Bioactive substance conjugate and its preparation method and use

Abstract

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

Description

[0001] This application is a divisional application of the application with Chinese national application number 201880069543.5, which entered the Chinese national phase on April 24, 2020, and whose invention name is “Bioactive 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 methods for preparing the same, and their use in preventing and / or treating diseases associated with abnormal cell activity, 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 accidentally kill normal cells, causing serious side effects. Targeted anti-tumor drugs have become a hot topic in today's tumor research because they have both targeting and anti-tumor activity. Since the 20th century, breakthroughs have been made in the development of anti-tumor drugs and targeted tumor treatment using biomacromolecular drugs (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, although biomacromolecular drugs have strong targeting, their therapeutic effects on solid tumors are limited; and although bioactive molecules have a high degree of killing efficacy against cancer cells, they often lack targeting and often accidentally kill normal cells, causing serious toxic side effects.

[0004] Recent research has revealed that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting properties of antibodies with the activity of bioactive molecules, becoming a kind of "biological missile." Antibodies guide ADCs to target cells, where they are then internalized, releasing the drug and treating the disease. Because antibodies are specific and targeted to tumor cell-associated targets, their application value lies not only in therapeutic applications but also as ideal carriers for targeted drug delivery, reducing drug side effects. Small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs) share the same design principles: bioactive molecules are chemically coupled to small molecule ligands that selectively bind to tumor cell surface receptors, thereby enhancing the targeting of the effector molecule to tumor cells. The difference between SMDCs and ADCs lies in the use of small molecule ligands in place of antibodies.

[0005] Currently, there are no SMDCs listed.

[0006] 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). Typically, ADC drugs consist of an antibody, a bioactive molecule, and a linker. The bioactive molecule is covalently coupled to the antibody via a linker; the antibody (e.g., a monoclonal antibody) can specifically recognize specific targets on the surface of tumor cells, thereby guiding the ADC to the surface of the cancer cell and allowing the ADC to enter the cancer cell through endocytosis; the bioactive molecule is then released inside the cancer cell, achieving the effect of specifically killing the cancer cell without damaging normal tissue cells.

[0007] Lysine is the most common attachment site in antibodies, and its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond. Currently, technologies exist for site-specific conjugation, whereby the carboxyl group of the linker is activated with an activating group, which then forms an amide bond with the specific ε-amino group of lysine in the antibody to complete the conjugation. However, these amide bonds are susceptible to hydrolysis by enzymes in the body, leading to the detachment of the bioactive molecule from the antibody before it reaches the target cell. This compromises the ADC's targeting ability and increases toxicity.

[0008] Typically, the sulfur groups of antibody cysteine ​​exist in the form of disulfide bonds. Opening the disulfide bonds in antibodies can provide multiple free sulfhydryl groups as coupling sites. One method of coupling with antibody sulfhydryl groups is to react the free sulfhydryl groups on the antibody with maleimide through Michael addition, or a specific substrate can react with the free sulfhydryl groups on the antibody through two Michael addition reactions to form a unique structural sulfur bridge. However, there are many literature reports that ADCs obtained by the sulfhydryl Michael addition method will undergo reverse Michael addition in the systemic circulation, resulting in toxic reactions. In patent WO2016142049, a bioactive molecule containing a methanesulfonyl-substituted benzoxadiazole structure and a linker structure is disclosed, but there is no specific description of the coupling with the antibody. Summary of the Invention

[0009] The present invention has discovered a novel class of bioactive conjugates, obtained by improving the conjugation method between the drug and the targeting moiety in ADC or SMDC drugs. The conjugates have high stability, extremely high conjugation efficiency (90%), and high drug loading (DAR value of 5-8). The present disclosure is based on these findings. Through in-depth research, we surprisingly found that after intravenous administration of the ADC of the present invention, such as BT001021 (Example 32), the exposure of the bioactive small molecule toxin in tumor tissue is significantly higher than that in plasma tissue. In contrast, the plasma exposure of Immu-132 ADC is significantly higher than that in tumor tissue under the same administration route. Therefore, the ADC of the present invention has a better therapeutic window than Immu-132. We also surprisingly found that the ADC of the present invention has better therapeutic effects than Immu-132 in animal models of gastric cancer, breast cancer, and non-small cell lung cancer.

[0010] The first aspect of the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0011] T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]-G

[0012] Formula (I)

[0013] Wherein, T is a biologically active molecular fragment, preferably a molecular fragment having anti-tumor biological activity;

[0014] L1 is selected from amino acids, peptides consisting of 2-10 amino acids, oligosaccharides, -(CH2) t1 -、-(CH2CH2O) t1 -(CH2) t2 -、

[0015]

[0016]

[0017] 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 are independently 0, 1, 2, 3, 4, 5 or 6, each x1 and x2 are 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;

[0018] L2 is selected from amino acids, peptides consisting of 2-10 amino acids, oligosaccharides, -(CH2) t1 -、-(CH2CH2O) t1 -(CH2) t2 -、

[0019]

[0020] 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;

[0021] 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 spiroheterocyclic 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 Alkyl, and the 1 position of L3 is connected to L2;

[0022] L4 is selected from Among them, Z5 is preferably selected from C 2-6 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;

[0023] E is selected from the group consisting of one or more R 12 The following groups substituted: 6-10 membered aryl, 5-14 membered heteroaryl; wherein, R 12 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl and halogenated C 1-6 alkyl;

[0024] G is a leaving group of a nucleophilic substitution reaction, such as a halogen, a sulfonyl group, a sulfonate group, a nitro group, etc.;

[0025] m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0026] 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,

[0027] 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 3-6Cycloalkyl, 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.

[0028] 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.

[0029] 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.

[0030] In some preferred embodiments, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val,

[0031] In some preferred embodiments, L1 is selected from

[0032] 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,

[0033] 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;

[0034] m1 is 0, 1, 2, or 3.

[0035] 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;

[0036] m1 is 0, 1, or 2.

[0037] 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;

[0038] m1 is 1.

[0039] In some preferred embodiments, L2 is selected from

[0040] In some preferred embodiments, L2 is selected from

[0041] 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;

[0042] m2 is 0, 1, 2 or 3.

[0043] 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 2-6Alkynyl; 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;

[0044] m2 is 0, 1 or 2.

[0045] 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.

[0046] 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.

[0047] In some preferred embodiments, L3 is selected from triazole; m2 is 0 or 1.

[0048] In some preferred embodiments, L3 is selected from m2 is 0 or 1; preferably, the 1 position of L3 is connected to L2.

[0049] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7: amino,

[0050]

[0051] R7 is independently selected from H (hydrogen), D (deuterium), =O, CN, CH2CN, methyl, CF3;

[0052] 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.

[0053] In some preferred embodiments, L3 is selected from

[0054]

[0055] 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.

[0056] In some preferred embodiments, L3 is selected from

[0057] 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 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;

[0058] m3 is selected from 0, 1, 2 or 3.

[0059] 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;

[0060] m3 is selected from 0, 1, 2 or 3.

[0061] In some preferred embodiments, L4 is selected from Wherein Z4 is a 5-6 membered heteroaryl group; R 10Selected 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;

[0062] m3 is selected from 0, 1, 2 or 3.

[0063] In some preferred embodiments, L4 is selected from

[0064] m3 is 1.

[0065] In some preferred embodiments, L4 is selected from

[0066] m3 is 1.

[0067] In some preferred embodiments, L4 is selected from

[0068] m3 is 1.

[0069] In some preferred embodiments, E is selected from the group consisting of 12 substituted 5-10 membered heteroaryl; wherein R 12 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and halogenated C 1-4 alkyl.

[0070] 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 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl and halogenated C 1-2 alkyl.

[0071] 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).

[0072] 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 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 alkoxy, 6-10 membered aryl and 5-12 membered heteroaryl.

[0073] In some preferred embodiments, G is selected from F, Cl, Br, I, Oms, Ots, Otf, methylsulfonyl, ethylsulfonyl, p-toluenesulfonyl, and naphthylsulfonyl.

[0074] In some preferred embodiments, G is selected from F, Cl, Br, Oms, Ots, methanesulfonyl, and p-toluenesulfonyl.

[0075] In some preferred embodiments, G is selected from Cl and mesyl.

[0076] In some preferred embodiments, In the embodiment, G is preferably methylsulfonyl, E is preferably pyrimidine, and m3 is 1.

[0077] 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.

[0078] 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.

[0079] In some preferred embodiments, for Here, 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.

[0080] 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.

[0081] 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.

[0082] 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 methylsulfonyl are substituents of the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0083] In some preferred embodiments, for where m 10 is an integer selected from 0-6, Z4 is selected from a 5-6 membered heteroaryl group; and the methylsulfonyl group is a substituent of a carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0084] In some preferred embodiments, for Z4 is selected from pyridine, pyrimidine, pyrazole, thiazole, oxazole and triazole, and the methylsulfonyl 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.

[0085] In some preferred embodiments, for Z4 is selected from pyridine, pyrimidine, pyrazole and triazole. More preferably, m 10 An integer selected from 0-6.

[0086] In some preferred embodiments, for Z4 is selected from oxazole and thiazole, and the methylsulfonyl group is a substituent of a carbon atom adjacent to the nitrogen atom in the pyrimidine ring. More preferably, m 10 An integer selected from 0-6.

[0087] In some preferred embodiments, for where m 10 is an integer selected from 0 to 6, Z4 is selected from a 6-10 membered aryl group; and the methylsulfonyl group is a substituent of a carbon atom adjacent to the nitrogen atom. More preferably, m 10 An integer selected from 0-6.

[0088] In some preferred embodiments, for where m 10 An integer selected from 0-6, Z4 is a benzene ring.

[0089] In some preferred embodiments, for

[0090] In some preferred embodiments, in formula (I) Selected from the following structural fragments:

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] 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 microtubule 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, promote tumor cell apoptosis or necrosis.

[0097] In some preferred embodiments, the bioactive molecule is selected from

[0098] where R 14 Selected from R 15 Substituted acyl or sulfonyl, R 15 Selected from C 1-6 Alkyl, halogenated C1-6 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.

[0099] 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.

[0100] In some preferred embodiments, the bioactive molecule is selected from

[0101] In some preferred embodiments, the bioactive molecule is selected from

[0102] In some preferred embodiments, the bioactive molecule is selected from

[0103] In some preferred embodiments, the bioactive molecule is selected from

[0104]

[0105] In some preferred embodiments, the bioactive molecule is selected from

[0106] In some preferred embodiments, T is selected from

[0107]

[0108]

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

[0110]

[0111] In some preferred embodiments, T is selected from

[0112]

[0113]

[0114] In some preferred embodiments, T is selected from

[0115]

[0116] In some preferred embodiments, T is selected from

[0117]

[0118] In some preferred embodiments, the compound represented by formula (I) is selected from

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] In some preferred embodiments, the compound is selected from

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] 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 linked to the linker via its active group (eg, sulfhydryl) to form the conjugate.

[0137] In some preferred embodiments, the conjugate structure is shown in formula (II):

[0138] {T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A

[0139] Formula (II)

[0140] 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);

[0141] The remaining groups are as described in the first aspect of the present disclosure.

[0142] 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. .

[0143] In some preferred embodiments, A is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an arylsulfonamide derivative (such as a carbonic anhydrase IX inhibitor), a polyene connecting two aliphatic indoles, a cyanine dye, or IR-783 or its derivatives.

[0144] In some preferred embodiments, A is selected from

[0145]

[0146] In some preferred embodiments, A is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody or multispecific antibody.

[0147] 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.

[0148] In some preferred embodiments, A is an anti-Trop-2 monoclonal antibody, such as M1, M2, and M3.

[0149]

[0150] 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.

[0151] 1. Hydrophobicity-engineered heavy and light chain sequences of antibody M1

[0152] Amino acid sequence of the M1 heavy chain variable region: (121aa)

[0153] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG WINTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSY WYFDVWGQGSLVTVSS(SEQ ID No.:11)

[0154] Amino acid sequence of the M1 light chain variable region: (107aa)

[0155] DIQLTQSPSSSLSASVGDRVSITCKASQDVSSAVAWYQQKPGKAPKLLIYSASYR YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSTPLTFGAGTKVEIK(SEQ ID No.:12)

[0156] 2. Hydrophobicity-engineered heavy and light chain sequences of antibody M2

[0157] Amino acid sequence of the M2 heavy chain variable region: (121aa)

[0158] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG WINTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSY WYFDVWGQGSLVTVSS(SEQ ID No.:13)

[0159] Amino acid sequence of the M2 light chain variable region: (107aa)

[0160] DIQLTQSPSSSLSASVGDRVSITCKASQDVSSAVAWYQQKPGKAPKLLIYSASYR YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK(SEQ ID No.:14)

[0161] 3. Hydrophobicity-engineered heavy and light chain sequences of antibody M3

[0162] Amino acid sequence of the M3 heavy chain variable region: (121aa)

[0163] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG WINTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSY WYFDVWGQGSLVTVSS(SEQ ID No.:15)

[0164] Amino acid sequence of the M3 light chain variable region: (107aa)

[0165] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYR YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSTPLTFGAGTKVEIK(SEQ ID No.:16)

[0166] M1, M2, M3 light chain constant region sequence: (107aa)

[0167] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID No.:9)

[0168] M1, M2, M3 heavy chain constant region sequence: (330aa)

[0169] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.:10)

[0170] The terminal Lys residue of the heavy chain can be easily deleted, but this deletion does not affect biological activity (see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143). The aforementioned M1, M2, and M3 monoclonal antibodies and sequences or fragments thereof with the terminal Lys residue of the heavy chain deleted are all considered the M1, M2, and M3 monoclonal antibodies of the present invention.

