Bioactive conjugate, preparation method therefor and use thereof

Abstract

The present invention relates to a bioactive conjugate, a preparation method therefor, and use thereof. In particular, the present invention relates to a novel type of bioactive molecular conjugates obtained by improving the coupling of a drug to a targeted moiety in ADC or SMDC, a preparation method therefor, and use thereof in the preparation of drugs for treating diseases associated with abnormal cellular activity.

Description

Bioactive compound conjugates, their preparation methods and uses Technical Field

[0001] This disclosure pertains to the field of pharmaceutical technology and relates to bioactive conjugates, methods for their preparation, and their use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to their use in the prevention and / or treatment of tumor diseases. Background Technology

[0002] Chemotherapy was once the standard treatment for cancer, but highly toxic bioactive molecules can mistakenly kill normal cells, causing serious side effects. Targeted antitumor drugs, possessing both targeting and antitumor activity, have become a hot topic in current cancer research. Since the 20th century, breakthroughs have been made in the development of antitumor drugs and targeted cancer therapy using macromolecular drugs (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, while macromolecular drugs have strong targeting capabilities, their efficacy against solid tumors is limited; and while bioactive molecules have high killing power against cancer cells, they often lack targeting specificity, frequently damaging normal cells and causing serious toxic side effects.

[0003] Recent studies have discovered that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting ability of antibodies with the activity of bioactive molecules, becoming a kind of "biological missile." Antibodies guide ADCs to bind to target cells, where they are then internalized, releasing drugs to treat diseases. Because antibodies have specificity and targeting for tumor cell-related targets, their application value lies not only in treatment but also in serving as ideal carriers for targeted drug delivery, reducing drug side effects. Small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs) share the same design principle: chemically conjugating bioactive molecules with small molecule ligands that selectively bind to receptors on the surface of tumor cells, thereby enhancing the targeting of effector molecules to tumor cells. The difference between SMDCs and ADCs is that SMDCs use small molecule ligands instead of antibodies. Currently, no SMDCs are commercially available.

[0004] Currently, there are four marketed ADCs: Mylotarg (Gemtuzumab Ozogamicin), Adcetris (Brentuximab Vedotin, CD30 monoclonal antibody-MMAE), Kadcyla (Trastuzumab Emtansine, trastuzumab-matansine alkaloid), and Besponsa (Inotuzumab ozogamicin, CD22 monoclonal antibody-kazidromycin). Typically, ADC drugs consist of an antibody, a bioactive molecule, and a linker. The bioactive molecule is covalently coupled to the antibody via the linker; the antibody (e.g., a monoclonal antibody) specifically recognizes specific targets on the surface of tumor cells, thereby guiding the ADC to the surface of cancer cells and allowing the ADC to enter the cancer cells through endocytosis; then, the bioactive molecule is released within the cancer cells, achieving the effect of specifically killing cancer cells without damaging normal tissue cells.

[0005] Lysine is the most common linker site in antibodies. Its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond. Currently, there are techniques for site-specific coupling, where the carboxyl group of the linker is activated by an activating group, which then forms an amide bond with a specific lysine ε-amino group in the antibody, completing the coupling. However, these amide bonds are easily hydrolyzed by enzymes in vivo, causing the bioactive molecule to detach from the antibody before reaching the target cells. This results in the loss of the ADC's targeting ability and increased toxicity.

[0006] Typically, the thiols on antibody cysteine ​​residues exist as disulfide bonds. Breaking these disulfide bonds in an antibody provides multiple free thiol groups as coupling sites. Coupling with antibody thiol groups can occur through either a Michael addition reaction between the free thiol group on the antibody and maleimide, or through two Michael addition reactions between a specific substrate and the free thiol group on the antibody, forming a uniquely structured thiolated bridge. However, numerous studies have reported that ADCs obtained via the thiol Michael addition method undergo reverse Michael addition in systemic circulation, resulting in toxic reactions. Patent WO2016142049 discloses a bioactive molecule and linker structure containing a methanesulfonyl-substituted phenylbenzadiazole, using amatoxins as the bioactive molecule, but does not specifically describe the antibody coupling process.

[0007] Summary of the Invention

[0008] This invention discovers a novel class of bioactive conjugates obtained by improving the conjugation method between the drug and the target moiety in ADCs or SMDCs. These conjugates exhibit high stability, extremely high conjugation efficiency (90%), and high drug loading (DAR value 5-8), and this disclosure is based on these findings. Through in-depth research, we surprisingly found that the ADCs of this invention, such as BT001021 (Example 32), after intravenous administration, show significantly higher exposure of the bioactive small molecule toxin in tumor tissue than in plasma tissue. Furthermore, Immu-132 ADC, under the same administration route, shows significantly higher plasma exposure than tumor tissue exposure, thus the ADCs of this invention have a better therapeutic window than Immu-132. We also surprisingly found that the ADCs of this invention have better therapeutic effects than Immu-132 in animal models of gastric cancer, breast cancer, and non-small cell lung cancer.

[0009] The first aspect of this disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof.

