Bioactive matter conjugate as well as preparation method and application thereof

Abstract

The invention relates to a bioactive substance conjugate and a preparation method and application thereof, in particular to a novel bioactive molecule conjugate obtained by improving the coupling mode of a drug in ADC or SMDC and a targeting part, a preparation method of the novel bioactive molecule conjugate and application of the novel bioactive molecule conjugate to preparation of drugs for treating cell activity abnormity related diseases.

Description

[0001] This application is a divisional application of the Chinese national application No. 201880069543.5, the date of entering the Chinese national phase is April 24, 2020, and the invention title is "Bioactive Conjugates and Their Preparation Methods and Uses". Technical Field

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

[0003] Chemotherapy was once the standard treatment for cancer, but highly cytotoxic bioactive molecules can kill normal cells by mistake, causing serious side effects. Targeted anti-tumor drugs have become a hot topic in the field of tumor research today due to their simultaneous targeting and anti-tumor activity. Since the 20th century, significant breakthroughs have been made in the development of anti-tumor drugs and tumor targeted therapy using biologic macromolecules (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, although biologic macromolecules have strong targeting properties, their therapeutic effects on solid tumors are limited; while bioactive molecules have a high killing effect on cancer cells, they often lack targeting properties and often accidentally damage normal cells, thus causing serious toxic side effects.

[0004] In recent years, it has been found that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting effect of antibodies and the activity of bioactive molecules, becoming a kind of "biological missile". The antibody guides the ADC to bind to the target cell, and then it is internalized by the cell, releasing the drug to treat the disease. Due to the specificity and targeting of antibodies to tumor cell-related targets, their application value is not only reflected in treatment, but also becomes an ideal carrier for targeted drug delivery, reducing the side effects of drugs. The design principle of small molecule drug conjugates (SMDCs) is the same as that of antibody-drug conjugates (ADCs), that is, bioactive molecules are conjugated to small molecule ligands that can selectively bind to receptors on the surface of tumor cells by chemical methods, thereby improving the targeting of effector molecules to tumor cells. The difference between SMDCs and ADCs is that SMDCs use small molecule ligands instead of antibodies.

[0005] Currently, no SMDC has been marketed.

[0006] Currently, there are four marketed ADCs: Mylotarg (Gemtuzumab Ozogamicin), Adcetris (Brentuximab Vedotin), Kadcyla (Trastuzumab Emtansine), and Besponsa (Inotuzumab ozogamicin). Generally, an ADC drug consists of an antibody, a bioactive molecule, and a linker. The bioactive molecule is covalently conjugated to the antibody through the linker; the antibody (such as a monoclonal antibody) can specifically recognize a specific target on the surface of tumor cells, and then can guide the ADC to the surface of cancer cells and enable the ADC to enter cancer cells through the endocytosis effect; then the bioactive molecule is released inside the cancer cells, achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

[0007] Lysine is the most common conjugation site in antibodies, and its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond. Currently, there are technologies that can achieve site-specific conjugation, that is, the carboxyl group of the linker is activated with an activating group, and then an amide bond is formed with the specific lysine ε-amino group in the antibody to complete the conjugation. However, such amide bonds are prone to hydrolysis under the action of enzymes in the body, resulting in the detachment of the bioactive molecule and the antibody before reaching the target cells, losing the targeting of the ADC and increasing toxicity at the same time.

[0008] Generally, the sulfhydryl groups of antibody cysteines exist in the form of disulfide bonds. Opening the disulfide bonds in the antibody can provide multiple free sulfhydryl groups as conjugation sites. For conjugation with antibody sulfhydryl groups, one method is that the free sulfhydryl groups on the antibody undergo a Michael addition reaction with maleimide, or a specific substrate undergoes two Michael addition reactions with the free sulfhydryl groups on the antibody to form a uniquely structured sulfur bridge bond. However, many literatures report 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 phenylenedioxazole structure substituted with a mesyl group and a linker structure are disclosed with an amanitin as the bioactive molecule, but there is no specific description of the conjugation with the antibody. Summary of the Invention

[0009] The present invention has discovered a novel class of bioactive conjugates, which are obtained by improving the conjugation method of 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 is 5-8). This disclosure is completed based on the above discoveries. Through in-depth research, we surprisingly found that for the ADCs of the present invention, such as BT001021 (Example 32), after intravenous administration, the exposure amount of the bioactive small molecule toxin in tumor tissues is significantly higher than that in plasma tissues. While for Immu-132 ADC under the same administration route, the plasma exposure amount is significantly higher than that in tumor tissues. Therefore, the ADCs of the present invention have a better therapeutic window than Immu-132. At the same time, we also surprisingly found that the ADCs of the present invention have better therapeutic effects than Immu-132 in tumor animal models of gastric cancer, breast cancer, and non-small cell lung cancer.

[0010] The first aspect of this disclosure provides a compound of 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 bioactive molecular fragment, preferably a molecular fragment with anti-tumor bioactivity;

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

[0015]

[0016]

[0017] Wherein, each R, R’, R1 and R2 are each independently H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, cyano, C 1-6 alkyl, halo C 1-6 alkyl (such as -CF3), cyano-substituted C 1-6 alkyl (such as -CH2CN), C 1-6 alkoxy, C 2-10 alkenyl, C 2-10 alkynyl, C 3-6a cycloalkyl group, a 6- to 10-membered aryl group, or a 5- to 12-membered heteroaryl group, each Z1 is independently an amino acid or a peptide composed of 2 to 10 amino acids, each t1 and t2 is independently 0, 1, 2, 3, 4, 5, or 6, each x1 and x2 is independently 0, 1, 2, 3, 4, 5, or 6, each x3 is independently 0, 1, 2, 3, or 4, and the 1-position of L1 is connected to T;

[0018] L2 is selected from an amino acid, a peptide composed of 2 to 10 amino acids, an oligosaccharide, -(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, halo 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- to 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- to 8-membered cycloalkyl, 3- to 8-membered heteroalicyclic, 6- to 12-membered bridged heteroalicyclic, 6- to 12-membered spiroheterocyclic, 6- to 12-membered fused heteroalicyclic, 6- to 10-membered aryl (such as phenyl or naphthyl), 5- to 12-membered heteroaryl, and 3- to 8-membered cycloalkyl-W-; wherein W is oxygen or NR8, R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CN, carboxyl, sulfonic acid group, C 1-6 alkyl, halo 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 wherein, Z5 is preferably selected from C 2-6 alkene, C 2-6 alkyne, amide group, sulfone group, sulfoxide group, 6-10 membered aryl group, 5-6 membered heteroaryl group; Z2 is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 3-8 cycloalkylene, 6-10 membered aryl group and 5-14 membered heteroaryl group; R9 is selected from H (hydrogen), C 1-6 alkyl; Z3 is absent or selected from C 1-6 alkylene, halo 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 following groups optionally substituted by one or more R 12 substituted: 6-10 membered aryl group, 5-14 membered heteroaryl group; wherein, R 12 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 alkyl and halo C 1-6 alkyl;

[0024] G is a leaving group for nucleophilic substitution reaction; such as halogen, sulfonyl group, sulfonate group, nitro, 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, peptides composed of 2-5 amino acids,

[0027] wherein, each 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 3-6The cycloalkyl group, 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 group, 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, and 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 group, 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 composed 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 L2 is connected to L1 at the 1-position;

[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 L2 is connected to L1 at the 1-position;

[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 L2 is connected to L1 at the 1-position;

[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 which are optionally substituted by one or more R7: amino, 3-8 membered cycloalkyl, 3-8 membered heteroalkyl, 6-12 membered bridged heteroaryl, 6-12 membered spiroheteroaryl, 6-12 membered fused heteroaryl, 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 group, C 1-4 alkyl, C 1-4 alkoxy, C 2-6 alkenyl and C 2-6 alkynyl; preferably, the 3-8 membered heteroalkyl, 6-12 membered bridged heteroaryl, 6-12 membered spiroheteroaryl or 6-12 membered fused heteroaryl contains one or more nitrogen atoms; preferably, the 3-8 membered heteroalkyl, 6-12 membered bridged heteroaryl, 6-12 membered spiroheteroaryl or 6-12 membered fused heteroaryl contains one or more quaternized nitrogen atoms; preferably, the 3-8 membered heteroalkyl, 6-12 membered bridged heteroaryl, 6-12 membered spiroheteroaryl or 6-12 membered fused heteroaryl 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 which are optionally substituted by one or more R7: amino, 3-6 membered heteroalkyl and 5-10 membered heteroaryl; wherein, R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C 1-4 alkyl, C 1-4 alkoxy, C 2-6 alkenyl and C 2-6Alkynyl; preferably, the 3- to 6-membered heteroalicyclic group contains one or more nitrogen atoms; preferably, the 3- to 6-membered heteroalicyclic group contains one or more quaternized nitrogen atoms; preferably, the 3- to 6-membered heteroalicyclic 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- to 6-membered heteroaryl; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C 1-4 alkyl, C 1-4 alkoxy, C 2-6 alkenyl 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 group, 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, L3 is connected to L2 at the 1-position.

[0049] In some preferred embodiments, L3 is selected from the following groups optionally substituted by 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, and 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 is 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- to 10-membered aryl group, a 5- to 6-membered heteroaryl group; R 10 is selected from H (hydrogen), C 1-6 alkyl; Z2 is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, 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- to 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 wherein, Z4 is a benzene ring, R 10 is selected from H (hydrogen), C 1-6 alkyl; Z2 is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, 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- to 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- or 6-membered heteroaryl; R 10 is selected from H (hydrogen), C 1-6 alkyl; Z2 is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, 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- to 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 5- to 10-membered heteroaryls optionally substituted with one or more R 12 ; wherein, R 12 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 alkyl and halo C 1-4 alkyl.

[0070] In some preferred embodiments, E is selected from the following groups optionally substituted with one or more R 12 : pyrimidine, quinazoline and pyrrolo[2,3-d]pyrimidine; wherein, R 12 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 alkyl and halo C 1-2 alkyl.

[0071] In some preferred embodiments, E is selected from pyrimidines optionally substituted with one or more R 12 ; wherein R 12 is independently selected from H (hydrogen) and D (deuterium).

[0072] In some preferred embodiments, G is selected from halogen, OMs, OTs, OTf, nitro, and the following groups optionally substituted with one or more R 13 : alkylthio, arylthio, heteroarylthio, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl; wherein R 13 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 alkyl, halo-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, mesyl, ethylsulfonyl, tosyl, and naphthalenesulfonyl.

[0074] In some preferred embodiments, G is selected from F, Cl, Br, OMs, OTs, mesyl, and tosyl.

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

[0076] In some preferred embodiments, in, G is preferably mesyl, E is preferably pyrimidine, and m3 is 1.

[0077] In some preferred embodiments, is wherein, m4 is preferably an integer from 0 to 6, and mesyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0078] In some preferred embodiments, is wherein, m5 is preferably an integer from 0 to 6, and mesyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0079] In some preferred embodiments, is wherein, m6 is preferably an integer selected from 0 to 6, and mesyl is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0080] In some preferred embodiments, is wherein m7 is an integer selected from 1 - 5, and the mesyl group is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0081] In some preferred embodiments, is wherein m8 is an integer selected from 1 - 5, and the mesyl group is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0082] In some preferred embodiments, is wherein m9 is an integer selected from 1 - 5, and R 13 is selected from hydrogen, C 1-6 alkyl, and the mesyl group is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0083] In some preferred embodiments, is wherein m 10 is selected from integers 0 - 6, and Z4 is selected from 5 - 6 membered heteroaryl groups; the mesyl group is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0084] In some preferred embodiments, is Z4 is selected from pyridine, pyrimidine, pyrazole, thiazole, oxazole and triazole, and the mesyl group is a substituent on the carbon atom adjacent to the nitrogen atom in the pyrimidine ring. More preferably, m 10 is selected from integers 0 - 6.

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

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

[0087] In some preferred embodiments, is wherein m 10An integer selected from 0 - 6, Z4 is selected from 6 - 10 aryl groups; a mesyl group is a substituent on the carbon atom adjacent to the nitrogen atom. More preferably, m 10 is an integer selected from 0 - 6.

[0088] In some preferred embodiments, is wherein m 10 is an integer selected from 0 - 6 and Z4 is a benzene ring.

[0089] In some preferred embodiments, is

[0090] In some preferred embodiments, in formula (I) is 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 (e.g., oxaliplatin), metal gold complexes; glycopeptide antibiotics, such as bleomycin or pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan or rubitecan), topoisomerase II inhibitors (e.g., actinomycin D, doxorubicin, docarubicin, 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 tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartic kinase inhibitors or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin 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] wherein R 14 is selected from R 15 substituted acyl or sulfonyl, R 15 is selected from C 1-6 alkyl, halo C 1-6 alkyl, 6-10 membered aryl and 5-12 membered heteroaryl; R 16 is selected from H (hydrogen), D (deuterium), C 1-6 alkyl, R 17 substituted C 1-6 alkyl, R 17 is selected from aryl, heteroaryl, including but not limited to phenyl, pyridyl, m 11 is selected from 0, 1, 2.

[0099] In some preferred embodiments, the bioactive molecule is selected from wherein R 14 is selected from R 15 substituted acyl or sulfonyl, and R 15 is selected from C 1-6 alkyl, halo C 1-6 alkyl, 6-10 membered aryl and 5-12 membered heteroaryl; and R 16 is selected from H (hydrogen), D (deuterium), C 1-6 alkyl, R 17 substituted C 1-6 alkyl, and R 17 is selected from aryl, heteroaryl, including but not limited to phenyl, pyridyl, and m 11 is selected 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. The targeting moiety is linked to the linker through its reactive group (such as a thiol group) to form a conjugate.

[0137] In some preferred embodiments, the conjugate has a structure as 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 (such as a small molecule ligand, a protein, a polypeptide, a non-protein reagent (such as a sugar, RNA or DNA)); γ is an integer or a decimal between 1 and 10; preferably, γ is an integer or a decimal between 5 and 8 (such as 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 targets of A are 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, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2, BMPR1B, Tyro7, c-Met, ApoE, CD11c, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD11b, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and BCMA.

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

[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 the 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 a monoclonal antibody against Her 2, such as trastuzumab, pertuzumab, or a monoclonal antibody against Trop-2, such as sacituzumab.

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

[0149]

[0150] The distribution of amino acids in each region or domain can follow the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0151] 1. Hydrophobic modification of the heavy and light chain sequences of antibody M1

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

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

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

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

[0156] 2. Hydrophobic modification of the heavy and light chain sequences of antibody M2

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

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

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

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

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

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

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

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

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

[0166] Constant region sequences of the light chains of M1, M2, and M3: (107aa)

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

[0168] Constant region sequences of the heavy chains of M1, M2, and M3: (330aa)

[0169] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No.:10)

[0170] The C-terminal Lys of the heavy chain is prone to deletion, but such deletion does not affect biological activity. See Dick, L.W. et al., Biotechnol. Bioeng., 100:1132 - 1143. The above-mentioned M1, M2, M3 monoclonal antibodies and the sequences or fragments thereof with the C-terminal Lys of the heavy chain deleted all belong to the M1, M2, M3 monoclonal antibodies described in 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 activators, bombesin, bradykinin, somatostatin or prostate-specific membrane antigen receptors.

[0172] In some preferred embodiments, A is selected from 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] Among them, γ is selected from an integer or a decimal between 1 and 10, and mAb is a monoclonal antibody against Trop-2 or a monoclonal antibody against Her 2; preferably, the monoclonal antibody against Trop-2 is selected from Sacituzumab, M1, M2 and M3 antibodies, and the monoclonal antibody against Her 2 is selected from trastuzumab and pertuzumab; preferably, γ is selected from an integer or a decimal between 5 and 8 (such as 5, 6, 7 or 8).

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

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Among them, γ is selected from an integer or a decimal between 1 and 10, and mAb is a monoclonal antibody against Trop-2 or a monoclonal antibody against Her 2; preferably, the monoclonal antibody against Trop-2 is selected from Sacituzumab, and the monoclonal antibody against Her 2 is selected from trastuzumab and pertuzumab; preferably, γ is selected from an integer or a decimal between 5 and 8 (such as 5, 6, 7 or 8).

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

[0194]

[0195]

[0196]

[0197]

[0198] Among them, A1 is the Sacituzumab antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A1 is the Sacituzumab antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A1 is the Sacituzumab antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A1 is a fragment of the Sacituzumab antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A2 is trastuzumab, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A2 is trastuzumab, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A2 is trastuzumab, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A3 is pertuzumab, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals between 6 - 7, 6 - 7.5, 6 - 8, 6.5 - 7, 6.5 - 7.5, 6.5 - 8, 7 - 8 or 7.5 - 8.

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

[0229]

[0230]

[0231] Among them, A3 is pertuzumab, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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] Among them, A4 is the M1 antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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.

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

[0239]

[0240] Among them, A4 is the M1 antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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.

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

[0242]

[0243]

[0244]

[0245]

[0246] Among them, A5 is the M2 antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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.

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

[0248]

[0249] Among them, A5 is the M2 antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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.

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

[0251]

[0252]

[0253]

[0254] Among them, A6 is the M3 antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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.

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

[0256]

[0257] Among them, A6 is the M3 antibody, and γ is selected from integers or decimals between 1 and 10; preferably, γ is selected from integers or decimals between 5 and 8, such as integers or decimals 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.

[0258] In another aspect, the present disclosure provides a method for preparing the conjugate described in the second aspect, which includes the step of coupling the linker of the compound of formula (I) with the active group of the targeting moiety.

[0259] In some preferred embodiments, the method includes using a reducing agent (such as TCEP) to open the disulfide bond of the targeting moiety to obtain a thiol group.

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

[0261] In some preferred embodiments, the targeting moiety is a monoclonal antibody against Her 2 (such as trastuzumab, Pertuzumab), or a monoclonal antibody against Trop-2 (such as Sacituzumab, M1, M2 or M3), or an active fragment or variant thereof.

[0262] In some preferred embodiments, the molar ratio of the targeting moiety to the compound of formula (I) is 1:(1 - 20); preferably, the conjugation 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 conjugate product; preferably, the conjugate product is purified by a chromatography method (such as 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 the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof, or the conjugate described in the second aspect in the preparation of a medicament for treating a disease associated with abnormal cell activity (such as a cancer disease).

