0.17 mmol) was dissolved in dichloromethane (6 mL). After complete dissolution, m-chloroperbenzoic acid (29 mg, 0.17 mmol) was added. After addition, the mixture was stirred at 25°C for 14 h. The reaction solution was concentrated and the residue was purified by preparative HPLC (method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 269.9 [M+H] + .

[0607] Step 4: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazole-4-carboxamide

[0608] At 25°C, compound 50-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (4 mL), and O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (42 mg, 0.11 mmol) and N,N-diisopropylethylamine (19 mg, 0.15 mmol) were added in sequence, stirred for 5 min, propargylamine (5.0 mg, 0.09 mmol) was added, and stirred at room temperature for 30 min after the addition was completed. 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 mg. ESI-MS (m / z): 306.9 [M+H] + .

[0609] Step 5: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)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-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatricarboxamido)benzyl)carbonate

[0610] At 25°C, compound 50-4 (6.0 mg, 0.02 mmol) and compound 33-1 (30 mg, 0.02 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (2 mL / 0.5 mL), and cuprous bromide (5.0 mg, 0.04 mmol) was added at once. After addition, the mixture was reacted at room temperature for 2 h. 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] + .

[0611] Step 6: Synthesis of 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)benzyl-((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

[0612] Compound 50-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2.0 mL) at 25°C. After complete dissolution, trifluoroacetic acid (0.2 mL) was added to the reaction solution and reacted at 25°C for 10 min. The reaction solution was concentrated and the residue was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 3.0 mg. ESI-MS (m / z): 816.5 [M / 2+H] + .

[0613] 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-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- (methyl)-3-phenylpropanamide (methyl)-phenylpropanamide (methyl)-carbamoyl (9-isopropyl)-1,8,11,15-tetraoxy-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

[0614]

[0615] Step 1: Synthesis of ((S)-1-(((S)-1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyryl)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionyl)-3-phenylpropionyl)methyl)phenyl)amino)-1-oxo-5-pentylurea-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino-(9H-fluoren-9-yl)methyl-formate

[0616] At room temperature, compound 51-1 (100 mg, 0.17 mmol) and ((S)-1-(((S)-1-((4-(((S)-2-amino-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-pentylurea-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino-(9H-fluoren-9-yl)methyl-formate trifluoroacetate (144 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (2 mL), cooled to 0°C, and 1-hydroxybenzotriazole (34 mg, 0.25 mmol) and N-methylmorpholine (51 mg, 0.51 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (48 mg, 0.25 mmol) were added in sequence. After the addition was completed, the reaction solution was stirred at 0°C for 5 h. The reaction solution was poured into water (20 mL) to precipitate a white solid, which was filtered, and the filter cake was washed with water and dried to obtain the title compound, 200 mg. ESI-MS (m / z): 1329.2 [M+H] + .

[0617] Step 2: Synthesis of (S)-2-((S)-2-amino-3-butyrylamide)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamide)-N,3-dimethylbutyrylamide)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide

[0618] At room temperature, compound 51-2 (200 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (5 mL), piperidine (0.5 mL) was added, and the reaction solution was stirred at room temperature for 2 h. The reaction solution was purified by preparative HPLC (method D) to obtain the title compound, 65 mg. ESI-MS (m / z): 1107.2 [M+H] + .

[0619] Step 3: Synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontriadecylamino)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanamide)methyl)phenyl)-5-ureidopentanamide

[0620] 32-Azide-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatricarboxylic acid (33.1 mg, 0.06 mmol) was dissolved in N,N-dimethylformamide (5 mL), and O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (38 mg, 0.10 mmol) and N,N-diisopropylethylamine (26 mg, 0.20 mmol) were added. The reaction solution was stirred at room temperature for 10 min, 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 h. The reaction solution was purified by preparative HPLC (method D) to obtain the title compound, 56 mg. ESI-MS (m / z): 821.8 [M / 2+H] + .

[0621] 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-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropane Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (3-phenylpropanamide, methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxy-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

[0622] At room temperature, compound 51-4 (56 mg, 0.04 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)-5-hexynamide (16 mg, 0.05 mmol) were dissolved in a mixed solution of dimethyl sulfoxide and water (2 mL / 0.5 mL), and copper bromide (10 mg, 68.17 umol) was added and stirred for 2 h. Filtered, the filtrate was purified by preparative high performance liquid chromatography (method D) to obtain the title compound, 50 mg. ESI-MS (m / z): 974.3 [M / 2+H] + .

[0623] 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-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatetradecanoyl)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

[0624]

[0625] Step 1: 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatetradecanoyl)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R Synthesis of (((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate

[0626] At room temperature, compound 53-1 (100 mg, 0.09 mmol) was dissolved in N, N-dimethylformamide (3 mL), 1-hydroxybenzotriazole (13 mg, 0.09 mmol) and N, N-diisopropylethylamine (36 mg, 0.28 mmol) were added, and then compound 52-1 (67 mg, 0.09 mol) was added. The reaction solution was stirred at room temperature for 16 h. The reaction solution was 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] + .

[0627] Step 2: 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-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatetradecanoyl)benzyl-((S)-1-(( Synthesis of (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-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0628] 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 cuprous bromide (18 mg, 0.13 mmol) was added and stirred for 1 h. Filtered, the filtrate was purified by preparative HPLC (method D) to obtain the title compound, 92 mg. ESI-MS (m / z): 982.8 [M / 2+H] + .

[0629] Example 23 (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-(methylsulfonyl)pyrimidin-5-yl)hexane-5-carbamoyl)methyl)-1H- Synthesis of (1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriacontamido)butyrylamino)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0630]

[0631] Step 1: Synthesis of 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonyloxy-3,6,12-triazatriadecanoylamide)benzyl-(4-nitrophenyl)-carbonate

[0632] At 25°C, compound 29-1 (500 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (10 mL), N,N-diisopropylethylamine (141 mg, 1.09 mmol) was added, and a solution of di(p-nitrobenzene) carbonate (332 mg, 1.09 mmol) in dichloromethane (1 mL) was added dropwise. After the addition was completed, the mixture was stirred at 25°C for 3 h. The reaction solution was purified by reverse column (C18) chromatography (acetonitrile / water = 1:2) to obtain the title compound, 400 mg. ESI-MS (m / z): 1081.9 [M+H] + .

[0633] Step 2: Synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((((4-((S)-2-((S)-2-(3-(2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriacontamido)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0634] At 25°C, compound 53-1 (60 mg, 0.06 mmol), ((2R)-3-((2S)-1-((3R,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (41 mg, 0.06 mmol) were dissolved in N,N-dimethylformamide (2 mL). After complete dissolution, 1-hydroxybenzotriazole (8 mg, 0.06 mmol) was added. After addition, the mixture was stirred at 25°C for 16 h. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound, 38 mg. ESI-MS (m / z): 837.2 [M / 2+H] + .

[0635] 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-(32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-carbamoyl)methyl)-1H-1 Synthesis of (1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriacontamido)butyrylamino)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0636] At 25°C, 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazole-4-carboxamide (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, cuprous bromide (11 mg, 0.08 mmol) was added. After addition, the mixture was stirred under N2 protection for 1 h. Filtered, the filtrate was purified by preparative HPLC (method D) to obtain the title compound, 25 mg. ESI-MS (m / z): 989.9 [M / 2+H] + .