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

[0011] Formula (I)

[0012] Wherein, T is a bioactive molecular fragment, preferably a molecular fragment with antitumor bioactivity;

[0013] L1 is selected from amino acids, peptides composed of 2-10 amino acids, oligosaccharides, and -(CH2). t1 -、-(CH2CH2O) t1 -(CH2) t2 -、 In this context, each of R, R', R1, and R2 is independently H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, cyano group, or C. 1-6 Alkyl, Halogenated C 1-6 Alkyl (e.g., -CF3), cyano-substituted C 1-6 Alkyl groups (e.g., -CH2CN), C 1-6 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl group, C 3-6Cycloalkyl, 6-10 aryl or 5-12 heteroaryl, each Z1 is independently an amino acid or a peptide composed of 2-10 amino acids, each t1 and t2 is independently 0, 1, 2, 3, 4, 5 or 6, each x1 and x2 is independently 0, 1, 2, 3, 4, 5 or 6, each x3 is independently 0, 1, 2, 3 or 4, and the 1 position of L1 is connected to T;

[0014] L2 is selected from amino acids, peptides composed of 2-10 amino acids, oligosaccharides, and -(CH2). t1 -、-(CH2CH2O) t1 -(CH2) t2 -、 Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CN, and C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl group and C 3-6 Cycloalkyl, or R3, R4 or R5, R6 or R3, R5 together with the carbon atom to which they are attached to form a 3-8 membered ring, t1 and t2 are each independently 0, 1, 2, 3, 4, 5 or 6, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L2 is connected to L1 at position 1;

[0015] L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, 3-8 membered cycloalkyl, 3-8 membered alicyclic, 6-12 membered bridged heterocyclic, 6-12 membered spirocyclic, 6-12 membered fused heterocyclic, 6-10 membered aryl (e.g., phenyl or naphthyl), 5-12 membered heteroaryl, and 3-8 membered cycloalkyl-W-; wherein W is oxygen or NR8, and R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CN, carboxyl, sulfonic acid, C 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 alkenyl and C 2-10 The alkynyl group, R8, is independently selected from H (hydrogen), D (deuterium), C (hydrogen), D (deuterium), and C (hydrogen). 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Alkoxy and cyano C 1-2 Alkyl group, and L3 is connected to L2 at position 1;

[0016] L4 is selected from Among them, Z5 is preferred from C 2-6 Olefins, C 2-6 Alkynes, amides, sulfones, sulfoxides, 6-10 aryl groups, 5-6 heteroaryl groups; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene, 6-10 aryl and 5-14 heteroaryl; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene, Halogenated C 1-6 alkylene and alkoxy-substituted C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6; and L4 is attached to E at the 2 position;

[0017] E is selected from one or more R. 12 The following groups are substituted: 6-10 aryl, 5-14 heteroaryl; wherein, R 12 Independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl and Halogenated C 1-6 alkyl;

[0018] G is the leaving group for nucleophilic substitution reactions; such as halogen, sulfonyl, sulfonate, nitro, etc.

[0019] m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0020] In some preferred embodiments, L1 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, and peptides composed of 2-5 amino acids. In this context, each of R, R', R1, and R2 is independently H (hydrogen), D (deuterium), and C (carbon). 1-6 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 3-6Cycloalkyl, Z1 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x1 is 0, 1, 2 or 3, x3 is 0, 1, 2, 3 or 4.

[0021] 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, Where R, R', and R1 are each independently H (hydrogen), D (deuterium), and C (carbon). 1-6 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 3-6 Cycloalkyl, Z1 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x1 and x3 are each independently 0, 1, 2 or 3.

[0022] In some preferred embodiments, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, In this context, 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 0, 1 or 2 independently.

[0023] In some preferred embodiments, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val,

[0024] In some preferred embodiments, L1 is selected from

[0025] In some preferred embodiments, L2 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, and peptides composed of 2-5 amino acids. Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CF3, CN, CH2CN, and C. 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group and C 3-6 The cycloalkyl group, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is connected to L1 at position 1;

[0026] m1 can be 0, 1, 2 or 3.

[0027] 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, Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CF3, CN, CH2CN, and C. 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group and C 3-6 The cycloalkyl group, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is connected to L1 at position 1;

[0028] m1 can be 0, 1, or 2.

[0029] In some preferred embodiments, L2 is selected from Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), and C. 1-4 Alkyl groups, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is connected to L1 at position 1;

[0030] m1 is 1.

[0031] In some preferred embodiments, L2 is selected from

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

[0033] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, 3-8 membered cycloalkyl, 3-8 membered alicyclic, 6-12 membered bridged heterocyclic, 6-12 membered spirocyclic, 6-12 membered fused heterocyclic, 6-10 membered aryl, 5-12 membered heteroaryl, and 3-8 membered cycloalkyl-W-; wherein W is oxygen or NR8, and R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 Alkyne group; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spirocyclic group, or 6-12 membered fused heterocyclic group contains one or more nitrogen atoms; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spirocyclic group, or 6-12 membered fused heterocyclic group contains one or more quaternized nitrogen atoms; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spirocyclic group, or 6-12 membered fused heterocyclic group contains one or more nitrogen atoms, wherein at least one nitrogen atom is substituted with =O; R8 is independently selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, C 2-6 alkenyl, C 3-6 alkynyl group, C 3-6 cycloalkyl, C 1-6 Alkoxy and cyano C 1-2 alkyl;

[0034] m2 can be 0, 1, 2, or 3.

[0035] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, 3-6 membered alicyclic groups, and 5-10 membered heteroaryl groups; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 Alkyne group; preferably, the 3-6 membered alicyclic group contains one or more nitrogen atoms; preferably, the 3-6 membered alicyclic group contains one or more quaternized nitrogen atoms; preferably, the 3-6 membered alicyclic group contains one or more nitrogen atoms, wherein at least one nitrogen atom is substituted with =O;

[0036] m2 can be 0, 1, or 2.