[0266] In some preferred embodiments, the cancer disease is a solid tumor or a non-solid tumor, such as selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as 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 lymphoma, central nervous system tumors (such as glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

[0267] In another aspect, the present disclosure provides the use of the compound described in the first aspect or a pharmaceutically acceptable salt thereof, or the conjugate described in the second aspect, or the pharmaceutical composition in the treatment of a disease associated with abnormal cell activity (such as a cancer disease).

[0268] In another aspect, the present disclosure provides a method for treating a disease associated with abnormal cell activity (such as a cancer disease), comprising administering to an individual in need thereof an effective dose of the compound described in the first aspect of the present disclosure, or a pharmaceutically acceptable salt thereof, or the conjugate or pharmaceutical composition described in the second aspect.

[0269] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in the respective fields. Meanwhile, to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0270] In the present disclosure, the pharmaceutical excipients refer to excipients and additives used in the production of drugs and the formulation of prescriptions. They are substances that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations in addition to the active ingredients. In addition to shaping, acting as a carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization assistance, sustained and controlled release, etc. They are important components that may affect the quality, safety, and effectiveness of drugs. According to their sources, they can be divided into natural products, semi-synthetic products, and fully synthetic products. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, solubilization aids, emulsifiers, colorants, binders, 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, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.; according to their routes of administration, they can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ophthalmic administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0271] The pharmaceutical composition can be made into various suitable dosage forms according to the route of administration. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder aerosols, sprays, etc. Among them, the pharmaceutical composition or the suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure, or a pharmaceutically acceptable salt or conjugate thereof, preferably contains 0.1 mg to 800 mg, more preferably contains 0.5 - 500 mg, more preferably contains 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 injection solutions, sterile powders for injection, and concentrated solutions for injection. Among them, the carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.

[0273] In the present disclosure, the term "individual" includes human or non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the diseases described herein) (referred to as patients) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates, such as non-mammals (such as birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and / or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).

[0274] In the present disclosure, the term "effective dose" refers to the amount of a compound that, when administered, will relieve one or more symptoms of the disorder being treated to a certain extent.

[0275] In the present disclosure, the term "conjugate" refers to a substance obtained by linking a bioactive molecule to a targeting moiety. In some embodiments of the present disclosure, the bioactive molecule is linked to the targeting moiety through a linker. The linker can be cleaved in a specific environment (such as an intracellular low pH environment) or under a specific action (such as the action of lysosomal proteases), so that the bioactive molecule is separated from the targeting moiety. In some embodiments of the present disclosure, the linker comprises cleavable or non-cleavable units, such as peptides or disulfide bonds. In some embodiments of the present disclosure, the bioactive molecule is directly linked to the targeting moiety through a covalent bond, and the covalent bond can be cleaved in a specific environment or under a specific action, so that the bioactive molecule is separated from the targeting moiety.

[0276] In the present disclosure, the terms "bioactive substance" and "bioactive molecule" refer to substances that inhibit or prevent the functions of cells 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 bioactivity. 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 212Radioactive isotopes of He and Lu; metal complexes, such as metal platinum complexes, metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin, docamicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs interfering with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, etc.; drugs acting on structural proteins, such as tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, etc.; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartic kinase inhibitors or histidine kinase inhibitors, etc.; also including proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cyclin inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, Pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances inhibiting tumor cell growth, promoting tumor cell apoptosis and necrosis; enzymes and their fragments, such as nuclease; antibiotics; toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including their fragments and / or variants; growth inhibitors; drug modules. The term "toxin" refers to a substance that can have a harmful effect on the growth or proliferation of cells.

[0277] In the present disclosure, the term "small molecule" refers to a small molecule drug with biological activity.

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

[0279] In the present disclosure, the term "targeting moiety" refers to the part in the conjugate that can specifically bind to a target (or a part of the target) on the cell surface. Through the interaction between the targeting moiety and 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 can be referred to as a "drug-antibody conjugate". In the present disclosure, "drug-antibody conjugate" and "immunoconjugate" can be used interchangeably.

[0281] In the present disclosure, the term "antibody" is taken in its broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed by at least two intact antibodies, provided that they have the desired biological activity. In the present disclosure, "antibody" and "immunoglobulin" are used interchangeably.

[0282] In the present disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., each antibody constituting the population is identical, except for possible minor natural variations. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, contain different antibodies directed against different determinants (epitopes). In addition to specificity, the advantage of monoclonal antibodies is that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring a particular method of production.

[0283] In some embodiments of the present disclosure, monoclonal antibodies specifically include chimeric antibodies, i.e., a portion of the heavy and / or light chain is identical or homologous to an antibody of a certain species, class, or subclass, and the remaining portion is identical or homologous to another antibody of a different species, class, or subclass, provided that they have the desired biological activity (see, e.g., US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies useful in the present disclosure include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., Old World 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" is also referred to as "bifunctional antibody conjugate" and refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a linker arm, which retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0286] The term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies having three different antigen-binding specificities and the latter being antibodies having four different antigen-binding specificities.

[0287] The term "intact antibody" refers to an antibody that comprises an antigen-binding variable region and light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be of natural sequence (e.g., human natural constant region sequences) or amino acid sequence variants thereof. The intact antibody is preferably an intact antibody having one or more effector functions.

[0288] The term "Probody" is a modified antibody that includes an antibody or an antibody fragment that specifically binds to its target and is capable of being coupled to a masking group, where the masking group refers to a cleavage constant of the binding ability of the antibody or antibody fragment to its target that is at least 100-fold or 1000-fold, or 10000-fold greater than the cleavage constant of the binding ability of the antibody or antibody fragment without the coupled masking group to its target.

[0289] In the present disclosure, a "humanized" form of a non-human (e.g., murine) 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 the human recipient immunoglobulin are replaced with hypervariable region residues of a non-human (e.g., mouse, rat, rabbit, or non-human primate) (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, a humanized antibody can also contain residues not present in the recipient antibody or donor antibody. These modifications are made to further optimize the performance of the antibody. A humanized antibody generally contains at least one, and usually two, variable regions in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, while the FRs are entirely or substantially entirely of human immunoglobulin sequence. A humanized antibody can also contain at least a portion of the immunoglobulin constant region (Fc, usually a human immunoglobulin Fc). For details, see, e.g., Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Bwl 2:593-596.

[0290] Intact antibodies can be classified into different "classes" based on the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes can be 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 designated 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] In the present disclosure, although in most cases the amino acid substitutions in the antibody are substitutions with L - amino acids, this is not limited thereto. In some embodiments, one or more D - amino acids may be included in the antibody peptide chain. Peptides containing D - amino acids are more stable and less prone to degradation in the oral cavity, intestine, 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 the hybridoma method using cells from many species (including mouse, hamster, rat, and human cells) (see, e.g., Kohler et al., 1975, Nature, 256:495), or prepared by recombinant DNA technology (see, e.g., US 4,816,567), or isolated from a phage antibody library (see, e.g., Clackson et al., 1991, Nature, 352:624 - 628; and Marks et al., 1991, Journal of Molecular Biology, 222:581 - 597). Monoclonal antibodies useful in the present disclosure include, but are not limited to: monoclonal antibodies against Her 2, such as trastuzumab, pertuzumab, or monoclonal antibodies against Trop - 2, such as sacituzumab (i.e., isactuzumab or hRS7 antibody), M1, M2, or M3.

[0293] In some embodiments of the present disclosure, the targets of the targeting moiety A are 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 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, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD11c, CD40, CD45 (PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPIN1, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD11b, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, and BCMA.

[0294] In some embodiments of the present disclosure, the target of the targeting moiety 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 activators, 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: 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 a monoclonal antibody against Her2, such as trastuzumab, pertuzumab; or the targeting moiety is a monoclonal antibody against Trop-2, such as sacituzumab, M1, M2, or M3.

[0297] In some embodiments of the present disclosure, the targeting moiety is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her2, and its amino acid sequence is known to those skilled in the art. For its schematic sequence, reference can be made to, for example, CN103319599.

[0298] In some embodiments of the present disclosure, the C-terminal Lys of the heavy chain of the targeting moiety is prone to deletion without affecting biological activity. See Dick, L.W. et al., Biotechnol. Bioeng., 100: 1132-1143. For example, the targeting moiety is a monoclonal antibody against Trop-2, such as Sacituzumab, M1, M2 or M3 with deletion of the C-terminal Lys of the heavy chain. For example, the targeting moiety is a monoclonal antibody against Her2, such as trastuzumab or pertuzumab with deletion of the C-terminal Lys of the heavy chain.

[0299] Exemplary heavy chain sequences and light chain sequences of trastuzumab can be found, for example, in SEQ ID No.: 17 and SEQ ID No.: 18. In the present disclosure, when referring to or involving the heavy chain sequence and light chain sequence of trastuzumab, the sequences shown in SEQ ID No.: 17 and SEQ ID No.: 18 are used for description respectively. Exemplary heavy chain sequences and light chain sequences of pertuzumab can be found in SEQ ID No.: 16 and SEQ ID No.: 15 of US7560111.

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

[0301] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)

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

[0303] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0304] In some embodiments of the present disclosure, the targeting moiety anti-Trop-2 antibody is RS7 (i.e., Sacituzumab of the present disclosure) described in U.S. Patent No. 7,517,964; and hRS7 (i.e., Sacituzumab of the present disclosure) described in US2012 / 0237518. The anti-Trop-2 antibodies that can be used in the present disclosure can also be obtained by screening through the methods of vector design, construction, and construction of antibody libraries displaying antibodies disclosed in CN103476941A, or can also be screened through the G- library of Sorrento Therapeutics, Inc.

[0305] The heavy chain sequence and light chain amino acid sequence of Sacituzumab 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] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)

[0308] The C-terminal K (or lys) of the heavy chain is prone to deletion, but this deletion does not affect biological activity. See Dick, L.W. et al., Biotechnol. Bioeng., 100: 1132-1143.

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

[0310] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[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 refers to the gene encoding human Her2, and neu refers to the gene encoding rat p185neu. In some embodiments, the compounds or conjugates of the present disclosure are capable of inhibiting or killing cells expressing the ErbB2 receptor, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, 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, also known as TACSTD2, M1S1, GA733-1, EGP-1, which is a cell surface receptor expressed by many human tumors such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer. In some embodiments, the compounds or conjugates of the present disclosure are capable of inhibiting or killing 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] As used herein, when appears in the conjugates of the present invention,

[0314] As used herein, the term "C 1-6 alkyl" refers to a straight-chain or branched-chain alkyl group containing 1-6 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] As used herein, the term "C 2-6 alkenyl" refers to a straight-chain, branched-chain, or cyclic alkenyl having at least one double bond and 2 to 6 carbon atoms, including, for example, "C 2-4 alkenyl", etc. Examples thereof include, but are not limited to: vinyl, 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, etc.

[0316] As used herein, the term "C 2-6 alkynyl" refers to a straight-chain or branched-chain alkynyl having at least one triple bond and 2 to 6 carbon atoms, including, for example, "C 2-4 alkynyl", etc. Examples thereof 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, etc.

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

[0318] As used herein, the term "3- to 8-membered cycloalkyl" or "C 3-8 cycloalkyl" refers to a saturated cyclic alkyl having 3 to 8 carbon atoms, including, for example, "3- to 6-membered cycloalkyl", "4- to 6-membered cycloalkyl", "5- to 7-membered cycloalkyl", or "5- to 6-membered cycloalkyl", etc. Specific examples include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0319] As used herein, the term "C 1-6 alkoxy" refers to a group having a C 1-6 alkyl-O- structure, wherein 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, etc.

[0320] As used herein, the term "3- to 8-membered heteroalicyclic group" refers to a cyclic group containing 3 to 8 ring atoms, where 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 oxo-substituted. The "3- to 8-membered heteroalicyclic group" includes, for example, "3- to 8-membered nitrogen-containing heteroalicyclic group", "3- to 8-membered oxygen-containing heteroalicyclic group", "3- to 6-membered heteroalicyclic group", "3- to 6-membered oxygen-containing heteroalicyclic group", "4- to 7-membered heteroalicyclic group", "4- to 6-membered heteroalicyclic group", "5- to 7-membered heteroalicyclic group", "5- to 6-membered heteroalicyclic group", "5- to 6-membered nitrogen-containing heteroalicyclic group", including but not limited to oxiranyl, oxocyclobutyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.

[0321] As used herein, the term "6- to 12-membered spirocyclic group" refers to a cyclic structure formed by two or more cyclic structures sharing one carbon atom with each other and containing 6 to 12 ring carbon atoms. Optionally, the carbon atoms in the cyclic structure can be oxo-substituted. The "6- to 12-membered spirocyclic group" includes, for example, "6- to 11-membered spirocyclic group", "6- to 10-membered spirocyclic group", "7- to 10-membered spirocyclic group", "7- to 9-membered spirocyclic group", "7- to 8-membered spirocyclic group", "9- to 10-membered spirocyclic group", "3- to 10-membered spirocyclic group", etc. Specific examples include but are not limited to:

[0322] etc.

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

[0324] etc.

[0325] As used herein, the term "6- to 12-membered fused cyclic group" refers to a cyclic structure formed by two or more cyclic structures sharing two adjacent atoms with each other and containing 6 to 12 ring carbon atoms, including "6- to 11-membered fused cyclic group", "6- to 10-membered fused cyclic group", "6- to 8-membered fused cyclic group", "10- to 12-membered fused cyclic group", "7- to 10-membered fused cyclic group". Examples thereof include but are not limited to:

[0326] etc.

[0327] As used herein, the term "6-12 membered spiroheterocyclic group" refers to a cyclic structure formed by two or more cyclic structures sharing one ring atom with each other and 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 in the cyclic structure (such as carbon atoms, nitrogen atoms or sulfur atoms) can be oxo-substituted. "6-12 membered spiroheterocyclic group" includes, for example, "6-11 membered spiroheterocyclic group", "5-10 membered spiroheterocyclic group", "7-11 membered spiroheterocyclic group", "7-10 membered spiroheterocyclic group", "7-9 membered spiroheterocyclic group", "7-8 membered spiroheterocyclic group", "9-10 membered spiroheterocyclic group", "3-10 membered spiroheterocyclic group", etc. Specific examples include but are not limited to: etc.

[0329] As used herein, the term "6-12 membered bridged heterocyclic group" refers to a cyclic structure formed by two or more cyclic structures sharing two non-adjacent ring atoms with each other and 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 in the cyclic structure (such as carbon atoms, nitrogen atoms or sulfur atoms) can be oxo-substituted. "6-12 membered bridged heterocyclic group" includes, for example, "6-11 membered bridged heterocyclic group", "6-9 membered bridged heterocyclic group", "6-10 membered bridged heterocyclic group", "7-10 membered bridged heterocyclic group", "7-9 membered bridged heterocyclic group", "7-8 membered bridged heterocyclic group", "8 membered bridged heterocyclic group", "9-10 membered bridged heterocyclic group", "3-10 membered bridged heterocyclic group", etc. Specific examples include but are not limited to:

[0330] etc.

[0331] As used herein, the term "6- to 12-membered fused heterocyclic group" refers to a cyclic structure formed by two or more cyclic structures sharing two adjacent atoms with each other and containing 6 to 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 may be oxo-substituted. "6- to 12-membered fused heterocyclic group" includes, for example, "6- to 11-membered fused heterocyclic group", "5- to 10-membered fused heterocyclic group", "7- to 10-membered fused heterocyclic group", "3- to 10-membered fused heterocyclic group", "3- to 10-membered nitrogen-containing fused heterocyclic group", "9- to 10-membered fused heterocyclic group", "9- to 10-membered nitrogen-containing fused heterocyclic group", "6- to 12-membered oxygen-containing fused heterocyclic group", 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, dihydroindolyl, dihydroisoindolyl, benzoxazolidinyl, benzothiazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl, 4H-1,3-benzoxazinyl, etc.

[0332] As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity, such as 6- to 20-membered aryl, 6- to 10-membered aryl, 5- to 8-membered aryl, etc. Specific examples include but are not limited to phenyl, naphthyl, anthryl, phenanthryl, etc. The "6- to 20-membered aryl" refers to an aryl containing 6 to 20 ring atoms.

[0333] As used herein, the term "heteroaryl" refers to a cyclic group having 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 cyclic structure may be oxo-substituted. Specific examples include, but are not limited to, 5- to 10-membered heteroaryl, 5- to 10-membered nitrogen-containing heteroaryl, 6- to 10-membered oxygen-containing heteroaryl, 6- to 8-membered nitrogen-containing heteroaryl, 5- to 8-membered oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridone, 4-pyridone, pyrimidinyl, 1,4-dioxacyclohexadienyl, 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, azepinyl, 1,3-diazepinyl, azocinyl, etc.

[0334] Advantages of the Invention

[0335] The present disclosure obtains a novel class of bioactive molecule conjugates by improving the conjugation method of the drug and the targeting moiety in the ADC or SMDC. In some embodiments of the present disclosure, the bioactive molecule conjugate is obtained by a nucleophilic substitution reaction of the heteroaromatic ring on the ADC drug linker with the free thiol group in the antibody molecule. The conjugate obtained by using the above conjugation method can achieve at least one of the following technical effects:

[0336] (1) Having high stability;

[0337] (2) Having a high drug loading capacity. In some embodiments, the DAR value of the conjugate can reach 5-8;

[0338] (3) Having extremely high conjugation efficiency. In some embodiments, the conjugation efficiency can reach 90%;

[0339] (4) The conjugate obtained according to the above conjugation method can effectively improve the stability of the drug molecule in circulation and reduce the shedding of non-targeted drugs in non-target cells;

[0340] (5) The conjugate can also increase the effective release of the bioactive molecule intracellularly, achieving the purpose of enhancing efficacy and reducing toxicity;

[0341] (6) The conjugate has good tumor tissue targeting property; and

[0342] (7) The conjugate has a good therapeutic effect in tumor animal models.

[0343] In addition, the conjugation method described in the present disclosure has a wide range of applications and can be widely used for the conjugation of bioactive molecules with antibodies or targeting small molecule ligands. Description of the Drawings

[0344] Figure 1 It is the TIC (Total Ion Chromatogram) of BT001002.

[0345] Figure 2 It is the deconvolution map of the light chain conjugated with BT001002.

[0346] Figure 3 It is the deconvolution map of the heavy chain conjugated with BT001002.

[0347] Figure 4 It is the TIC (Total Ion Chromatogram) of BT001004.

[0348] Figure 5 It is the deconvolution map of the light chain conjugated with BT001004.

[0349] Figure 6 It is the deconvolution map of the heavy chain conjugated with BT001004.