[0637] 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-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontriacontylamino)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-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0638]

[0639] Step 1: Synthesis of (S)-4-(35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentacontriadecylamino)benzyl-(4-nitrophenyl)-carbonate

[0640] At room temperature, compound 54-1 (1 g, 0.95 mmol) was dissolved in dichloromethane (20 mL), N, N-diisopropylethylamine (488 mg, 3.77 mmol) was added, and then a solution of di-(p-nitrobenzene)-carbonate (860 mg, 2.83 mmol) in dichloromethane (10 mL) was added dropwise, and the reaction solution was stirred at room temperature for 6 h. Purification by silica gel column (dichloromethane / methanol = 40 / 1) gave the title compound, 900 mg. ESI-MS (m / z): 953.0 [M+H-273] + .

[0641] Step 2: 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butanoyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatridecylamino)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1 Synthesis of (R,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-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0642] At room temperature, compound 54-2 (2 mL) was added with 1-hydroxybenzotriazole (33 mg, 0.25 mmol) and N,N-diisopropylethylamine (48 mg, 0.37 mmol), and then compound 52-1 (88 mg, 0.12 mmol) was added. The reaction solution was stirred at room temperature for 16 h. The reaction solution was purified by preparative HPLC (method B) to obtain the title compound, 150 mg. ESI-MS (m / z): 1803.6 [M+H] + .

[0643] Step 3: 4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-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-diazapentacontriacontylamino)benzyl ((S)-1-(((S)-1- Synthesis of (((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-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0644] At room temperature, compound 54-3 (100 mg, 0.06 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)-5-hexynamide (26 mg, 0.08 mmol) were dissolved in dimethyl sulfoxide (2 mL) and water (0.5 mL), and cuprous bromide (16 mg, 0.11 mmol) was added. The mixture was stirred for 2 h. Filtered, the filtrate was purified by preparative HPLC (method B) to obtain the title compound, 70 mg. ESI-MS (m / z): 1936.6 [M+H-273] + .

[0645] Step 4: 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-diazapentacontriacontylamino)benzyl-((S)-1-(((3R,4S, Synthesis of 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-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0646] Compound 54-4 (70 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL) at room temperature, and trifluoroacetic acid (0.2 mL) was added dropwise. The reaction solution was stirred at room temperature for 20 min. The reaction solution was concentrated, and the residue was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 55 mg. ESI-MS (m / z): 918.8 [M / 2+H] + .

[0647] Example 25 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)benzyl((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

[0648]

[0649] Step 1: Synthesis of methyl 4-(2-(methylthio)pyrimidin-5-yl)benzoate

[0650] At 25°C, compound 49-1 (252 mg, 1.0 mmol), water (3 mL), Pd(dppf)Cl2 (40 mg, 0.05 mmol) and potassium carbonate (277 mg, 2.0 mmol) were added to a solution of methyl p-bromobenzoate (215 mg, 1.0 mmol) in 1,4-dioxane (5 mL) in sequence, and stirred at 80°C for 4 h. The mixture was extracted with ethyl acetate (30 mL × 3), the organic phases were combined, dried, the insoluble matter was filtered out, 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] + .

[0651] Step 2: Synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic acid

[0652] At 25°C, lithium hydroxide monohydrate (322 mg, 7.68 mmol) and water (3 mL) were added to a solution of compound 55-1 (500 mg, 1.92 mmol) in tetrahydrofuran (3 mL) respectively, and the mixture was stirred for 4 h. The pH of the reaction solution was adjusted to 3-4 with 1N hydrochloric acid, extracted with ethyl acetate (20 mL×3), the organic phases were combined, dried, and the insoluble matter was filtered off. The residue was purified by preparative high performance liquid chromatography (method D) to obtain the title compound, 430 mg. ESI-MS (m / z): 246.9 [M+H] + .

[0653] Step 3: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid

[0654] At 25°C, m-chloroperbenzoic acid (420 mg, 2.44 mmol) was added to a solution of compound 55-2 (200 mg, 0.81 mmol) in dichloromethane (5 mL) and stirred for 5 h. The title compound was purified by silica gel column chromatography, 180 mg. ESI-MS (m / z): 279.0 [M+H] + .

[0655] Step 4: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)benzamide

[0656] At 25°C, add benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (100 mg, 0.26 mmol) to a solution of compound 55-3 (50 mg, 0.18 mmol) in dichloromethane (10 mL), stir and react for 30 min, then add propargylamine (10 mg, 0.2 mmol) and N,N-diisopropylethylamine (70 mg, 0.5 mmol) to the reaction solution, stir and react for 2.5 h. Purify by silica gel column chromatography to obtain the title compound, 20 mg. ESI-MS (m / z): 316.0 [M+H] + .

[0657] Step 5: (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)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-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazole- Synthesis of 1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentacontanamide)benzyl)carbonate At 25°C, under N2 protection, cuprous iodide (10 mg, 0.05 mmol) and water (2 mL) were added to a solution of compound 55-4 (16 mg, 0.05 mmol) and compound 33-1 (80 mg, 0.05 mmol) in dimethyl sulfoxide (2 mL) in sequence, and the mixture was stirred for 1 h. Purification (method B) gave the title compound, 79 mg. ESI-MS (m / z): 1641.5 [M-273+H] + .

[0658] Step 6: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)benzyl((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

[0659] At 25°C, compound 55-5 (55 mg, 0.029 mmol) was added to a mixed solvent of trifluoroacetic acid (0.5 mL) in water / acetonitrile (0.1 mL / 0.5 mL). The mixture was stirred for 15 min, and the reaction solution was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 42 mg. ESI-MS (m / z): 821.0 [M / 2+H] + .

[0660] 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-methylpropionamido)propyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonyloxy-2,7,10,16-tetraazatetradodecane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0661]

[0662] Step 1: Synthesis of 3-(2-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriacontanoic acid

[0663] 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 cuprous bromide (465 mg, 3.21 mmol) was added at once. After the addition was completed, the reaction was stirred for 12 h. 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] + .

[0664] Step 2: Synthesis of (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-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)amino)-1-oxo-5-ureidopentanamido-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0665] At 25°C, (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-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3 -Dimethylbutyrylamino (185 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), HATU (137 mg, 0.36 mmol) was added, and the mixture was stirred for 5 min. (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanic acid (131 mg, 0.26 mmol) was added, and the mixture was stirred at room temperature for 30 min. The reaction solution was directly used for the next step. ESI-MS (m / z): 626.0 [M / 2+H] + .

[0666] 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-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidopentanamide

[0667] At 25°C, diethylamine (0.5 mL) was added to the reaction solution of step 2. After the addition was complete, the reaction was stirred for 30 min. The reaction solution was purified by preparative HPLC (method D) to obtain the title compound, 70 mg. ESI-MS (m / z): 515.0 [M / 2+H] + .

[0668] Step 4: 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 Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-4-yl)-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonyloxy-2,7,10,16-tetraazatetradodecane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0669] 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-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidopentanamide (95 mM) was added. g, 0.092mmol), 32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriacontanoic acid (79mg, 0.092mmol) was dissolved in N,N-dimethylformamide (4mL), HATU (70mg, 0.184mmol) was added at one time, and the reaction was stirred at room temperature for 1h. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound, 30mg. ESI-MS (m / z): 935.8[M / 2+H] + .