[0037] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino or 5-6 heteroaryl; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 Alkyne group; m2 is 0 or 1.

[0038] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, N-methylpiperidine, pyrazole, and triazole; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 Alkyne group; m2 is 0 or 1.

[0039] In some preferred embodiments, L3 is selected from triazole; m2 is 0 or 1.

[0040] In some preferred embodiments, L3 is selected from m2 is 0 or 1; preferably, the 1 position of L3 is connected to L2.

[0041] In some preferred embodiments, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino,

[0042]

[0043] R7 is independently selected from H (hydrogen), D (deuterium), =O, CN, CH2CN, methyl, CF3;

[0044] W is NR8, and R8 is selected from H (hydrogen), D (deuterium), and C. 1-6 Alkyl, C 2-6 alkenyl, C 3-6 alkynyl group and C 3-6 Cycloalkyl.

[0045] In some preferred embodiments, L3 is selected from

[0046]

[0047] Among them, R q Each is independently selected from C 1-6 Alkyl, C 2-6 alkenyl, C 3-6 alkynyl group and C3-8 Cycloalkyl; β1 is 0, 1 or 2; β2 is 1, 2 or 3.

[0048] In some preferred embodiments, L3 is selected from

[0049] In some preferred embodiments, L4 is selected from Z4 is a 6-10 aryl group or a 5-6 heteroaryl group; R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position;

[0050] m3 is selected from 0, 1, 2 or 3.

[0051] In some preferred embodiments, L4 is selected from Where Z4 is a benzene ring, R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene, or R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position;

[0052] m3 is selected from 0, 1, 2 or 3.

[0053] In some preferred embodiments, L4 is selected from Z4 is a 5-6 member heteroaryl group; R 10 Selected from H (hydrogen), C 1- 6-alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position;

[0054] m3 is selected from 0, 1, 2 or 3.

[0055] In some preferred embodiments, L4 is selected from

[0056] m3 is 1.

[0057] In some preferred embodiments, L4 is selected from

[0058] m3 is 1.

[0059] In some preferred embodiments, L4 is selected from

[0060] m3 is 1.

[0061] In some preferred embodiments, E is selected from one or more R. 12 Substituted 5-10 aryl groups; of which, R 12 Independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and Halogenated C 1-4 alkyl.

[0062] In some preferred embodiments, E is selected from one or more R. 12 The following groups are substituted: pyrimidine, quinazoline, and pyrrolo[2,3-d]pyrimidine; wherein, R 12 Independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl and Halogenated C 1-2 alkyl.

[0063] In some preferred embodiments, E is selected from one or more R. 12 Substituted pyrimidines; wherein, R 12 It is independently selected from H (hydrogen) and D (deuterium).

[0064] In some preferred embodiments, G is selected from halogens, OMs, OTs, OTf, nitro, and optionally oxidized by one or more Rs. 13 The following groups are substituted: alkyl thioether group, aryl thioether group, heteroaryl thioether group, alkyl sulfoxide group, aryl sulfoxide group, heteroaryl sulfoxide group, alkyl sulfonyl group, aryl sulfonyl group, heteroaryl sulfonyl group; wherein, R13 Independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Alkoxy, 6-10 aryl and 5-12 heteroaryl.

[0065] In some preferred embodiments, G is selected from F, Cl, Br, I, OMs, OTs, OTf, methanesulfonyl, ethanesulfonyl, p-toluenesulfonyl, and naphthalenesulfonyl.

[0066] In some preferred embodiments, G is selected from F, Cl, Br, OMs, OTs, methanesulfonyl and p-toluenesulfonyl.

[0067] In some preferred embodiments, G is selected from Cl and methanesulfonyl.

[0068] In some preferred embodiments, In this context, G is preferably methanesulfonyl, E is preferably pyrimidine, and m3 is 1.

[0069] In some preferred embodiments, for Wherein, m4 is preferably an integer from 0 to 6, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0070] In some preferred embodiments, for Wherein, m5 is preferably an integer from 0 to 6, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0071] In some preferred embodiments, for Wherein, m6 is preferably an integer from 0 to 6, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0072] In some preferred embodiments, for Where m7 is selected from integers from 1 to 5, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0073] In some preferred embodiments, for Where m8 is selected from integers from 1 to 5, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0074] In some preferred embodiments, for Where m9 is selected from integers from 1 to 5, R13 Selected from hydrogen, C 1-6 Alkyl and methanesulfonyl groups are substituents for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0075] In some preferred embodiments, for Where m 10 The integers are selected from 0 to 6, and Z4 is selected from 5-6 heteroaryl groups; methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

[0076] In some preferred embodiments, for Z4 is derived from pyridine, pyrimidine, pyrazole, thiazole, oxazole, and triazole, wherein the methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring. More preferably, m 10 Integers selected from 0 to 6.

[0077] In some preferred embodiments, for Z4 is derived from pyridine, pyrimidine, pyrazole, and triazole. More preferably, m 10 Integers selected from 0 to 6.

[0078] In some preferred embodiments, for Z4 is an oxazole and a thiazole, where the methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring. More preferably, m 10 Integers selected from 0 to 6.