[0350] Figure 7 It is the SEC chromatogram of BT001002.

[0351] Figure 8 It is the SEC chromatogram of the molecular weight Marker of BT001002.

[0352] Figure 9 It is the SEC chromatogram of BT001004.

[0353] Figure 10 It is the deconvolution map of the light chain conjugated with BT001012.

[0354] Figure 11 It is the deconvolution map of the heavy chain conjugated with BT001012.

[0355] Figure 12 It is the deconvolution map of the light chain conjugated with BT001013.

[0356] Figure 13 It is the deconvolution map of the heavy chain conjugated with BT001013.

[0357] Figure 14 It is the deconvolution map of the light chain conjugated with BT001018.

[0358] Figure 15 It is the deconvolution map of the heavy chain conjugated with BT001018.

[0359] Figure 16 Deconvolution graph of the conjugate light chain of BT001021.

[0360] Figure 17 Deconvolution graph of the conjugate heavy chain of BT001021.

[0361] Figure 18 Deconvolution graph of the conjugate light chain of BT001023.

[0362] Figure 19 Deconvolution graph of the conjugate heavy chain of BT001023.

[0363] Figure 20 Deconvolution graph of the conjugate light chain of BT001040.

[0364] Figure 21 Deconvolution graph of the conjugate heavy chain of BT001040.

[0365] Figure 22 Deconvolution graph of the conjugate light chain of BT001041.

[0366] Figure 23 Deconvolution graph of the conjugate heavy chain of BT001041.

[0367] Figure 24 Deconvolution graph of the conjugate light chain of BT001042.

[0368] Figure 25 Deconvolution graph of the conjugate heavy chain of BT001042.

[0369] Figure 26 Deconvolution graph of the conjugate light chain of BT001043.

[0370] Figure 27 Deconvolution graph of the conjugate heavy chain of BT001043.

[0371] Figure 28 Deconvolution graph of the conjugate light chain of BT001044.

[0372] Figure 29 Deconvolution graph of the conjugate heavy chain of BT001044.

[0373] Figure 30 Deconvolution graph of the conjugate light chain of BT001046.

[0374] Figure 31 Deconvolution graph of the conjugate heavy chain of BT001046.

[0375] Figure 32 Deconvolution graph of the conjugate light chain of BT001047.

[0376] Figure 33 Deconvolution graph of the conjugate heavy chain of BT001047.

[0377] Figure 34 SEC chromatogram of BT001012.

[0378] Figure 35 SEC chromatogram of BT001013.

[0379] Figure 36 SEC chromatogram of BT001018.

[0380] Figure 37 SEC chromatogram of BT001021.

[0381] Figure 38 SEC chromatogram of BT001023.

[0382] Figure 39 SEC chromatogram of BT001042.

[0383] Figure 40 SEC chromatogram of BT001043.

[0384] Figure 41 SEC chromatogram of BT001044.

[0385] Figure 42 SEC chromatogram of BT001046.

[0386] Figure 43 SEC chromatogram of BT001047.

[0387] Figure 44 Growth changes in tumor volume of mice in each group in the NCI-N87 human gastric cancer model.

[0388] Figure 45 Changes in body weight of mice in each group in the NCI-N87 human gastric cancer model.

[0389] Figure 46 Growth changes in tumor volume of mice in each group in the HCC1806 human breast cancer model.

[0390] Figure 47A Growth changes in tumor volume of mice in each group in the HCC827 human non-small cell lung cancer xenograft model.

[0391] Figure 47B Changes in body weight of mice in each group in the HCC827 human non-small cell lung cancer xenograft model.

[0392] Figure 48AGrowth changes of tumor volume in mice of each group in the NCI-N87 human gastric cancer xenograft model.

[0393] Figure 48B Changes in body weight of mice in each group in the NCI-N87 human gastric cancer xenograft model.

[0394] Figure 49A .Growth changes of tumor volume in mice of each group in the MDA-MB-231 human breast cancer-bearing mouse model.

[0395] Figure 49B .Changes in body weight of mice in each group in the MDA-MB-231 human breast cancer-bearing mouse model. Detailed implementation manners

[0396] The present disclosure is further described below through the description of detailed implementation manners, but this is not a limitation to the present disclosure. Those skilled in the art can make various modifications or improvements according to the teachings of the present disclosure without departing from the basic idea and scope of the present disclosure.

[0397] The abbreviations in the present invention have the following meanings:

[0398]

[0399] Preparation Scheme

[0400] The structures of the compounds recorded in the following examples are determined by nuclear magnetic resonance ( 1 1H NMR) or mass spectrometry (MS).

[0401] Nuclear magnetic resonance ( 1 1H NMR) measurements are performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer; the measurement solvents are deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6); the internal standard substance is tetramethylsilane (TMS).

[0402] The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

[0403] s: singlet, d: doublet, t: triplet, q: quartet, dd: doubledoublet, qd: quartet doublet, ddd: double doubledoublet, ddt: double double triplet, dddd: double doubledoubledoublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. The δ value is expressed in ppm values.

[0404] The measuring instrument for mass spectrometry (MS) used an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0405] The preparative liquid chromatography method is as follows:

[0406] Method A:

[0407] Chromatographic column: Daisogel C18 10μm 100x250mm

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

[0409]

[0410] Method B:

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

[0412] Mobile phase A: water; Mobile phase B: acetonitrile

[0413]

[0414] Method C:

[0415] Chromatographic column: Daisogel C18 10μm 50x250mm

[0416] Mobile phase A: water containing 0.1% trifluoroacetic acid; Mobile phase B: acetonitrile

[0417]

[0418] Method D: Chromatographic column: Waters SunFire C18 5μm 19x250mm

[0419] Mobile phase A: acetonitrile; Mobile phase B: water containing 0.05% formic acid

[0420] Time: 0 min - 16 min; Mobile phase A: 10% - 90%; Flow rate: 28 mL / min

[0421] I. Synthesis of bioactive molecules

[0422] 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)

[0423]

[0424] 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-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)carbamate

[0425] At room temperature, dissolve 1-hydroxybenzotriazole (2.0 mg, 14.74 μmol) in N,N-dimethylformamide (4 mL), cool to 0 °C, and successively add tert-butyl (4-methylaminobenzyl)carbamate (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-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine (10.0 mg, 13.5 μmol, commercially available), stir for 5 min, add 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (10.0 mg, 20.1 μmol), and stir the reaction at 0 °C for 1 h after addition. Monitor the reaction of the raw materials to completion by high performance liquid chromatography - mass spectrometry, and purify the reaction solution 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] + 。

[0426] 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

[0427] At room temperature, dissolve 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) in 1,4-dioxane (0.5 mL), cool the temperature to 0 °C, add hydrochloric acid-dioxane solution (1 mL, 4.0 M), stir the reaction at room temperature for 3 h after addition. Monitor the complete reaction of the raw materials by high performance liquid chromatography-mass spectrometry, distill off the solvent under reduced pressure, and purify the crude product by preparative liquid chromatography (Method C) to obtain the trifluoroacetate salt of the title compound, which is 5.0 mg of white solid. ESI-MS (m / z): 850.5 [M+H] + 。

[0428] 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-methylbutanamido)-N,3-dimethylbutanamide (T011)

[0429]

[0430] Step 1: Synthesis of (S)-(4-((2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropanamido)methyl)phenyl)carbamate

[0431] 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-phenylpropanoic 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 added successively. The reaction was carried out at 0 °C overnight. The reaction solution was poured into water (50 mL), and a white solid precipitated. The solid was filtered, and the filter cake was washed with water (20 mL × 3). The solid was purified by silica gel column chromatography to obtain the title compound as a white solid, 380 mg. ESI-MS (m / z): 592.3 [M+H] + 。

[0432] Step 2: Synthesis of tert-butyl (S)-(4-((2-amino-3-phenylpropanamido)methyl)phenyl)carbamate

[0433] 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-phenylpropanamido)methyl)phenyl)carbamate (102 mg, 0.17 mmol) in tetrahydrofuran (2 mL). The reaction was carried out 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, and the solvent was evaporated under reduced pressure. The residue was purified by preparative liquid chromatography (Method D) to obtain the title compound as a white solid, 65 mg. ESI-MS (m / z): 370.2 [M+H] + 。

[0434] Step 3: Synthesis of (4-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-(methylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)carbamate

[0435] At 0 °C, (S)-(4-((2-Amino-3-phenylpropanamido)methyl)phenyl)carbamic acid tert-butyl ester (15 mg, 0.04 mmol) and N-methylmorpholine (12 mg, 0.12 mmol) were added to a solution of (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-methylpropanoic acid (24 mg, 0.04 mmol) in N,N-dimethylformamide (2 mL). 1-Hydroxybenzotriazole (8 mg, 0.06 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12 mg, 0.06 mmol) were added successively. The reaction was carried out 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] + 。

[0436] 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-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide

[0437] 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-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)carbamate (14.0 mg, 0.015 mmol) in dichloromethane (1.5 mL). The reaction was carried out at room temperature for 1 h. The solvent was removed 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] + 。

[0438] The following molecules can be synthesized using a similar synthetic method:

[0439]

[0440] Synthesis of (S)-N-(2-(4-Ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylethanamide in Example 3

[0441]

[0442] At room temperature, belotecan hydrochloride (1.0 g, 2.13 mmol) and triethylamine (0.65 g, 0.9 mL) were dissolved in dichloromethane (50 mL), and acetic anhydride (0.22 g, 2.13 mmol) was slowly added dropwise. The reaction was carried out at room temperature for 1 h. The organic phase was washed with water (10 mL×2) and dried over anhydrous sodium sulfate. The insoluble matter was filtered off, the solvent was evaporated, and the 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] + 。

[0443] Synthesis of (S)-N-(2-(4-Ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide in Example 4

[0444]

[0445] Methanesulfonyl chloride (462 mg, 12.77 mmol, purity about 70%) was added dropwise to a solution of belotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in dichloromethane (40 mL). The reaction was carried out at room temperature for 2 h. The mixture was filtered by suction, and the filter cake was washed three times with dichloromethane (3 mL) to obtain 2.2 g of the title compound.

[0446] The structure characterization data are as follows:

[0447] 11H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 8.4 Hz, 1H), 8.20 (dd, J = 8.4, 1.2 Hz, 1H), 7.93 - 7.84 (m, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.35 (s, 1H), 6.56 (s, 1H), 5.44 (d, J = 9.2 Hz, 4H), 3.98 (p, J = 6.7 Hz, 1H), 3.50 (t, J = 8.0 Hz, 2H), 3.42 - 3.35 (m, 2H), 3.00 (s, 3H), 1.93 - 1.82 (m, 2H), 1.15 (d, J = 6.7 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m / z): 512.2 [M+H] + . [α] D 20 was +28.19° (c = 0.101 g / 100 mL, CH3CN).

[0448] The remaining bioactive molecules whose synthesis methods are not described are commercially available or prepared by methods disclosed in the prior art.

[0449] II. Synthesis of Compounds Containing Cell Bioactive Molecules and Linkers

[0450] 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-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidovaleramide

[0451]

[0452] Step 1: Synthesis of tert-butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyrate (Compound 1-2)

[0453] 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 added slowly in portions, and the mixture was stirred at room temperature for 10 min. 4-Bromobutanoic acid tert-butyl ester (725 mg, 3.27 mmol) was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 2 h, quenched with saturated aqueous ammonium chloride solution, and extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was removed under reduced pressure to obtain the title compound, 500 mg. ESI-MS (m / z): 296.1 [M+H] + 。

[0454] Step 2: Synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (Compound 1-3)

[0455] At room temperature, compound 1-2 (500 mg, 1.69 mmol) was dissolved in dichloromethane (6 mL). Trifluoroacetic acid (3 mL) was added. After the addition was complete, the reaction was carried out at room temperature for 4 h, and the solvent was removed under reduced pressure to obtain the title compound, 400 mg. ESI-MS (m / z): 240.1 [M+H] + 。

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

[0457] 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 carried out for 8.0 h while maintaining the temperature. After cooling to room temperature, a large amount of solid precipitated. The solid was filtered by suction to obtain the title compound, 3.65 g. ESI-MS (m / z): 701.4 [M+H] + 。

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

[0459] At room temperature, trifluoroacetic acid (15 mL) was added to compound 1-5 (3.0 g, 4.29 mmol), and the mixture was stirred at room temperature for 1.0 h. The solvent was removed under reduced pressure to obtain a yellow oil, and anhydrous ether (20 mL) was added. A large amount of solid precipitated out. The mixture was vigorously stirred for 0.5 h and then filtered by suction to obtain the trifluoroacetate salt of the title compound, 3.06 g. ESI-MS (m / z): 601.3 [M+H] + 。

[0460] Step 5: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-7)

[0461] At room temperature, Boc-D-phenylalanine (1.1 g, 4.2 mmol) and the trifluoroacetate salt of compound 1-6 (3.0 g, 4.2 mmol) were dissolved in N,N-dimethylformamide (40 mL). 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 successively. The reaction system was stirred at a constant temperature for 1.0 h. The reaction solution was added dropwise to ice water (400 mL), and the mixture was vigorously stirred for 0.5 h. A large amount of solid precipitated out, and the solid was filtered by suction to obtain the title compound, 3.3 g. ESI-MS (m / z): 848.4 [M+H] + 。

[0462] Step 6: Synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-amino-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (Compound 1-8)

[0463] At room temperature, compound 1-7 (3.0 g, 3.3 mmol) was dissolved in trifluoroacetic acid (30 mL), and the mixture was stirred at room temperature for 1.0 h. The solvent was removed under reduced pressure to obtain a yellow oil, and anhydrous ether (100 mL) was added. The mixture was vigorously stirred for 0.5 h. A large amount of solid precipitated out, and the solid was filtered by suction to obtain the trifluoroacetate salt of the title compound, 2.1 g. ESI-MS (m / z): 748.4 [M+H] + 。

[0464] 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-butanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (Compound 1-9)

[0465] At room temperature, dissolve (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-(dimethylamino)-3-butanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropanoic acid (1.3 g, 2.17 mmol) and the trifluoroacetate of Compound 1-8 (1.8 g, 2.17 mmol) in N,N-dimethylformamide (20 mL), cool to 0 °C, successively add 1-hydroxybenzotriazole (440 mg, 3.26 mmol) and N-methylmorpholine (658 mg, 6.51 mmol), and finally add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (624 mg, 1.38 mmol). After adding, stir the reaction mixture at 0 °C for 5 h. Purify by preparative liquid chromatography (Method D) to obtain the title compound, 1.8 g. ESI-MS (m / z): 1329.2 [M+H] + 。

[0466] Step 8: Synthesis of (S)-2-((S)-2-amino-3-butanamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidopentanamide (Compound 1-10)

[0467] At room temperature, dissolve Compound 1-9 (500 mg, 0.38 mmol) in N,N-dimethylformamide (5 mL), add piperidine (324 mg, 3.8 mmol), and stir at room temperature for 3 h. Purify by preparative liquid chromatography (Method D) to obtain the title compound, 350 mg. ESI-MS (m / z): 1107.2 [M+H] + 。

[0468] Step 9: Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanamido)-3-methylbutanamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidopentanamide (Compound TL001)

[0469] At room temperature, dissolve Compound 1-10 (60 mg, 0.054 mmol) and 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (26 mg, 0.066 mmol) in N,N-dimethylformamide (3 mL). Cool the solution to 0 °C, and successively add N,N-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazole-1-oxide tripyrrolidinophosphonium hexafluorophosphate (281 mg, 0.54 mmol). After the addition, stir the reaction mixture at room temperature for 3 h. Purify by preparative liquid chromatography (Method D) to obtain the title compound, 30 mg. ESI-MS (m / z): 664.5 [M / 2 + H] + 。

[0470] 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-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butanamido)-butanamido)-5-ureidopentanamide

[0471]

[0472] Step 1: Synthesis of 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (Compound 2-2)

[0473] At room temperature, 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butanoic acid (300 mg, 1.25 mmol) was dissolved in methanol (8 mL), sodium thiomethoxide (351 mg, 5.02 mmol) was added at once, and the temperature was raised to 50°C for overnight reaction. Purification by preparative liquid chromatography (method D) gave the title compound, 120 mg. ESI-MS (m / z): 252.1 [M+H] + .

[0474] Step 2: Synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butyramido)-butyramido)-5-ureidopentanamide (Compound 2-3)

[0475] A similar procedure as described in step nine of Example 5 was used, with 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid replacing 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid, and purification by preparative liquid chromatography (method D) was performed to obtain the title compound, 20 mg. ESI-MS (m / z): 670.5 [M / 2+H] + .

[0476] Step 3: Synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butanamido)-butanamido)-5-ureidopentanamide (Compound TL002)

[0477] At room temperature, compound 2-3 (20 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL), and m-chloroperbenzoic acid (4.0 mg, 0.022 mmol) was added. After addition, the mixture was reacted at room temperature for 2 h. Purification by preparative liquid chromatography (method D) gave the title compound, 5.0 mg. ESI-MS (m / z): 686.5 [M / 2+H]+ .

[0478] 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-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0479]

[0480] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (Compound 3-2)

[0481] At room temperature, methyl 5-hexynoate (500 mg, 3.97 mmol) and 5-bromo-2-(methylthio)pyrimidine were dissolved in N,N-dimethylformamide (3 mL). Triethylamine (3 mL), copper(I) iodide (75 mg, 0.4 mmol), and bis(triphenylphosphine)palladium(II) dichloride (279 mg, 0.4 mmol) were added successively. The reaction mixture was heated to 95 °C and stirred under nitrogen protection for 6 h. The reaction was quenched by adding water, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed under reduced pressure. The residue was purified by preparative liquid chromatography (Method D) to obtain the title compound, 300 mg. ESI-MS (m / z): 251.3 [M+H] + .

[0482] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-3)

[0483] At room temperature, Compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixed solution of tetrahydrofuran and water (4 mL / 4 mL). Lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added, and the reaction mixture was stirred at room temperature for 4 h. The reaction was diluted with water and extracted with ethyl acetate (20 mL × 2). The aqueous phase was adjusted to pH = 3 with 1 N hydrochloric acid and then extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed under reduced pressure to obtain the title compound, 120 mg.