[0171] 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.

[0172] 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.

[0173] In some preferred embodiments, the conjugate is selected from:

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] wherein γ is selected from an integer or decimal between 1 and 10, and the 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 and 8 (e.g., 5, 6, 7, or 8).

[0184] In some preferred embodiments, the conjugate is selected from:

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] wherein γ is selected from an integer or decimal between 1 and 10, and 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 and 8 (e.g., 5, 6, 7 or 8).

[0193] In some preferred embodiments, the conjugate is:

[0194]

[0195]

[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, 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.

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

[0200]

[0201]

[0202] 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, 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.

[0203] In some preferred embodiments, the conjugate is:

[0204]

[0205] 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, 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.

[0206] In some preferred embodiments, the conjugate is:

[0207]

[0208] 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.

[0209] In some preferred embodiments, the conjugate is:

[0210]

[0211]

[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]

[0218] 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.

[0219] In some preferred embodiments, the conjugate is:

[0220]

[0221] 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.

[0222] In some preferred embodiments, the conjugate is:

[0223]

[0224]

[0225]

[0226]

[0227] wherein A3 is pertuzumab, and 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, such as an integer or decimal between 6 and 7, 6 and 7.5, 6 and 8, 6.5 and 7, 6.5 and 7.5, 6.5 and 8, 7 and 8, or 7.5 and 8.

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

[0229]

[0230]

[0231] wherein A3 is pertuzumab, and 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, such as an integer or decimal between 6 and 7, 6 and 7.5, 6 and 8, 6.5 and 7, 6.5 and 7.5, 6.5 and 8, 7 and 8, or 7.5 and 8.

[0232] In some preferred embodiments, the conjugate is:

[0233]

[0234]

[0235]

[0236]

[0237] 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, 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.

[0238] In some preferred embodiments, the conjugate is:

[0239]

[0240] 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, 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.

[0241] In some preferred embodiments, the conjugate is:

[0242]

[0243]

[0244]

[0245]

[0246] 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, 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.

[0247] In some preferred embodiments, the conjugate is:

[0248]

[0249] 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, 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.

[0250] In some preferred embodiments, the conjugate is:

[0251]

[0252]

[0253]

[0254] 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, 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.

[0255] In some preferred embodiments, the conjugate is:

[0256]

[0257] 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, 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.

[0258] In another aspect, the present disclosure provides a method for preparing the conjugate according to the second aspect, comprising the step of conjugating a linker of the compound of formula (I) to an active group of a targeting moiety.

[0259] 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.

[0260] In some preferred embodiments, the method comprises the step of forming a C—S bond between a linker of the compound of formula (I) and a sulfhydryl group of the targeting moiety.

[0261] In some preferred embodiments, the targeting moiety is an anti-Her 2 monoclonal antibody (e.g., Trastuzumab, Pertuzumab) or an anti-Trop-2 monoclonal antibody (e.g., Sacituzumab, M1, M2 or M3), or an active fragment or variant thereof.

[0262] 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.

[0263] In some preferred embodiments, the method further comprises the step of purifying the coupled product; preferably, the coupled product is purified by a chromatography method (eg, one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography or affinity chromatography).

[0264] 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.

[0265] In another aspect, the present disclosure provides use of the compound or pharmaceutically acceptable salt thereof according to the first aspect or the conjugate according to the second aspect in the preparation of a medicament for treating diseases associated with abnormal cell activity (eg, cancer).

[0266] 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.

[0267] 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).

[0268] In another aspect, 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 pharmaceutically acceptable salt thereof described in the first aspect or the conjugate or pharmaceutical composition described in the second aspect provided by the present disclosure.

[0269] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0270] For the purposes of this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These substances, in addition to the active ingredients, have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients also perform important functions such as solubilization, dissolution enhancement, and sustained-release control. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their source, they can be categorized as natural, semi-synthetic, or fully synthetic. 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, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0271] The pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, 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 can 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.

[0272] The pharmaceutical composition can be administered in the form of an injection, including an injection solution, 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, sterile fixed oils such as monoglycerides or diglycerides can also be used as solvents or suspending media.

[0273] In the present disclosure, the term "individual" includes humans and non-human animals. Exemplary human individuals include human individuals suffering from a disease (e.g., a disease described herein) (referred to as a patient) or normal individuals. 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.).

[0274] 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.

[0275] In the present disclosure, the term "conjugate" refers to a substance obtained by connecting a bioactive molecule to a targeting portion. In some embodiments of the present disclosure, the bioactive molecule and the targeting portion are connected via a linker. The linker can be broken in a specific environment (e.g., a low pH environment within the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive molecule from the targeting portion. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive molecule and the targeting portion are directly connected via a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive molecule from the targeting portion.

[0276] 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 but is not limited to: selenolide, cabazitaxel, etc.; 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, pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell growth, promote tumor cell apoptosis and necrosis; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; growth inhibitors; drug moieties. The term "toxin" refers to a substance that can produce a deleterious effect on cell growth or proliferation.

[0277] In this disclosure, the term "small molecule" refers to small molecule drugs with biological activity.

[0278] In the present disclosure, the term "linker" refers to a fragment that connects a biologically active molecule to a targeting moiety.

[0279] In this disclosure, the term "targeting moiety" refers to a portion of a conjugate that is capable of specifically binding to a target (or 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.

[0280] 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.

[0281] In this disclosure, the term "antibody" is used 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 this disclosure, "antibody" and "immunoglobulin" can be used interchangeably.

[0282] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. Monoclonal antibodies have high specificity for a single determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage of being 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 specific method.

[0283] 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., monkeys, orangutans, etc.) and human constant region sequences.

[0284] The term "antibody fragment" refers to a portion of an antibody, preferably the antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single-chain antibody molecules.

[0285] 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. The conjugate retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0286] 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.

[0287] The term "intact 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 may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions.

[0288] The term "probody" refers to 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.

[0289] In the present disclosure, the "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody 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, typically two, variable regions in which all or nearly all of the hypervariable loops correspond to those of a non-human immunoglobulin, while the FRs are entirely or almost entirely human immunoglobulin sequences. The humanized antibody may also include at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details, see, for example, 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.

[0290] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different antibody classes are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0291] Although amino acid substitutions in antibodies are generally made with L-amino acids in the present disclosure, this is not limiting. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less susceptible to degradation in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0292] 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 cells of many species (including mice, hamsters, rats and people) (see, for example, Kohler et al., 1975, Nature, 256:495), or made 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). 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.

[0293] 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. .

[0294] 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.

[0295] 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.

[0296] 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.

[0297] In some embodiments of the present disclosure, the targeting moiety is trastuzumab or pertuzumab. Trastuzumab is an anti-Her 2 monoclonal antibody, and its amino acid sequence is known to those skilled in the art. Its schematic sequence can be found in, for example, CN103319599.

[0298] 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 (Pertuzumab) heavy chain last Lys deleted.

[0299] Exemplary heavy and light chain sequences of trastuzumab can be found, for example, in SEQ ID No.: 17 and SEQ ID No.: 18. In this disclosure, when referring to or relating to the heavy and light chain sequences of trastuzumab, the sequences set forth in SEQ ID No.: 17 and SEQ ID No.: 18, respectively, are used for description. Exemplary heavy and light chain sequences of pertuzumab can be found in SEQ ID No.: 16 and SEQ ID No.: 15 of US Pat. No. 7,560,111.

[0300] SEQ ID No.: 17 (heavy chain sequence)

[0301] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)

[0302] SEQ ID No.: 18 (light chain sequence)

[0303] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0304] 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 antibodies that can be used in the present disclosure can also be obtained by screening the vector design, construction, and antibody library construction methods disclosed in CN103476941A, or by G- The library was screened.

[0305] 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.

[0306] SEQ ID No.: 19 (heavy chain sequence)

[0307] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQ GSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)

[0308] The last K (or lys) of the heavy chain can be easily deleted, but this deletion does not affect the biological activity, see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143.

[0309] SEQ ID No.: 20 (light chain sequence)

[0310] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0311] In the present disclosure, ErbB2 and Her2 / neu are used interchangeably, and both refer to the native sequence human Her2 protein (Genebank accession number: X03363, see, for example, 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 are capable of inhibiting or killing 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.

[0312] In the present disclosure, Trop-2 or TROP2 refers to human trophoblast cell-surface antigen 2 (TACSTD2), also known as M1S1, GA733-1, and EGP-1, which is a cell surface receptor expressed by many human tumor cells (e.g., breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, and cervical cancer). In some embodiments, the compounds or conjugates of the present disclosure can inhibit or kill cells expressing the TROP2 receptor, such as breast cancer cells, colorectal cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, prostate cancer cells, or cervical cancer cells.

[0313] 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.

[0314] 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 alkyl”, etc., 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, etc.

[0315] 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.

[0316] 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.

[0317] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0318] In this context, 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". Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0319] 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.

[0320] As used herein, the term "3-8 membered alicyclic heterocyclic 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 may be oxo-substituted. "3-8 membered alicyclic heterocyclic group" includes, for example, "3-8 membered nitrogen-containing alicyclic heterocyclic group", "3-8 membered oxygen-containing alicyclic heterocyclic group", "3-6 membered alicyclic heterocyclic group", "3-6 membered oxygen-containing alicyclic heterocyclic group", "4-7 membered alicyclic heterocyclic group", "4-6 membered alicyclic heterocyclic group", "5-7 membered alicyclic heterocyclic group", "5-6 membered alicyclic heterocyclic group", "5-6 membered nitrogen-containing alicyclic heterocyclic group", including but not limited to oxiranyl, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.

[0321] As used herein, 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 may 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" and the like. Specific examples include, but are not limited to:

[0322] wait.

[0323] As used herein, the term "6-12 membered bridged cyclic group" refers to a cyclic structure containing 6-12 ring carbon atoms formed by two or more cyclic structures sharing two non-adjacent carbon atoms. Optionally, the carbon atoms in the cyclic structure may be oxo-substituted. "6-12 membered bridged cyclic group" includes, for example, "6-11 membered bridged cyclic group", "5-10 membered bridged cyclic group", "7-10 membered bridged cyclic group", "7-9 membered bridged cyclic group", "7-8 membered bridged cyclic group", "9-10 membered bridged cyclic group", "3-10 membered bridged cyclic group" and the like. Specific examples include, but are not limited to:

[0324] wait.

[0325] As used herein, 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," and "7-10-membered fused cyclic group." Examples include, but are not limited to:

[0326] wait.

[0327] In this article, the term "6-12 yuan spiro heterocyclic radical" refers to a cyclic structure formed by two or more cyclic structures sharing a ring atom with each other, 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). 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 radical" includes, for example, "6-11 yuan spiro heterocyclic radical", "5-10 yuan spiro heterocyclic radical", "7-11 yuan spiro heterocyclic radical", "7-10 yuan spiro heterocyclic radical", "7-9 yuan spiro heterocyclic radical", "7-8 yuan spiro heterocyclic radical", "9-10 yuan spiro heterocyclic radical", "3-10 yuan spiro heterocyclic radical" etc. Specific examples include but are not limited to: wait.

[0328] As used herein, the term "6-12 membered bridged heterocyclic group" refers to a cyclic 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 cyclic structures sharing two non-adjacent ring atoms. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may 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", etc. Specific examples include, but are not limited to:

[0329] wait.

[0330] As used herein, the term "6-12 membered fused heterocyclic radical" refers to a cyclic 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 cyclic structures sharing two adjacent atoms. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may be oxoed. "6-12 membered fused heterocyclic radical" includes, for example, "6-11 membered fused heterocyclic radical", "5-10 membered fused heterocyclic radical", "7-10 membered fused heterocyclic radical", "3-10 membered fused heterocyclic radical", "3-10 membered nitrogen-containing fused heterocyclic radical", "9-10 membered fused heterocyclic radical", "9-10 membered nitrogen-containing fused heterocyclic radical", "6-12 membered oxygen-containing fused heterocyclic radical", etc. 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, hexahydrofuroimidazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, octahydrocyclopenta[c]pyrrolyl, dihydroindolinyl, dihydroisoindolinyl, benzoxazolidinyl, benzothiazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl, 4H-1,3-benzoxazinyl, and the like.

[0331] As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromatic properties, 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.

[0332] In this article, 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, 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-pyridonyl, 4-pyridonyl, 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.

[0333] Advantageous Effects of the Invention

[0334] This disclosure improves the coupling method between the drug and the targeting moiety in an ADC or SMDC to obtain a novel class of bioactive molecule conjugates. In some embodiments of the disclosure, the bioactive molecule conjugates are obtained by a nucleophilic substitution reaction between a heteroaromatic ring on the ADC drug linker and a free thiol group in an antibody molecule. The conjugates obtained using this coupling method can achieve at least one of the following technical effects:

[0335] (1) High stability;

[0336] (2) having a high drug loading capacity. In some embodiments, the DAR value of the conjugate can reach 5-8;

[0337] (3) having extremely high coupling efficiency. In some embodiments, the coupling efficiency can reach 90%;

[0338] (4) The conjugate obtained by the above-mentioned 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;

[0339] (5) The conjugate can also increase the effective release of bioactive molecules into cells, thereby achieving the purpose of enhancing efficacy and reducing toxicity;

[0340] (6) The conjugate has good tumor tissue targeting ability; and

[0341] (7) The conjugate has a good therapeutic effect on tumor animal models.