[0079] In some preferred embodiments, for Where m 10 The group is selected from integers between 0 and 6, and Z4 is selected from 6-10 aryl groups; the methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom. More preferably, m 10 Integers selected from 0 to 6.

[0080] In some preferred embodiments, for Where m 10 Integers selected from 0 to 6, Z4 is a benzene ring.

[0081] In some preferred embodiments, for

[0082] In some preferred embodiments, in formula (I) Selected from the following structural fragments:

[0083]

[0084]

[0085]

[0086]

[0087] In some preferred embodiments, T represents a bioactive molecular fragment. In some preferred embodiments, the bioactive molecule is selected from metal complexes, such as platinum complexes (e.g., oxaliplatin), gold complexes; glycopeptide antibiotics, such as bleomycin or bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan), topoisomerase II inhibitors (e.g., actinomycin D, doxorubicin, doxorubicin, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); and DNA synthesis interfering drugs, such as methotrexate, 5-fluorouracil, and cytarabine. Glycosides, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nerabine; drugs acting on structural proteins, such as microtubule inhibitors, vinblastine alkaloids, vincristine, paclitaxel, docetaxel, or cabazitaxel; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; maytansine derivatives; calichiomycin derivatives; olrital statin derivatives; pyrrolobenzodiazepine dimers (PBD) derivatives; melphalan; mitomycin C; chlorambucil; and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis, or necrosis.

[0088] In some preferred embodiments, the bioactive molecule is selected from... Where R 14 Selected from R 15 Substituted acyl or sulfonyl group, R 15 Selected from C 1-6 Alkyl, Halogenated C 1-6 Alkyl, 6-10 aryl and 5-12 heteroaryl; R 16 Selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, R 17 Replacement C 1-6 alkyl, R 17 Selected from aryl and heteroaryl groups, including but not limited to phenyl and pyridyl groups, m 11Selected from 0, 1, 2.

[0089] In some preferred embodiments, the bioactive molecule is selected from... Where R 14 Selected from R 15 Substituted acyl or sulfonyl group, R 15 Selected from C 1-6 Alkyl, Halogenated C 1-6 Alkyl, 6-10 aryl and 5-12 heteroaryl; R 16 Selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, R 17 Replacement C 1-6 alkyl, R 17 Selected from aryl and heteroaryl groups, including but not limited to phenyl and pyridyl groups, m 11 Selected from 0, 1, 2.

[0090] In some preferred embodiments, the bioactive molecule is selected from...

[0091] In some preferred embodiments, the bioactive molecule is selected from...

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

[0093] In some preferred embodiments, the bioactive molecule is selected from...

[0094]

[0095] In some preferred embodiments, the bioactive molecules are selected from...

[0096]

[0097] In some preferred embodiments, T is selected from

[0098]

[0099] In some preferred embodiments, T is selected from

[0100]

[0101] In some preferred embodiments, T is selected from

[0102]

[0103]

[0104] In some preferred embodiments, T is selected from

[0105]

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

[0107]

[0108] In some preferred embodiments, the compound represented by formula (I) is selected from...

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] In some preferred embodiments, the compound is selected from...

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] In a second aspect, this disclosure provides a conjugate comprising a bioactive molecule, a linker, and a targeting moiety. The targeting moiety is connected to the linker via its active group (e.g., a thiol group) to form the conjugate.

[0127] In some preferred embodiments, the coupling structure is as shown in formula (II):

[0128] {T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A

[0129] Equation (II)

[0130] Wherein, A is the target portion (e.g., small molecule ligand, protein, peptide, non-protein reagent (e.g., sugar, RNA or DNA)); γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8 (e.g., 5, 6, 7 or 8);

[0131] The remaining groups are as described in the first aspect of this disclosure.

[0132] In some preferred embodiments, the target of A is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, O772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2. BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, 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 , ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR 1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA and TENB2. .

[0133] In some preferred embodiments, A is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aryl sulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linking two aliphatic indoles, an anthocyanin dye, or IR-783 or a derivative thereof.

[0134] In some preferred embodiments, A is selected from

[0135]

[0136]

[0137] In some preferred embodiments, A is an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment 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.

[0138] 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.

[0139] In some preferred embodiments, A is a monoclonal antibody against Trop-2, such as M1, M2, and M3.

[0140]

[0141] The distribution of amino acids in different regions or domains can be followed according to the definitions in Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 878-883.

[0142] 1. Heavy and light chain sequences of hydrophobic modified antibody M1

[0143] The amino acid sequence of the M1 heavy chain variable region is: (121aa)

[0144]

[0145] The amino acid sequence of the M1 light chain variable region is: (107 aa)

[0146]

[0147] 2. Heavy and light chain sequences of hydrophobic modified antibody M2

[0148] The amino acid sequence of the variable region of the M2 heavy chain is: (121aa)

[0149]

[0150] The amino acid sequence of the M2 light chain variable region is: (107aa)

[0151]

[0152] 3. Heavy and light chain sequences of hydrophobic modified antibody M3

[0153] The amino acid sequence of the M3 heavy chain variable region is: (121aa)

[0154]

[0155] The amino acid sequence of the M3 light chain variable region is: (107aa)

[0156]

[0157] M1, M2, M3 light chain constant region sequence: (107aa)

[0158]

[0159] M1, M2, M3 heavy chain constant region sequence: (330aa)

[0160]

[0161] The Lys terminus of the heavy chain is prone to deletion, but this deletion does not affect biological activity; see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143. The aforementioned M1, M2, and M3 monoclonal antibodies, and their sequences or fragments with the Lys terminus deleted from the heavy chain, all belong to the M1, M2, and M3 monoclonal antibodies described in this invention.