[0484] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-4)

[0485] At room temperature, dissolve compound 3-3 (20 mg, 0.085 mmol) in dichloromethane (4 mL), add m-chloroperoxybenzoic acid (22 mg, 0.127 mmol). After addition, stir the reaction mixture at room temperature overnight. Purify by preparative liquid chromatography (Method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 269.1 [M+H] + 。

[0486] 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-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (Compound TL003)

[0487] Using a similar operation described in Step 9 of Example 5, replace 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid with 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid. Purify by preparative liquid chromatography (Method D) to obtain the title compound, 14 mg. ESI-MS (m / z): 679.0 [M / 2+H] + 。

[0488] Example 8 (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-nonadecyloxy-3,9,36-triazahentetracontan-41-amido)benzyl) carbonate

[0489]

[0490] Step 1: Synthesis of (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)-(9H-fluorenyl)carbamate (Compound 19-2)

[0491] At room temperature, Fmoc-L-citrulline (5.0 g, 12.58 mmol), 4-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 the temperature was raised to 45 °C and reacted for 6 h. The reaction solution was concentrated under reduced pressure, and the title compound was obtained by trituration with anhydrous diethyl ether (100 mL), 6.0 g. ESI-MS (m / z): 503.3 [M+H] + 。

[0492] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide (Compound 19-3)

[0493] At room temperature, Compound 19-2 (1.0 g, 1.99 mmol) was dissolved in N,N-dimethylformamide (8 mL), piperidine (339 mg, 3.98 mmol) was added dropwise, and after the addition was complete, the reaction was carried out at room temperature for 30 min. Dichloromethane (10 mL) was added, and stirring was continued for 10 min. The reaction solution was concentrated under reduced pressure and purified by flash silica gel column chromatography to obtain the title compound, 400 mg. ESI-MS (m / z): 281.2 [M+H] + 。

[0494] Step 3: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatritriacontanoylamino)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide (Compound 19-4)

[0495] Compound 19-3 (150 mg, 0.54 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxy-6-azatricycloundecanoic acid (296 mg, 0.54 mmol) were dissolved in dichloromethane (10 mL), cooled to 0 °C, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (145 mg, 0.58 mmol) was added, and the mixture was allowed to react at room temperature overnight. The reaction solution was concentrated under reduced pressure and purified by flash silica gel column chromatography to obtain the title compound, 200 mg. ESI-MS (m / z): 817.5 [M+H] + 。

[0496] Step 4: Synthesis of 4-((S)-35-azido-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diazatetracosane)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 19-5)

[0497] At room temperature, dissolve (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 (200 mg, 0.39 mmol) in dichloromethane (10 mL). Cool the solution to 0 °C, add a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL), and then slowly add a solution of triphosgene (116 mg, 0.39 mmol) in dichloromethane (1.0 mL). After addition, stir the reaction mixture at 0 °C for 1 h. Add a solution of Compound 19-4 (159 mg, 0.18 mmol) in dichloromethane (2.0 mL) to the reaction mixture. After addition, stir the reaction mixture at room temperature for 1 h. Purify the reaction mixture by preparative high performance liquid chromatography (Method D) to obtain the title compound, 160 mg. ESI-MS (m / z): 678.0 [M / 2 + H] + 。

[0498] Step 5: Synthesis of 4-((S)-35-amino-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diazatetracosane)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 19-6)

[0499] At room temperature, dissolve Compound 19-5 (80 mg, 0.059 mmol) in tetrahydrofuran (1.0 mL). Cool the solution to 0 °C, add a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL), and add platinum dioxide (15 mg, 0.059 mmol) in one portion under nitrogen protection. After addition, displace the air with hydrogen three times, and stir the reaction mixture at room temperature for 6 h. Filter the reaction mixture, concentrate the filtrate to obtain the crude product, and purify the crude product by preparative high performance liquid chromatography (Method D) to obtain the title compound, 40 mg. ESI-MS (m / z): 665.0 [M / 2 + H]+ 。

[0500] Step 6: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-4-yl (4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonadecyloxy-3,9,36-triazahentetracontan-41-amido)benzyl) carbonate (Compound TL019)

[0501] Dissolve Compound 19-6 (30 mg, 0.016 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-5-hexynoic acid (6.4 mg, 0.024 mmol) in N,N-dimethylformamide (1 mL), cool the temperature to 0 °C, and successively add benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (16.5 mg, 0.032 mmol) and N,N-diisopropylethylamine (6.2 mg, 0.047 mmol). After addition, react at room temperature for 2 h. Purify by preparative high performance liquid chromatography (Method D) to obtain the title compound, 10 mg. ESI-MS (m / z): 790.0 [M / 2 + H] + 。

[0502] Example 9 (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-4-yl-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl))-5-hexynamido)butyramido)-5-ureidopentanamido)benzyl) carbonate

[0503]

[0504] Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentanamido-2-yl)amino)-3-methyl-butyramido-2-yl)-(9H-fluorenyl)methyl-carbamate

[0505] Perform an operation similar to that in Step 1 of Example 8, using Compound 28-1 to replace Compound 19-1, to obtain the title compound, 310 mg. ESI-MS (m / z): 602.3 [M + H] + 。

[0506] Step 2: Synthesis of (S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 28-2)

[0507] Using an operation similar to that in Step 2 of Example 8, replacing Compound 19-2 with Compound 28-2, the title compound, 150 mg, was obtained. ESI-MS (m / z): 380.3 [M+H] + 。

[0508] Step 3: Synthesis of N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (Compound 28-4)

[0509] At room temperature, benzotriazol-1-yloxytris(pyrrolidino)phosphonium 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-methylbutanoyl)amino)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (190 mg, 0.5 mmol) in N,N-dimethylformamide (10 mL). The reaction was stirred at room temperature for 3 h. Purification by preparative high performance liquid chromatography (Method D) gave the title compound, 78 mg. ESI-MS (m / z): 630.3 [M+H] + 。

[0510] 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)

[0511] Using an operation similar to that in Step 4 of Example 8, replacing Compound 19-4 with Compound 28-4, the title compound, 1.76 mg, was obtained. ESI-MS (m / z): 1167.4 [M+H] + 。

[0512] Example 10 (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-nonaoxy-3,6,12-triaza-tritriacontanamido)benzyl) carbonate

[0513]

[0514] Step 1: Synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-azatetracosane)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Compound 29-1)

[0515] Using an operation similar to that in 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] + 。

[0516] 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-nonaoxy-3,6,12-triaza-tritriacontanamido)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)

[0517] Using an operation similar to that in Step 4 of Example 8, replacing Compound 19-4 with Compound 29-1, the title compound, 30 mg, was obtained. ESI-MS (m / z): 727.5 [M / 2+H] + 。

[0518] 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-nonaoxy-3,6,12-triaza-33-yl)benzyl) carbonate (Compound TL029)

[0519] At room temperature, dissolve Compound 29-2 (20 mg, 0.014 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(2-propyn-1-yl)-5-hexynamide (4.3 mg, 0.014 mmol) in a mixed solvent of dimethyl sulfoxide and water (1 mL / 0.25 mL), add copper(I) bromide (3.95 mg, 0.027 mmol), and stir the reaction for 1 h. Purify by high performance liquid chromatography (Method D) to obtain the title compound, 15 mg. ESI-MS (m / z): 880.0 [M / 2 + H] + 。

[0520] Example XI (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-nonaoxy-3,6,12,39-tetraaza-45-yl)carbamoyl)benzyl) carbonate

[0521]

[0522] Step 1: Synthesis of (S)-2-((S)-35-amino-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diaza-35-yl)carbamoyl)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide

[0523] At 20 °C, compound 29-1 (400 mg, 0.44 mmol) was dissolved in methanol and tetrahydrofuran (2.0 mL:4.0 mL). After complete dissolution, platinum dioxide (40 mg) was added in one portion under nitrogen protection. The mixture was purged with hydrogen three times and then subjected to a hydrogenation reaction at 20 °C for 2 h. The mixture was filtered, and the filter cake was washed with methanol. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 200 mg. ESI-MS (m / z): 890.4 [M+H] + 。

[0524] 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-nonaoxy-2,7,10,16-tetraazatetracontane-42-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0525] At 20 °C, compound 22-1 (250 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (1.0 mL). HATU (160 mg, 0.42 mmol) and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were added successively. After addition, the mixture was stirred at room temperature overnight. The mixture was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 250 mg.

[0526] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]pyridoindolo[1,2-b]quinolin-4-yl (4-((2S,5S)-5-isopropyl-4,5-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,40-tetraoxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxy-3,6,12,39-tetraazatetracontane-44-carbamoyl)benzyl) carbonate (Compound TL022)

[0527] At 20 °C, dissolve (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 (70 mg, 0.14 mmol) in dichloromethane (4.0 mL), cool the temperature to 0 °C, add a solution of 4-dimethylaminopyridine (200 mg, 1.64 mmol) in dichloromethane (1.0 mL), and then slowly dropwise add a solution of triphosgene (40.6 mg, 0.14 mmol) in dichloromethane (1.0 mL). After addition, stir the reaction at 0 °C for 1 h. Blow away the unreacted phosgene with nitrogen, add a solution of compound 22-2 (139 mg, 0.12 mmol) in dichloromethane (2.0 mL) to the reaction solution, and stir the reaction at 0 °C for 1 h. Purify by preparative high performance liquid chromatography (Method D) to obtain the title compound, 1.5 mg. ESI-MS (m / z): 839.5 [M / 2 + H] + 。

[0528] Example XII 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-triaza-42-yl)-41-ynylcarbonylamino)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0529]

[0530] Step 1: 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-(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-triaza-42-yl)-41-ynylcarbonylamino)benzyl carbonate

[0531] 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-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.2 mg, 0.056 mmol) and N,N-diisopropylethylamine (8.6 mg, 0.067 mmol) were added, and the mixture was stirred for 10 min. Compound 24-1 (35 mg, 0.022 mmol) was added, and the reaction was stirred for 1 h. After purification by preparative high performance liquid chromatography (Method B), the title compound, 20 mg, was obtained. ESI-MS (m / z): 1821.8 [M+H] + 。

[0532] 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-triaza-42-yl-41-ynylcarboxamido)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 TL024)

[0533] At room temperature, Compound 24-2 (20 mg, 0.011 mmol) was dissolved in acetonitrile (1 mL), and a solution of trifluoroacetic acid (0.5 mL) in acetonitrile (0.5 mL) was added dropwise, and the mixture was stirred for 20 min. After purification by preparative high performance liquid chromatography (Method C), the trifluoroacetate salt of the title compound, 12 mg, was obtained. ESI-MS (m / z): 1549.6 [M+H] + 。

[0534] 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-2-methyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)yl)hex-5-ynylcarboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triaza-30-ylcarboxamido)benzyl carbonate

[0535]

[0536] Step 1: Preparation of (S)-(9H-Fluoren-9-yl)-methyl (1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)carbamate

[0537] At room temperature, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.31 g, 5.30 mmol) and 4-aminobenzyl alcohol (593 mg, 4.82 mmol) were added to a dichloromethane (35 mL) solution of compound 30-1 (1.5 g, 4.82 mmol), and the mixture was stirred for 3 h. Purification by silica gel column chromatography gave the title compound, 1.8 g. ESI-MS (m / z): 417.2 [M+H] +

[0538] Step 2: Preparation of (S)-2-Amino-N-(4-(hydroxymethyl)phenyl)propanamide

[0539] At room temperature, ethylenediamine (5 mL) was added to a dichloromethane (20 mL) solution of compound 30-2 (1.8 g, 4.32 mmol), and the reaction was carried out for 2 h. Purification by silica gel column chromatography gave the title compound, 820 mg. ESI-MS (m / z): 195.1 [M+H]+

[0540] 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

[0541] At room temperature, (2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic 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 successively added to a dichloromethane (2 mL) solution of compound 30-3 (503 mg, 2.58 mmol), and the mixture was stirred for 4 h. Purification by silica gel column chromatography gave the title compound, 1.1 g. ESI-MS (m / z): 516.2 [M+H]+

[0542] Step 4: Preparation of (S)-2-Amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0543] At room temperature, ethylenediamine (2 mL) was added to a solution of compound 30-4 (1.1 g, 2.13 mmol) in dichloromethane (8 mL). The mixture was stirred for 1 h. Purification by silica gel column chromatography gave the title compound, 610 mg. ESI-MS (m / z): 294.2 [M+H] +

[0544] Step 5: Preparation of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxo-6-diazahentriacontanamido)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0545] At room temperature, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (160 mg, 0.42 mmol), 1-hydroxybenzotriazole (57 mg, 0.42 mmol), N,N-diisopropylethylamine (109 mg, 0.84 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxo-6-azatricyclodecane-1-carboxylic acid (156 mg, 0.28 mmol) were added to compound 30-5 (84 mg, 0.28 mmol) in dichloromethane (3 mL). The mixture was stirred for 4 h. Purification by silica gel column chromatography gave the title compound, 163 mg. ESI-MS (m / z): 830.4 [M+H] +

[0546] 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-nonaoxo-3,6,12-triazatricontanamido)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]quinoline-4-yl) carbonate

[0547] Under nitrogen protection, a solution of triphosgene (16 mg, 0.05 mmol) in dichloromethane (0.3 mL) was added dropwise to a mixed solution of 4-dimethylaminopyridine (65 mg, 0.53 mmol) and (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (45 mg, 0.09 mmol) in dichloromethane (0.7 mL) at 0 °C, and the reaction was carried out at 0 °C for 1 h. A solution of compound 30-6 (73 mg, 0.09 mmol) in dichloromethane (1 mL) was added dropwise to the reaction solution, and the reaction was carried out at 0 °C for 1 h. Purification by silica gel column chromatography gave the title compound, 33 mg. ESI-MS (m / z): 1367.6 [M + H] +

[0548] 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)amino)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonaoxy-3,6,12-triazahentriacontanamido)benzyl carbonate (Compound TL030)

[0549] At room temperature, copper(I) bromide (5 mg, 0.04 mmol) and compound 30-7 (20 mg, 15 μmol) were added dropwise to a mixed solution of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (9 mg, 0.007 mmol) in water and N,N-dimethylformamide (0.2 mL:0.8 mL), and the reaction was stirred for 4 h. Purification by preparative high performance liquid chromatography (Method D) gave the title compound, 4.15 mg. ESI-MS (m / z): 1672.7 [M + H] +

[0550] Example 14 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-diazahentriacontanamido)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

[0551]

[0552] Step 1: Synthesis of 6-(2-(Methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide

[0553] At 25 °C, prop-2-yn-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL). 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 successively added, and the mixture was stirred for 2 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by 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] + .

[0554] 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-diazahentriacontanamido)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

[0555] Under nitrogen protection at 25 °C, dissolve T-030 (250 mg, 0.49 mmol) in dichloromethane (10 mL), cool the temperature to 0 °C, add a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL), and then slowly dropwise add a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL). After addition, stir the reaction at 0 °C for 20 min, and blow the reaction solution with nitrogen for 20 min. Add a solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-aza-triglycinamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL). After addition, stir the reaction at 0 °C for 1 h. Concentrate the reaction solution under reduced pressure, and purify the residue by preparative high performance liquid chromatography (Method A) to obtain the title compound, 500 mg. ESI-MS (m / z): 1597.5 [M + H] + 。

[0556] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-4-yl (4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-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-nonaoxy-3,9-diaza-35-pentacosanamide)benzyl) carbonate

[0557] At room temperature, dissolve compound 33-1 (14 mg, 0.05 mmol) in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), add cuprous bromide (11 mg, 0.08 mmol), and stir the reaction for 1 h. Purify by preparative high performance liquid chromatography (Method B) to obtain the title compound, 30 mg. ESI-MS (m / z): 815.9 [(M - 273) / 2 + H] + 。

[0558] 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-diazahentriacontanamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indeno[1,2-b]quinolin-4-yl) carbonate (Compound TL033)

[0559] Dissolve Compound 33-2 (30 mg, 0.02 mmol) in dichloromethane (1.0 mL). Add trifluoroacetic acid (0.2 mL) to the reaction solution and react at room temperature for 30 min. Purify by preparative high performance liquid chromatography (Method C) to obtain the trifluoroacetate salt of the title compound, 20.0 mg. Its structural characterization is as follows:

[0560] 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).

[0561] 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-nonaoxy-3,9-diazahentriacontanamido)benzyl ((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-4-carbonate

[0562]

[0563] 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-nonaoxy-((S)-9-((tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1,2,3,4-tetrahydroquinoline-1H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-4-yl) carbonate

[0564] Dissolve compound 34-1 (100 mg, 0.2 mmol) in dry dichloromethane (2 mL) under nitrogen protection at room temperature, cool to 0 °C, add a solution of 4-dimethylaminopyridine (144 mg, 1.18 mmol) in dry dichloromethane (0.5 mL), and then slowly add a solution of triphosgene (41 mg, 0.14 mmol) in dry dichloromethane (0.5 mL). After addition, stir the reaction at 0 °C for 1 h. Add a solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-aza-triamide)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (160 mg, 0.15 μmol) in dry dichloromethane (0.5 mL) to the reaction solution. After addition, react at room temperature for 1 h. Purify by preparative high-performance liquid chromatography (Method B) to obtain the title compound, 60 mg. ESI-MS (m / z): 1592.7 [M+H] + 。

[0565] 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]indazino[1,2-b]quinolin-4-yl-4-((S)-2-(4-(((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27,30,33-decaoxo-3,9-diazahentriacontanamido)carbonate

[0566] At room temperature, compound 34-2 (40 mg, 0.03 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (11.50 mg, 0.04 mmol) were dissolved in dimethyl sulfoxide and water (0.5 mL: 0.1 mL), and copper(I) bromide (9.01 mg, 0.06 mmol) was added. The mixture was stirred for 1 h. After purification by preparative high performance liquid chromatography (Method B), the title compound, 20 mg, was obtained. ESI-MS (m / z): 1897.5 [M+H].

[0567] Step 3: Synthesis of 4-((S)-2-(4-aminobutyl)-3,6,9,12,15,18,21,24,27,30,33-decaoxo-3,9-diazahentriacontanamido)benzyl ((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-4-carboxylate (Compound TL034)

[0568] At room temperature, compound 34-3 (30 mg, 0.018 mmol) was dissolved in acetonitrile and water (0.4 mL: 0.1 mL), and a mixed solution of trifluoroacetic acid and acetonitrile (0.5 mL: 0.5 mL) was added dropwise. The mixture was stirred at room temperature for 2 h. After purification by preparative high performance liquid chromatography (Method C), the trifluoroacetate salt of the title compound, 12 mg, was obtained. ESI-MS (m / z): 1511.5 [M+H] + 。

[0569] Synthesis of Example XVI 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-diazahentriacontanamido)benzyl ((S)-4-Ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indazino[1,2-b]quinoline-4-yl) Carbonate

[0570]

[0571] 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-nonaoxa-3,9-diazahentriacontanamido)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]indazino[1,2-b]quinoline-4-yl) Carbonate

[0572] Using a synthetic method similar to that of Step 1 in Example XV, replacing compound 34-1 with compound 35-1, the title compound, 60 mg, was obtained. ESI-MS (m / z): 1561.5 [M+H] + 。

[0573] 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]indazino[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-nonaoxa-3,9-diazahentriacontanamido] Carbonate

[0574] Using a synthetic method similar to that of Step 2 in Example XV, replacing compound 34-2 with compound 35-2, the title compound, 20 mg, was obtained. ESI-MS (m / z): 1866.5 [M+H].