[0342] In addition, the coupling method disclosed in the present invention has a wide range of applications and can be widely used for coupling bioactive molecules with antibodies or targeted small molecule ligands. BRIEF DESCRIPTION OF THE DRAWINGS

[0343] Figure 1 This is the TIC diagram (total ion current diagram) of BT001002.

[0344] Figure 2 This is the deconvolution image of BT001002 coupled light chain.

[0345] Figure 3 This is the deconvoluted image of BT001002 coupled heavy chain.

[0346] Figure 4 This is the TIC diagram (total ion current diagram) of BT001004.

[0347] Figure 5 This is the deconvoluted image of BT001004 coupled light chain.

[0348] Figure 6 This is the deconvoluted image of BT001004 coupled heavy chain.

[0349] Figure 7 This is the SEC chromatogram of BT001002.

[0350] Figure 8 This is the molecular weight marker SEC chromatogram of BT001002.

[0351] Figure 9 This is the SEC chromatogram of BT001004.

[0352] Figure 10 This is the deconvoluted image of BT001012 coupled light chain.

[0353] Figure 11 This is the deconvoluted image of BT001012 coupled heavy chain.

[0354] Figure 12 This is the deconvoluted image of BT001013 coupled light chain.

[0355] Figure 13 This is the deconvoluted image of BT001013 coupled heavy chain.

[0356] Figure 14 This is the deconvoluted image of BT001018 coupled light chain.

[0357] Figure 15 This is the deconvoluted image of BT001018 coupled heavy chain.

[0358] Figure 16 This is the deconvoluted image of BT001021 coupled light chain.

[0359] Figure 17 This is the deconvoluted image of BT001021 coupled heavy chain.

[0360] Figure 18 This is the deconvoluted image of BT001023 coupled light chain.

[0361] Figure 19 This is the deconvoluted image of BT001023 coupled heavy chain.

[0362] Figure 20 This is the deconvoluted image of BT001040 coupled light chain.

[0363] Figure 21 This is the deconvoluted image of BT001040 coupled heavy chain.

[0364] Figure 22 This is the deconvoluted image of BT001041 coupled light chain.

[0365] Figure 23 This is the deconvoluted image of BT001041 coupled heavy chain.

[0366] Figure 24 This is the deconvoluted image of BT001042 coupled light chain.

[0367] Figure 25 This is the deconvoluted image of BT001042 coupled heavy chain.

[0368] Figure 26 This is the deconvoluted image of BT001043 coupled light chain.

[0369] Figure 27 This is the deconvoluted image of BT001043 coupled heavy chain.

[0370] Figure 28 This is the deconvoluted image of BT001044 coupled light chain.

[0371] Figure 29 Deconvoluted image of BT001044 coupled heavy chain.

[0372] Figure 30 This is the deconvoluted image of BT001046 coupled light chain.

[0373] Figure 31 This is the deconvoluted image of BT001046 coupled heavy chain.

[0374] Figure 32 This is the deconvoluted image of BT001047 coupled light chain.

[0375] Figure 33 Deconvoluted image of BT001047 coupled heavy chain.

[0376] Figure 34 This is the SEC chromatogram of BT001012.

[0377] Figure 35 SEC chromatogram was obtained for BT001013.

[0378] Figure 36 SEC chromatogram was obtained for BT001018.

[0379] Figure 37 This is the SEC chromatogram of BT001021.

[0380] Figure 38 This is the SEC chromatogram of BT001023.

[0381] Figure 39 This is the SEC chromatogram of BT001042.

[0382] Figure 40 This is the SEC chromatogram of BT001043.

[0383] Figure 41 This is the SEC chromatogram of BT001044.

[0384] Figure 42 This is the SEC chromatogram of BT001046.

[0385] Figure 43 This is the SEC chromatogram of BT001047.

[0386] Figure 44 Growth changes of tumor volume in each group of mice in the NCI-N87 human gastric cancer model.

[0387] Figure 45 Changes in body weight of mice in each group in the NCI-N87 human gastric cancer model.

[0388] Figure 46 Growth changes of tumor volume in each group of mice in the HCC1806 human breast cancer model.

[0389] Figure 47A Growth changes of tumor volume in each group of mice in the HCC827 human non-small cell lung cancer xenograft model.

[0390] Figure 47B Changes in body weight of mice in each group in the HCC827 human non-small cell lung cancer xenograft model.

[0391] Figure 48AGrowth changes of tumor volume in each group of mice in the NCI-N87 human gastric cancer xenograft model.

[0392] Figure 48B Changes in body weight of mice in each group in the NCI-N87 human gastric cancer transplant tumor model.

[0393] Figure 49A .The growth changes of tumor volume in each group of mice in the MDA-MB-231 human breast cancer tumor-bearing mouse model.

[0394] Figure 49B .Changes in body weight of mice in each group in the MDA-MB-231 human breast cancer tumor-bearing mouse model. DETAILED DESCRIPTION

[0395] The present disclosure is further described below by describing specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the present disclosure without departing from the basic idea and scope of the present disclosure.

[0396] The abbreviations used in this invention have the following meanings:

[0397]

[0398] Preparation plan

[0399] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0400] Nuclear magnetic resonance (NMR) 1 H NMR was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0401] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0402] s: singlet, d: doublet, t: triplet, q: quartet, dd: doubledoublet, qd: quartet doublet, ddd: doubledoubledoublet, ddt: doubledoubletriplet, dddd: doubledoubledoubledoublet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0403] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0404] The preparative liquid chromatography method is as follows:

[0405] Method A:

[0406] Chromatographic column: Daisogel C18 10μm 100x250mm

[0407] Mobile phase A: water; mobile phase B: acetonitrile

[0408]

[0409] Method B:

[0410] Chromatographic column: Daisogel C18 10μm 50x250mm

[0411] Mobile phase A: water; mobile phase B: acetonitrile

[0412]

[0413] Method C:

[0414] Chromatographic column: Daisogel C18 10μm 50x250mm

[0415] Mobile phase A: water containing 0.1% trifluoroacetic acid; Mobile phase B: acetonitrile

[0416]

[0417] Method D: Column: Waters SunFire C18 5μm 19x250mm

[0418] Mobile phase A: acetonitrile; mobile phase B: water containing 0.05% formic acid

[0419] Time: 0 min-16 min; Mobile phase A: 10%-90%; Flow rate: 28 mL / min

[0420] 1. Synthesis of Bioactive Molecules

[0421] Example 1 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 (T001)

[0422]

[0423] Step 1: Synthesis of tert-butyl (4-((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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)carbamate

[0424] 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-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)-pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (10.0 mg, 13.5 μmol, commercially available) was stirred for 5 minutes, and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (10.0 mg, 20.1 μmol) was added. After addition, the reaction was stirred at 0°C for 1 hour. The reaction was monitored for complete reaction by HPLC-MS / MS. 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] + .

[0425] 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

[0426] 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, and hydrochloric acid-dioxane solution (1 mL, 4.0 M) was added. The reaction was stirred at room temperature for 3 h. The reaction of the starting material was monitored for completeness 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] + .

[0427] 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)

[0428]

[0429] Step 1: Synthesis of tert-butyl (S)-(4-((2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido)methyl)phenyl)carbamate

[0430] 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). 1-Hydroxybenzotriazole (203 mg, 1.5 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (288 mg, 1.5 mmol) were then added sequentially. The reaction was allowed to react 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 x 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] + .

[0431] Step 2: Synthesis of tert-butyl (S)-(4-((2-amino-3-phenylpropionamido)methyl)phenyl)carbamate

[0432] 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). The reaction was allowed to react at room temperature for 2 h. Water (20 mL) was added to the reaction solution, and the mixture was 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] + .

[0433] 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

[0434] At 0°C, tert-butyl (S)-(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- To a solution of 3-methoxy-2-methylpropionic acid (24 mg, 0.04 mmol) in N,N-dimethylformamide (2 mL) was added 1-hydroxybenzotriazole (8 mg, 0.06 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12 mg, 0.06 mmol) in sequence. The reaction was allowed to react 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] + .

[0435] 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

[0436] 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 the mixture was allowed to react 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]. + .

[0437] The following molecules can be synthesized using similar synthetic methods:

[0438]

[0439] Example 3 Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-diketo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide

[0440]

[0441] 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). Acetic anhydride (0.22 g, 2.13 mmol) was slowly added dropwise and allowed to react at room temperature for 1 h. The organic phase was washed with water (10 mL x 2) and dried over anhydrous sodium sulfate. The insoluble matter was filtered off, the solvent was evaporated, and the product 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] + .

[0442] Example 4 Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-diketo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide

[0443]

[0444] Methanesulfonyl chloride (462 mg, 12.77 mmol, approximately 70% purity) 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). The mixture was allowed to react at room temperature for 2 h. The mixture was filtered and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound, 2.2 g.

[0445] The structural characterization data are as follows:

[0446] 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 The concentration is +28.19° (c = 0.101 g / 100 mL, CH3CN).

[0447] The remaining biologically active molecules whose synthesis methods are not described are commercially available or prepared by methods disclosed in the prior art.

[0448] 2. Synthesis of compounds containing cell-bioactive molecules and linkers

[0449] Example 5 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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide

[0450]

[0451] Step 1: Synthesis of tert-butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyrate (Compound 1-2)

[0452] 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 portionwise and stirred at room temperature for 10 min. tert-Butyl 4-bromobutyrate (725 mg, 3.27 mmol) was added dropwise. After addition, the mixture was allowed to react at room temperature for 2 h. The mixture was quenched with saturated aqueous ammonium chloride and 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 off, and the solvent was evaporated under reduced pressure to obtain the title compound, 500 mg. ESI-MS (m / z): 296.1 [M+H] + .

[0453] Step 2: Synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid (Compound 1-3)

[0454] Compound 1-2 (500 mg, 1.69 mmol) was dissolved in dichloromethane (6 mL) at room temperature, and trifluoroacetic acid (3 mL) was added. After addition, the mixture was reacted at room temperature for 4 h. The solvent was evaporated under reduced pressure to obtain the title compound, 400 mg. ESI-MS (m / z): 240.1 [M+H] + .

[0455] 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)

[0456] At room temperature, 4-(N-Boc-aminomethyl)-aniline (6.0 g, 27 mmol), compound 1-4 (3.35 g, 6.75 mmol), and 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). The temperature was raised to 45°C and the reaction was maintained 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] + .

[0457] Step 4: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(aminomethyl)phenyl)amino)-1-oxo-5-ureidopentanyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-6)

[0458] Trifluoroacetic acid (15 mL) was added to compound 1-5 (3.0 g, 4.29 mmol) 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 (20 mL) was added, and a large amount of solid precipitated. 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] + .

[0459] 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)

[0460] At room temperature, Boc-D-phenylalanine (1.1 g, 4.2 mmol) and the trifluoroacetic acid salt of compound 1-6 (3.0 g, 4.2 mmol) were dissolved in N,N-dimethylformamide (40 mL). The temperature was lowered 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 sequentially. The reaction system was stirred for 1.0 h. The reaction solution was added dropwise into ice water (400 mL) and stirred vigorously for 0.5 h. A large amount of solid precipitated, which was filtered to obtain the title compound (3.3 g). ESI-MS (m / z): 848.4 [M+H] + .

[0461] 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)

[0462] 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 and was filtered to obtain the trifluoroacetic acid salt of the title compound (2.1 g). ESI-MS (m / z): 748.4 [M+H] + .

[0463] 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-butyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (Compound 1-9)

[0464] At room temperature, (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-(dimethylamino)-3-butyramido)-N,3-dimethylbutyramido)-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). The temperature was cooled to 0°C, and 1-hydroxybenzotriazole (440 mg, 3.26 mmol) and N-methylmorpholine (658 mg, 6.51 mmol) were added in sequence. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (624 mg, 1.38 mmol) was added. After the addition was complete, the reaction solution was stirred at 0°C for 5 h. Purification by preparative liquid chromatography (method D) afforded the title compound, 1.8 g. ESI-MS (m / z): 1329.2 [M+H] + .

[0465] Step 8: Synthesis of (S)-2-((S)-2-amino-3-butyramido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide (Compound 1-10)

[0466] Compound 1-9 (500 mg, 0.38 mmol) was dissolved in N,N-dimethylformamide (5 mL) at room temperature, and piperidine (324 mg, 3.8 mmol) was added. The mixture was stirred at room temperature for 3 h. Purification by preparative liquid chromatography (Method D) afforded the title compound, 350 mg. ESI-MS (m / z): 1107.2 [M+H] + .

[0467] 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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide (Compound TL001)

[0468] At room temperature, compound 1-10 (60 mg, 0.054 mmol) and 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid (26 mg, 0.066 mmol) were dissolved in N,N-dimethylformamide (3 mL). The temperature was cooled to 0°C, and N,N-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazole-1-oxytripyrrolidone hexafluorophosphate (281 mg, 0.54 mmol) were added sequentially. After addition, the reaction system was stirred at room temperature for 3 h. Purification by preparative liquid chromatography (Method D) afforded the title compound, 30 mg. ESI-MS (m / z): 664.5 [M / 2+H]. + .