[0162] In some preferred embodiments, A is selected from RGD peptides that recognize cell surface integrin receptors; growth factors such as EGF, PDGF, or VEGF that recognize cell surface growth factor receptors; and peptides that can recognize functional cell surface plasminogen activator, dermalin, bradykinin, somatostatin, or prostate-specific membrane antigen receptors.

[0163] 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.

[0164] In some preferred embodiments, the coupling agent is selected from:

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Wherein, γ is selected from an integer or 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 decimal between 5 and 8 (e.g. 5, 6, 7 or 8).

[0175] In some preferred embodiments, the coupling agent is selected from:

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] Wherein, γ is selected from an integer or 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 or pertuzumab; preferably, γ is selected from an integer or decimal between 5 and 8 (e.g., 5, 6, 7 or 8).

[0184] In some preferred embodiments, the coupling agent is:

[0185]

[0186]

[0187]

[0188]

[0189] Wherein, A1 is the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0190] In some preferred embodiments, the coupling agent is:

[0191]

[0192]

[0193] Wherein, A1 is the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0194] In some preferred embodiments, the coupling agent is:

[0195]

[0196] Wherein, A1 is the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0197] In some preferred embodiments, the coupling agent is:

[0198]

[0199] Wherein, A1 is a fragment of the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0200] In some preferred embodiments, the coupling agent is:

[0201]

[0202]

[0203]

[0204] Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0205] In some preferred embodiments, the coupling agent is:

[0206]

[0207]

[0208] Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 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 coupling agent is:

[0210]

[0211] Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0212] In some preferred embodiments, the coupling agent is:

[0213]

[0214]

[0215]

[0216] Wherein, A3 is pertuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0217] In some preferred embodiments, the coupling agent is:

[0218]

[0219]

[0220] Wherein, A3 is pertuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0221] In some preferred embodiments, the coupling agent is:

[0222]

[0223]

[0224]

[0225]

[0226] Wherein, A4 is the M1 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0227] In some preferred embodiments, the coupling agent is:

[0228]

[0229] Wherein, A4 is the M1 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0230] In some preferred embodiments, the coupling agent is:

[0231]

[0232]

[0233]

[0234]

[0235] Wherein, A5 is the M2 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0236] In some preferred embodiments, the coupling agent is:

[0237]

[0238] Wherein, A5 is the M2 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0239] In some preferred embodiments, the coupling agent is:

[0240]

[0241]

[0242]

[0243] Wherein, A6 is the M3 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0244] In some preferred embodiments, the coupling agent is:

[0245]

[0246] Wherein, A6 is the M3 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

[0247] In another aspect, this disclosure provides a method for preparing the conjugate described in the second aspect, comprising the step of coupling a linker of a compound of formula (I) to an active group of a target moiety.

[0248] In some preferred embodiments, the method includes using a reducing agent (e.g., TCEP) to open the disulfide bonds of the target moiety to obtain a thiol group.

[0249] In some preferred embodiments, the method includes the step of forming a CS bond between the linker of the compound of formula (I) and the thiol group of the target moiety.

[0250] In some preferred embodiments, the targeting portion is an anti-Her 2 monoclonal antibody (e.g., trastuzumab, pertuzumab) or an anti-Trop-2 monoclonal antibody (e.g., sacituzumab, M1, M2 or M3), or an active fragment or variant thereof.

[0251] In some preferred embodiments, the molar ratio of the target portion to the compound of formula (I) is 1:(1-20); preferably, the coupling is carried out in water and / or an organic solvent; preferably, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol) and any combination thereof.

[0252] In some preferred embodiments, the method further includes a step of purifying the coupling product; preferably, the coupling product is purified by a chromatographic method (e.g., one or more of ion exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, or affinity chromatography).

[0253] In another aspect, this disclosure provides a pharmaceutical composition comprising the compound described in the first aspect of this disclosure or a pharmaceutically acceptable salt thereof, or a conjugate described in the second aspect, and one or more pharmaceutical excipients.

[0254] In another aspect, this disclosure provides the use of the compounds described in the first aspect or pharmaceutically acceptable salts thereof or conjugates described in the second aspect in the preparation of medicaments for treating diseases associated with abnormal cellular activity, such as cancer.

[0255] In some preferred embodiments, the cancer is a solid tumor or a non-solid tumor, such as those selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumors, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, and thyroid cancer.

[0256] In another aspect, this disclosure provides the use of the compounds described in the first aspect or pharmaceutically acceptable salts thereof, or conjugates or pharmaceutical compositions described in the second aspect, in the treatment of diseases (e.g., cancer) associated with abnormal cellular activity.

[0257] On the other hand, this disclosure provides methods for treating diseases associated with abnormal cellular activity (such as cancer), including administering an effective dose of the compound provided in the first aspect of this disclosure or a pharmaceutically acceptable salt thereof, or a conjugate or pharmaceutical composition provided in the second aspect, to an individual in need of such treatment.

[0258] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0259] In this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0260] The pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound disclosed herein or its pharmaceutically acceptable salts or conjugates, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5-350 mg, and particularly preferably 1-250 mg.