[0575] 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-diazahentriacontanamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indeno[1,2-b]quinolin-4-yl) carbonate (Compound TL035)

[0576] Using a synthetic method similar to that in Step 3 of Example 15, with Compound 35-3 replacing Compound 34-3, the trifluoroacetate salt of the title compound, 4.9 mg, was obtained. ESI-MS (m / z): 1594.5 [M+H] + 。

[0577] Synthesis of Example 17 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-triazatetracont-41-enamido)benzyl -((S)-4-ethyl-11-(2-(N-isopropylethanamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate

[0578]

[0579] Step 1: Synthesis of (Z)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enoic acid

[0580] At 20 °C, Compound 3-4 (200 mg, 0.67 mmol) was dissolved in methanol (8.0 mL). Lindlar catalyst (20 mg) was added under nitrogen protection, and the mixture was purged with hydrogen three times. The hydrogenation reaction was carried out at 20 °C for 3 h. The mixture was filtered, and the filtrate was concentrated to dryness to obtain the title compound, 150 mg. ESI-MS (m / z): 271.1 [M+H] + 。

[0581] Step 2: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylethanamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 4-((S,Z)-2-(4-(((4-methoxyphenyl)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-triazahentetracont-41-enamide)benzyl carbonate

[0582] At room temperature, dissolve compound 45-2 (8 mg, 0.030 mmol) in dichloromethane (2 mL), add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (14.9 mg, 0.039 mmol) and N,N-diisopropylethylamine (8.8 mg, 0.068 mmol). Stir the reaction mixture at room temperature for 10 min, add compound 48-1 (30 mg, 0.020 mmol), and stir the reaction mixture at room temperature for 1 h. Purify by preparative high performance liquid chromatography (Method B) to obtain the title compound, 30 mg. ESI-MS (m / z): 1787.8 [M+H] + 。

[0583] 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-triazahentetracont-41-enamide)benzyl ((S)-4-ethyl-11-(2-(N-isopropylethanamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate (Compound TL045)

[0584] At room temperature, dissolve compound 45-3 (30 mg, 0.017 mmol) in acetonitrile (1 ml), and dropwise add a solution of trifluoroacetic acid (0.5 ml) in acetonitrile (0.5 ml). Stir the reaction mixture at room temperature for 20 min. Purify by preparative high performance liquid chromatography (Method C) to obtain the title compound trifluoroacetate salt, 9 mg. ESI-MS (m / z): 1515.6 [M+H] + 。

[0585] Example XVIII 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-triazahentetracont-41-ynylcarboxamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylethanamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate

[0586]

[0587] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylethanamido)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)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazahentetracont-41-ynylcarboxamido)benzyl carbonate

[0588] Using a synthetic method similar to that of Step 1 in Example XII, replacing compound 24-1 with compound 48-1. The title compound, 15 mg, was obtained. ESI-MS (m / z): 1785.8 [M+H] + 。

[0589] 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-triazahentetracont-41-ynylcarboxamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylethanamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate (Compound TL048)

[0590] Using a synthetic method similar to that of Step 2 in Example XII, replacing compound 24-2 with compound 48-2. The trifluoroacetate salt of the title compound, 11.35 mg, was obtained. ESI-MS (m / z): 1513.7 [M+H] + 。

[0591] 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-diazahentriacontylamino)benzyl-((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) carbonate

[0592]

[0593] Step 1: Synthesis of 2-(2-(Methylthio)pyrimidin-5-yl)thiazole-4-carboxylic acid

[0594] Compound 49-1 (100 mg, 0.40 mmol), 2-bromo-4-thiazolecarboxylic acid (99.01 mg, 0.48 mmol), potassium carbonate (137.03 mg, 0.99 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (29.02 mg, 0.04 mmol) were placed in N,N-dimethylformamide (4 mL) and water (1 mL), under nitrogen protection, and the reaction system was heated to 100 °C and stirred for 4 hours. The reaction solution was cooled to room temperature, dropped into water, filtered by suction, the filtrate was collected, extracted with ethyl acetate (10 mL × 3), the aqueous phase was collected, the pH was adjusted to 3 with dilute hydrochloric acid, a solid was precipitated, filtered by suction, and the filter cake was collected to obtain the title compound, 70 mg. ESI-MS (m / z): 254.0 [M+H] + 。

[0595] Step 2: Synthesis of 2-(2-(Methylsulfonyl)pyrimidin-5-yl)thiazole-4-carboxylic acid

[0596] Compound 49-2 (73 mg, 0.29 mmol) was dissolved in dichloromethane (15 mL), m-chloroperbenzoic acid (175.53 mg, 0.87 mmol, 85%) was added, and the reaction system was stirred at room temperature overnight. The solvent was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 286.0 [M+H] + 。

[0597] Step 3: Synthesis of 2-(2-(Methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)thiazole-4-carboxamide

[0598] Dissolve compound 49-3 (20 mg, 0.07 mmol) in dichloromethane (2 mL), add O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (39.98 mg, 0.11 mmol), cool the reaction system to 0 °C, add N,N-diisopropylethylamine (22.65 mg, 0.18 mmol) and propargylamine (4.63 mg, 0.09 mmol), and stir the reaction solution at room temperature for 3 h. Purify by preparative high performance liquid chromatography (Method D) to obtain the title compound, 10 mg. ESI-MS (m / z): 323.0 [M+H] + 。

[0599] 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-(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-diazapentatriacontylamino)benzyl) carbonate

[0600] At room temperature, dissolve compound 33-1 (30 mg, 0.02 mmol) and compound 49-4 (9.08 mg, 0.03 mmol) in dimethyl sulfoxide and water (2 mL / 0.5 mL), add copper(I) bromide (5.39 mg, 0.04 mmol), and stir the reaction for 2 h. Filter, and purify the filtrate by preparative high performance liquid chromatography (Method B) to obtain the title compound, 20 mg. ESI-MS (m / z): 1647.3 [M+H-273] + 。

[0601] 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-diazapentatriacontylamino)benzyl-((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) carbonate

[0602] At room temperature, compound 49-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 min. The reaction mixture was concentrated, and the residue was purified by preparative high performance liquid chromatography (Method C) to obtain the trifluoroacetate salt of the title compound, 8 mg. ESI-MS (m / z): 1647.9 [M+H] + 。

[0603] Example 20 4-((S)-2-(4-Aminobutyric acid)-35-(4-((2-(2-(Methylsulfonyl)pyrimidin-5-yl)-oxazol-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diazahentriacontanamido)benzyl-((S)-4-Ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indeno[1,2-b]quinolin-4-yl) carbonate

[0604]

[0605] Step 1: Synthesis of ethyl 2-(2-(methylthio)pyrimidin-5-yl)oxazole-4-carboxylate

[0606] At 25 °C, ethyl 2-bromooxazole-4-carboxylate (100 mg, 0.45 mmol) and compound 49-1 (126 mg, 0.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane and water (4 mL / 2 mL). Potassium carbonate (125 mg, 0.9 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (33 mg, 0.05 mmol) were added successively. Under N2 protection, the temperature was raised to 90 °C and the reaction was carried out for 3 h. The reaction mixture was filtered through diatomaceous earth, the filtrate was diluted with water (50 mL), and extracted with ethyl acetate (30 mL×3). The organic phases were combined and dried. The desiccant was filtered off, 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] + 。

[0607] Step 2: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)oxazole-4-carboxylic acid

[0608] At 25 °C, compound 50-1 (50 mg, 0.19 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (4 mL / 2 mL). After complete dissolution, lithium hydroxide monohydrate (40 mg, 0.94 mmol) was added. After addition, the reaction was carried out at 25 °C for 1 h. The reaction solution was diluted with water (15 mL), extracted with ethyl acetate (20 mL × 2). The aqueous phase was adjusted to pH = 2 - 3 with 1 N dilute hydrochloric acid, and extracted with a mixed solvent of dichloromethane / methanol (v:v = 10:1) (20 mL × 3). The organic phases were combined, washed with saturated brine (30 mL × 1), and dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated to obtain the title compound, 40 mg. It was used directly in the next reaction without purification. ESI-MS (m / z): 238.1 [M+H] + 。

[0609] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole-4-carboxylic acid

[0610] At 25 °C, compound 50-2 (40 mg, 0.17 mmol) was dissolved in dichloromethane (6 mL). After complete dissolution, m-chloroperbenzoic acid (29 mg, 0.17 mmol) was added. After addition, the reaction was stirred at 25 °C for 14 h. The reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 20 mg. ESI-MS (m / z): 269.9 [M+H] + 。

[0611] Step 4: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)oxazole-4-carboxamide

[0612] At 25 °C, compound 50-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (4 mL). 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 successively, and the mixture was stirred for 5 min. Propargylamine (5.0 mg, 0.09 mmol) was added. After addition, the reaction was stirred at room temperature for 30 min. The reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 5.0 mg. ESI-MS (m / z): 306.9 [M+H] + 。

[0613] Step 5: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazole-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentatricarboxamido)benzyl)carbonate

[0614] At 25°C, compound 50-4 (6.0 mg, 0.02 mmol) and compound 33-1 (30 mg, 0.02 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (2 mL / 0.5 mL), and cuprous bromide (5.0 mg, 0.04 mmol) was added at once. After addition, the mixture was reacted at room temperature for 2 h. The reaction solution was filtered and purified by preparative high performance liquid chromatography (method B) to obtain the title compound, 25 mg. ESI-MS (m / z): 1631.3 [(M-273+H] + .

[0615] 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

[0616] Compound 50-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2.0 mL) at 25°C. After complete dissolution, trifluoroacetic acid (0.2 mL) was added to the reaction solution and reacted at 25°C for 10 min. The reaction solution was concentrated and the residue was purified by preparative HPLC (method C) to obtain the trifluoroacetate salt of the title compound, 3.0 mg. ESI-MS (m / z): 816.5 [M / 2+H] + .

[0617] 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-methylpropanamide)-3-phenylpropanamide)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonaoxa-2,7,10,16-tetraazatetracontane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0618]

[0619] 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-butanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrol-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-pentylurea-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino-(9H-fluoren-9-yl)methyl formate

[0620] At room temperature, dissolve compound 51-1 (100 mg, 0.17 mmol) and ((S)-1-(((S)-1-((4-(((S)-2-amino-3-phenylpropanamido)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) in N,N-dimethylformamide (2 mL), cool to 0 °C, and successively add 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). After addition, stir the reaction mixture at 0 °C for 5 h. Pour the reaction mixture into water (20 mL), precipitate a white solid, filter by suction, wash the filter cake with water, and dry to obtain the title compound, 200 mg. ESI-MS (m / z): 1329.2 [M+H] + 。

[0621] 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-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidovaleramide

[0622] At room temperature, dissolve compound 51-2 (200 mg, 0.12 mmol) in N,N-dimethylformamide (5 mL), add piperidine (0.5 mL), and stir the reaction mixture at room temperature for 2 h. The reaction mixture was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 65 mg. ESI-MS (m / z): 1107.2 [M+H] + 。

[0623] 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-diazapentatriacontylamino)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidovaleramide

[0624] Dissolve 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatricarboxylic acid (33.1 mg, 0.06 mmol) in N,N-dimethylformamide (5 mL), add O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (38 mg, 0.10 mmol) and N,N-diisopropylethylamine (26 mg, 0.20 mmol), stir the reaction mixture at room temperature for 10 min, cool to 0 °C, add compound 51-3 (55 mg, 0.05 mmol), and stir the reaction mixture at room temperature for 2 h. The reaction mixture was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 56 mg. ESI-MS (m / z): 821.8 [M / 2+H] + 。

[0625] Step 4: Synthesis of 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-methylpropanamide)-3-phenylpropanamide)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonaoxa-2,7,10,16-tetraazatetracontane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0626] At room temperature, dissolve compound 51-4 (56 mg, 0.04 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-5-hexynamide (16 mg, 0.05 mmol) in a mixed solution of dimethyl sulfoxide and water (2 mL / 0.5 mL), add copper(I) bromide (10 mg, 68.17 μmol), and stir the reaction for 2 h. Filter, and purify the filtrate by preparative high performance liquid chromatography (Method D) to obtain the title compound, 50 mg. ESI-MS (m / z): 974.3 [M / 2 + H] + 。

[0627] 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-triazatetracontanyl)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0628]

[0629] 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-nonaoxa-3,6,12-triazatetracontanyl)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0630] At room temperature, dissolve compound 53-1 (100 mg, 0.09 mmol) in N,N-dimethylformamide (3 mL), add 1-hydroxybenzotriazole (13 mg, 0.09 mmol) and N,N-diisopropylethylamine (36 mg, 0.28 mmol), then add compound 52-1 (67 mg, 0.09 mol). Stir the reaction mixture at room temperature for 16 h. Purify the reaction mixture by preparative high performance liquid chromatography (Method D) to obtain the title compound, 120 mg. ESI-MS (m / z): 830.1 [M / 2 + H] + 。

[0631] Step 2: Synthesis of 4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazatetracontanyl)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0632] At room temperature, compound 52-2 (22 mg, 0.07 mmol) was dissolved in a mixed solution of dimethyl sulfoxide and water (3 mL / 0.3 mL), and copper(I) bromide (18 mg, 0.13 mmol) was added. The mixture was stirred for 1 h. After filtration, the filtrate was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 92 mg. ESI-MS (m / z): 982.8 [M / 2 + H] + 。

[0633] Example 23 Synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylcarbamoyl)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxo-6-aza-30-alkylcarboxamido)butanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0634]

[0635] 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-nonaoxo-3,6,12-triaza-38-alkylcarboxamide)benzyl-(4-nitrophenyl)-carbonate

[0636] At 25 °C, compound 29-1 (500 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (10 mL), N,N-diisopropylethylamine (141 mg, 1.09 mmol) was added, and a solution of bis(4-nitrophenyl) 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 mixture was purified by reverse column (C18) chromatography (acetonitrile / water = 1:2) to obtain the title compound, 400 mg. ESI-MS (m / z): 1081.9 [M + H] + 。

[0637] Step 2: Synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((((4-((S)-2-((S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxo-6-azahentriacontanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0638] At 25 °C, 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)butanamido)butanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (41 mg, 0.06 mmol) were dissolved in N,N-dimethylformamide (2 mL). After complete dissolution, 1-hydroxybenzotriazole (8 mg, 0.06 mmol) was added. After addition, the reaction mixture was stirred at 25 °C for 16 h. The reaction solution was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 38 mg. ESI-MS (m / z): 837.2 [M / 2 + H] + 。

[0639] Step 3: Synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl 2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-ylcarbamoyl)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxo-6-azahentriacontanamido)butanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0640] At 25 °C, 2-(2-(Methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazole-4-carboxamide (9 mg, 0.03 mmol) and compound 53-2 (50 mg, 0.03 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (1 mL / 0.25 mL). After complete dissolution, copper(I) bromide (11 mg, 0.08 mmol) was added. After addition, the reaction mixture was stirred under N2 protection for 1 h. The mixture was filtered, and the filtrate was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 25 mg. ESI-MS (m / z): 989.9 [M / 2 + H] + 。

[0641] 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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0642]

[0643] Step 1: Synthesis of (S)-4-(35-Azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butanoyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontylamino)benzyl-(4-nitrophenyl)-carbonate

[0644] At room temperature, compound 54-1 (1 g, 0.95 mmol) was dissolved in dichloromethane (20 mL). N,N-Diisopropylethylamine (488 mg, 3.77 mmol) was added, and then a solution of bis-(4-nitrophenyl)-carbonate (860 mg, 2.83 mmol) in dichloromethane (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 6 h. Purification by silica gel column chromatography (dichloromethane / methanol = 40 / 1) gave the title compound, 900 mg. ESI-MS (m / z): 953.0 [M + H - 273] + 。

[0645] Step 2: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butanoyl)-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0646] At room temperature, 1-hydroxybenzotriazole (33 mg, 0.25 mmol) and N,N-diisopropylethylamine (48 mg, 0.37 mmol) were added to compound 54-2 (2 mL), and then compound 52-1 (88 mg, 0.12 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by preparative high performance liquid chromatography (Method B) to obtain the title compound, 150 mg. ESI-MS (m / z): 1803.6 [M+H] + 。

[0647] Step 3: Synthesis of 4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butanoyl)-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0648] At room temperature, compound 54-3 (100 mg, 0.06 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-5-hexynamide (26 mg, 0.08 mmol) were dissolved in dimethyl sulfoxide (2 mL) and water (0.5 mL). Copper(I) bromide (16 mg, 0.11 mmol) was added, and the mixture was stirred for 2 h. After filtration, the filtrate was purified by preparative high performance liquid chromatography (method B) to obtain the title compound, 70 mg. ESI-MS (m / z): 1936.6 [M+H-273] + 。

[0649] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-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)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0650] At room temperature, compound 54-4 (70 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL). 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 high performance liquid chromatography (method C) to obtain the trifluoroacetate salt of the title compound, 55 mg. ESI-MS (m / z): 918.8 [M / 2+H] + 。

[0651] 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-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indeno[1,2-b]quinolin-4-yl) carbonate

[0652]

[0653] Step 1: Synthesis of methyl 4-(2-(methylthio)pyrimidin-5-yl)benzoate

[0654] At 25 °C, compound 49-1 (252 mg, 1.0 mmol), water (3 mL), Pd(dppf)Cl2 (40 mg, 0.05 mmol) and potassium carbonate (277 mg, 2.0 mmol) were successively added to a solution of methyl p-bromobenzoate (215 mg, 1.0 mmol) in 1,4-dioxane (5 mL). The mixture was stirred at 80 °C for 4 h. It was extracted with ethyl acetate (30 mL × 3), and the organic phases were combined, dried, and the insoluble matters were 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] + 。