[0469] Example 6 (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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butanamido)-butanamido)-5-ureidopentanamide

[0470]

[0471] Step 1: Synthesis of 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid (Compound 2-2)

[0472] 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 all at once. The temperature was raised to 50°C and the reaction was allowed to proceed overnight. Purification by preparative liquid chromatography (Method D) afforded the title compound, 120 mg. ESI-MS (m / z): 252.1 [M+H] + .

[0473] Step 2: 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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butanamido)-butanamido)-5-ureidopentanamide (Compound 2-3)

[0474] A similar procedure as described in Step 9 of Example 5 was used, substituting 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid for 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid. Purification by preparative liquid chromatography (Method D) afforded the title compound, 20 mg. ESI-MS (m / z): 670.5 [M / 2+H]. + .

[0475] 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-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butanamido)-butanamido)-5-ureidopentanamide (Compound TL002)

[0476] 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 allowed to react at room temperature for 2 h. Purification by preparative liquid chromatography (Method D) afforded the title compound, 5.0 mg. ESI-MS (m / z): 686.5 [M / 2+H]+ .

[0477] 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-methylbutanamido)-N,3-dimethylbutanamido)-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-oxybutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0478]

[0479] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (Compound 3-2)

[0480] At room temperature, methyl 5-hexynoate (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 sequentially. The mixture was heated to 95°C and stirred under nitrogen for 6 h. The mixture was quenched with water and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, and 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]. + .

[0481] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-3)

[0482] 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), and lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added. The reaction was stirred at room temperature for 4 h, diluted with water, and extracted with ethyl acetate (20 mL × 2). The aqueous phase was adjusted to pH = 3 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL × 3). 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.

[0483] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-4)

[0484] 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 addition, 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] + .

[0485] 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-methylpropionylamino)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (Compound TL003)

[0486] A similar procedure as described in Step 9 of Example 5 was used, substituting 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid for 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid. Purification by preparative liquid chromatography (Method D) afforded the title compound, 14 mg. ESI-MS (m / z): 679.0 [M / 2+H]. + .

[0487] 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-amido)benzyl)carbonate

[0488]

[0489] 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)

[0490] At room temperature, Fmoc-L-citrulline (5.0 g, 12.58 mmol), p-aminobenzyl alcohol (6.20 g, 50.32 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (6.22 g, 25.16 mmol) were dissolved in dichloromethane (100 mL) and heated 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] + .

[0491] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 19-3)

[0492] Compound 19-2 (1.0 g, 1.99 mmol) was dissolved in N,N-dimethylformamide (8 mL) at room temperature, and piperidine (339 mg, 3.98 mmol) was added dropwise. After addition, the mixture was allowed to react at room temperature for 30 min. Dichloromethane (10 mL) was added and stirring continued for 10 min. The reaction solution was concentrated under reduced pressure and purified on a flash silica gel column to obtain the title compound, 400 mg. ESI-MS (m / z): 281.2 [M+H] + .

[0493] 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)

[0494] Compound 19-3 (150 mg, 0.54 mmol) and 3,2-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). The temperature was lowered to 0°C, and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (145 mg, 0.58 mmol) was added. The mixture was allowed to react at room temperature overnight. The reaction solution was concentrated under reduced pressure and purified on a flash silica gel column to obtain the title compound (200 mg). ESI-MS (m / z): 817.5 [M+H] + .

[0495] 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-azatetracosane)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)

[0496] (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) at room temperature and cooled to 0°C. A solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL) was added, followed by a solution of triphosgene (116 mg, 0.39 mmol) in dichloromethane (1.0 mL) which 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 allowed to react 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] + .

[0497] 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-azatetracosane)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)

[0498] Compound 19-5 (80 mg, 0.059 mmol) was dissolved in tetrahydrofuran (1.0 mL) at room temperature and cooled to 0°C. 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 in one portion under nitrogen. After addition, the air was replaced with hydrogen three times and the mixture was allowed to react at room temperature for 6 h. The reaction mixture was filtered and the filtrate was concentrated to obtain the crude product, which was purified by preparative HPLC (Method D) to obtain the title compound, 40 mg. ESI-MS (m / z): 665.0 [M / 2+H].+ .

[0499] Step 6: 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-((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-amido)benzyl)carbonate (Compound TL019)

[0500] 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). The temperature was lowered to 0°C, and benzotriazol-1-yl-oxytripyrrolidino hexafluorophosphate (16.5 mg, 0.032 mmol) and N,N-diisopropylethylamine (6.2 mg, 0.047 mmol) were added sequentially. The mixture was reacted at room temperature for 2 h. Purification by preparative HPLC (Method D) afforded the title compound (10 mg). ESI-MS (m / z): 790.0 [M / 2+H]. + .

[0501] Example 9 (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)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)butanamido)-5-ureidopentanamido)benzyl)carbonate

[0502]

[0503] Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentanamido-2-yl)amino)-3-methyl-butanamido-2-yl)-(9H-fluorenyl)methyl-carbamate

[0504] Using a procedure similar to that of step 1 of Example 8, replacing compound 19-1 with compound 28-1, the title compound (310 mg) was obtained. ESI-MS (m / z): 602.3 [M+H] + .

[0505] Step 2: Synthesis of (S)-2-((S)-2-amino-3-methylbutanamide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 28-2)

[0506] Using a procedure similar to that of step 2 of Example 8, replacing compound 19-2 with compound 28-2, the title compound was obtained, 150 mg. ESI-MS (m / z): 380.3 [M+H] + .

[0507] 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)

[0508] Benzotriazol-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. The reaction was stirred at room temperature for 3 h. Purification by preparative HPLC (Method D) afforded the title compound, 78 mg. ESI-MS (m / z): 630.3 [M+H]. + .

[0509] 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)butanamido)-5-ureidopentanamido)benzyl)carbonate (Compound TL028)

[0510] Using a procedure similar to that of 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] + .

[0511] 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-nonyloxy-3,6,12-triazatricarboxamido)benzyl)carbonate

[0512]

[0513] 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)

[0514] Using a procedure similar to that of step 3 of Example 8, replacing compound 19-3 with compound 28-3, the title compound (180 mg) was obtained. ESI-MS (m / z): 916.5 [M+H] + .

[0515] 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)

[0516] A similar procedure was used as in step 4 of Example 8, replacing compound 19-4 with compound 29-1 to obtain the title compound, 30 mg. ESI-MS (m / z): 727.5 [M / 2+H] + .

[0517] 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-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)

[0518] 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 mixture of dimethyl sulfoxide and water (1 mL / 0.25 mL) at room temperature. Cuprous bromide (3.95 mg, 0.027 mmol) was added and stirred for 1 h. The title compound was purified by preparative HPLC (Method D) to obtain 15 mg of the title compound. ESI-MS (m / z): 880.0 [M / 2+H]. + .

[0519] Example 11 (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-tetraazapentatetradecane-44-carbamoyl)benzyl)carbonate

[0520]

[0521] Step 1: Synthesis of (S)-2-((S)-35-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

[0522] Compound 29-1 (400 mg, 0.44 mmol) was dissolved in methanol and tetrahydrofuran (2.0 mL:4.0 mL) at 20°C. After complete dissolution, platinum dioxide (40 mg) was added all at once under nitrogen. The atmosphere was replaced with hydrogen three times, and the reaction was hydrogenated at 20°C for 2 h. The mixture was filtered, the filter cake was washed with methanol, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (Method D) to obtain the title compound, 200 mg. ESI-MS (m / z): 890.4 [M+H] + .

[0523] 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-tetraazatetradodec-42-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0524] Compound 22-1 (250 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (1.0 mL) at 20°C. HATU (160 mg, 0.42 mmol) and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were added sequentially. The mixture was stirred at room temperature overnight. Purification by preparative HPLC (Method D) afforded the title compound (250 mg).

[0525] 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-tetraazapentatetradecane-44-carbamoyl)benzyl)carbonate (Compound TL022)

[0526] 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), and the temperature was lowered 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 complete, the reaction was stirred at 0°C for 1 h. The unreacted phosgene 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 mixture. The mixture was stirred at 0°C for 1 h. Purification by preparative HPLC (Method D) afforded the title compound, 1.5 mg. ESI-MS (m / z): 839.5 [M / 2+H] + .

[0527] 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-triazatetradodecyl-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

[0528]

[0529] 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)diphenylmethyl)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

[0530] 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. The mixture was stirred for 10 min, and compound 24-1 (35 mg, 0.022 mmol) was added. The reaction was stirred for 1 h. Purification by preparative HPLC (Method B) gave the title compound, 20 mg. ESI-MS (m / z): 1821.8 [M+H]. + .

[0531] 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)

[0532] Compound 24-2 (20 mg, 0.011 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 mixture was stirred for 20 min. Purification by preparative HPLC (Method C) afforded the trifluoroacetate salt of the title compound (12 mg). ESI-MS (m / z): 1549.6 [M+H] + .

[0533] 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

[0534]

[0535] Step 1: Preparation of (S)-(9H-fluoren-9-yl)-methyl(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate

[0536] 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 afforded the title compound, 1.8 g. ESI-MS (m / z): 417.2 [M+H] +

[0537] Step 2: Preparation of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)propionamide

[0538] At room temperature, ethylenediamine (5 mL) was added to a solution of compound 30-2 (1.8 g, 4.32 mmol) in dichloromethane (20 mL) and the reaction was allowed to proceed for 2 h. Purification by silica gel column chromatography afforded the title compound, 820 mg. ESI-MS (m / z): 195.1 [M+H]+

[0539] 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

[0540] At room temperature, (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoic 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 sequentially to a solution of compound 30-3 (503 mg, 2.58 mmol) in dichloromethane (2 mL). The mixture was stirred for 4 h. Purification by silica gel column chromatography afforded the title compound, 1.1 g. ESI-MS (m / z): 516.2 [M+H]+

[0541] Step 4: Preparation of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0542] 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 reaction was stirred for 1 h. Purification by silica gel column chromatography afforded the title compound, 610 mg. ESI-MS (m / z): 294.2 [M+H] +

[0543] Step 5: Preparation of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-diazapentatriacontamide)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0544] 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 3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatricyclodecane-1-oic acid (156 mg, 0.28 mmol) were added to a solution of compound 30-5 (84 mg, 0.28 mmol) in dichloromethane (3 mL). The mixture was stirred for 4 hours. Purification by silica gel column chromatography afforded the title compound, 163 mg. ESI-MS (m / z): 830.4 [M+H]. +

[0545] 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

[0546] Under nitrogen, 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. The mixture was allowed to react 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 allowed to react for 1 h at 0°C. The mixture was purified by silica gel column chromatography to obtain the title compound, 33 mg. ESI-MS (m / z): 1367.6 [M+H] +

[0547] Step 7: Preparation 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-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)

[0548] At room temperature, cuprous bromide (5 mg, 0.04 mmol) and compound 30-7 (20 mg, 15 μmol) were added dropwise to a mixture 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). The reaction was stirred for 4 h. Purification by preparative HPLC (Method D) afforded the title compound, 4.15 mg. ESI-MS (m / z): 1672.7 [M+H]. +

[0549] 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

[0550]

[0551] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide

[0552] 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) at 25°C. N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were added sequentially. The mixture was stirred for 2 h. The reaction solution was concentrated under reduced pressure, and the residue was purified on a flash silica gel column (ethyl acetate / petroleum ether = 3 / 1) to obtain the title compound, 700 mg. ESI-MS (m / z): 306.1 [M+H] + .

[0553] Step 2: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanoylamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)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

[0554] Under nitrogen at 25°C, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, and a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added. Then, a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the addition, the mixture was stirred at 0°C for 20 min, and the reaction solution was purged with nitrogen for 20 min. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added. The reaction mixture was stirred at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC (Method A) to obtain the title compound, 500 mg. ESI-MS (m / z): 1597.5 [M+H] + .

[0555] 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

[0556] Compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL:0.5 mL) at room temperature. Cuprous bromide (11 mg, 0.08 mmol) was added and the mixture was stirred for 1 h. Purification by preparative HPLC (Method B) afforded the title compound (30 mg). ESI-MS (m / z): 815.9 [(M-273) / 2+H] + .

[0557] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-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 (Compound TL033)

[0558] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL). Trifluoroacetic acid (0.2 mL) was added to the reaction mixture and allowed to react at room temperature for 30 min. Purification by preparative HPLC (Method C) afforded the trifluoroacetate salt of the title compound (20.0 mg). Its structural characterization is as follows:

[0559] 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).

[0560] 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

[0561]

[0562] 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-tetrahydroquinolin-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate

[0563] Compound 34-1 (100 mg, 0.2 mmol) was dissolved in dry dichloromethane (2 mL) under nitrogen protection at room temperature, 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 complete, the reaction was stirred at 0°C for 1 h. To the reaction mixture was added a solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (160 mg, 0.15 μmol) in dry dichloromethane (0.5 mL). The reaction was allowed to react at room temperature for 1 h. Purification by preparative HPLC (Method B) afforded the title compound, 60 mg. ESI-MS (m / z): 1592.7 [M+H]. + .