[0261] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0262] In this disclosure, the term "individual" includes humans or non-human animals. Exemplary human individuals include human individuals suffering from a disease (such as the disease described herein) (referred to as patients) or normal individuals. The term "non-human animal" in this disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0263] In this disclosure, the term "effective dose" refers to the amount of a compound that, when administered, will alleviate one or more symptoms of the treated condition to a certain extent.

[0264] In this disclosure, the term "conjugate" refers to a substance obtained by linking a bioactive molecule to a target moiety. In some embodiments of this disclosure, the bioactive molecule and the target moiety are linked via a linker. The linker is capable of cleaving under specific environmental conditions (e.g., intracellular low pH) or specific actions (e.g., the action of lysosomal proteases), thereby separating the bioactive molecule from the target moiety. In some embodiments of this disclosure, the linker comprises cleavable or cleavable units, such as peptides or disulfide bonds. In some embodiments of this disclosure, the bioactive molecule and the target moiety are directly linked by a covalent bond, which is capable of cleaving under specific environmental conditions or actions, thereby separating the bioactive molecule from the target moiety.

[0265] In this disclosure, the terms "bioactive substance" and "bioactive molecule" refer to substances that inhibit or prevent cell function and / or cause cell death or damage. In some embodiments of this disclosure, the bioactive substance or bioactive molecule in the conjugate is a molecule with antitumor biological activity. For example, a radioactive isotope, such as At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212Radioactive isotopes of Lu; metal complexes, such as platinum complexes, gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin and bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan; topoisomerase II inhibitors, actinomycin D, doxorubicin, doxorubicin, docalimicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelabine, etc.; drugs that act on structural proteins, such as microtubule inhibitors, vinblastine alkaloids, vincristine, vinblastine, paclitaxel, etc. Sitaxel, cabazitaxel, etc.; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; also including proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cyclin inhibitors, maytansine derivatives, calichiomycin derivatives, olistatin derivatives, pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell growth and promote tumor cell apoptosis and necrosis; enzymes and their fragments, such as ribolysins; 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 detrimental effect on cell growth or proliferation.

[0266] In this disclosure, the term "small molecule" refers to a small molecule drug with biological activity.

[0267] In this disclosure, the term "connector" refers to a segment that links a bioactive molecule to a target region.

[0268] In this disclosure, the term "targeting portion" refers to a portion of the conjugate that can specifically bind to a target (or a portion of the target) on the cell surface. Through the interaction between the targeting portion and the target, the conjugate can be delivered to a specific cell population.

[0269] In this disclosure, when the targeting portion of the conjugate is an antibody, the conjugate may be referred to as a "drug-antibody conjugate". In this disclosure, "drug-antibody conjugate" and "immunoconjugate" are used interchangeably.

[0270] In this disclosure, the term "antibody" is used in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, provided they possess the desired biological activity. In this disclosure, "antibody" and "immunoglobulin" are used interchangeably.

[0271] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the cluster are identical except for a small number of possible naturally occurring mutations. Monoclonal antibodies possess high specificity against a single determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, comprise different antibodies targeting different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage of being synthesized without contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a substantially homogeneous group of antibodies, and should not be construed as requiring special methods for preparation.

[0272] In some embodiments of this disclosure, monoclonal antibodies further include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to one, a class, or a subclass of antibody, while the remainder is identical or homologous to another, a different class, or a different subclass of antibody, provided they possess the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies that can be used in this disclosure include primatized antibodies, which comprise a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

[0273] The term "antibody fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity-determining region fragments, diabody, linear antibody, and single-chain antibody molecules.

[0274] The term "bispecific antibody," also known as "bifunctional antibody-drug conjugate," refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a conjugate arm. This conjugate retains the activity of each antibody and thus has both bifunctionality and bispecificity.

[0275] The term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies. The former is an antibody that has the binding specificity of three different antigens, while the latter is an antibody that has the binding specificity of four different antigens.

[0276] The term "intact antibody" refers to an antibody that contains an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be natural sequences (e.g., human natural constant region sequences) or amino acid sequence variants thereof. Intact antibodies are preferably intact antibodies with one or more effector functions.

[0277] The term "probody" is a modified antibody, including an antibody or antibody fragment that is specifically designed to bind to its target and can be coupled with a masking group, wherein the masking group is defined as having a cleavage constant for the binding ability of the antibody or antibody fragment to its target that is at least 100 times, 1000 times, or 10000 times greater than the cleavage constant for the binding ability of an antibody or antibody fragment without a coupled masking group to its target.

[0278] In this disclosure, the “humanized” form of a nonhuman (e.g., mouse) antibody refers to a chimeric antibody containing a minimal amount of nonhuman immunoglobulin sequence. Most humanized antibodies are donor antibodies (e.g., mouse, rat, rabbit, or nonhuman primate) hypervariable region residues of human recipient immunoglobulins that have been replaced with nonhuman (e.g., mouse, rat, rabbit, or nonhuman primate) hypervariable region residues having the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with nonhuman residues. Furthermore, humanized antibodies may also contain residues not present in the recipient or donor antibody. These modifications are intended to further optimize antibody performance. Humanized antibodies generally contain at least one, typically two, variable regions, where all or almost all hypervariable loops correspond to those of the nonhuman immunoglobulin, while the FR is entirely or almost entirely a sequence of human immunoglobulin. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc, typically human immunoglobulin Fc). For details, see, for example, Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-329; and Presta, 1992, Curr Op Struct Bwl 2: 593-596.