[0655] Step 2: Synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic acid

[0656] At 25 °C, lithium hydroxide monohydrate (322 mg, 7.68 mmol) and water (3 mL) were respectively added to a solution of compound 55-1 (500 mg, 1.92 mmol) in tetrahydrofuran (3 mL). The mixture was stirred for 4 h. The pH of the reaction solution was adjusted to 3 - 4 with 1N hydrochloric acid. It was extracted with ethyl acetate (20 mL × 3), and the organic phases were combined, dried, and the insoluble matters were filtered off. The residue was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 430 mg. ESI-MS (m / z): 246.9 [M+H] + 。

[0657] Step 3: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid

[0658] At 25 °C, m-chloroperoxybenzoic acid (420 mg, 2.44 mmol) was added to a solution of compound 55-2 (200 mg, 0.81 mmol) in dichloromethane (5 mL). The mixture was stirred for 5 h. It was purified by silica gel column chromatography to obtain the title compound, 180 mg. ESI-MS (m / z): 279.0 [M+H] + 。

[0659] Step 4: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)benzamide

[0660] At 25 °C, benzotriazol - 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). The mixture was stirred for 30 min, then propargylamine (10 mg, 0.2 mmol) and N,N-diisopropylethylamine (70 mg, 0.5 mmol) were added to the reaction solution, and the mixture was stirred for 2.5 h. Purification by silica gel column chromatography gave the title compound, 20 mg. ESI-MS (m / z): 316.0 [M + H] + 。

[0661] Step 5: 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-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-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-nonaoxy-3,9-diazahentriacontanamido)benzyl) carbonate. Under N2 protection at 25 °C, copper(I) iodide (10 mg, 0.05 mmol) and water (2 mL) were successively added to a solution of compound 55-4 (16 mg, 0.05 mmol) and compound 33-1 (80 mg, 0.05 mmol) in dimethyl sulfoxide (2 mL). The mixture was stirred for 1 h. Purification (Method B) gave the title compound, 79 mg. ESI-MS (m / z): 1641.5 [M - 273 + H] + 。

[0662] 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-nonaoxy-3,9-diazahentriacontanamido)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

[0663] At 25 °C, compound 55-5 (55 mg, 0.029 mmol) was added to a mixed solvent of water / acetonitrile (0.1 mL / 0.5 mL) of trifluoroacetic acid (0.5 mL). The reaction mixture was stirred for 15 min, and the reaction solution was purified by preparative high performance liquid chromatography (Method C) to obtain the trifluoroacetate salt of the title compound, 42 mg. ESI-MS (m / z): 821.0 [M / 2 + H] + 。

[0664] 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-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)propyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonaoxy-2,7,10,16-tetraazatetracontane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0665]

[0666] Step 1: Synthesis of 32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxy-6-aza-32-oic acid

[0667] At 20 °C, compound 56-1 (750 mg, 1.28 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (496 mg, 1.54 mmol) were dissolved in dimethyl sulfoxide (10 mL), and cuprous bromide (465 mg, 3.21 mmol) was added in one portion. After addition, the reaction mixture was stirred for 12 h. The reaction solution was filtered, and the filtrate was purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 500 mg. ESI-MS (m / z): 860.4 [M + H] + 。

[0668] 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-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)propyl)phenyl)amino)-1-oxo-5-ureidopentanamido-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0669] At 25 °C, dissolve (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(4-aminophenyl)-3-azidopropyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide (185 mg, 0.24 mmol) in N,N-dimethylformamide (5 mL), add HATU (137 mg, 0.36 mmol), stir the reaction for 5 min, add (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidovaleric acid (131 mg, 0.26 mmol), and stir at room temperature for 30 min. The reaction solution is directly used for the next step. ESI-MS (m / z): 626.0 [M / 2 + H] + 。

[0670] Step 3: Synthesis of (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)propyl)phenyl)-5-ureidopentanamide

[0671] At 25 °C, add diethylamine (0.5 mL) to the reaction solution of Step 2. After addition, stir the reaction for 30 min. The reaction solution is purified by preparative high performance liquid chromatography (Method D) to obtain the title compound, 70 mg. ESI-MS (m / z): 515.0 [M / 2 + H] + 。

[0672] Step 4: N-((1-((6S,9S)-1-amino-6-((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-4-yl)-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonyloxy-2,7,10,16-tetraazatetradodecane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0673] At 25°C, (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-(4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidopentanamide (95 mM) was added. g, 0.092mmol), 32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatriacontanoic acid (79mg, 0.092mmol) was dissolved in N,N-dimethylformamide (4mL), HATU (70mg, 0.184mmol) was added at one time, and the reaction was stirred at room temperature for 1h. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound, 30mg. ESI-MS (m / z): 935.8[M / 2+H] + .

[0674] 3. Conjugation of compounds containing cell bioactive molecules and linkers to antibodies

[0675] Example 27 Preparation of BT001002

[0676] Take 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg / mL), dilute it with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6), then add 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) and mix well. Adjust the pH to 7.4 with 1 M K2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution and mix well. Let it stand at room temperature for 30 min. Add 15-fold molar amount of TL003 dissolved in dimethyl sulfoxide to the above solution system, mix well, and let it stand at room temperature for 2 h. After completion, add 6.1 μL of 100 mM cysteine to terminate the reaction. Finally, use a G-25 gel column to replace the buffer with a 20 mM PB buffer solution at pH 6.44 to obtain the conjugate product of TL003 and Sacituzumab antibody, named BT001002.

[0677]

[0678] Example 28 Preparation of BT001004

[0679] Take 0.285 mL of Sacituzumab antibody (anti-Trop-2, 17.6 mg / mL), dilute it with 0.095 mL of diluent (a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate, pH 7.6); then adjust the pH to 7.4 with 1 M Na2HPO4 solution, add 10 mM TCEP solution and mix well. Let it stand at room temperature for 30 min; add 9-fold molar amount of TL019 dissolved in dimethyl sulfoxide to the above solution system, mix well, and let it stand at room temperature for 2 h. Finally, use a G-25 gel column to replace the buffer with a PBS buffer solution at pH 6.5 to obtain the conjugate product of TL019 and Sacituzumab antibody, named BT001004.

[0680]

[0681] Example 29 Preparation of BT001012

[0682] Using a method similar to that in Example 27, replace TL003 with the trifluoroacetate salt of TL024 to obtain the conjugate product of TL024 and Sacituzumab antibody, named BT001012.

[0683]

[0684] Example 30 Preparation of BT001013

[0685] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL048 to obtain a product of the conjugate of TL048 and Sacituzumab antibody, named BT001013.

[0686]

[0687] Preparation of Example 31: BT001018

[0688] Using a method similar to that of Example 27, replace TL003 with TL030 to obtain a product of the conjugate of TL030 and Sacituzumab antibody, named BT001018.

[0689]

[0690] Preparation of Example 32: BT001021

[0691] Take 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg / mL), dilute it with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate (pH 7.6), then add 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) and mix well. Adjust the pH to 7.4 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution and mix well. Let it stand at room temperature for 30 min. Add 10 times the amount of the trifluoroacetate salt of TL033 dissolved in dimethyl sulfoxide to the above solution system, mix well, and let it stand at room temperature for 2 h. After completion, add 6.1 μL of 100 mM cysteine to terminate the reaction. Finally, replace the buffer with PBS buffer solution at pH 6.5 using a G-25 gel column to obtain a product of the conjugate of TL033 and Sacituzumab antibody, named BT001021.

[0692]

[0693] Preparation of Example 33: BT001022

[0694] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL034 to obtain a product of the conjugate of TL034 and Sacituzumab antibody, named BT001022.

[0695]

[0696] Preparation of Example 34: BT001023

[0697] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL035 to obtain a product of the conjugate of TL035 and the Sacituzumab antibody, named BT001023.

[0698]

[0699] Preparation of Example 35, BT001032

[0700] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL045 to obtain a product of the conjugate of TL045 and the Sacituzumab antibody, named BT001032.

[0701]

[0702] Preparation of Example 36, BT001033

[0703] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL033 and replace the Sacituzumab antibody with the M1 antibody to obtain a product of the conjugate of TL033 and the M1 antibody, named BT001033.

[0704]

[0705] Preparation of Example 37, BT001034

[0706] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL033 and replace the Sacituzumab antibody with the M2 antibody to obtain a product of the conjugate of TL033 and the M2 antibody, named BT001034.

[0707]

[0708] Preparation of Example 38, BT001035

[0709] Take 0.3 mL of M3 antibody (anti-Trop-2, 33.5 mg / mL), dilute it with 0.25 mL of a solution containing 20 mM PB, 150 mM NaCl, and 20 mM sodium edetate (pH 7.6), then add 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) and mix well. Adjust the pH to 7.4 with 1 M Na2HPO4 solution, add 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution and mix well. Let it stand at room temperature for 30 min. Add 10 times the molar amount of the trifluoroacetate salt of TL033 dissolved in dimethyl sulfoxide to the above solution system, mix well, and let it stand at room temperature for 2 h. After completion, add 6.1 μL of 100 mM cysteine to terminate the reaction. Finally, use a G-25 gel column to replace the buffer with PBS buffer solution at pH 6.5 to obtain the conjugate product of TL033 and M3 antibody, named BT001035.

[0710]

[0711] Example 39 Preparation of BT001036

[0712] Using a method similar to that in Example 27, replace TL003 with the trifluoroacetate salt of TL033 and replace the Sacituzumab antibody with the trastuzumab antibody to obtain the conjugate product of TL033 and trastuzumab antibody, named BT001036.

[0713]

[0714] Example 40 Preparation of BT001040

[0715] Using a method similar to that in Example 27, replace TL003 with the trifluoroacetate salt of TL049 to obtain the conjugate product of TL049 and Sacituzumab antibody, named BT001040.

[0716]

[0717] Example 41 Preparation of BT001041

[0718] Using a method similar to that in Example 27, replace TL003 with the trifluoroacetate salt of TL050 to obtain the conjugate product of TL050 and Sacituzumab antibody, named BT001041.

[0719]

[0720] Example 42 Preparation of BT001042

[0721] Using a method similar to that of Example 27, replace TL003 with TL051 to obtain a product of the conjugate of TL051 and Sacituzumab antibody, named BT001042.

[0722]

[0723] Preparation of Example 43, BT001043

[0724] Using a method similar to that of Example 27, replace TL003 with TL052 to obtain a product of the conjugate of TL052 and Sacituzumab antibody, named BT001043.

[0725]

[0726] Preparation of Example 44, BT001044

[0727] Using a method similar to that of Example 27, replace TL003 with TL053 to obtain a product of the conjugate of TL053 and Sacituzumab antibody, named BT001044.

[0728]

[0729] Preparation of Example 45, BT001045

[0730] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL054 to obtain a product of the conjugate of TL054 and Sacituzumab antibody, named BT001045.

[0731]

[0732] Preparation of Example 46, BT001046

[0733] Using a method similar to that of Example 27, replace TL003 with the trifluoroacetate salt of TL055 to obtain a product of the conjugate of TL055 and Sacituzumab antibody, named BT001046.

[0734]

[0735] Preparation of Example 47, BT001047

[0736] Using a method similar to that of Example 27, replace TL003 with TL056 to obtain a product of the conjugate of TL056 and Sacituzumab antibody, named BT001047.

[0737]

[0738] Example 48. Determination of the molecular weight of BT001002 by LC-MS

[0739] Perform LCMS molecular weight analysis on the conjugated BT001002.

[0740] Chromatographic determination conditions:

[0741] Liquid chromatography column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0742] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0743] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg;

[0744] 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

[0745] Mass spectrometry determination conditions:

[0746] Mass spectrometry model: Triple TOF 5600+;

[0747] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m / z600 - 5000;

[0748] The results are as Figures 1 - 3 shown.

[0749] The theoretical and measured molecular weights of BT001002

[0750]

[0751] In the table, mAb represents monoclonal antibody; LC represents antibody light chain; HC represents antibody heavy chain; DAR1 represents a conjugate containing one antibody light chain / heavy chain and one cell bioactive molecule; DAR2 represents a conjugate containing one antibody light chain / heavy chain and two cell bioactive molecules; DAR3 represents a conjugate containing one antibody light chain / heavy chain and three cell bioactive molecules; DAR4 represents a conjugate containing one antibody light chain / heavy chain and four cell bioactive molecules; Glycoform represents the sugar chain structure on two heavy chains: G0F represents fucosylated and galactose-free. In the following text, mAb, LC, HC, DAR1, DAR2, DAR3, DAR4, and G0F are as described above.

[0752] FromFigures 1 - 3 It can be seen that after the antibody is conjugated with TL003, the molecular weights of both the light chain and the heavy chain change. One cell bioactive molecule is conjugated to the light chain and three cell bioactive molecules are conjugated to the heavy chain. It can be inferred therefrom that the conjugation ratio (DAR) of the entire antibody to the cell bioactive molecule is 8.

[0753] Example 49: Determination of the molecular weight of BT001004 by LC-MS

[0754] Perform LC-MS molecular weight analysis on the conjugated BT001004.

[0755] Chromatographic determination conditions:

[0756] Liquid chromatography column: ACQUITU Protein BEH C18 1.7μm, 2.1mm×100mm;

[0757] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0758] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg;

[0759] 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

[0760] Mass spectrometry determination conditions:

[0761] Mass spectrometry model: Triple TOF 5600+;

[0762] GS1 60; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m / z 600 - 5000;

[0763] The results are as Figures 4 - 6 shown.

[0764] The theoretical and measured molecular weights of BT001004

[0765]

[0766] LC represents the antibody light chain and HC represents the antibody heavy chain.

[0767] From Figures 4 - 6It can be known that in BT001004, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, with DAR1 ratios of 14% and 86% respectively), and the heavy chain is conjugated with 1 to 3 cell bioactive molecules (DAR1, DAR2, and DAR3 ratios of 13%, 19%, and 68% respectively). From this, the conjugation ratio (DAR) of the entire antibody with the cell bioactive molecule is calculated to be 7.0.

[0768] Example 50 LC-MS determination of the molecular weight of BT001012

[0769] Using a method similar to that in Example 48, the results are as Figure 10 and 11 shown.

[0770] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001012 obtained after conjugating TL024 with the antibody are shown in the following table:

[0771]

[0772] From Figure 10 and 11 it can be known that in BT001012, the antibody light chain is conjugated with 0 to 1 toxin (LC, with DAR1 ratios of 12.9% and 87.1% respectively), and the heavy chain is conjugated with 1 to 3 toxins (DAR1, DAR2, and DAR3 ratios of 13.4%, 10.8%, and 75.8% respectively). From this, the conjugation ratio (DAR) of the antibody with the toxin is calculated to be 7.0.

[0773] Example 51 LC-MS determination of the molecular weight of BT001013

[0774] Using a method similar to that in Example 48, the results are as Figure 12 and 13 shown.

[0775] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001013 obtained after conjugating TL048 with the antibody are shown in the following table:

[0776]

[0777] From Figure 12 and 13 it can be known that in BT001013, the antibody light chain is conjugated with 0 to 1 toxin (LC, with DAR1 ratios of 6.8% and 93.2% respectively), and the heavy chain is conjugated with 1 to 4 toxins (DAR1, DAR2, DAR3, and DAR4 ratios of 12.8%, 12.8%, 64.9%, and 9.5% respectively). From this, the conjugation ratio (DAR) of the antibody with the toxin is calculated to be 7.3.

[0778] Example 52. Determination of the molecular weight of BT001018 by LC-MS

[0779] Using a method similar to that in Example 48, the results are as Figure 14 and 15 shown.

[0780] The theoretical and measured molecular weights of the light and heavy chains of BT001018 obtained after coupling TL030 with the antibody (calculated based on the main glycoform G0F) are shown in the following table:

[0781]

[0782] From Figure 14 and 15 it can be seen that in BT001018, the antibody light chain is coupled with 0 to 1 toxin (LC, with DAR1 ratios of 55.3% and 44.7% respectively), and the heavy chain is coupled with 1 to 3 toxins (DAR1, DAR2, DAR3 ratios of 19.6%, 23.3%, and 49.6% respectively). From this, the coupling ratio (DAR) of the antibody to the toxin is calculated to be 5.2.

[0783] Example 53. Determination of the molecular weight of BT001021 by LC-MS

[0784] Perform LCMS molecular weight analysis on the coupled BT001021.

[0785] Chromatographic determination conditions:

[0786] Liquid chromatography column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0787] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0788] Flow rate: 0.25 mL / min; Sample chamber temperature: 8°C; Column temperature: 60°C; Injection volume: 1 μg;

[0789] 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

[0790] Mass spectrometry determination conditions:

[0791] Mass spectrometry model: Triple TOF 5600+;

[0792] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m / z600 - 5000;

[0793] The results are asFigure 16 and 17 as shown

[0794] The theoretical and measured molecular weights of the light and heavy chains of BT001021 obtained after coupling TL033 with an antibody (calculated based on the main glycoform G0F) are shown in the following table:

[0795]

[0796]

[0797] From Figure 16 and 17 it can be seen that in BT001021, the antibody light chain is coupled with 0 - 1 toxin (LC, DAR1 ratios are 4.5% and 95.5% respectively), and the heavy chain is coupled with 1 - 3 toxins (DAR1, DAR2, DAR3 ratios are 15.3%, 17.6%, and 67.1% respectively). From this, the antibody-to-toxin conjugation ratio (DAR) is calculated to be 6.9.

[0798] Example Fifty-Four: LC-MS Determination of the Molecular Weight of BT001023

[0799] Using a method similar to that in Example Forty-Eight, the results are as Figure 18 and 19 shown

[0800] The theoretical and measured molecular weights of the light and heavy chains of BT001023 obtained after coupling TL035 with an antibody (calculated based on the main glycoform G0F) are shown in the following table:

[0801]

[0802] From Figure 18 and 19 it can be seen that in BT001023, the antibody light chain is coupled with 0 - 1 toxin (LC, DAR1 ratios are 15% and 85% respectively), and the heavy chain is coupled with 0 - 3 toxins (HC, DAR1, DAR2, DAR3 ratios are 6.7%, 16.7%, 12.7%, and 63.9% respectively). From this, the antibody-to-toxin conjugation ratio (DAR) is calculated to be 6.4.