[0670] 3. Conjugation of compounds containing cell bioactive molecules and linkers to antibodies

[0671] Example 27 Preparation of BT001002

[0672] Take 0.3mL of Sacituzumab antibody (anti-Trop-2, 33.5mg / mL), dilute with 0.25mL of solution (pH 7.6) containing 20mM PB, 150mM NaCl and 20mM sodium edetate, then add 0.45mL of solution (pH 7.6) containing 20mM PB and 150mM NaCl, mix well, adjust pH to 7.4 with 1M K2HPO4 solution, add 10mM TCEP (tris(2-carboxyethyl)phosphine) solution, mix well, and let stand at room temperature for 30min. Add 15 times the amount of TL003 dissolved in dimethyl sulfoxide to the above solution system, mix well, let stand at room temperature for 2h, and add 6.1μl of 100mM cysteine ​​to terminate the reaction after completion. Finally, the buffer was replaced with a 20 mM PB buffer solution at pH 6.44 using a G-25 gel column to obtain a product of TL003 coupled with Sacituzumab antibody, named BT001002.

[0673]

[0674] Example 28 Preparation of BT001004

[0675] Take 0.285mL Sacituzumab antibody (anti-Trop-2, 17.6mg / mL), dilute with 0.095mL diluent (containing 20mM PB, 150mM NaCl and 20mM sodium edetate solution, pH 7.6); then adjust the pH to 7.4 with 1M Na2HPO4 solution, add 10mM TCEP solution and mix well, and place at room temperature for 30min; add 9 times the amount of TL019 dissolved in dimethyl sulfoxide to the above solution system, mix well, and stand at room temperature for 2h. Finally, use G-25 gel column to replace the buffer solution with PBS buffer solution of pH 6.5 to obtain the product of TL019 and Sacituzumab antibody coupling, named BT001004.

[0676]

[0677] Example 29 Preparation of BT001012

[0678] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL024 to obtain a product of TL024 coupled with Sacituzumab antibody, which was named BT001012.

[0679]

[0680] Example 30 Preparation of BT001013

[0681] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL048 to obtain a product of TL048 coupled with Sacituzumab antibody, which was named BT001013.

[0682]

[0683] Example 31 Preparation of BT001018

[0684] A method similar to that in Example 27 was used to replace TL003 with TL030 to obtain a product of TL030 coupled with Sacituzumab antibody, which was named BT001018.

[0685]

[0686] Example 32 Preparation of BT001021

[0687] Take 0.3mL Sacituzumab antibody (anti-Trop-2, 33.5mg / mL), dilute with 0.25mL solution (pH7.6) containing 20mM PB, 150mM NaCl and 20mM sodium edetate, then add 0.45mL solution (pH7.6) containing 20mM PB and 150mM NaCl, mix well, adjust pH to 7.4 with 1M Na2HPO4 solution, add 10mM TCEP (tris(2-carboxyethyl)phosphine) solution, mix well, and let stand at room temperature for 30min. Add 10 times the amount of TL033 trifluoroacetate dissolved in dimethyl sulfoxide to the above solution system, mix well, let stand at room temperature for 2h, and add 6.1μl of 100mM cysteine ​​to terminate the reaction after completion. Finally, the buffer solution was replaced with a PBS buffer solution at pH 6.5 using a G-25 gel column to obtain a product of TL033 coupled with Sacituzumab antibody, which was named BT001021.

[0688]

[0689] Example 33 Preparation of BT001022

[0690] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL034 to obtain a product of TL034 coupled with Sacituzumab antibody, which was named BT001022.

[0691]

[0692] Example 34 Preparation of BT001023

[0693] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL035 to obtain a product of TL035 coupled with Sacituzumab antibody, which was named BT001023.

[0694]

[0695] Example 35 Preparation of BT001032

[0696] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL045 to obtain a product of TL045 coupled with Sacituzumab antibody, which was named BT001032.

[0697]

[0698] Example 36 Preparation of BT001033

[0699] A method similar to that of Example 27 was used, whereby TL003 was replaced by the trifluoroacetate salt of TL033, and the Sacituzumab antibody was replaced by the M1 antibody, to obtain a product of TL033 coupled with the M1 antibody, which was named BT001033.

[0700]

[0701] Example 37 Preparation of BT001034

[0702] A method similar to that in Example 27 was used, whereby TL003 was replaced by the trifluoroacetate salt of TL033, and the Sacituzumab antibody was replaced by the M2 antibody, to obtain a product of TL033 and M2 antibody coupling, which was named BT001034.

[0703]

[0704] Example 38 Preparation of BT001035

[0705] Take 0.3mL M3 antibody (anti-Trop-2, 33.5mg / mL), dilute with 0.25mL solution (pH7.6) containing 20mM PB, 150mM NaCl and 20mM sodium edetate, then add 0.45mL solution (pH7.6) containing 20mM PB and 150mM NaCl, mix well, adjust pH to 7.4 with 1M Na2HPO4 solution, add 10mM TCEP (tri(2-carboxyethyl)phosphine) solution, mix well, and let stand at room temperature for 30min. Add 10 times the amount of trifluoroacetate of TL033 dissolved in dimethyl sulfoxide to the above solution system, mix well, let stand at room temperature for 2h, and add 6.1μl of 100mM cysteine ​​to terminate the reaction. Finally, use G-25 gel column to replace the buffer with pH6.5 PBS buffer solution to obtain the product of TL033 and M3 antibody coupling, named BT001035.

[0706]

[0707] Example 39 Preparation of BT001036

[0708] A method similar to that in Example 27 was used, whereby TL003 was replaced by the trifluoroacetate salt of TL033, and the Sacituzumab antibody was replaced by the trastuzumab antibody, to obtain a product of TL033 and trastuzumab coupled thereto, which was named BT001036.

[0709]

[0710] Example 40 Preparation of BT001040

[0711] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL049 to obtain a product of TL049 coupled with Sacituzumab antibody, which was named BT001040.

[0712]

[0713] Example 41 Preparation of BT001041

[0714] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL050 to obtain a product of TL050 coupled with Sacituzumab antibody, which was named BT001041.

[0715]

[0716] Example 42 Preparation of BT001042

[0717] A method similar to that in Example 27 was used to replace TL003 with TL051 to obtain a product of TL051 coupled with Sacituzumab antibody, which was named BT001042.

[0718]

[0719] Example 43 Preparation of BT001043

[0720] A method similar to that in Example 27 was used to replace TL003 with TL052 to obtain a product of TL052 coupled with Sacituzumab antibody, which was named BT001043.

[0721]

[0722] Example 44 Preparation of BT001044

[0723] A method similar to that in Example 27 was used to replace TL003 with TL053 to obtain a product of TL053 coupled with Sacituzumab antibody, which was named BT001044.

[0724]

[0725] Example 45 Preparation of BT001045

[0726] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL054 to obtain a product of TL054 coupled with Sacituzumab antibody, which was named BT001045.

[0727]

[0728] Example 46 Preparation of BT001046

[0729] A method similar to that in Example 27 was used to replace TL003 with the trifluoroacetate salt of TL055 to obtain a product of TL055 coupled with Sacituzumab antibody, which was named BT001046.

[0730]

[0731] Example 47 Preparation of BT001047

[0732] A method similar to that in Example 27 was used to replace TL003 with TL056 to obtain a product of TL056 coupled with Sacituzumab antibody, which was named BT001047.

[0733]

[0734] Example 48 LC-MS determination of BT001002 molecular weight

[0735] The molecular weight of the coupled BT001002 was analyzed by LCMS.

[0736] Chromatographic conditions:

[0737] Liquid chromatography column: Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0738] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0739] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg;

[0740] Time (minutes) 1 7 8 9 13 Mobile phase A (volume %) 90 20 20 90 90 Mobile phase B (volume %) 10 80 80 10 10

[0741] Mass spectrometry conditions:

[0742] Mass spectrometer model: Triple TOF 5600+;

[0743] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m / z600-5000;

[0744] The results are as follows Figure 1-3 shown.