[0564] 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-diazapentatriacontamido)carbonate

[0565] 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) at room temperature. Cuprous bromide (9.01 mg, 0.06 mmol) was added and the mixture was stirred for 1 h. Purification by preparative HPLC (Method B) afforded the title compound, 20 mg. ESI-MS (m / z): 1897.5 [M+H].

[0566] 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)

[0567] Compound 34-3 (30 mg, 0.018 mmol) was dissolved in acetonitrile and water (0.4 mL:0.1 mL) at room temperature. A mixture of trifluoroacetic acid and acetonitrile (0.5 mL:0.5 mL) was added dropwise and stirred at room temperature for 2 h. Purification by preparative HPLC (Method C) afforded the title compound as a trifluoroacetate salt (12 mg). ESI-MS (m / z): 1511.5 [M+H] + .

[0568] 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

[0569]

[0570] 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-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)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

[0571] A similar synthesis method to that in Step 1 of Example 15 was used, substituting Compound 35-1 for Compound 34-1, to obtain the title compound, 60 mg. ESI-MS (m / z): 1561.5 [M+H] + .

[0572] Step 2: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)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-diazapentatriacontamido]carbonate

[0573] A similar synthesis method to that of Step 2 of Example 15 was used, substituting Compound 35-2 for Compound 34-2, to obtain the title compound (20 mg). ESI-MS (m / z): 1866.5 [M+H].

[0574] 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-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)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)

[0575] A similar synthesis method to that in Step 3 of Example 15 was used, substituting Compound 35-3 for Compound 34-3, to obtain the trifluoroacetic acid salt of the title compound, 4.9 mg. ESI-MS (m / z): 1594.5 [M+H] + .

[0576] 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-triazatetradodecyl-41-enamido)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

[0577]

[0578] Step 1: Synthesis of (Z)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enoic acid

[0579] Compound 3-4 (200 mg, 0.67 mmol) was dissolved in methanol (8.0 mL) at 20°C. Lindela's catalyst (20 mg) was added under nitrogen. The atmosphere was replaced with hydrogen three times and hydrogenated at 20°C for 3 h. The filtrate was filtered and dried to give the title compound (150 mg). ESI-MS (m / z): 271.1 [M+H] + .

[0580] 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)diphenylmethyl)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-enamido)benzyl carbonate

[0581] Compound 45-2 (8 mg, 0.030 mmol) was dissolved in dichloromethane (2 mL) at room temperature. 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 mixture was stirred at room temperature for 10 minutes. Compound 48-1 (30 mg, 0.020 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. The title compound was purified by preparative HPLC (Method B) to obtain 30 mg of the title compound. ESI-MS (m / z): 1787.8 [M+H] + .

[0582] 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-enamido)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)

[0583] 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 mixture was stirred at room temperature for 20 minutes. Purification by preparative HPLC (Method C) afforded the title compound, trifluoroacetate salt, 9 mg. ESI-MS (m / z): 1515.6 [M+H] + .

[0584] 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-triazatetradodecyl-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

[0585]

[0586] 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)diphenylmethyl)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

[0587] A similar synthesis method was used as in step 1 of Example 12, with compound 48-1 replacing compound 24-1. The title compound was obtained, 15 mg. ESI-MS (m / z): 1785.8 [M+H] + .

[0588] 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)

[0589] A similar synthesis method was used as in step 2 of Example 12, but compound 48-2 was substituted for compound 24-2. The trifluoroacetic acid salt of the title compound was obtained, 11.35 mg. ESI-MS (m / z): 1513.7 [M+H] + .

[0590] 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-diazapentatriacontylamino)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

[0591]

[0592] Step 1: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)thiazole-4-carboxylic acid

[0593] 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]palladium dichloride (29.02 mg, 0.04 mmol) were dissolved in N,N-dimethylformamide (4 mL) and water (1 mL) under nitrogen. The reaction system was heated to 100°C and stirred for 4 hours. The reaction solution was cooled to room temperature, added dropwise to water, and filtered. The filtrate was collected and extracted with ethyl acetate (10 mL x 3). The aqueous phase was collected and the pH was adjusted to 3 with dilute hydrochloric acid. A solid precipitated and the filter cake was filtered to obtain the title compound (70 mg). ESI-MS (m / z): 254.0 [M+H]. + .

[0594] Step 2: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxylic acid

[0595] 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 HPLC (Method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 286.0 [M+H] + .

[0596] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)thiazole-4-carboxamide

[0597] 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) afforded the title compound (10 mg). ESI-MS (m / z): 323.0 [M+H] + .

[0598] Step 4: 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)diphenylmethyl)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-diazapentatridecylamino)benzyl)carbonate

[0599] 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) at room temperature. Cuprous bromide (5.39 mg, 0.04 mmol) was added and stirred for 2 h. The mixture was filtered and 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]. + .

[0600] 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-diazapentatridecylamino)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

[0601] 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 mixture was stirred at room temperature for 20 min. The reaction mixture 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] + .

[0602] 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

[0603]

[0604] Step 1: Synthesis of ethyl 2-(2-(methylthio)pyrimidin-5-yl)oxazole-4-carboxylate

[0605] 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 mixture 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 sequentially. Under N2 protection, the temperature was raised to 90°C and the reaction was allowed to react for 3 h. The reaction solution was filtered through a pad of Celite, and the filtrate was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The organic phases were combined and dried. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. 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]. + .

[0606] Step 2: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)-oxazole-4-carboxylic acid

[0607] Compound 50-1 (50 mg, 0.19 mmol) was dissolved in a mixture of tetrahydrofuran and water (4 mL / 2 mL) at 25°C. After complete dissolution, lithium hydroxide monohydrate (40 mg, 0.94 mmol) was added. The mixture was allowed to react at 25°C for 1 h. The reaction solution was diluted with water (15 mL) and extracted with ethyl acetate (20 mL x 2). The aqueous phase was adjusted to pH 2-3 with 1N dilute hydrochloric acid and extracted with a dichloromethane / methanol mixture (v:v = 10:1) (20 mL x 3). The organic phases were combined, washed with saturated brine (30 mL x 1), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated to obtain the title compound (40 mg). This was used directly in the next step without purification. ESI-MS (m / z): 238.1 [M+H]. + .

[0608] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole-4-carboxylic acid

[0609] Compound 50-2 (40 mg, 0.17 mmol) was dissolved in dichloromethane (6 mL) at 25°C. After complete dissolution, m-chloroperbenzoic acid (29 mg, 0.17 mmol) was added. 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] + .

[0610] Step 4: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazole-4-carboxamide

[0611] Compound 50-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (4 mL) at 25°C. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (42 mg, 0.11 mmol) and N,N-diisopropylethylamine (19 mg, 0.15 mmol) were added sequentially. The mixture was stirred for 5 minutes. Propargylamine (5.0 mg, 0.09 mmol) was added. After addition, the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (Method D) to obtain the title compound, 5.0 mg. ESI-MS (m / z): 306.9 [M+H] + .

[0612] Step 5: Synthesis of (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 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-diazapentatriacontamido)benzyl)carbonate

[0613] Compound 50-4 (6.0 mg, 0.02 mmol) and compound 33-1 (30 mg, 0.02 mmol) were dissolved in a mixture of dimethyl sulfoxide and water (2 mL / 0.5 mL) at 25°C. Cuprous bromide (5.0 mg, 0.04 mmol) was added all at once. The mixture was allowed to react at room temperature for 2 h. The reaction mixture was filtered and purified by preparative HPLC (Method B) to obtain the title compound (25 mg). ESI-MS (m / z): 1631.3 [(M-273+H]] + .

[0614] 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

[0615] 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 allowed to react 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] + .

[0616] 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)propanamide-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-tetraazaanthan-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0617]

[0618] 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-butyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-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

[0619] 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). The temperature was cooled to 0°C, and 1-hydroxybenzotriazole (34 mg, 0.25 mmol), 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 complete, 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, washed with water, and dried to obtain the title compound, 200 mg. ESI-MS (m / z): 1329.2 [M+H] + .

[0620] Step 2: Synthesis of (S)-2-((S)-2-amino-3-butyramido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidopentanamide

[0621] Compound 51-2 (200 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (5 mL) at room temperature, and piperidine (0.5 mL) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative HPLC (Method D) to obtain the title compound, 65 mg. ESI-MS (m / z): 1107.2 [M+H] + .

[0622] 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-diazapentatridecylamino)-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-methylpropionamide)-3-phenylpropionamide)methyl)phenyl)-5-ureidopentanamide

[0623] 3,2-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). 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 mixture was stirred at room temperature for 10 minutes, cooled to 0°C, and compound 51-3 (55 mg, 0.05 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by preparative HPLC (Method D) to obtain the title compound, 56 mg. ESI-MS (m / z): 821.8 [M / 2+H]. + .

[0624] 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-tetraazaanthan-42-yl)-1H-1,2,3-triazol-4-yl) (methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0625] 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 mixture of dimethyl sulfoxide and water (2 mL / 0.5 mL) at room temperature. Cuprous bromide (10 mg, 68.17 μmol) was added and stirred for 2 h. The mixture was filtered, and the filtrate was purified by preparative HPLC (Method D) to obtain the title compound, 50 mg. ESI-MS (m / z): 974.3 [M / 2+H]. + .

[0626] 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)(methyl)carbamate

[0627]

[0628] 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-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0629] 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, followed by compound 52-1 (67 mg, 0.09 mol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by preparative HPLC (Method D) to obtain the title compound (120 mg). ESI-MS (m / z): 830.1 [M / 2+H]. + .

[0630] 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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0631] Compound 52-2 (22 mg, 0.07 mmol) was dissolved in a mixture of dimethyl sulfoxide and water (3 mL / 0.3 mL) at room temperature. Cuprous bromide (18 mg, 0.13 mmol) was added and stirred for 1 h. The mixture was filtered, and 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] + .

[0632] 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-(3-(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

[0633]

[0634] 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-triazatriacontamido)benzyl-(4-nitrophenyl)-carbonate

[0635] Compound 29-1 (500 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (10 mL) at 25°C. 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 addition, the mixture was stirred at 25°C for 3 h. The reaction solution was purified by reverse-phase column (C18) chromatography (acetonitrile / water = 1:2) to obtain the title compound, 400 mg. ESI-MS (m / z): 1081.9 [M+H] + .

[0636] 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

[0637] Compound 53-1 (60 mg, 0.06 mmol) and ((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) at 25°C. 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 HPLC (Method D) to obtain the title compound, 38 mg. ESI-MS (m / z): 837.2 [M / 2+H]. + .

[0638] Step 3: (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((S)-2-((S)-3-methyl-2-(3-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-carbamoyl)methyl)-1H-1 Synthesis of L-phenylalanine

[0639] 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 mixture of dimethyl sulfoxide and water (1 mL / 0.25 mL). After complete dissolution, cuprous bromide (11 mg, 0.08 mmol) was added. The mixture was stirred under nitrogen for 1 h. The mixture was filtered, and 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]. + .

[0640] 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-diazapentatriacontylamino)benzyl-((S)-1-(((S)-1-(((3R, 4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0641]

[0642] Step 1: Synthesis of (S)-4-(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-(4-nitrophenyl)-carbonate

[0643] 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, followed by a solution of di-(p-nitrobenzene) carbonate (860 mg, 2.83 mmol) in dichloromethane (10 mL) added dropwise. The reaction mixture was stirred at room temperature for 6 h. Purification by silica gel column (dichloromethane / methanol = 40 / 1) afforded the title compound, 900 mg. ESI-MS (m / z): 953.0 [M+H-273]. + .

[0644] 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-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0645] 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), followed by compound 52-1 (88 mg, 0.12 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by preparative HPLC (Method B) to obtain the title compound (150 mg). ESI-MS (m / z): 1803.6 [M+H] + .

[0646] 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-diazapentatridecylamino)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0647] 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) at room temperature. Cuprous bromide (16 mg, 0.11 mmol) was added and the mixture was stirred for 2 h. The mixture was filtered and 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]. + .

[0648] 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-diazapentatridecylamino)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-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0649] 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 mixture was stirred at room temperature for 20 min. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (Method C) to obtain 55 mg of the trifluoroacetate salt of the title compound. ESI-MS (m / z): 918.8 [M / 2+H] + .

[0650] 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

[0651]

[0652] Step 1: Synthesis of methyl 4-(2-(methylthio)pyrimidin-5-yl)benzoate

[0653] 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 sequentially to a solution of methyl p-bromobenzoate (215 mg, 1.0 mmol) in 1,4-dioxane (5 mL) at 25°C. The mixture was stirred at 80°C for 4 h. Extraction was performed with ethyl acetate (30 mL x 3). The organic phases were combined, dried, and the insoluble matter was filtered off. The residue was purified by silica gel column chromatography to obtain the title compound, 220 mg. ESI-MS (m / z): 261.0 [M+H] + .

[0654] Step 2: Synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic acid

[0655] 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) at 25°C and stirred for 4 h. The pH of the reaction solution was adjusted to 3-4 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried, and the insoluble material was filtered off. The residue was purified by preparative HPLC (Method D) to obtain the title compound, 430 mg. ESI-MS (m / z): 246.9 [M+H] + .

[0656] Step 3: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid

[0657] 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. Purification by silica gel column chromatography afforded the title compound (180 mg). ESI-MS (m / z): 279.0 [M+H] + .