[0803] Example Fifty-Five: LC-MS Determination of the Molecular Weight of BT001040

[0804] Perform LC-MS molecular weight analysis on the conjugated BT001040

[0805] Liquid chromatography column: Thermo MabPacTM RP 4μm, 3.0mm * 100mm

[0806] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN

[0807] Flow rate: 0.25 mL / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 1 μg

[0808] 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

[0809] Mass spectrometry conditions:

[0810] Mass spectrometry model: Triple TOF 5600+

[0811] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5000; DP 250; m / z 600 - 5000

[0812] The theoretical and measured molecular weights of the light and heavy chains of BT001040 obtained after coupling TL049 with the antibody (calculated based on the main glycoform G0F) are shown in the following table:

[0813]

[0814] From Figure 20 and 21 it can be seen that in BT001040, the antibody light chain is coupled with 0 - 1 cell bioactive molecules (the proportions of LC and DAR1 are 4.9% and 95.1% respectively), and the heavy chain is coupled with 1 - 4 cell bioactive molecules (the proportions of DAR1, DAR2, DAR3, and DAR4 are 16.5%, 14.3%, 52.6%, and 16.6% respectively). From this, the coupling ratio (DAR) of the antibody with the cell bioactive molecule is calculated to be 7.3.

[0815] Example Fifty-Six LC-MS Determination of the Molecular Weight of BT001041

[0816] Using a method similar to that in Example Fifty-Five, the results are as Figure 22 and 23 shown.

[0817] The theoretical and measured molecular weights of the light and heavy chains of BT001041 obtained after coupling TL050 with the antibody (calculated based on the main glycoform G0F) are shown in the following table:

[0818]

[0819] From Figure 22 and 23It can be seen that in BT001041, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, with DAR1 ratios of 10.5% and 89.5% respectively), and the heavy chain is conjugated with 1 to 4 cell bioactive molecules (DAR1, DAR2, DAR3, DAR4 ratios of 21.3%, 14.8%, 57.9%, and 6.0% respectively). From this, the conjugation ratio (DAR) of the antibody to the cell bioactive molecule is calculated to be 6.8.

[0820] Example Fifty-Seven: Determination of the Molecular Weight of BT001042 by LC-MS

[0821] Using a method similar to that in Example Fifty-Five, the results are as Figure 24 and 25 shown.

[0822] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001042 obtained after conjugation of TL051 with the antibody are shown in the following table:

[0823]

[0824] From Figure 24 and 25 it can be seen that in BT001042, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, with DAR1 ratios of 14.9% and 85.1% respectively), and the heavy chain is conjugated with 1 to 3 cell bioactive molecules (DAR1, DAR2, DAR3 ratios of 19.7%, 9.4%, and 70.9% respectively). From this, the conjugation ratio (DAR) of the antibody to the cell bioactive molecule is calculated to be 6.7.

[0825] Example Fifty-Eight: Determination of the Molecular Weight of BT001043 by LC-MS

[0826] Using a method similar to that in Example Fifty-Five, the results are as Figure 26 and 27 shown.

[0827] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001043 obtained after conjugation of TL052 with the antibody are shown in the following table:

[0828]

[0829]

[0830] From Figure 26 and 27It is known that in BT001043, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, with DAR1 ratios of 9.1% and 90.9% respectively), and the heavy chain is conjugated with 1 to 3 cell bioactive molecules (DAR1, DAR2, DAR3 ratios of 20.1%, 11.4%, and 68.4% respectively). From this, the conjugation ratio (DAR) of the antibody to the cell bioactive molecule is calculated to be 6.8.

[0831] Example Fifty-Nine: Determination of the Molecular Weight of BT001044 by LC-MS

[0832] Using a method similar to that in Example Fifty-Five, the results are as Figure 28 and 29 shown.

[0833] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001044 obtained after conjugation of TL053 with the antibody are shown in the following table:

[0834]

[0835] From Figure 28 and 29 it is known that in BT001044, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, with DAR1 ratios of 23.0% and 77.0% respectively), and the heavy chain is conjugated with 1 to 3 cell bioactive molecules (DAR1, DAR2, DAR3 ratios of 19.4%, 11.4%, and 69.3% respectively). From this, the conjugation ratio (DAR) of the antibody to the cell bioactive molecule is calculated to be 6.5.

[0836] Example Sixty: Determination of the Molecular Weight of BT001046 by LC-MS

[0837] Using a method similar to that in Example Fifty-Five, the results are as Figure 30 and 31 shown.

[0838] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001046 obtained after conjugation of TL055 with the antibody are shown in the following table:

[0839]

[0840]

[0841] From Figure 30 and 31It can be seen that in BT001046, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, the DAR1 ratios are 33.8% and 66.2% respectively), and the heavy chain is conjugated with 0 to 3 cell bioactive molecules (the DAR0, DAR1, DAR2, and DAR3 ratios are 21.9%, 6.1%, 9.6%, and 62.3% respectively). From this, the conjugation ratio (DAR) of the antibody to the cell bioactive molecule is calculated to be 5.6.

[0842] Example 61 Determination of the molecular weight of BT001047 by LC-MS

[0843] Using a method similar to that in Example 55, the results are as Figure 32 and 33 shown.

[0844] The theoretical and measured molecular weights (calculated based on the main glycoform G0F) of the light and heavy chains of BT001047 obtained after conjugation of TL056 with the antibody are shown in the following table:

[0845]

[0846] From Figure 32 and 33 it can be seen that in BT001047, the antibody light chain is conjugated with 0 to 1 cell bioactive molecule (LC, the DAR1 ratios are 13.7% and 86.3% respectively), and the heavy chain is conjugated with 1 to 3 cell bioactive molecules (the DAR1, DAR2, and DAR3 ratios are 22.2%, 13.5%, and 64.3% respectively). From this, the conjugation ratio (DAR) of the antibody to the cell bioactive molecule is calculated to be 6.6.

[0847] Example 62 Size-exclusion chromatography analysis

[0848] The conjugation reaction was monitored by SEC-HPLC, and the conjugate was detected by SEC.

[0849] Chromatographic conditions:

[0850] Liquid chromatography column: TOSOH TSKgel SuperSW mAb, 4μm, 7.8mm x 300mm;

[0851] Mobile phase: 100 mmol / L Na2HPO4, 100 mmol / L NaCl, 5% isopropanol, pH 7.0;

[0852] Flow rate: 0.5 ml / min; Detection wavelength: 280 nm; Column temperature: room temperature; Sample chamber temperature: 8°C;

[0853] Injection volume: 30 μg; Isocratic elution: 30 min.

[0854] The SEC chromatograms of the antibody conjugated with TL003 to obtain BT001002 and the SEC chromatogram of the molecular weight marker are shown in Figure 7 and 8 respectively. According to the molecular weight markers of the molecular weight marker, it is confirmed that the molecular weight corresponding to the main peak of the conjugated product is approximately 150 kD, that is, BT001002 obtained by conjugating the antibody with TL003, and the heavy and light chains are not separated, and the antibody still maintains its overall structure.

[0855] The SEC chromatogram of BT001004 obtained by conjugating TL019 with the antibody is shown in Figure 9 As shown. According to the SEC retention time and the peak area ratio, it is confirmed that the molecular weight of the main conjugated product is approximately 150 kD, that is, BT001004 obtained by conjugating TL019 with the antibody, and the complete structure of the antibody is still maintained.

[0856] The SEC chromatogram of BT001012 obtained by conjugating TL024 with the antibody is shown in Figure 34 As shown. According to the SEC retention time and the peak area ratio, it is confirmed that the molecular weight of the main conjugated product is approximately 150 kD, that is, BT001012 obtained by conjugating TL024 with the antibody, and the complete structure of the antibody is still maintained.

[0857] The SEC chromatogram of BT001013 obtained by conjugating TL048 with the antibody is shown in Figure 35 As shown. According to the SEC retention time and the peak area ratio, it is confirmed that the molecular weight of the main conjugated product is approximately 150 kD, that is, BT001013 obtained by conjugating TL048 with the antibody, and the complete structure of the antibody is still maintained.

[0858] The SEC chromatogram of BT001018 obtained by conjugating TL030 with the antibody is shown in Figure 36 As shown. According to the SEC retention time and the peak area ratio, it is confirmed that the molecular weight of the main conjugated product is approximately 150 kD, that is, BT001018 obtained by conjugating TL030 with the antibody, and the complete structure of the antibody is still maintained.

[0859] The SEC chromatogram of BT001021 obtained by conjugating TL033 with the antibody is shown in Figure 37 As shown. According to the SEC retention time and the peak area ratio, it is confirmed that the molecular weight of the main conjugated product is approximately 150 kD, that is, BT001021 obtained by conjugating TL033 with the antibody, and the complete structure of the antibody is still maintained.

[0860] The SEC chromatogram of BT001023 obtained by conjugating TL035 with the antibody is shown in Figure 38As shown, based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main conjugate product was approximately 150 kD, that is, BT001023 obtained by conjugating TL035 with the antibody, still maintained the intact structure of the antibody.

[0861] The SEC chromatogram of BT001042 obtained by conjugating TL051 with the antibody is as Figure 39 shown. Based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main conjugate product was approximately 150 kD, that is, BT001042 obtained by conjugating TL051 with the antibody, still maintained the intact structure of the antibody.

[0862] The SEC chromatogram of BT001043 obtained by conjugating TL052 with the antibody is as Figure 40 shown. Based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main conjugate product was approximately 150 kD, that is, BT001043 obtained by conjugating TL052 with the antibody, still maintained the intact structure of the antibody.

[0863] The SEC chromatogram of BT001044 obtained by conjugating TL053 with the antibody is as Figure 41 shown. Based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main conjugate product was approximately 150 kD, that is, BT001044 obtained by conjugating TL053 with the antibody, still maintained the intact structure of the antibody.

[0864] The SEC chromatogram of BT001046 obtained by conjugating TL055 with the antibody is as Figure 42 shown. Based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main conjugate product was approximately 150 kD, that is, BT001046 obtained by conjugating TL055 with the antibody, still maintained the intact structure of the antibody.

[0865] The SEC chromatogram of BT001047 obtained by conjugating TL056 with the antibody is as Figure 43 shown. Based on the SEC retention time and peak area ratio, it was confirmed that the molecular weight of the main conjugate product was approximately 150 kD, that is, BT001047 obtained by conjugating TL056 with the antibody, still maintained the intact structure of the antibody.

[0866] Example 63: Detection of the inhibitory effects of bioactive molecules and antibody-drug conjugates on in vitro cell viability

[0867] First, culture tumor cells MDA-MB-468 (Trop-2 positive cell line) and HCC1806 (Trop-2 positive cell line); co-culture the bioactive molecules and ADC molecules of the present disclosure with the tumor cells, then add CCK8 reagent (Dongren Chemical Technology Co., Ltd., Cat: CK04, Lot: JJ744), and read the value with an enzyme-linked immunosorbent assay instrument (manufacturer: Molecular Devices, model: SpectraMax M2) (the detection wavelength is 450 nm) to detect the activity of dehydrogenase in mitochondria, so as to evaluate the inhibitory effect of ADC on cell proliferation. The sources of the tumor cells are shown in Table 1.

[0868] Table 1.

[0869] Cell Name Tumor Type Source MDA - MB - 468 Breast Cancer Kangnuotai HCC1806 Breast Cancer Nanjing Kebai Biotechnology

[0870] In vitro cell activity detection: Dilute the bioactive molecules or ADC (12 concentration gradients) with the corresponding detection medium (containing 2% FBS). Digest the tumor cells by the conventional method using trypsin, collect the cells and count them, and resuspend them with the corresponding detection medium (containing 2% FBS). Add the diluted bioactive molecules or ADC into a 96-well plate, and then add the cells. Then add 20 μL of CCK8 reagent to each well, react for 4 hours, and read the value with an enzyme-linked immunosorbent assay instrument (the detection wavelength is 450 nm). The experimental conditions and detection results are shown in Tables 2 and 3.

[0871] Table 2. Cell killing results of bioactive molecules

[0872]

[0873]

[0874] The test results show that the bioactive molecules all have the effect of killing tumor cells.

[0875] Table 3. Cell line killing results of conjugates (ADC)

[0876]

[0877] The test results show that the ADC molecules obtained by the new conjugation method have the effect of killing tumor cells. It shows that the ADC formed by the new conjugation method can produce a killing effect on tumor cells, and the new conjugation method is effective when applied to ADC molecules.

[0878] Example Sixty - Four In vivo pharmacodynamic tests of antibody-drug conjugates and bioactive molecules

[0879] Tested drugs

[0880] Drug name, source, and preparation method:

[0881] BT001021, with a liquid concentration of 5.44 mg / ml, is aliquoted and stored at -20°C. When used, it is diluted with normal saline according to the dose to obtain a test solution.

[0882] Immu-132 (prepared according to Example 2 of WO2015 / 012904A2, DAR = 5.4, also denoted as IMMU-132), with a liquid concentration of 13.158 mg / ml, is aliquoted and stored at -20°C. When used, it is diluted with normal saline according to the dose to obtain a test solution.

[0883] T-030, a solid powder, is formulated into a concentration of 5.2 mg / ml with 100% DMSO (Sigma), aliquoted and stored at -20°C. When used, it is diluted with normal saline according to the dose to obtain a test solution.

[0884] SN-38 (also denoted as SN38), a solid powder, is formulated into a concentration of 3.23 mg / ml with 100% DMSO (Sigma), aliquoted and stored at -20°C. When used, it is diluted with normal saline according to the dose to obtain a test solution.

[0885] Note: The dosage of the toxin is formulated according to the equimolar ratio of the ADC sample.

[0886] The structures of T-030, SN-38, and Immu-132 are as follows:

[0887]

[0888] Experimental animals and cell lines

[0889] Balb / c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK(Jing)2016-0011); gastric cancer cell line NCI-N87 (ATCC), breast cancer cell line HCC1806 (Nanjing Kebai).

[0890] Experimental grouping and evaluation methods

[0891] Tumor-bearing mice with a tumor volume of 100 - 200 mm 3 selected by random grouping (the number of groups is determined according to the number of samples), 6 mice per group. The administration volume is 10 mL / kg, and the administration route is intravenous injection via the tail vein. The tumor diameter is measured with a vernier caliper twice a week, and the tumor volume is calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the major and minor axes of the tumor, respectively. The death of animals is observed and recorded daily.

[0892] The tumor growth inhibition rate TGI(%) is calculated using the following formula to evaluate the antitumor efficacy of the antibody-drug conjugate:

[0893] TGI(%) = [1 - (VT 末 - VT 始 ) / (VC 末 - VC 始 )] * 100%

[0894] Where V T末 : The mean tumor volume at the end of the experiment in the treatment group

[0895] V T始 : The mean tumor volume at the start of drug administration in the treatment group

[0896] V C末 : The mean tumor volume at the end of the experiment in the solvent control group

[0897] V C始 : The mean tumor volume at the start of drug administration in the solvent control group

[0898] The following Experimental Examples 1 and 2 evaluate the inhibition of tumor proliferation in tumor-bearing mice constructed by subcutaneous transplantation of human tumor cells by the antibody conjugate BT001021. Specifically, in Experimental Examples 1 and 2 of this study, tumor-bearing mouse models were constructed by subcutaneous transplantation of human gastric cancer cell line NCI-N87 and human triple-negative breast cancer cell line HCC1806. When the tumor volume grew to about 100 mm 3 or so, they were randomly grouped, and after grouping, BT001021 was administered intravenously 2 times a week for a total of 6 times. The tumor volume and animal body weight changes were measured 2 times a week to evaluate the pharmacodynamic effect (antitumor efficacy) of the antibody-drug conjugate on tumor-bearing mice.

[0899] Experimental Example 1. Inhibition of antibody-drug conjugate and bioactive molecule on NCI-N87

[0900] Experimental method:

[0901] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer model. When the average tumor volume was about 90 mm 3When the tumor size reached a certain value, the mice were randomly grouped into a normal saline group, a BT001021 group (3 mg / kg, intravenous injection, twice a week for 3 weeks), a positive drug Immu-132 group (3 mg / kg, intravenous injection, twice a week for 3 weeks), a T030 group, and an SN38 group. After grouping, the corresponding drugs were injected via 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 4. Figure 44 and 45 .

[0902] Experimental conclusion:

[0903] In this experimental example, the human gastric cancer cell line NCI-N87 was used to establish a subcutaneous xenograft tumor model of human gastric cancer, and the efficacy of BT001021 in the NCI-N87 human gastric cancer-bearing mouse model was evaluated.

[0904] It can be seen from the experimental results that BT001021 (3 mg / kg, intravenous injection, twice a week for 3 weeks) can significantly inhibit the tumor growth of mice in the NCI-N87 gastric cancer xenograft tumor model, and tumor regression occurred at the end of the dosing period. Its anti-tumor activity is superior to that of the positive drug Immu-132. No animal deaths or significant decreases in animal body weight were observed in all treatment groups during the observation period, indicating that BT001021 has no obvious toxicity.

[0905] Table 4. Gastric cancer NCI-N87 model

[0906]

[0907] Experimental Example 2. Inhibition of antibody-drug conjugate on HCC1806

[0908] Experimental method:

[0909] HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC1806 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a breast cancer model. When the average tumor volume reached approximately 130 mm 3 When the tumor size reached a certain value, the mice were randomly grouped into a normal saline group, a BT001021 group (10 mg / kg, intravenous injection, twice a week for 3 weeks), and a positive drug Immu-132 group (10 mg / kg, intravenous injection, twice a week for 3 weeks). After grouping, the corresponding drugs were injected via the tail vein, twice a week for a total of 5 times. After administration, the tumor volume of the mice was observed and measured regularly. The specific results are shown in Table 5. Figure 46 .

[0910] Experimental conclusion:

[0911] In this experimental example, the human breast cancer cell line HCC1806 was used to establish a subcutaneous xenograft model of human breast cancer, and the efficacy of BT001021 administration was evaluated in the HCC1806 human breast cancer-bearing mouse model.

[0912] It can be seen from the experimental results that BT001021 (10 mg / kg, intravenous injection, twice a week for 3 weeks) significantly inhibited the tumor growth in mice of the HCC1806 breast cancer xenograft model, and its anti-tumor activity was superior to that of the positive control Immu-132.

[0913] Table 5: Breast cancer HCC1806 model

[0914]

[0915]

[0916] From Table 4, Table 5 and Figures 44 - 46 It can be seen that the antibody drug of the present invention, BT001021, can significantly inhibit tumor growth in the mouse NCI-N87 model, is significantly superior to Immu-132 at the same dose, and has no obvious weight loss and no obvious drug toxicity. In the mouse HCC1806 model, due to the high malignancy of the tumor, when the dose was increased to 10 mg / kg, Immu-132 did not show obvious inhibitory activity, while BT001021 could significantly inhibit tumor growth. The above results indicate that BT001021 of the present invention has good efficacy and excellent safety.