[0745] BT001002 Theoretical and Measured Molecular Weight

[0746]

[0747] In the table, mAb means monoclonal antibody; LC means antibody light chain; HC means antibody heavy chain; DAR1 means a conjugate comprising one antibody light chain / heavy chain and one cell bioactive molecule; DAR2 means a conjugate comprising one antibody light chain / heavy chain and two cell bioactive molecules; DAR3 means a conjugate comprising one antibody light chain / heavy chain and three cell bioactive molecules; DAR4 means a conjugate comprising one antibody light chain / heavy chain and four cell bioactive molecules; glycoform means the sugar chain structure on the two heavy chains: G0F means fucosylated without galactose. mAb, LC, HC, DAR1, DAR2, DAR3, DAR4 and G0F are described above below.

[0748] Depend on Figure 1-3It can be seen that after the antibody was coupled to TL003, the molecular weight of the light and heavy chains changed, with the light chain coupled to 1 cell bioactive molecule and the heavy chain coupled to 3 cell bioactive molecules. It can be inferred that the coupling ratio (DAR) of the entire antibody to the cell bioactive molecules is 8.

[0749] Example 49 LC-MS determination of BT001004 molecular weight

[0750] The molecular weight of the coupled BT001004 was analyzed by LC-MS.

[0751] Chromatographic conditions:

[0752] Liquid chromatography column: Protein BEH C18 1.7μm, 2.1mm×100mm;

[0753] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0754] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg;

[0755] Time (minutes) 2 20 22 25 26 30 Mobile phase A (volume %) 80 60 10 10 80 80 Mobile phase B (volume %) 20 40 90 90 20 20

[0756] Mass spectrometry conditions:

[0757] Mass spectrometer model: Triple TOF 5600+;

[0758] GS1 60; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m / z 600-5000;

[0759] The results are as follows Figure 4-6 shown.

[0760] BT001004 theoretical molecular weight and measured molecular weight

[0761]

[0762] LC stands for antibody light chain, and HC stands for antibody heavy chain.

[0763] Depend on Figure 4-6 It can be seen that the antibody light chain in BT001004 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 14% and 86% respectively), and the heavy chain is coupled to 1-3 cellular bioactive molecules (DAR1, DAR2, DAR3 ratios are 13%, 19%, 68% respectively). The coupling ratio (DAR) of the entire antibody to cellular bioactive molecules is calculated to be 7.0.

[0764] Example 50 LC-MS determination of BT001012 molecular weight

[0765] Using a method similar to Example 48, the results are as follows Fig.10 and 11 shown.

[0766] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001012 light chain and heavy chain obtained after TL024 was coupled to the antibody are shown in the following table:

[0767]

[0768] Depend on Fig.10 and 11 It can be seen that the antibody light chain in BT001012 is coupled to 0 to 1 toxin (LC and DAR1 ratios are 12.9% and 87.1% respectively), and the heavy chain is coupled to 1 to 3 toxins (DAR1, DAR2, DAR3 ratios are 13.4%, 10.8%, 75.8% respectively). The antibody-to-toxin coupling ratio (DAR) is calculated to be 7.0.

[0769] Example 51 LC-MS determination of BT001013 molecular weight

[0770] Using a method similar to Example 48, the results are as follows Fig.12 and 13 shown.

[0771] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001013 light chain and heavy chain obtained after TL048 was coupled to the antibody are shown in the following table:

[0772]

[0773] Depend on Fig.12 and 13 It can be seen that the antibody light chain in BT001013 is coupled to 0 to 1 toxin (LC and DAR1 ratios are 6.8% and 93.2% respectively), and the heavy chain is coupled to 1 to 4 toxins (DAR1, DAR2, DAR3, DAR4 ratios are 12.8%, 12.8%, 64.9%, 9.5% respectively). The coupling ratio (DAR) of the antibody to toxin is calculated to be 7.3.

[0774] Example 52 LC-MS determination of molecular weight of BT001018

[0775] Using a method similar to Example 48, the results are as follows Fig.14 and 15 shown.

[0776] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001018 light chain and heavy chain obtained after TL030 was coupled with the antibody are shown in the following table:

[0777]

[0778] Depend on Fig.14 and 15 It can be seen that the antibody light chain in BT001018 is coupled to 0 to 1 toxin (LC and DAR1 ratios are 55.3% and 44.7% respectively), and the heavy chain is coupled to 1 to 3 toxins (DAR1, DAR2, DAR3 ratios are 19.6%, 23.3%, 49.6% respectively). The antibody-to-toxin coupling ratio (DAR) is calculated to be 5.2.

[0779] Example 53 LC-MS determination of BT001021 molecular weight

[0780] The molecular weight of the coupled BT001021 was analyzed by LCMS.

[0781] Chromatographic conditions:

[0782] HPLC column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0783] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0784] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg;

[0785] Time (minutes) 1 7 8 9 13 Mobile phase A (volume %) 90 20 20 90 90 Mobile phase B (volume %) 10 80 80 10 10

[0786] Mass spectrometry conditions:

[0787] Mass spectrometer model: Triple TOF 5600+;

[0788] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m / z600-5000;

[0789] The results are as follows Fig.16 and 17 shown.

[0790] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001021 light chain and heavy chain obtained after TL033 was coupled with the antibody are shown in the following table:

[0791]

[0792]

[0793] Depend on Fig.16 and 17 It can be seen that the antibody light chain in BT001021 is coupled to 0 to 1 toxin (LC and DAR1 ratios are 4.5% and 95.5% respectively), and the heavy chain is coupled to 1 to 3 toxins (DAR1, DAR2, DAR3 ratios are 15.3%, 17.6%, 67.1% respectively). The antibody-to-toxin coupling ratio (DAR) is calculated to be 6.9.

[0794] Example 54 LC-MS determination of BT001023 molecular weight

[0795] Using a method similar to Example 48, the results are as follows Fig.18 and 19 shown.

[0796] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001023 light chain and heavy chain obtained after TL035 was coupled with the antibody are shown in the following table:

[0797]

[0798] Depend on Fig.18 and 19 It can be seen that the antibody light chain in BT001023 is coupled to 0 to 1 toxin (LC, DAR1 ratios are 15% and 85% respectively), and the heavy chain is coupled to 0 to 3 toxins (HC, DAR1, DAR2, DAR3 ratios are 6.7%, 16.7%, 12.7%, 63.9% respectively). The coupling ratio (DAR) of the antibody to toxin is calculated to be 6.4.

[0799] Example 55 LC-MS determination of BT001040 molecular weight

[0800] LC-MS molecular weight analysis of BT001040 after coupling

[0801] Liquid chromatography column: Thermo MabPacTM RP 4μm, 3.0mm*100mm

[0802] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN

[0803] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg

[0804] Time (minutes) 2 20 22 25 26 30 Mobile phase A (volume %) 80 60 10 10 80 80 Mobile phase B (volume %) 20 40 90 90 20 20

[0805] Mass spectrometry conditions:

[0806] Mass spectrometer model: Triple TOF 5600+

[0807] GS1 35; GS2 35; CUR30; TEM 350; ISVF5000; DP250; m / z 600-5000

[0808] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001040 light chain and heavy chain obtained after TL049 was coupled to the antibody are shown in the following table:

[0809]

[0810] Depend on Fig. 20 and 21 It can be seen that the antibody light chain in BT001040 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 4.9% and 95.1% respectively), and the heavy chain is coupled to 1-4 cellular bioactive molecules (DAR1, DAR2, DAR3, DAR4 ratios are 16.5%, 14.3%, 52.6%, 16.6% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 7.3.