[0658] Step 4: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)benzamide

[0659] Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (100 mg, 0.26 mmol) was added to a solution of compound 55-3 (50 mg, 0.18 mmol) in dichloromethane (10 mL) at 25°C and stirred for 30 minutes. Propargylamine (10 mg, 0.2 mmol) and N,N-diisopropylethylamine (70 mg, 0.5 mmol) were then added to the reaction mixture and stirred for 2.5 hours. Purification by silica gel column chromatography afforded the title compound (20 mg). ESI-MS (m / z): 316.0 [M+H] + .

[0660] 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-amino-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatriacontamido)benzyl carbonate: At 25°C, under nitrogen protection, cuprous iodide (10 mg, 0.05 mmol) and water (2 mL) were added sequentially 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). The mixture was stirred for 1 hour. Purification (Method B) afforded the title compound, 79 mg. ESI-MS (m / z): 1641.5 [M-273+H]. + .

[0661] 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

[0662] Compound 55-5 (55 mg, 0.029 mmol) was added to a mixture of trifluoroacetic acid (0.5 mL) and water / acetonitrile (0.1 mL / 0.5 mL) at 25°C. The reaction was stirred for 15 minutes. The reaction solution was purified by preparative HPLC (Method C) to obtain 42 mg of the trifluoroacetate salt of the title compound. ESI-MS (m / z): 821.0 [M / 2+H] + .

[0663] 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-tetraazatetradodec-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0664]

[0665] 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

[0666] 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) at 20°C. Cuprous bromide (465 mg, 3.21 mmol) was added all at once and the reaction was stirred for 12 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (Method D) to obtain the title compound, 500 mg. ESI-MS (m / z): 860.4 [M+H]. + .

[0667] 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

[0668] 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 1-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 used directly in the next reaction. ESI-MS (m / z): 626.0 [M / 2+H] + .

[0669] 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

[0670] Diethylamine (0.5 mL) was added to the reaction mixture from step 2 at 25°C. After addition, the mixture was stirred for 30 min. The reaction mixture was purified by preparative HPLC (Method D) to obtain the title compound, 70 mg. ESI-MS (m / z): 515.0 [M / 2+H] + .

[0671] 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 (1,2,3-triazol-4-yl)-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-tetraazatetradodec-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0672] 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 (95m g, 0.092 mmol), 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 (79 mg, 0.092 mmol) was dissolved in N,N-dimethylformamide (4 mL), and HATU (70 mg, 0.184 mmol) was added all at once. The reaction was stirred at room temperature for 1 hour. The reaction solution was purified by preparative HPLC (Method D) to obtain the title compound, 30 mg. ESI-MS (m / z): 935.8 [M / 2+H] + .

[0673] 3. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies

[0674] Example 27 Preparation of BT001002

[0675] 0.3 mL of sacituzumab antibody (anti-Trop-2, 33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6). 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added and mixed thoroughly. The pH was adjusted to 7.4 with 1 M K2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 30 minutes. A 15-fold amount of TL003 dissolved in dimethyl sulfoxide was added to the above solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours. After completion, 6.1 μL of 100 mM cysteine ​​was added to terminate the reaction. 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.

[0676]

[0677] Example 28 Preparation of BT001004

[0678] 0.285 mL of sacituzumab antibody (anti-Trop-2, 17.6 mg / mL) was diluted with 0.095 mL of diluent (a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate, pH 7.6). The pH was then adjusted to 7.4 with 1 M Na₂HPO₄ solution, and 10 mM TCEP solution was added, mixed, and allowed to stand at room temperature for 30 minutes. To this solution, a 9-fold amount of TL019 dissolved in dimethyl sulfoxide was added, mixed, and allowed to stand at room temperature for 2 hours. Finally, the buffer was exchanged with PBS buffer (pH 6.5) using a G-25 gel column to obtain the TL019-sacituzumab antibody conjugate, designated BT001004.

[0679]

[0680] Example 29 Preparation of BT001012

[0681] A method similar to that of 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.

[0682]

[0683] Example 30 Preparation of BT001013

[0684] A method similar to that of Example 27 was used to replace TL003 with the trifluoroacetate salt of TL048 to obtain a product of TL048 coupled with the sacituzumab antibody, which was named BT001013.

[0685]

[0686] Example 31 Preparation of BT001018

[0687] A method similar to that of Example 27 was used, except that TL003 was replaced by TL030 to obtain a product of TL030 coupled with Sacituzumab antibody, which was named BT001018.

[0688]

[0689] Example 32 Preparation of BT001021

[0690] 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6). 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added and mixed thoroughly. The pH was adjusted to 7.4 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 30 minutes. A 10-fold amount of TL033 trifluoroacetate dissolved in dimethyl sulfoxide was added to the above solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours. After completion, 6.1 μL of 100 mM cysteine ​​was added to terminate the reaction. Finally, the buffer solution was replaced with a pH 6.5 PBS buffer solution using a G-25 gel column to obtain a product conjugated with TL033 and Sacituzumab antibody, named BT001021.

[0691]

[0692] Example 33 Preparation of BT001022

[0693] A method similar to that of 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.

[0694]

[0695] Example 34 Preparation of BT001023

[0696] A method similar to that of Example 27 was used to replace TL003 with the trifluoroacetate salt of TL035 to obtain a product of TL035 coupled with the sacituzumab antibody, which was named BT001023.

[0697]

[0698] Example 35 Preparation of BT001032

[0699] A method similar to that of 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.

[0700]

[0701] Example 36 Preparation of BT001033

[0702] A method similar to that of Example 27 was used, except that 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.

[0703]

[0704] Example 37 Preparation of BT001034

[0705] A method similar to that of Example 27 was used, except that 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 conjugation, which was named BT001034.

[0706]

[0707] Example 38 Preparation of BT001035

[0708] 0.3 mL of M3 antibody (anti-Trop-2, 33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6). 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was added and mixed thoroughly. The pH was adjusted to 7.4 with 1 M Na₂HPO₄ solution, and 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 30 minutes. A 10-fold amount of TL033 trifluoroacetate dissolved in dimethyl sulfoxide was added to the above solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours. After completion, 6.1 μL of 100 mM cysteine ​​was added to terminate the reaction. Finally, the buffer was exchanged with PBS (pH 6.5) using a G-25 gel column to obtain the TL033-M3 antibody conjugated product, designated BT001035.

[0709]

[0710] Example 39 Preparation of BT001036

[0711] A method similar to that of Example 27 was used, except that 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.

[0712]

[0713] Example 40 Preparation of BT001040

[0714] A method similar to that of 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.

[0715]

[0716] Example 41 Preparation of BT001041

[0717] A method similar to that of 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.

[0718]

[0719] Example 42 Preparation of BT001042

[0720] A method similar to that of Example 27 was used, and TL003 was replaced with TL051 to obtain a product of TL051 coupled with Sacituzumab antibody, which was named BT001042.

[0721]

[0722] Example 43 Preparation of BT001043

[0723] A method similar to that of Example 27 was used, except that TL003 was replaced by TL052 to obtain a product of TL052 coupled with Sacituzumab antibody, which was named BT001043.

[0724]

[0725] Example 44 Preparation of BT001044

[0726] A method similar to that of Example 27 was used, except that TL003 was replaced by TL053 to obtain a product of TL053 coupled with Sacituzumab antibody, which was named BT001044.

[0727]

[0728] Example 45 Preparation of BT001045

[0729] A method similar to that of 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.

[0730]

[0731] Example 46 Preparation of BT001046

[0732] A method similar to that of Example 27 was used to replace TL003 with the trifluoroacetate salt of TL055 to obtain a product of TL055 coupled with the sacituzumab antibody, which was named BT001046.

[0733]

[0734] Example 47 Preparation of BT001047

[0735] A method similar to that of Example 27 was used, and TL003 was replaced with TL056 to obtain a product of TL056 coupled with Sacituzumab antibody, which was named BT001047.

[0736]

[0737] Example 48 LC-MS determination of BT001002 molecular weight

[0738] The molecular weight of the coupled BT001002 was analyzed by LCMS.

[0739] Chromatographic determination conditions:

[0740] Liquid chromatography column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0741] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0742] Flow rate: 0.25 mL / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μg;

[0743] 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

[0744] Mass spectrometry conditions:

[0745] Mass spectrometer model: Triple TOF 5600+;

[0746] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m / z600-5000;

[0747] The results are as follows Figure 1-3 shown.

[0748] BT001002 theoretical molecular weight and measured molecular weight

[0749]

[0750] In the table, mAb stands for monoclonal antibody; LC stands for antibody light chain; HC stands for antibody heavy chain; DAR1 stands for a conjugate comprising one antibody light chain / heavy chain and one cellular bioactive molecule; DAR2 stands for a conjugate comprising one antibody light chain / heavy chain and two cellular bioactive molecules; DAR3 stands for a conjugate comprising one antibody light chain / heavy chain and three cellular bioactive molecules; and DAR4 stands for a conjugate comprising one antibody light chain / heavy chain and four cellular bioactive molecules. Glycoforms represent the carbohydrate structure of the two heavy chains; G0F stands for fucosylated and galactose-free. In the following, mAb, LC, HC, DAR1, DAR2, DAR3, DAR4, and G0F are as described above.

[0751] Depend on Figure 1-3 It can be seen that after the antibody is conjugated to TL003, the molecular weights of both the light and heavy chains change, with the light chain conjugated to one cellular bioactive molecule and the heavy chain conjugated to three cellular bioactive molecules. It can be inferred that the coupling ratio (DAR) of the entire antibody to cellular bioactive molecules is 8.

[0752] Example 49 LC-MS determination of BT001004 molecular weight

[0753] The molecular weight of the coupled BT001004 was analyzed by LC-MS.

[0754] Chromatographic determination conditions:

[0755] Liquid chromatography column: ACQUITU Protein BEH C18 1.7μm, 2.1mm×100mm;

[0756] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0757] Flow rate: 0.25 mL / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μg;

[0758] 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

[0759] Mass spectrometry conditions:

[0760] Mass spectrometer model: Triple TOF 5600+;

[0761] GS1 60; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m / z 600-5000;

[0762] The results are as follows Figure 4-6 shown.

[0763] BT001004 theoretical molecular weight and measured molecular weight

[0764]

[0765] LC represents the antibody light chain, and HC represents the antibody heavy chain.

[0766] Depend on Figure 4-6It 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%, and 68% respectively). The coupling ratio (DAR) of the entire antibody to cellular bioactive molecules is calculated to be 7.0.

[0767] Example 50 Determination of Molecular Weight of BT001012 by LC-MS

[0768] Using a method similar to Example 48, the results are as follows Figure 10 and 11 shown.

[0769] The theoretical and measured molecular weights of the BT001012 light and heavy chains obtained after conjugation of TL024 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0770]

[0771] Depend on Figure 10 and 11 It can be seen that the light chain of the antibody in BT001012 is coupled to 0 to 1 toxin (the ratios of LC and DAR1 are 12.9% and 87.1% respectively), and the heavy chain is coupled to 1 to 3 toxins (the ratios of DAR1, DAR2, and DAR3 are 13.4%, 10.8%, and 75.8% respectively). The coupling ratio (DAR) of the antibody to toxin is calculated to be 7.0.

[0772] Example 51 Determination of Molecular Weight of BT001013 by LC-MS

[0773] Using a method similar to Example 48, the results are as follows Figure 12 and 13 shown.

[0774] The theoretical and measured molecular weights of the BT001013 light and heavy chains obtained after conjugation of TL048 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0775]

[0776] Depend on Figure 12 and 13 It can be seen that the light chain of the antibody in BT001013 is coupled to 0 to 1 toxin (the ratios of LC and DAR1 are 6.8% and 93.2% respectively), and the heavy chain is coupled to 1 to 4 toxins (the ratios of DAR1, DAR2, DAR3, and DAR4 are 12.8%, 12.8%, 64.9%, and 9.5% respectively). The coupling ratio (DAR) of the antibody to toxin is calculated to be 7.3.

[0777] Example 52: Determination of Molecular Weight of BT001018 by LC-MS

[0778] Using a method similar to Example 48, the results are as follows Figure 14 and 15 shown.

[0779] The theoretical and measured molecular weights of the BT001018 light and heavy chains obtained after conjugation of TL030 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0780]

[0781] Depend on Figure 14 and 15 It can be seen that the light chain of the antibody in BT001018 is coupled to 0 to 1 toxin (the ratios of LC and DAR1 are 55.3% and 44.7% respectively), and the heavy chain is coupled to 1 to 3 toxins (the ratios of DAR1, DAR2, and DAR3 are 19.6%, 23.3%, and 49.6% respectively). The coupling ratio (DAR) of the antibody to toxin is calculated to be 5.2.

[0782] Example 53 LC-MS determination of BT001021 molecular weight

[0783] The molecular weight of the coupled BT001021 was analyzed by LCMS.

[0784] Chromatographic determination conditions:

[0785] Liquid chromatography column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0786] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0787] Flow rate: 0.25 mL / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μg;

[0788] 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

[0789] Mass spectrometry conditions:

[0790] Mass spectrometer model: Triple TOF 5600+;

[0791] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m / z600-5000;

[0792] The results are as follows Figure 16 and 17 shown.