[0917] In the subcutaneous xenograft models of Experimental Examples 1 and 2, the anti-tumor activity of BT001021 was significantly superior to that of Immu-132 at the same dose. It is speculated that BT001021 has the potential to treat solid tumors, and BT001021 is expected to benefit more patients clinically compared with Immu-132.

[0918] Experimental Example 3. Inhibition of antibody-drug conjugate on HCC827

[0919] Example 3 evaluated the inhibitory effects of BT001021 and BT001035 on the proliferation of a mouse model bearing human tumors constructed by subcutaneous transplantation of HCC827 non-small cell lung cancer cells. Specifically, in this experiment, a mouse model bearing human non-small cell lung cancer cell line HCC827 was established by subcutaneous transplantation. After the tumor volume grew to about 100 mm 3 or so, the mice were randomly grouped. After grouping, BT001021 and BT001035 were administered 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 inhibitory efficacy) of BT001021 and BT001035 on the tumor-bearing mice was calculated.

[0920] Experimental method:

[0921] HCC827 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C under 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 xenograft model. When the average tumor volume reached approximately 80 mm3, the mice were randomly grouped according to tumor size into a normal saline group, a positive drug Immu-132 (10 mg / kg, IV, BIW×3W) group, a BT001021 (10 mg / kg, IV, BIW×3W) group, and a BT001035 (10 mg / kg, IV, BIW×3W) group. After grouping, the corresponding drugs were injected via the tail vein, administered twice a week for a total of 6 times. After drug 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 .

[0922] Experimental conclusion:

[0923] It can be seen from the experimental results that BT001021 and BT001035 can significantly inhibit the tumor growth of mice in the HCC827 non-small cell lung cancer xenograft model, and tumor regression occurred at the end of drug administration. The anti-tumor activity was superior to that of the positive group Immu-132. No animal deaths or significant reduction in animal body weight occurred in all treatment groups during the observation period, and no obvious drug toxicity was shown. The mice had good tolerance to each evaluated drug during the treatment period.

[0924] Table 6. HCC827 model of lung cancer

[0925]

[0926] From Table 6, Figure 47A and Figure 47B it can be seen that both BT001021 and BT001035 had significant anti-tumor growth activities during the evaluation period. At the same dose, their anti-tumor activities were significantly superior to those of Immu-132. During drug administration, there was no obvious weight loss or obvious drug toxicity in each group of animals. The above results indicate that BT001021 and BT001035 have excellent anti-tumor activities.

[0927] In this subcutaneous xenograft model, the anti-tumor activities of BT001021 and BT001035 were both significantly superior to those of Immu-132 at the same dose. It is speculated that BT001021 and BT001035 have the potential to treat solid tumors, and compared with Immu-132, BT001021 and BT001035 are expected to benefit more patients clinically.

[0928] Experimental Example 4.Inhibition of Antibody-Drug Conjugate on NCI-N87

[0929] Experimental Example 4 evaluated the inhibition of the antibody conjugate BT001036 on the tumor proliferation of tumor-bearing mice constructed by subcutaneous transplantation of human tumor cells. Specifically, in this experiment, a tumor-bearing mouse model was constructed by subcutaneous transplantation of the human gastric cancer cell line NCI-N87. When the tumor volume grew to about 140 mm 3 or so, the mice were randomly grouped. After grouping, BT001036 was administered intravenously 2 times a week for a total of 6 times. The tumor volume and animal body weight were measured 2 times a week to evaluate the efficacy (tumor inhibition effect) of the antibody-drug conjugate on the tumor-bearing mice.

[0930] Experimental method:

[0931] NCI-N87 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a gastric cancer xenograft model. When the average tumor volume was about 140 mm 3 , the mice were randomly grouped according to the tumor size into a normal saline group, a BT001036 (1.5 mg / kg, IV, BIW×3W) group, and a BT001036 (3 mg / kg, IV, BIW×3W) group. After grouping, the corresponding drugs were injected into the tail vein 2 times a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The specific results are shown in Table 7, Figure 48A , Figure 48B .

[0932] Table 7. Gastric Cancer NCI-N87 Model

[0933]

[0934] Experimental conclusion:

[0935] In this experimental example, a human gastric cancer subcutaneous xenograft model was constructed by subcutaneous transplantation of the human gastric cancer cell line NCI-N87 to evaluate the efficacy of BT001036 administration in the NCI-N87 human gastric cancer tumor-bearing mouse model.

[0936] It can be seen from the experimental results that both high and low doses (1.5 mg / kg, 3 mg / kg) of BT001036 can significantly inhibit the tumor growth of mice in the NCI-N87 gastric cancer xenograft model, and tumor regression occurred at the end of drug administration, with excellent anti-tumor activity. There were no animal deaths and significant reduction in animal body weight in all treatment groups during the observation period, and no obvious drug toxicity was shown. The mice had good tolerance to each evaluated drug during the treatment period.

[0937] Experimental Example 5.Inhibition of Antibody-Drug Conjugate on MDA-MB-231

[0938] Experimental Example 5 evaluated the inhibitory effect of BT001021 on the proliferation of tumor-bearing mice models constructed by subcutaneous transplantation of human tumor cells of MDA-MB-231 breast cancer. Specifically, in this experiment, a tumor-bearing mouse model was constructed by subcutaneous transplantation of human breast cancer cell line MDA-MB-231. After the tumor volume grew to about 130 mm 3 or so, the mice were randomly grouped. After grouping, BT001021 was administered intravenously 2 times a week for a total of 6 times. At the same time, the tumor volume and animal body weight changes were measured, and the pharmacodynamic effect (tumor inhibitory effect) of BT001021 on tumor-bearing mice was calculated.

[0939] Experimental method:

[0940] NCI-MDA-MB-231 cells were cultured in RPMI1640 culture medium containing 10% fetal bovine serum under the conditions of 37 °C and 5% CO2. MDA-MB-231 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a breast cancer xenograft model. When the average tumor volume was about 130 mm 3 , the mice were randomly grouped according to the tumor size into a normal saline group and a BT001021 (3 mg / kg) group. After grouping, the corresponding drugs were injected into the tail vein 2 times a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The specific results are shown in Table 8, Figure 49A and 49B .

[0941] Experimental conclusion:

[0942] It can be seen from the experimental results that BT001021 can significantly inhibit the tumor growth of mice in the MDA-MB-231 breast cancer xenograft model, and tumor regression occurred at the end of drug administration. There were no animal deaths and significant reduction in animal body weight in all treatment groups during the observation period, and no obvious drug toxicity was shown. The mice had good tolerance to each evaluated drug during the treatment period.

[0943] Table 8. Breast cancer MDA-MB-231 model

[0944]

[0945] In the subcutaneous xenograft model, BT001021 had significant anti-tumor activity. There were no animal deaths and significant reduction in animal body weight in all treatment groups during the observation period, and no obvious drug toxicity was shown. The mice had good tolerance to each evaluated drug during the treatment period.

[0946] Example Sixty - Five Pharmacokinetics Test of Antibody-Drug Conjugate and Active Biomolecule in Vivo

[0947] Experimental Example 6 evaluated the in vivo pharmacokinetics of antibody-drug conjugates and active biomolecules. Specifically, 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. When the tumor volume grew to 100-200 mm 3 , they were randomly grouped. After grouping, BT001021 and T-030 were administered intravenously once. The concentration of T-030 in tumor tissues and sera was measured to evaluate the in vivo pharmacokinetic behavior of antibody-conjugated drug BT001021 and active biomolecule T-030 in tumor-bearing mice.

[0948] Tested drugs

[0949] Drug names and preparation methods:

[0950] BT001021, with a liquid concentration of 20 mg / ml, was aliquoted and stored at -20 °C. When used, it was diluted with physiological saline according to the dose to obtain a test solution;

[0951] T-030 was prepared into 1 mg / ml with dimethyl sulfoxide and diluted with physiological saline according to the dose to obtain a test solution.

[0952] Experimental animals and cell lines:

[0953] Balb / c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011); gastric cancer cell line NCI-N87 (ATCC).

[0954] Experimental grouping and evaluation methods:

[0955] Tumor-bearing mice with a tumor volume of 100-200 mm selected by random grouping (the number of groups was determined according to the number of samples), 4 mice per group, and the administration route was single intravenous injection via the tail vein. 3

[0956] Experimental Example 6. In vivo pharmacokinetic test of BT001021 and T-030 in tumor-bearing mice

[0957] Experimental methods:

[0958] NCI-N87 cells were cultured in 1640 medium containing 10% heat-inactivated fetal bovine serum at 37 °C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into Balb / c-nu mice to establish a gastric cancer xenograft model. When the average tumor volume was about 100-200 mm 3 ​At that time, according to the tumor size, the mice were randomly grouped into a normal saline group, a T-030 (0.23 mg / kg, intravenous injection, single dose) group, and a BT001021 (10 mg / kg, intravenous injection, single dose) group. After grouping, the corresponding drugs were injected via the tail vein. In the T-030 group, serum and tumor tissues were collected at 1 h, 2 h, 4 h, 8 h, 24 h, and 72 h after drug administration (at 72 h after drug administration, T-030 could not be detected in both serum and tumor tissues, so serum and tumor tissues were not collected at 168 h after drug administration). In the BT001021 group, serum and tumor tissues were collected at 1 h, 2 h, 4 h, 8 h, 24 h, 72 h, and 168 h after drug administration. The concentration of T-030 in serum and tumor was detected by LC-MS / MS method, and the specific results are shown in Table 9. The administration dose of T-030 (0.23 mg / kg) was converted in an equimolar manner according to the administration dose of BT001021 (10 mg / kg).

[0959] Table 9. Pharmacokinetic parameters of T-030 in tumor and serum after intravenous administration of T-030 and BT001021 to tumor-bearing mice

[0960]

[0961] Experimental conclusion:

[0962] The AUC of BT001021 (10 mg / kg) administration group in tumor and serum last was 850.1 h*ng / ml and 174.97 h*ng / ml respectively. The AUC of T-030 administration group in tumor and serum last was 3.85 h*ng / ml and 5.58 h*ng / ml respectively. By comparing the three groups, it can be seen that the exposure of T-030 in the BT001021 administration group was significantly increased compared with that in the T-030 administration group. Moreover, the exposure of the active biomolecule T-030 in the tumor of the BT001021 administration group was significantly higher than that in the serum, while the exposure of the active biomolecule in the serum and tumor of the T-030 administration group was basically the same, indicating that the antibody-drug conjugate (BT001021) has strong tumor tissue targeting.

[0963] The C of the active biomolecule T-030 in tumor and serum of the BT001021 (10 mg / kg) administration group max was 7.82 ng / ml and 11.7 ng / ml respectively. The C of the active biomolecule T-030 in tumor and serum of the T-030 administration group max was 1.20 ng / ml and 1.81 ng / ml respectively, indicating that the antibody-drug conjugate (BT001021) has higher concentrations of the active biomolecule (T-030) in tumor tissue and serum.

[0964] In the BT001021 (10 mg / kg) administration group, the T of the active biomolecule T-030 in the tumor 1 / 2 was 93.14 h, and the T of the active biomolecule T-030 in the tumor in the T-030 administration group 1 / 2 was 2.55 h. This indicates that the antibody-drug conjugate (BT001021) has a longer half-life in tumor tissues.

[0965] In summary, compared with the corresponding active biomolecule (T-030), BT001021 has significant tumor tissue targeting and good pharmacokinetic properties.

[0966] Experimental Example 7. In vivo pharmacokinetic test of antibody-drug conjugates BT001021 and Immu-132.

[0967] In this experiment, a tumor-bearing mouse model was constructed by subcutaneously transplanting the human gastric cancer cell line NCI-N87 into Balb / c-nu mice. When the tumor volume grew to 100 - 200 mm 3 , they were randomly grouped, and after grouping, BT001021 and Immu-132 were administered intravenously once. The concentrations of the active biomolecules T-030 and SN-38 corresponding to BT001021 and Immu-132 in tumor tissues and serum were measured to evaluate the in vivo pharmacokinetic behavior of the antibody-conjugated drugs BT001021 and Immu-132 in tumor-bearing mice.

[0968] Tested drugs

[0969] Drug name, preparation method:

[0970] BT001021, with a liquid concentration of 20 mg / ml, was aliquoted and stored at -20 °C. When used, it was diluted with normal saline according to the dose to obtain the test solution;

[0971] Immu-132 was diluted with normal saline according to the dose to obtain the test solution.

[0972] Experimental animals and cell lines:

[0973] Balb / c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016 - 0011); gastric cancer cell line NCI-N87 (ATCC).

[0974] Experimental grouping and evaluation method:

[0975] Tumor-bearing mice with a tumor volume of 100 - 200 mm 3 selected by random grouping (the number of groups is determined according to the number of samples), 4 mice per group, and the administration route was a single intravenous injection via the tail vein.

[0976] Experimental method:

[0977] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 37 °C under 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into Balb / c-nu mice to establish a gastric cancer xenograft model. When the average tumor volume reached about 100 - 200 mm 3 3, the mice were randomly grouped according to tumor size into the BT001021 (5 mg / kg, intravenous injection, single dose) group and the Immu-132 (5 mg / kg, intravenous injection, single dose) group. After grouping, the corresponding drugs were injected via the tail vein. Serum and tumor tissues were collected at 2 h, 24 h, 48 h, and 72 h after drug administration, and the concentrations of T-030 or SN-38 in the serum and tumor were detected by LC-MS / MS method.

[0978] Table 10. Pharmacokinetic parameters of T-030 and SN-38 in tumors and sera of tumor-bearing mice after intravenous administration of BT001021 and Immu-132

[0979]

[0980] Experimental conclusion:

[0981] The AUCs of the toxin small molecule in the tumor and serum of the BT001021 administration group last were 427.2 h*ng / ml and 115.3 h*ng / ml respectively, and the AUCs of the toxin small molecule in the tumor and serum of the Immu-132 administration group last were 116.8 h*ng / ml and 422.7 h*ng / ml respectively. The C of the toxin small molecule in the tumor of the BT001021 administration group max was 6.8 ng / ml, and the C of the toxin small molecule in the tumor of the Immu-132 administration group max was 2.8 ng / ml. 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.

[0982] 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 that have been published, and these changes are within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein, The compound of formula (I) is as follows: 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, And the position 1 of L1 is connected to T; L2 is selected from and is connected to L1 at the 1-position of L2; L3 is a triazole optionally substituted by one or more R7; wherein, R7 is independently selected from hydrogen, deuterium, C 1-4 alkyl and C 1-4 alkoxy; L4 is selected from and is connected to E at the 2-position of L4; E is an optionally substituted pyrimidinyl group substituted by one or more R 12 ; wherein R 12 is independently selected from hydrogen, deuterium, halogen, C 1-6 alkyl and halo C 1-6 alkyl; G is selected from F, Cl, Br, Oms, Ots, mesyl group and tosyl group; m1 is 0, 1 or 2; m2 is 0 or 1; m3 is 1.

2. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from Cit-Val, 3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, wherein, L2 is selected from 4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, L3 is triazole.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, E is pyrimidinyl.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, G is mesyl group.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein, T is selected from 8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein, The compound of formula (I) is as follows:

9. The compound of formula (III) or a pharmaceutically acceptable salt thereof, Among them, R 14 Selected from R 15 Substituted acyl or sulfonyl, R 15 Selected from C 1-6 Alkyl, halo C 1-6 Alkyl, 6-10-membered aryl and 5-12-membered heteroaryl.

10. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein, R 15 selected from C 1-6 alkyl or halo C 1-6 alkyl.

11. The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein, R 15 Selected from C 1-6 alkyl groups.

12. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from 13. The compound of formula (IV) or a pharmaceutically acceptable salt thereof, Wherein R 16 is selected from H (hydrogen), D (deuterium), C 1-6 alkyl, R 17 substituted C 1-6 alkyl, R 17 is selected from aryl, heteroaryl, m 11 is selected from 0, 1, 2.

14. The compound according to claim 13 or a pharmaceutically acceptable salt thereof, wherein, R 16 Selected from R 17 Substituted C 1-6 Of the alkyl group, R 17 Selected from aryl, heteroaryl.

15. The compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein, R 17 Selected from phenyl and pyridyl.

16. The compound according to claim 14 or 15, or a pharmaceutically acceptable salt thereof, wherein, m 11 Selected from 0.

17. The compound or a pharmaceutically acceptable salt thereof according to claim 16, wherein, The compound is selected from:

18. The compound according to claim 17 or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from:

19. A compound or a pharmaceutically acceptable salt thereof according to claim 14 or 15, wherein, m 11 Selected from 1. The compound according to claim 19 or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from:

21. The compound according to claim 13 or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from:

22. A compound or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from:

23. A pharmaceutical composition, which comprises the compound according to any one of claims 9-22 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutical excipients.

24. Use of the compound according to any one of claims 9-22 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 15 in the preparation of a drug for treating a disease associated with abnormal cell activity (such as a cancer disease).

25. The use according to claim 24, wherein the cancer disease is selected from solid tumors or non-solid tumors; for example, selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as 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 lymphoma, central nervous system tumors (such as glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer and thyroid cancer.

26. A method for preparing a conjugate, wherein, The conjugate contains a bioactive molecular fragment, a linker and a targeting moiety, and the preparation method includes the step of preparing a linker using the following compound or a pharmaceutically acceptable salt thereof; The compound or a pharmaceutically acceptable salt thereof, which is selected from: (1) (2) 6-(2-(Mesyl)pyrimidin-5-yl)-5-hexynoic acid; (3) 6-(2-(Methylthio)pyrimidin-5-yl)-5-hexynoic acid; (4) Methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate; or (5) 5-Bromo-2-methylthiopyrimidine.

27. The preparation method according to claim 26, wherein The preparation method further includes the step of reacting the linker with a bioactive molecular fragment or a targeting moiety.

28. The preparation method according to claim 25 or 26, wherein The structure of the conjugate is as shown in formula (Ia): {T-[L1-(L2)m1-(L3)m2-(L4)m3-E]} γ -A Formula (Ia) Wherein, T, L1, L2, L3, L4, E, m1, m2 and m3 are as defined in any one of claims 1-8, A is a targeting moiety, and γ is an integer or a decimal between 5 and 8.

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