[0811] Example 56 LC-MS determination of molecular weight of BT001041

[0812] Using a method similar to Example 55, the results are as follows Fig. 22 and 23 shown.

[0813] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001041 light chain and heavy chain obtained after TL050 was coupled with the antibody are shown in the following table:

[0814]

[0815] Depend on Fig. 22 and 23 It can be seen that the antibody light chain in BT001041 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 10.5% and 89.5% respectively), and the heavy chain is coupled to 1-4 cellular bioactive molecules (DAR1, DAR2, DAR3, DAR4 ratios are 21.3%, 14.8%, 57.9%, 6.0% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.8.

[0816] Example 57 LC-MS determination of BT001042 molecular weight

[0817] Using a method similar to Example 55, the results are as follows Fig.24 and 25 shown.

[0818] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001042 light chain and heavy chain obtained after TL051 was coupled to the antibody are shown in the following table:

[0819]

[0820] Depend on Fig.24 and 25 It can be seen that the antibody light chain in BT001042 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 14.9% and 85.1% respectively), and the heavy chain is coupled to 1-3 cellular bioactive molecules (DAR1, DAR2, DAR3 ratios are 19.7%, 9.4%, 70.9% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.7.

[0821] Example 58 LC-MS determination of BT001043 molecular weight

[0822] Using a method similar to Example 55, the results are as follows Fig.26 and 27 shown.

[0823] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001043 light chain and heavy chain obtained after TL052 was coupled to the antibody are shown in the following table:

[0824]

[0825]

[0826] Depend on Fig.26 and 27 It can be seen that the antibody light chain in BT001043 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 9.1% and 90.9% respectively), and the heavy chain is coupled to 1-3 cellular bioactive molecules (DAR1, DAR2, DAR3 ratios are 20.1%, 11.4%, 68.4% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.8.

[0827] Example 59 LC-MS determination of BT001044 molecular weight

[0828] Using a method similar to Example 55, the results are as follows Fig.28 and 29 shown.

[0829] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001044 light chain and heavy chain obtained after TL053 was coupled with the antibody are shown in the following table:

[0830]

[0831] Depend on Fig.28 and 29 It can be seen that the antibody light chain in BT001044 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 23.0% and 77.0% respectively), and the heavy chain is coupled to 1-3 cellular bioactive molecules (DAR1, DAR2, DAR3 ratios are 19.4%, 11.4%, 69.3% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.5.

[0832] Example 60 LC-MS determination of BT001046 molecular weight

[0833] Using a method similar to Example 55, the results are as follows Fig.30 and 31 shown.

[0834] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001046 light chain and heavy chain obtained after TL055 was coupled with the antibody are shown in the following table:

[0835]

[0836]

[0837] Depend on Fig.30 and 31 It can be seen that the antibody light chain in BT001046 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 33.8% and 66.2% respectively), and the heavy chain is coupled to 0-3 cellular bioactive molecules (DAR0, DAR1, DAR2, DAR3 ratios are 21.9%, 6.1%, 9.6%, 62.3% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 5.6.

[0838] Example 61 Determination of Molecular Weight of BT001047 by LC-MS

[0839] Using a method similar to Example 55, the results are as follows Fig.32 and 33 shown.

[0840] The theoretical molecular weight and measured molecular weight (calculated based on the main glycoform G0F) of the BT001047 light chain and heavy chain obtained after TL056 was coupled to the antibody are shown in the following table:

[0841]

[0842] Depend on Fig.32 and 33 It can be seen that the antibody light chain in BT001047 is coupled to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 13.7% and 86.3% respectively), and the heavy chain is coupled to 1-3 cellular bioactive molecules (DAR1, DAR2, DAR3 ratios are 22.2%, 13.5%, 64.3% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.6.

[0843] Example 62 Size Exclusion Chromatography Analysis

[0844] The coupling reaction was monitored by SEC-HPLC and the conjugate was subjected to SEC detection.

[0845] Chromatographic conditions:

[0846] HPLC column: TOSOH TSKgel SuperSW mAb, 4 μm, 7.8 mm x 300 mm;

[0847] Mobile phase: 100mmol / L Na2HPO4, 100mmol / L NaCl, 5% isopropanol, pH 7.0;

[0848] Flow rate: 0.5 ml / min; Detection wavelength: 280 nm; Column temperature: room temperature; Sample chamber temperature: 8 °C;

[0849] Injection volume: 30 μg; isocratic run: 30 min.

[0850] The SEC chromatograms of BT001002 obtained by coupling the antibody with TL003 and the SEC chromatograms of the molecular weight marker are shown in Figure 1. Figure 7 and 8 As shown, according to the molecular weight marker, it is confirmed that the molecular weight corresponding to the main peak of the coupling product is about 150kD, that is, the light and heavy chains of BT001002 obtained by coupling the antibody with TL003 are not separated, and the antibody still maintains the overall structure.

[0851] The SEC chromatogram of BT001004 obtained by coupling TL019 with the antibody is shown in Fig. 9 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001004 obtained by coupling TL019 with the antibody still maintained the complete structure of the antibody.

[0852] The SEC chromatogram of BT001012 obtained by coupling TL024 with the antibody is shown in Fig.34As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001012 obtained by coupling TL024 with the antibody still maintained the complete structure of the antibody.

[0853] The SEC chromatogram of BT001013 obtained by coupling TL048 with the antibody is shown in Fig.35 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001013 obtained by coupling TL048 with the antibody still maintained the complete structure of the antibody.

[0854] The SEC chromatogram of BT001018 obtained by coupling TL030 with the antibody is shown in Fig.36 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001018 obtained by coupling TL030 with the antibody still maintained the complete structure of the antibody.

[0855] The SEC chromatogram of BT001021 obtained by coupling TL033 with the antibody is shown in Fig.37 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001021 obtained by coupling TL033 with the antibody still maintained the complete structure of the antibody.

[0856] The SEC chromatogram of BT001023 obtained by coupling TL035 with the antibody is shown in Fig.38 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001023 obtained by coupling TL035 with the antibody still maintained the complete structure of the antibody.

[0857] The SEC chromatogram of BT001042 obtained by coupling TL051 with the antibody is shown in Fig.39 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001042 obtained by coupling TL051 with the antibody still maintained the complete structure of the antibody.

[0858] The SEC chromatogram of BT001043 obtained by coupling TL052 with the antibody is shown in Fig.40 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001043 obtained by coupling TL052 with the antibody still maintained the complete structure of the antibody.

[0859] The SEC chromatogram of BT001044 obtained by coupling TL053 with the antibody is shown in Fig.41As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001044 obtained by coupling TL053 with the antibody still maintained the complete structure of the antibody.

[0860] The SEC chromatogram of BT001046 obtained by coupling TL055 with the antibody is shown in Fig.42 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001046 obtained by coupling TL055 with the antibody still maintained the complete structure of the antibody.

[0861] The SEC chromatogram of BT001047 obtained by coupling TL056 with the antibody is shown in Fig.43 As shown, according to the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was about 150kD, that is, BT001047 obtained by coupling TL056 with the antibody still maintained the complete structure of the antibody.