[0279] Intact antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant regions. The five main classes are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different antibody classes are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known in the art.

[0280] In this disclosure, although in most cases the amino acid substitutions in the antibody are L-amino acids, this is not the only possibility. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less prone to degradation in the oral cavity, intestines, or plasma than peptides containing only L-amino acids.

[0281] The monoclonal antibodies used in this disclosure can be produced by many methods. For example, the monoclonal antibodies used in this disclosure can be obtained by hybridoma methods using cells from many species, including mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256:495), or by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597). Monoclonal antibodies that can be used in this 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.

[0282] In some embodiments of this disclosure, the targets of the targeting portion A are selected from: epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, 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, STEAP1, O772P, MPF, Napi3b, Sema5b, PSCA. hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DL L3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2. BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, 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 , ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR 1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA and TENB2. .

[0283] In some embodiments of this disclosure, the target of the targeting portion A is selected from: RGD peptides that recognize cell surface integrin receptors; growth factors such as EGF, PDGF, and VEGF that recognize cell surface growth factor receptors; and peptides that can recognize functional cell surface plasminogen activator, dermalin, bradykinin, somatostatin, and prostate-specific membrane antigen receptors.

[0284] In some embodiments of this disclosure, the target of the targeting portion A is selected from: CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, and DR5 ligand.

[0285] In some embodiments of this disclosure, the targeting portion A is a monoclonal antibody against Her 2, such as trastuzumab or pertuzumab; or the targeting portion is a monoclonal antibody against Trop-2, such as sacituzumab, M1, M2, or M3.

[0286] In some embodiments of this disclosure, the target portion is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her 2, and its amino acid sequence is known to those skilled in the art; a schematic sequence can be found, for example, in CN103319599.

[0287] In some embodiments of this disclosure, the Lys terminus of the heavy chain of the targeting moiety is readily deleted without affecting biological activity; see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143. For example, the targeting moiety is an anti-Trop-2 monoclonal antibody, such as Sacituzumab, M1, M2, or M3, with a Lys terminus deleted from the heavy chain. Similarly, the targeting moiety is an anti-Her 2 monoclonal antibody, such as trastuzumab or pertuzumab, with a Lys terminus deleted from the heavy chain.

[0288] Exemplary heavy and light chain sequences of trastuzumab can be found, for example, in SEQ ID No.: 17 and SEQ ID No.: 18. In this disclosure, when references are made to or relating to the heavy and light chain sequences of trastuzumab, the sequences shown in SEQ ID No.: 17 and SEQ ID No.: 18 are used, respectively. Exemplary heavy and light chain sequences of pertuzumab can be found in SEQ ID No.: 16 and SEQ ID No.: 15 of US7560111.

[0289] SEQ ID No.: 17 (heavy chain sequence)

[0290]

[0291] SEQ ID No.: 18 (light chain sequence)

[0292]

[0293] In some embodiments of this disclosure, the targeted anti-Trop-2 antibody is RS7 (i.e., Sacituzumab of this disclosure) described in U.S. Patent No. 7,517,964; and hRS7 (i.e., Sacituzumab of this disclosure) described in US2012 / 0237518. The anti-Trop-2 antibody that can be used in this disclosure can also be obtained through screening using the vector design, construction, and antibody library construction methods disclosed in CN103476941A, or through Sorrento Therapeutics, Inc. The document was obtained through a screening process.

[0294] The heavy chain sequence and light chain amino acid sequence of the sacituzumab monoclonal antibody can be found, for example, in SEQ ID No.: 19 and SEQ ID No.: 20.

[0295] SEQ ID No.: 19 (heavy chain sequence)

[0296]

[0297]

[0298] The terminal K (or lys) of the heavy chain is prone to deletion, but this deletion does not affect biological activity. See Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143.

[0299] SEQ ID No.: 20 (light chain sequence)

[0300]

[0301] In this disclosure, ErbB2 and Her2 / neu are used interchangeably, both representing the native sequence of the human Her2 protein (Genebank accession number: X03363, see, for example, Semba et al., 1985, PNAS, 82: 6497-6501; and Yamamoto et al., 1986, Nature, 319: 230-234) and its functional derivatives, such as amino acid sequence variants. ErbB2 represents the gene encoding human Her2, and neu represents the gene encoding rat p185neu. In some embodiments, the compounds or conjugates of this 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, kidney cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells, or liver cancer cells.

[0302] In this disclosure, Trop-2 or TROP2 refers to human trophoblast cell-surface antigen 2, also known as TACSTD2, M1S1, GA733-1, or EGP-1, which is a cell surface receptor expressed by many human tumor cells (such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, and cervical cancer). In some embodiments, the compounds or conjugates of this disclosure can inhibit or kill cells expressing the TROP2 receptor, such as breast cancer cells, colorectal cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, prostate cancer cells, or cervical cancer cells.

[0303] In this document, the couplings of the present invention appear This indicates the specific way in which the thiol group in the antibody is linked to the linker when the target region is an antibody.

[0304] In this article, the term "C" 1-6 "Alkyl" refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, including, for example, "C". 1-4 Alkyl", C 1-3 Alkyl groups, 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.

[0305] In this article, the term "C" 2-6 "Alkenyl" refers to a straight-chain, branched, or cyclic alkenyl group containing at least one double bond and having 2-6 carbon atoms, including, for example, "C". 2-4 Examples of these compounds 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.