[0793] The theoretical and measured molecular weights of the BT001021 light and heavy chains obtained after conjugation of TL033 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0794]

[0795]

[0796] Depend on Figure 16 and 17 It can be seen that the light chain of the antibody in BT001021 is coupled to 0 to 1 toxin (the ratios of LC and DAR1 are 4.5% and 95.5% respectively), and the heavy chain is coupled to 1 to 3 toxins (the ratios of DAR1, DAR2, and DAR3 are 15.3%, 17.6%, and 67.1% respectively). The coupling ratio (DAR) of the antibody to toxin is calculated to be 6.9.

[0797] Example 54 LC-MS determination of BT001023 molecular weight

[0798] Using a method similar to Example 48, the results are as follows Figure 18 and 19 shown.

[0799] The theoretical and measured molecular weights of the BT001023 light and heavy chains obtained after conjugation of TL035 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0800]

[0801] Depend on Figure 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.

[0802] Example 55 LC-MS determination of BT001040 molecular weight

[0803] LC-MS molecular weight analysis of BT001040 after coupling

[0804] Liquid chromatography column: Thermo MabPacTM RP 4μm, 3.0mm*100mm

[0805] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN

[0806] Flow rate: 0.25 mL / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μg

[0807] 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

[0808] Mass spectrometry conditions:

[0809] Mass spectrometer model: Triple TOF 5600+

[0810] GS1 35; GS2 35; CUR30; TEM 350; ISVF5000; DP250; m / z 600-5000

[0811] The theoretical and measured molecular weights of the BT001040 light and heavy chains obtained after conjugation of TL049 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0812]

[0813] Depend on Figure 20 and 21 It can be seen that the light chain of the antibody 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.

[0814] Example 56 Determination of Molecular Weight of BT001041 by LC-MS

[0815] Using a method similar to Example 55, the results are as follows Figure 22 and 23 shown.

[0816] The theoretical and measured molecular weights of the BT001041 light and heavy chains obtained after conjugation of TL050 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0817]

[0818] Depend on Figure 22 and 23It 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.

[0819] Example 57 LC-MS determination of BT001042 molecular weight

[0820] Using a method similar to Example 55, the results are as follows Figure 24 and 25 shown.

[0821] The theoretical and measured molecular weights of the BT001042 light and heavy chains obtained after conjugation of TL051 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0822]

[0823] Depend on Figure 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.

[0824] Example 58 LC-MS determination of BT001043 molecular weight

[0825] Using a method similar to Example 55, the results are as follows Figure 26 and 27 shown.

[0826] The theoretical and measured molecular weights of the BT001043 light and heavy chains obtained after conjugation of TL052 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0827]

[0828]

[0829] Depend on Figure 26 and 27It can be seen that the light chain of the antibody 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%, and 68.4% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.8.

[0830] Example 59 LC-MS determination of BT001044 molecular weight

[0831] Using a method similar to Example 55, the results are as follows Figure 28 and 29 shown.

[0832] The theoretical and measured molecular weights of the BT001044 light and heavy chains obtained after conjugation of TL053 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0833]

[0834] Depend on Figure 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%, and 69.3% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.5.

[0835] Example 60 LC-MS determination of BT001046 molecular weight

[0836] Using a method similar to Example 55, the results are as follows Figure 30 and 31 shown.

[0837] The theoretical and measured molecular weights of the BT001046 light and heavy chains obtained after conjugation of TL055 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0838]

[0839]

[0840] Depend on Figure 30 and 31It can be seen that the light chain of the antibody 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.

[0841] Example 61 Determination of Molecular Weight of BT001047 by LC-MS

[0842] Using a method similar to Example 55, the results are as follows Figure 32 and 33 shown.

[0843] The theoretical and measured molecular weights of the BT001047 light and heavy chains obtained after conjugation of TL056 to the antibody (calculated based on the major glycoform G0F) are shown in the table below:

[0844]

[0845] Depend on Figure 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%, and 64.3% respectively). The coupling ratio (DAR) of the antibody to the cellular bioactive molecules is calculated to be 6.6.

[0846] Example 62 Size Exclusion Chromatography Analysis

[0847] The coupling reaction was monitored by SEC-HPLC, and the conjugate was detected by SEC.

[0848] Chromatographic conditions:

[0849] Liquid chromatography column: TOSOH TSKgel SuperSW mAb, 4 μm, 7.8 mm x 300 mm;

[0850] Mobile phase: 100 mmol / L Na2HPO4, 100 mmol / L NaCl, 5% isopropanol, pH 7.0;

[0851] Flow rate: 0.5 ml / min; detection wavelength: 280 nm; column temperature: room temperature; sample chamber temperature: 8°C;

[0852] Injection volume: 30 μg; isocratic run: 30 min.

[0853] The SEC chromatograms of BT001002 obtained by coupling the antibody with TL003 and the molecular weight marker SEC chromatograms are shown in Figure 2. Figure 7 and 8 As shown, according to the molecular weight marker, it is confirmed that the main peak of the coupling product corresponds to a molecular weight of approximately 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.

[0854] The SEC chromatogram of BT001004 obtained by coupling TL019 with the antibody is shown in the figure below. Figure 9 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001004 obtained by coupling TL019 with the antibody still maintained the complete structure of the antibody.

[0855] The SEC chromatogram of BT001012 obtained by coupling TL024 with the antibody is shown in the figure below. Figure 34 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150 kD, that is, BT001012 obtained by coupling TL024 with the antibody still maintained the complete structure of the antibody.

[0856] The SEC chromatogram of BT001013 obtained by coupling TL048 with antibody is shown in the figure below. Figure 35 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150 kD, that is, BT001013 obtained by coupling TL048 with the antibody still maintained the complete structure of the antibody.

[0857] The SEC chromatogram of BT001018 obtained by coupling TL030 with antibody is shown in the figure below. Figure 36 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001018 obtained by coupling TL030 with the antibody still maintained the complete structure of the antibody.

[0858] The SEC chromatogram of BT001021 obtained by coupling TL033 with antibody is shown in the figure below. Figure 37 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150 kD, that is, BT001021 obtained by coupling TL033 with the antibody still maintained the complete structure of the antibody.

[0859] The SEC chromatogram of BT001023 obtained by coupling TL035 with antibody is shown in the figure below. Figure 38As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150 kD, that is, BT001023 obtained by coupling TL035 with the antibody still maintained the complete structure of the antibody.

[0860] The SEC chromatogram of BT001042 obtained by coupling TL051 with the antibody is shown in the figure below. Figure 39 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001042 obtained by coupling TL051 with the antibody still maintained the complete structure of the antibody.

[0861] The SEC chromatogram of BT001043 obtained by coupling TL052 with antibody is shown in the figure below. Figure 40 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001043 obtained by coupling TL052 with the antibody still maintained the complete structure of the antibody.

[0862] The SEC chromatogram of BT001044 obtained by coupling TL053 with antibody is shown in the figure below. Figure 41 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001044 obtained by coupling TL053 with the antibody still maintained the complete structure of the antibody.

[0863] The SEC chromatogram of BT001046 obtained by coupling TL055 with the antibody is shown in the figure below. Figure 42 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001046 obtained by coupling TL055 with the antibody still maintained the complete structure of the antibody.

[0864] The SEC chromatogram of BT001047 obtained by coupling TL056 with the antibody is shown in the figure below. Figure 43 As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main coupling product was approximately 150kD, that is, BT001047 obtained by coupling TL056 with the antibody still maintained the complete structure of the antibody.

[0865] Example 63 Detection of the Inhibitory Effects of Bioactive Molecules and Antibody-Drug Conjugates on In Vitro Cell Activity

[0866] First, tumor cells (MDA-MB-468, a Trop-2-positive cell line) and HCC1806, a Trop-2-positive cell line, were cultured. The bioactive molecules and ADCs disclosed herein were then co-cultured with the tumor cells. CCK8 reagent (Dongren Chemical Technology Co., Ltd., Cat: CK04, Lot: JJ744) was then added. The activity of mitochondrial dehydrogenases was measured using a microplate reader (Molecular Devices, SpectraMax M2, 450 nm) to assess the inhibitory effect of the ADCs on cell proliferation. Tumor cell sources are shown in Table 1.

[0867] Table 1.

[0868] Cell name Tumor type source MDA-MB-468 Breast cancer Connaughty HCC1806 Breast cancer Nanjing Kebai Biological

[0869] In vitro cell viability assay: Bioactive molecules or ADCs were diluted in the corresponding assay medium (containing 2% FBS) (12 concentration gradients). Tumor cells were digested using trypsin using conventional methods, harvested and counted, and resuspended in the corresponding assay medium (containing 2% FBS). The diluted bioactive molecules or ADCs were added to a 96-well plate, followed by the cells. Then, 20 μL of CCK8 reagent was added to each well, the reaction was allowed to proceed for 4 hours, and the results were read using a microplate reader (detection wavelength was 450 nm). The experimental conditions and test results are shown in Tables 2 and 3.

[0870] Table 2. Cell killing results of bioactive molecules

[0871]

[0872]

[0873] The test results show that all biologically active molecules have tumor cell killing effects.

[0874] Table 3. Conjugate (ADC) cell line killing results

[0875]

[0876] The test results show that the ADC molecules obtained using the new conjugation method have tumor cell killing effects. This shows that the ADC formed using the new conjugation method can kill tumor cells and that the new conjugation method is effective in applying to ADC molecules.

[0877] Example 64 In vivo efficacy testing of antibody drug conjugates and active biomolecules

[0878] Test drug

[0879] Drug name, source, and preparation method:

[0880] BT001021, liquid concentration is 5.44 mg / ml, stored at -20°C, and diluted with normal saline according to the dose to obtain the test solution;

[0881] Immu-132 (prepared with reference to Example 2 of WO2015 / 012904A2, DAR = 5.4, also referred to as IMMU-132), liquid concentration 13.158 mg / ml, stored at -20°C, and diluted with normal saline according to the dose to obtain the test solution;

[0882] T-030, solid powder, was prepared with 100% DMSO (Sigma) to a concentration of 5.2 mg / ml, stored at -20°C, and diluted with normal saline according to the dose to obtain the test solution;

[0883] 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 the test solution.

[0884] Note: The dosage of toxin is prepared based on the equimolar ratio of ADC sample.

[0885] The structures of T-030, SN-38, and Immu-132 are as follows:

[0886]

[0887] Experimental animals and cell lines

[0888] Balb / c-nu mice (Beijing Weitonglihua Laboratory 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).

[0889] Experimental grouping and evaluation methods

[0890] The tumor volume was between 100 and 200 mm. 3 Tumor-bearing mice were divided into 6 groups. 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 and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0891] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy of antibody-drug conjugates:

[0892] TGI (%) = [1-(VT 末 -VT 始 ) / (VC 末 -VC 始 )]*100%

[0893] Where V T末 : Mean tumor volume at the end of the experiment in the treatment group

[0894] V T始 : Mean tumor volume at the start of drug administration in the treatment group

[0895] V C末 : Mean tumor volume at the end of the solvent control group experiment

[0896] V C始 : Mean tumor volume at the start of drug administration in the solvent control group

[0897] The following Experimental Examples 1 and 2 evaluate the inhibition of tumor proliferation of the antibody conjugate BT001021 in mice with subcutaneous transplantation of human tumor cells. Specifically, in Experimental Examples 1 and 2, human gastric cancer cell line NCI-N87 and human triple-negative breast cancer cell line HCC1806 were subcutaneously transplanted to construct tumor-bearing mouse models. 3 After about 1 hour, the mice were randomly divided into groups and intravenously administered BT001021 twice a week for a total of 6 times. Tumor volume and animal body weight changes were measured twice a week to evaluate the efficacy of the antibody drug conjugate in tumor-bearing mice (tumor inhibition effect).

[0898] Experimental Example 1. Inhibition of NCI-N87 by Antibody Drug Conjugates and Active Biomolecules

[0899] Experimental methods:

[0900] NCI-N87 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were collected during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer model. The average tumor volume was approximately 90 mm. 3At the time of the study, mice were randomly divided into groups according to tumor size: saline group, BT001021 (3 mg / kg, intravenous injection, twice weekly for 3 weeks), positive drug Immu-132 (3 mg / kg, intravenous injection, twice weekly for 3 weeks), T030 group, and SN38 group. After grouping, the corresponding drug was injected into the tail vein twice weekly 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 4. Figure 44 and 45 .

[0901] Experimental conclusion:

[0902] In this experiment, the human gastric cancer cell line NCI-N87 was used to construct a subcutaneous transplant tumor model of human gastric cancer, and the efficacy of BT001021 in the NCI-N87 human gastric cancer tumor-bearing mouse model was evaluated.

[0903] The experimental results show that BT001021 (3 mg / kg, intravenous injection, twice a week for 3 weeks) can significantly inhibit tumor growth in NCI-N87 gastric cancer transplanted tumor model mice, and tumor regression occurred at the end 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.

[0904] Table 4. Gastric cancer NCI-N87 model

[0905]

[0906] Experimental Example 2. Inhibition of HCC1806 by antibody-drug conjugates

[0907] Experimental methods:

[0908] HCC1806 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC1806 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a breast cancer model. The average tumor volume was approximately 130 mm. 3 At the time of the study, mice were randomly divided into three groups based on tumor size: a saline group, a BT001021 (10 mg / kg, intravenous injection, twice weekly for 3 weeks), and a positive drug Immu-132 (10 mg / kg, intravenous injection, twice weekly for 3 weeks) group. After grouping, the corresponding drug was injected into the tail vein twice weekly for a total of 5 times. After administration, the mice were observed and their tumor volumes were measured regularly. The specific results are shown in Table 5. Figure 46 .