[0862] Example 63 Detection of the inhibitory effect of bioactive molecules and antibody-drug conjugates on in vitro cell activity

[0863] First, tumor cells MDA-MB-468 (Trop-2 positive cell line) and HCC1806 (Trop-2 positive cell line) were cultured; the bioactive molecules and ADC molecules disclosed in the present invention were co-cultured with tumor cells, and then CCK8 reagent (Dongren Chemical Technology Co., Ltd., Cat: CK04, Lot: JJ744) was added, and the activity of dehydrogenase in mitochondria was detected using an ELISA reader (manufacturer: Molecular Devices, model: SpectraMax M2) (detection wavelength of 450nm) to evaluate the inhibitory effect of ADC on cell proliferation. The source of tumor cells is shown in Table 1.

[0864] Table 1.

[0865] Cell Name Tumor type source MDA-MB-468 Breast cancer Connaughty HCC1806 Breast cancer Nanjing Kebai Biological

[0866] In vitro cell activity detection: Dilute the bioactive molecule or ADC (12 concentration gradients) with the corresponding detection medium (containing 2% FBS). Use pancreatic enzymes to digest tumor cells by conventional methods, collect and count cells, and resuspend them with the corresponding detection medium (containing 2% FBS). Add the diluted bioactive molecule or ADC to a 96-well plate, and then add cells. Then add 20 μL of CCK8 reagent to each well, react for 4 hours, and read the microplate reader (detection wavelength is 450nm). The experimental conditions and test results are shown in Tables 2 and 3.

[0867] Table 2. Cell killing results of bioactive molecules

[0868]

[0869]

[0870] The test results showed that the biologically active molecules all had tumor cell killing effects.

[0871] Table 3. Conjugate (ADC) cell line killing results

[0872]

[0873] The test results show that the ADC molecules obtained by the new coupling method have a tumor cell killing effect. This shows that the ADC formed by the new coupling method can kill tumor cells, and the new coupling method is effective in the application of ADC molecules.

[0874] Embodiment 64 In vivo efficacy testing of antibody drug conjugates and active biomolecules

[0875] Test drug

[0876] Drug name, source, and preparation method:

[0877] BT001021, liquid concentration is 5.44 mg / ml, stored at -20°C, diluted with saline according to the dose to obtain the test solution;

[0878] Immu-132 (prepared with reference to Example 2 of WO2015 / 012904A2, DAR=5.4, also referred to as IMMU-132), liquid concentration of 13.158 mg / ml, stored at -20°C, diluted with physiological saline according to the dose to obtain a test solution when used;

[0879] T-030, solid powder, prepared with 100% DMSO (Sigma) to a concentration of 5.2 mg / ml, stored at -20°C, and diluted with physiological saline according to the dose to obtain the test solution when used;

[0880] SN-38 (also referred to as SN38), solid powder, was prepared with 100% DMSO (Sigma) to a concentration of 3.23 mg / ml, stored at -20°C, and diluted with physiological saline according to the dose to obtain a test solution.

[0881] Note: The toxin dosage is prepared according to the equimolar ratio of ADC sample.

[0882] The structures of T-030, SN-38 and Immu-132 are as follows:

[0883]

[0884] Experimental animals and cell lines

[0885] Balb / c-nu mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., production license number: SCXK(Beijing)2016-0011); gastric cancer cell line NCI-N87 (ATCC), breast cancer cell line HCC1806 (Nanjing Kebai).

[0886] Experimental grouping and evaluation methods

[0887] The tumor volume was 100-200 mm2 and the random grouping (the number of groups was determined according to the number of samples) was selected. 3 The tumor-bearing mice were 6 per group. The administration volume was 10 mL / kg, and the administration route was tail vein injection. The administration was given twice a week. The tumor diameter was measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0888] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition efficacy of antibody-drug conjugates:

[0889] TGI (%) = [1-(V T末 -V T始 ) / (V C末 -V C始 )]*100%

[0890] Where V T末 : Mean tumor volume at the end of the treatment group experiment

[0891] V T始 : Mean tumor volume of treatment group at the beginning of drug administration

[0892] V C末 : Mean tumor volume at the end of the experiment in the solvent control group

[0893] V C始 : Mean tumor volume at the start of drug administration in the solvent control group

[0894] The following Experimental Examples 1 and 2 evaluate the inhibition of tumor proliferation of tumor-bearing mice constructed by subcutaneous transplantation of human tumor cells by the antibody conjugate BT001021. Specifically, in Experimental Examples 1 and 2, subcutaneous transplantation of human gastric cancer cell line NCI-N87 and human triple-negative breast cancer cell line HCC1806 was used to construct a tumor-bearing mouse model. When the tumor volume grew to 100 mm 3After about 1 hour, the mice were randomly divided into groups and given BT001021 intravenously twice a week for a total of 6 times. The tumor volume and animal body weight changes were measured twice a week to evaluate the efficacy (tumor inhibition effect) of the antibody drug conjugate on tumor-bearing mice.

[0895] Experimental example 1. Inhibition of NCI-N87 by antibody-drug conjugates and active biomolecules

[0896] Experimental methods:

[0897] NCI-N87 cells were cultured in 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 a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a gastric cancer model. When the average tumor volume was about 90 mm 3 At the time of the experiment, the mice were randomly divided into normal saline group, BT001021 (3 mg / kg, intravenous injection, twice a week × 3 weeks), positive drug Immu-132 (3 mg / kg, intravenous injection, twice a week × 3 weeks), T030 group, and SN38 group according to the tumor size. After grouping, the corresponding drugs were injected into the tail vein twice a week for a total of 6 times. After the drug administration, the tumor volume and body weight of the mice were observed and measured regularly. The specific results are shown in Table 4. Fig.44 and 45 .

[0898] Experimental conclusion:

[0899] In this experimental example, human gastric cancer cell line NCI-N87 was used to construct a human gastric cancer subcutaneous transplant tumor model, and the efficacy of BT001021 in the NCI-N87 human gastric cancer tumor-bearing mouse model was evaluated.

[0900] The experimental results show that BT001021 (3 mg / kg, intravenous injection, twice a week for 3 weeks) can significantly inhibit the tumor growth of NCI-N87 gastric cancer transplanted tumor model mice, and tumor regression occurred at the endpoint of administration. The anti-tumor activity is better than that of positive Immu-132. There was no animal death or significant animal weight loss in all treatment groups during the observation period, indicating that BT001021 has no obvious toxicity.

[0901] Table 4. Gastric cancer NCI-N87 model

[0902]

[0903] Experimental example 2. Inhibition of HCC1806 by antibody-drug conjugates

[0904] Experimental methods:

[0905] HCC1806 cells were cultured in 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 to a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a breast cancer model. When the average tumor volume was about 130 mm 3 At the same time, the mice were randomly divided into normal saline group, BT001021 (10 mg / kg, intravenous injection, twice a week × 3 weeks) group and positive drug Immu-132 (10 mg / kg, intravenous injection, twice a week × 3 weeks) group according to the tumor size. After grouping, the corresponding drugs were injected into the tail vein twice a week for a total of 5 times. After drug administration, the tumor volume of mice was observed and measured regularly. The specific results are shown in Table 5. Fig.46 .

[0906] Experimental conclusion:

[0907] In this experiment, the human breast cancer cell line HCC1806 was used to construct a human breast cancer subcutaneous transplant tumor model, and the efficacy of BT001021 in the HCC1806 human breast cancer tumor-bearing mouse model was evaluated.

[0908] The experimental results show that BT001021 (10 mg / kg, intravenous injection, twice a week × 3 weeks) significantly inhibits tumor growth in HCC1806 breast cancer transplanted tumor model mice, and its anti-tumor activity is better than that of positive Immu-132.

[0909] Table 5: Breast cancer HCC1806 model

[0910]

[0911]

[0912] From Table 4, Table 5 and Figures 44-46 It can be seen that the antibody drug of the present invention, BT001021, can significantly inhibit tumor growth in the mouse NCI-N87 model, and is significantly better than Immu-132 at the same dose, without obvious weight loss and no obvious drug toxicity. In the mouse HCC1806 model, due to the high malignancy of the tumor, when the dose was increased to 10 mg / kg, Immu-132 did not show obvious inhibitory activity, while BT001021 can significantly inhibit tumor growth. The above results show that BT001021 of the present invention has good efficacy and excellent safety.

[0913] In the subcutaneous transplant tumor models of Experimental Examples 1 and 2, the anti-tumor activity of BT001021 was significantly better than that of Immu-132 at the same dose. It is speculated that BT001021 has the potential to treat solid tumors. Compared with Immu-132, BT001021 is expected to benefit more patients clinically.

[0914] Experimental Example 3. Inhibition of HCC827 by antibody-drug conjugates

[0915] Example 3 Evaluation of the effect of BT001021 and BT001035 on the proliferation inhibition of HCC827 non-small cell lung cancer subcutaneously transplanted human tumor cells to construct a tumor-bearing mouse model. Specifically, in this experiment, human non-small cell lung cancer cell line HCC827 was subcutaneously transplanted to construct a tumor-bearing mouse model. When the tumor volume grew to 100mm 3 The mice were randomly divided into groups and given BT001021 and BT001035 intravenously twice a week for a total of 6 times. The tumor volume and animal body weight changes were measured twice a week, and the efficacy (tumor inhibition effect) of BT001021 and BT001035 on tumor-bearing mice was calculated.

[0916] Experimental methods:

[0917] HCC827 cells were cultured in 1640 culture medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a lung cancer transplant tumor model. When the average tumor volume was about 80mm3, the mice were randomly divided into normal saline group, positive drug Immu-132 (10mg / kg, IV, BIW×3W) group, BT001021 (10mg / kg, IV, BIW×3W) group, and BT001035 (10mg / kg, IV, BIW×3W) group according to the tumor size. After grouping, 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 specific results are shown in Table 6. Fig.47A , Fig.47B .

[0918] Experimental conclusion:

[0919] The experimental results show that BT001021 and BT001035 can significantly inhibit the tumor growth of HCC827 non-small cell lung cancer transplanted tumor model mice, and tumor regression occurred at the end point of administration. The anti-tumor activity was better than that of the positive group Immu-132. There was no animal death or significant animal weight loss in all treatment groups during the observation period, and no obvious drug toxicity was shown. During the treatment period, the mice tolerated the evaluated drugs well.

[0920] Table 6. Lung cancer HCC827 model

[0921]

[0922] From Table 6, Fig.47A and Fig.47B It can be seen that BT001021 and BT001035 have significant tumor growth inhibition activity during the evaluation period, and at the same dose, their tumor inhibition activity is significantly better than Immu-132. During the administration period, there was no obvious weight loss and obvious drug toxicity in the animals in each group. The above results show that BT001021 and BT001035 have excellent anti-tumor activity.

[0923] In this subcutaneous transplant tumor model, the anti-tumor activities of BT001021 and BT001035 were significantly better than Immu-132 at the same dose. It is speculated that BT001021 and BT001035 have the potential to treat solid tumors. Compared with Immu-132, BT001021 and BT001035 are expected to benefit more patients clinically.

[0924] Experimental Example 4. Inhibition of NCI-N87 by Antibody Drug Conjugates

[0925] Experimental Example 4: Evaluation of the inhibitory effect of antibody conjugate BT001036 on tumor proliferation in mice with subcutaneous transplantation of human tumor cells. Specifically, in this experiment, a tumor-bearing mouse model was constructed by subcutaneous transplantation of human gastric cancer cell line NCI-N87. When the tumor volume grew to 140 mm 3 After about 1 hour, the mice were randomly divided into groups and given BT001036 intravenously twice a week for a total of 6 times. The tumor volume and animal body weight changes were measured twice a week to evaluate the efficacy (tumor inhibition effect) of the antibody drug conjugate on tumor-bearing mice.

[0926] Experimental methods:

[0927] NCI-N87 cells were cultured in 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 a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a gastric cancer xenograft model. When the average tumor volume was about 140 mm 3 At the same time, the mice were randomly divided into normal saline group, BT001036 (1.5 mg / kg, IV, BIW×3W) group and BT001036 (3 mg / kg, IV, BIW×3W) group according to the tumor size. The corresponding drugs were injected into the tail vein after grouping. The drugs were administered 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 specific results are shown in Table 7. Fig.48A , Fig.48B .

[0928] Table 7. Gastric Cancer NCI-N87 Model

[0929]

[0930] Experimental conclusion:

[0931] In this experimental example, the human gastric cancer cell line NCI-N87 was subcutaneously transplanted to construct a human gastric cancer subcutaneous transplant tumor model and to evaluate the efficacy of BT001036 in the NCI-N87 human gastric cancer tumor-bearing mouse model.

[0932] The experimental results show that both high and low doses of BT001036 (1.5 mg / kg, 3 mg / kg) can significantly inhibit tumor growth in NCI-N87 gastric cancer transplanted tumor model mice, and tumor regression occurred at the endpoint of dosing. The anti-tumor activity was excellent. There was no animal death or significant animal weight loss in all treatment groups during the observation period, and no obvious drug toxicity was shown. During the treatment period, the mice tolerated the evaluated drugs well.

[0933] Experimental Example 5. Inhibition of MDA-MB-231 by antibody drug conjugates

[0934] Experimental Example 5: Evaluation of the effect of BT001021 on the proliferation inhibition of a tumor-bearing mouse model constructed by subcutaneously transplanting human tumor cells of MDA-MB-231 breast cancer. Specifically, in this experiment, a tumor-bearing mouse model was constructed by subcutaneously transplanting human breast cancer cell line MDA-MB-231. When the tumor volume grew to 130 mm 3 The mice were randomly divided into groups and given BT001021 intravenously twice a week for a total of 6 times. The tumor volume and animal body weight changes were measured at the same time, and the efficacy (tumor inhibition effect) of BT001021 on tumor-bearing mice was calculated.

[0935] Experimental methods:

[0936] NCI-MDA-MB-231 cells were cultured in RPMI1640 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 to a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a breast cancer transplant model. When the average tumor volume was about 130 mm 3 At the same time, the mice were randomly divided into normal saline group and BT001021 (3 mg / kg) group according to the tumor size. The corresponding drugs were injected into the tail vein after grouping. The drugs were administered 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 specific results are shown in Table 8. Fig.49A and 49B .

[0937] Experimental conclusion:

[0938] The experimental results show that BT001021 can significantly inhibit tumor growth in mice with MDA-MB-231 breast cancer transplant tumor model, and tumor regression occurred at the endpoint of dosing. There was no animal death or significant animal weight loss in all treatment groups during the observation period, and no obvious drug toxicity was shown. During the treatment period, the mice tolerated all evaluated drugs well.

[0939] Table 8. MDA-MB-231 breast cancer model

[0940]

[0941] In the subcutaneous transplant tumor model, BT001021 showed significant anti-tumor activity. There was no animal death or significant animal weight loss in all treatment groups during the observation period, and no obvious drug toxicity was shown. During the treatment period, mice tolerated all evaluated drugs well.