[0909] Experimental conclusion:

[0910] This experimental example used the human breast cancer cell line HCC1806 to construct a human breast cancer subcutaneous transplant tumor model and evaluated the efficacy of BT001021 in the HCC1806 human breast cancer tumor-bearing mouse model.

[0911] The experimental results show that BT001021 (10 mg / kg, intravenous injection, twice a week for 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.

[0912] Table 5: Breast cancer HCC1806 model

[0913]

[0914]

[0915] From Table 4, Table 5 and Figures 44-46 As can be seen, the antibody drug of the present invention, BT001021, can significantly inhibit tumor growth in the NCI-N87 mouse model, significantly outperforming Immu-132 at the same dose, without significant weight loss or overt toxicity. In the HCC1806 mouse model, due to the high malignancy of the tumor, Immu-132 showed no significant inhibitory activity at a dose of 10 mg / kg, while BT001021 significantly inhibited tumor growth. These results demonstrate that BT001021 of the present invention has good efficacy and an excellent safety profile.

[0916] 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 in clinical practice.

[0917] Experimental Example 3. Inhibition of HCC827 by antibody-drug conjugates

[0918] 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 100 mm 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.

[0919] Experimental methods:

[0920] 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 80 mm3, the mice were randomly divided into normal saline group, positive drug Immu-132 (10 mg / kg, IV, BIW×3W) group, BT001021 (10 mg / kg, IV, BIW×3W), and BT001035 (10 mg / kg, IV, BIW×3W) group according to 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. Figure 47A , Figure 47B .

[0921] Experimental conclusion:

[0922] The experimental results show that BT001021 and BT001035 can significantly inhibit tumor growth in mice with HCC827 non-small cell lung cancer transplanted tumor models, and tumor regression occurred at the endpoint of dosing. The anti-tumor activity is 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.

[0923] Table 6. Lung cancer HCC827 model

[0924]

[0925] From Table 6, Figure 47A and Figure 47B As can be seen, both BT001021 and BT001035 showed significant tumor growth inhibition activity during the evaluation period. At the same dose, their tumor inhibition activity was significantly superior to that of Immu-132. During the dosing period, no significant weight loss or drug toxicity was observed in any group of animals. These results demonstrate that BT001021 and BT001035 have excellent anti-tumor activity.

[0926] In this subcutaneous transplant tumor model, the anti-tumor activity of BT001021 and BT001035 was significantly better than that of 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 in clinical practice.

[0927] Experimental Example 4.Inhibition of NCI-N87 by Antibody Drug Conjugates

[0928] Experimental Example 4 evaluates the inhibitory effect of antibody conjugate BT001036 on tumor proliferation in mice with subcutaneous transplantation of human tumor cells. Specifically, in this experiment, human gastric cancer cell line NCI-N87 was subcutaneously transplanted to construct a tumor-bearing mouse model. When the tumor volume grew to 140 mm 3 After about 1 hour, the mice were randomly divided into groups and intravenously administered BT001036 twice a week for a total of 6 times. Tumor volume and animal body weight changes were measured twice a week to evaluate the efficacy of the antibody drug conjugate in tumor-bearing mice (tumor inhibition effect).

[0929] Experimental methods:

[0930] NCI-N87 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer xenograft model. The average tumor volume was approximately 140 mm. 3 At 4 pm, mice were randomly divided into three groups according to tumor size: saline group, BT001036 (1.5 mg / kg, IV, BIW×3W) group, and BT001036 (3 mg / kg, IV, BIW×3W) group. 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 7. Figure 48A , Figure 48B .

[0931] Table 7. Gastric cancer NCI-N87 model

[0932]

[0933] Experimental conclusion:

[0934] In this experiment, 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.

[0935] 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 mice with NCI-N87 gastric cancer transplanted tumor model, and tumor regression occurred at the end of dosing, with excellent 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, the mice tolerated the evaluated drugs well.

[0936] Experimental Example 5.Inhibition of MDA-MB-231 by antibody-drug conjugates

[0937] 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. 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 of BT001021 on tumor-bearing mice (tumor inhibition effect) was calculated.

[0938] Experimental methods:

[0939] 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 were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a breast cancer xenograft model. The average tumor volume was approximately 130 mm. 3 At 37 ℃, the mice were randomly divided into two groups according to tumor size: normal saline group and BT001021 (3 mg / kg) group. The corresponding drugs were injected into the tail vein after grouping. The drugs were administered twice a week for a total of 6 times. The tumor volume and body weight of the mice were observed and measured regularly after administration. The specific results are shown in Table 8. Figure 49A and 49B .

[0940] Experimental conclusion:

[0941] The experimental results show that BT001021 can significantly inhibit tumor growth in mice with MDA-MB-231 breast cancer transplant tumor models, and tumor regression occurred at the endpoint of dosing. There were no animal deaths 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] Table 8. MDA-MB-231 breast cancer model

[0943]

[0944] 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.

[0945] Example 65 In vivo pharmacokinetic testing of antibody drug conjugates and active biomolecules

[0946] 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 dose of BT001021 and T-030. T-030 concentrations in tumor tissue and serum were measured to evaluate the pharmacokinetic behavior of the antibody-drug conjugate BT001021 and the active biomolecule T-030 in tumor-bearing mice.

[0947] Test drug

[0948] Drug name, preparation method:

[0949] BT001021, liquid concentration is 20 mg / ml, stored in aliquots at -20°C, and diluted with normal saline according to the dose to obtain the test solution when used;

[0950] T-030 was prepared with dimethyl sulfoxide at a concentration of 1 mg / ml and diluted with physiological saline according to the dose to obtain a test solution.

[0951] Experimental animals and cell lines:

[0952] Balb / c-nu mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011); gastric cancer cell line NCI-N87 (ATCC).

[0953] Experimental grouping and evaluation methods:

[0954] The tumor volume was between 100 and 200 mm. 3 Tumor-bearing mice were divided into 4 groups, and the drug was administered via a single tail vein injection.

[0955] Experimental Example 6. In vivo pharmacokinetic testing of BT001021 and T-030 in tumor-bearing mice

[0956] Experimental methods:

[0957] NCI-N87 cells were cultured in 1640 medium containing 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into Balb / c-nu mice to establish a gastric cancer xenograft model. The tumors were grown until the average volume reached approximately 100-200 mm. 3Patients were randomly divided according to tumor size into the normal saline group, the T-030 (0.23 mg / kg, intravenous injection, single dose) group, and the BT001021 (10 mg / kg, intravenous injection, single dose) group. The corresponding drug was injected into the tail vein after grouping. Serum and tumor tissue were collected from the T-030 group at 1, 2, 4, 8, 24, and 72 hours after administration (T-030 was undetectable in serum and tumor tissue by 72 hours after administration; therefore, serum and tumor tissue were not collected at 168 hours after administration). Serum and tumor tissue were collected from the BT001021 group at 1, 2, 4, 8, 24, 72, and 168 hours after administration. T-030 concentrations in serum and tumors were determined by LC-MS / MS. The results are shown in Table 9. The T-030 dose (0.23 mg / kg) was converted to the BT001021 dose (10 mg / kg) in equimolar terms.

[0958] Table 9. Pharmacokinetic parameters of T-030 in tumor and serum after intravenous administration of T-030 and BT001021 in tumor-bearing mice

[0959]

[0960] Experimental conclusion:

[0961] AUC of BT001021 (10 mg / kg) in tumor and serum last The AUC of the T-030 group in tumor and serum were 850.1h*ng / ml and 174.97h*ng / ml, respectively. last The three levels were 3.85h*ng / ml and 5.58h*ng / ml, respectively. A comparison of the three showed that the exposure of T-030 in the BT001021-treated group was significantly higher than that in the T-030-treated group. In addition, the exposure of the active biomolecule T-030 in the tumor of the BT001021-treated group was significantly higher than that in the serum. The exposure of the active biomolecule in the serum and tumor of the T-030-treated group was basically the same, indicating that the antibody drug conjugate (BT001021) has strong tumor tissue targeting.

[0962] The C level 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 T-030 administration group were 7.82ng / ml and 11.7ng / ml respectively. max The concentrations of active biomolecule (T-030) in tumor tissue and serum were 1.20 ng / ml and 1.81 ng / ml, respectively.

[0963] The T-030 expression of active biomolecule in tumors in the BT001021 (10 mg / kg) group was significantly decreased. 1 / 2 The T of the active biomolecule 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) was 2.55 h, indicating that the antibody drug conjugate (BT001021) has a longer half-life in tumor tissue.

[0964] In summary, BT001021 has significant tumor tissue targeting and good pharmacokinetic properties compared with the corresponding active biological molecule (T-030).

[0965] Experimental Example 7. In vivo pharmacokinetic testing of antibody drug conjugates BT001021 and Immu-132.

[0966] In this experiment, human gastric cancer cell line NCI-N87 was subcutaneously transplanted into Balb / c-nu mice to establish a tumor-bearing mouse model. 3 The mice were randomly divided into groups and given a single intravenous dose of BT001021 and Immu-132. The concentrations of the active biomolecules T-030 and SN-38 corresponding to BT001021 and Immu-132, respectively, were measured in tumor tissue and serum, and the pharmacokinetic behavior of the antibody-drug conjugates BT001021 and Immu-132 in tumor-bearing mice was evaluated in vivo.

[0967] Test drug

[0968] Drug name, preparation method:

[0969] BT001021, liquid concentration is 20 mg / ml, stored in aliquots at -20°C, and diluted with normal saline according to the dose to obtain the test solution when used;

[0970] Immu-132 was diluted with normal saline according to the dose to obtain the test solution.

[0971] Experimental animals and cell lines:

[0972] Balb / c-nu mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011); gastric cancer cell line NCI-N87 (ATCC).

[0973] Experimental grouping and evaluation methods:

[0974] The tumor volume was between 100 and 200 mm. 3 Tumor-bearing mice were divided into 4 groups, and the drug was administered via a single tail vein injection.

[0975] Experimental methods:

[0976] NCI-N87 cells were cultured in 1640 medium containing 10% heat-inactivated fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into Balb / c-nu mice to establish a gastric cancer xenograft model. The tumors were grown until the average volume reached approximately 100-200 mm. 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.

[0977] Table 10. Pharmacokinetic parameters of T-030 and SN-38 in tumors and serum of tumor-bearing mice after intravenous administration of BT001021 and Immu-132

[0978]

[0979] Experimental conclusion:

[0980] AUC of toxin small molecules in tumor and serum in the BT001021-administered group last The AUC of the toxin small molecule in the tumor and serum of the Immu-132 administration group were 427.2h*ng / ml and 115.3h*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 6.8ng / ml in the Immu-132 administration group. max The above results indicate that compared with Immu-132, BT001021 has better tumor tissue targeting and better pharmacokinetic properties, and has a better therapeutic window.

[0981] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein: The compound of formula (I) is shown below: T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]-G Formula (I) Wherein, T is a molecular fragment with anti-tumor biological activity; L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, 、 、 、 、 、 or , and the 1 position of L1 is connected to T; L2 is selected from 、 、 、 、 、 、 and , and the 1 position of L2 is connected to L1; L3 is triazole; L4 is selected from , and the 2 position of L4 is connected to E; E is a pyrimidinyl group; G is a methylsulfonyl group; m1 is 0, 1, or 2; m2 is 0 or 1; m3 is 1.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein L1 is selected from Cit-Val, or .

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein L2 is selected from or .

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein T is selected from 、 、 or .

5. A pharmaceutical composition comprising 0.01 mg to 800 mg of a conjugate and one or more pharmaceutical excipients, wherein the conjugate is: in, A1 is the sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 8.

6. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating a disease associated with abnormal cell activity, wherein the disease associated with abnormal cell activity is a cancer disease, and the cancer disease is selected from lung cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, cervical cancer and endometrial cancer.

7. The use according to claim 6, wherein the cancer disease is selected from small cell lung cancer or non-small cell lung cancer.

8. A method for preparing a conjugate, wherein: The conjugate contains a bioactive molecular fragment, a linker and a targeting portion, and the preparation method includes the steps of preparing the linker using the following compound or a pharmaceutically acceptable salt thereof; The compound or a pharmaceutically acceptable salt thereof is selected from: (1) 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid; (2) 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid; or (3) Methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate.

9. The preparation method according to claim 8, wherein The preparation method further comprises the step of reacting the linker with a bioactive molecule fragment or a targeting moiety.

10. The preparation method according to claim 8 or 9, wherein The structure of the conjugate is shown in formula (Ia): <h2 style=";text-align:left;direction:ltr">{T-[L1-(L2)m1-(L3)m2-(L4)m3-E]}<h2 style=";text-align:left;direction:ltr"> γ <h2 style=";text-align:left;direction:ltr"> -A Formula (Ia) wherein T, L1, L2, L3, L4, E, m1, m2 and m3 are as defined in any one of claims 1 to 4, A is a targeting moiety, and γ is selected from an integer or decimal between 5 and 8.

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