[0942] Embodiment 65 In vivo pharmacokinetic testing of antibody drug conjugates and active biomolecules

[0943] Experimental Example 6 Evaluation of the in vivo pharmacokinetics of antibody-drug conjugates and active biological molecules. Specifically, in this experiment, human gastric cancer cell line NCI-N87 was subcutaneously transplanted into Balb / c-nu mice to construct a tumor-bearing mouse model. When the tumor volume grew to 100-200 mm 3 The mice were randomly divided into groups and given a single intravenous injection of BT001021 and T-030. The concentration of T-030 in tumor tissue and serum was measured to evaluate the pharmacokinetic behavior of the antibody-drug conjugate BT001021 and the active biological molecule T-030 in tumor-bearing mice.

[0944] Test drug

[0945] Drug name, preparation method:

[0946] BT001021, liquid concentration is 20 mg / ml, stored at -20°C, diluted with saline according to the dose to obtain the test solution;

[0947] T-030 was prepared with dimethyl sulfoxide to 1 mg / ml and diluted with physiological saline according to the dose to obtain the test solution.

[0948] Experimental animals and cell lines:

[0949] Balb / c-nu mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., production license number: SCXK(Beijing)2016-0011); gastric cancer cell line NCI-N87 (ATCC).

[0950] Experimental grouping and evaluation method:

[0951] The tumor volume was 100-200 mm2 and the random grouping (the number of groups was determined according to the number of samples) was selected. 3 Tumor-bearing mice, 4 mice / group, were given a single dose via tail vein injection.

[0952] Experimental Example 6. In vivo pharmacokinetic testing of BT001021 and T-030 in tumor-bearing mice

[0953] Experimental methods:

[0954] NCI-N87 cells were cultured in 1640 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 to a suitable concentration, and inoculated subcutaneously in Balb / c-nu mice to establish a gastric cancer xenograft model. When the average tumor volume was about 100-200mm 3 At the time of administration, the patients were randomly divided into normal saline group, T-030 (0.23 mg / kg, intravenous injection, single dose) group and BT001021 (10 mg / kg, intravenous injection, single dose) group according to the tumor size. The corresponding drugs were injected into the tail vein after grouping. Serum and tumor tissues were collected from the T-030 group at 1h, 2h, 4h, 8h, 24h and 72h after administration (T-030 was no longer detected in serum and tumor tissues at 72h after administration, so serum and tumor tissues were not collected at 168h after administration). Serum and tumor tissues were collected from the BT001021 group at 1h, 2h, 4h, 8h, 24h, 72h and 168h after administration. The concentration of T-030 in serum and tumor was detected by LC-MS / MS method. The specific results are shown in Table 9. The T-030 dosage (0.23 mg / kg) was converted into equimolar ratio according to the BT001021 dosage (10 mg / kg).

[0955] Table 9. Pharmacokinetic parameters of T-030 in tumor and serum of tumor-bearing mice after intravenous administration of T-030 and BT001021

[0956]

[0957] Experimental conclusion:

[0958] AUC of BT001021 (10 mg / kg) in tumor and serum last The AUC of the T-030 group in tumor and serum were 850.1 h*ng / ml and 174.97 h*ng / ml, respectively. lastThey were 3.85h*ng / ml and 5.58h*ng / ml respectively. The comparison of the three showed that the exposure of T-030 in the BT001021 group was significantly higher than that in the T-030 group. In addition, the exposure of active biological molecules T-030 in tumors of the BT001021 group was significantly higher than that in serum, while the exposure of active biological molecules in serum and tumors of the T-030 group was basically the same, indicating that the antibody drug conjugate (BT001021) has strong tumor tissue targeting.

[0959] C of active biomolecule T-030 in tumor and serum of BT001021 (10 mg / kg) group max The C of active biomolecule T-030 in tumor and serum of the T-030 administration group was 7.82ng / ml and 11.7ng / ml respectively. max They were 1.20 ng / ml and 1.81 ng / ml, respectively, indicating that the antibody drug conjugate (BT001021) had a higher concentration of active biomolecule (T-030) in tumor tissue and serum.

[0960] The T-030 level of active biomolecule in tumors in the BT001021 (10 mg / kg) group 1 / 2 The T of the active biological molecule T-030 in the tumor of the T-030 administration group was 93.14h. 1 / 2 The half-life of the antibody-drug conjugate (BT001021) in tumor tissue was 2.55 h.

[0961] In summary, BT001021 has significant tumor tissue targeting and good pharmacokinetic properties compared with the corresponding active biological molecule (T-030).

[0962] Experimental Example 7. In vivo pharmacokinetic testing of antibody drug conjugates BT001021 and Immu-132.

[0963] In this experiment, human gastric cancer cell line NCI-N87 was subcutaneously transplanted into Balb / c-nu mice to construct a tumor-bearing mouse model. 3 The mice were randomly divided into groups and given a single intravenous injection of BT001021 and Immu-132. The concentrations of active biomolecules T-030 and SN-38 corresponding to BT001021 and Immu-132 in tumor tissue and serum were measured, and the pharmacokinetic behavior of antibody-drug conjugates BT001021 and Immu-132 in tumor-bearing mice was evaluated.

[0964] Test drug

[0965] Drug name, preparation method:

[0966] BT001021, liquid concentration is 20 mg / ml, stored at -20°C, diluted with saline according to the dose to obtain the test solution;

[0967] Immu-132 was diluted with normal saline according to the dose to obtain the test solution.

[0968] Experimental animals and cell lines:

[0969] Balb / c-nu mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., production license number: SCXK(Beijing)2016-0011); gastric cancer cell line NCI-N87 (ATCC).

[0970] Experimental grouping and evaluation method:

[0971] The tumor volume was 100-200 mm2 and the random grouping (the number of groups was determined according to the number of samples) was selected. 3 Tumor-bearing mice, 4 mice / group, were given a single dose via tail vein injection.

[0972] Experimental methods:

[0973] NCI-N87 cells were cultured in 1640 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 to a suitable concentration, and inoculated subcutaneously in Balb / c-nu mice to establish a gastric cancer xenograft model. When the average tumor volume was about 100-200mm 3 The patients were randomly divided into BT001021 (5 mg / kg, intravenous injection, single dose) group and Immu-132 (5 mg / kg, intravenous injection, single dose) group according to tumor size. The corresponding drugs were injected into the tail vein after grouping. Serum and tumor tissues were collected 2h, 24h, 48h and 72h after administration, and the concentrations of T-030 or SN-38 in serum and tumors were detected by LC-MS / MS method.

[0974] Table 10. Pharmacokinetic parameters of T-030 and SN-38 in tumor and serum of tumor-bearing mice after intravenous administration of BT001021 and Immu-132

[0975]

[0976] Experimental conclusion:

[0977] AUC of toxin small molecules in tumor and serum in the BT001021 administration group lastThe AUC of the toxin small molecule in the tumor and serum of the Immu-132 administration group were 427.2 h*ng / ml and 115.3 h*ng / ml, respectively. last The C of toxin small molecules in tumors in the BT001021 group were 116.8h*ng / ml and 422.7h*ng / ml respectively. max The C of the toxin small molecule in the tumor was 6.8 ng / ml. max The above results show that compared with Immu-132, BT001021 has better tumor tissue targeting and better pharmacokinetic properties, and has a better therapeutic window.

[0978] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A compound selected from: 6-(2-(Methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid; 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid; or Methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate.

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