[0306] In this article, the term "C" 2-6 "Alkyne group" refers to a straight-chain or branched alkynyl group containing at least one triple bond and having 2-6 carbon atoms, including, for example, "C". 2-4Examples of "alkynyl" 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.

[0307] In this article, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0308] In this document, the terms "3-8 membered cycloalkyl" or "C" are used. 3-8 "Cycloalkyl" refers to a saturated cyclic alkyl group containing 3-8 carbon atoms, including, for example, "3-6 membered cycloalkyl", "4-6 membered cycloalkyl", "5-7 membered cycloalkyl" or "5-6 membered cycloalkyl". Specific examples include, but are not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, etc.

[0309] In this article, the term "C" 1-6 "Alkoxy" refers to a group with a C 1-6 Alkyl-O- groups, wherein C 1-6 Alkyl groups are as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexoxy.

[0310] In this document, the term "3-8 membered alicyclic heterocyclic group" refers to a cyclic group containing 3-8 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the cyclic structure may be oxidized. "3-8 membered alicyclic heterocyclic group" includes, for example, "3-8 membered nitrogen-containing alicyclic heterocyclic group," "3-8 membered oxygen-containing alicyclic heterocyclic group," "3-6 membered alicyclic heterocyclic group," "3-6 membered oxygen-containing alicyclic heterocyclic group," "4-7 membered alicyclic heterocyclic group," "4-6 membered alicyclic heterocyclic group," "5-7 membered alicyclic heterocyclic group," "5-6 membered alicyclic heterocyclic group," and "5-6 membered nitrogen-containing alicyclic heterocyclic group," including but not limited to ethylene oxide, oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.

[0311] In this document, the term "6-12 membered spirocyclic group" refers to a cyclic structure containing 6-12 cyclic carbon atoms, formed by two or more cyclic structures sharing a single carbon atom. Optionally, the carbon atom in the cyclic structure may be substituted with oxygen. "6-12 membered spirocyclic group" includes, for example, "6-11 membered spirocyclic group," "6-10 membered spirocyclic group," "7-10 membered spirocyclic group," "7-9 membered spirocyclic group," "7-8 membered spirocyclic group," "9-10 membered spirocyclic group," "3-10 membered spirocyclic group," etc. Specific examples include, but are not limited to: wait.

[0312] In this document, the term "6-12-membered bridged ring group" refers to a ring structure containing 6-12 ring carbon atoms, formed by two or more ring structures sharing two non-adjacent carbon atoms. Optionally, the carbon atoms in the ring structure may be substituted with oxygen. "6-12-membered bridged ring group" includes, for example, "6-11-membered bridged ring group," "5-10-membered bridged ring group," "7-10-membered bridged ring group," "7-9-membered bridged ring group," "7-8-membered bridged ring group," "9-10-membered bridged ring group," "3-10-membered bridged ring group," etc. Specific examples include, but are not limited to: wait.

[0313] In this paper, the term "6-12 fused ring group" refers to a cyclic structure containing 6-12 ring carbon atoms, formed by two or more ring structures sharing two adjacent atoms, including "6-11 fused ring group", "6-10 fused ring group", "6-8 fused ring group", "10-12 fused ring group", and "7-10 fused ring group". Examples include, but are not limited to: wait.

[0314] In this document, the term "6-12 spiroheterocyclic group" refers to a cyclic structure containing 6-12 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom) formed by two or more cyclic structures sharing a single ring atom. Optionally, the ring atom (e.g., a carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may be substituted with oxygen. "6-12 spiroheterocyclic group" includes, for example, "6-11 spiroheterocyclic group," "5-10 spiroheterocyclic group," "7-11 spiroheterocyclic group," "7-10 spiroheterocyclic group," "7-9 spiroheterocyclic group," "7-8 spiroheterocyclic group," "9-10 spiroheterocyclic group," "3-10 spiroheterocyclic group," etc. Specific examples include, but are not limited to: wait.

[0315] In this document, the term "6-12-membered bridged heterocyclic group" refers to a ring structure containing 6-12 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom), formed by two or more ring structures sharing two non-adjacent ring atoms. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the ring structure may be substituted with oxygen. "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: wait.

[0316] In this document, the term "6-12 fused heterocyclic group" refers to a ring structure containing 6-12 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom), formed by two or more ring structures sharing two adjacent atoms. Optionally, the ring atoms (e.g., carbon atoms, nitrogen atoms, or sulfur atoms) in the ring structure may be substituted with oxygen. "6-12 fused heterocyclic group" includes, for example, "6-11 fused heterocyclic group", "5-10 fused heterocyclic group", "7-10 fused heterocyclic group", "3-10 fused heterocyclic group", "3-10 nitrogen-containing fused heterocyclic group", "9-10 fused heterocyclic group", "9-10 nitrogen-containing fused heterocyclic group", "6-12 oxygen-containing fused heterocyclic group", etc. Specific examples include, but are not limited to: tetrahydroimidazo[4,5-c]pyridyl, 3,4-dihydroquinazolinyl, 1,2-dihydroquinoxalinyl, benzo[d][1,3]dioxacyclopentenyl, 1,3-dihydroisobenzofuranyl, 4H-1,3-benzooxazinyl, 4,6-dihydro-1H-furano[3,4-d]imidazoyl, 3a,4,6,6a-tetrahydro-1H-furano[3,4-d]imidazoyl, 4,6-dihydro-1H-thieno[3,4-d]imidazoyl, 4,6-Dihydro-1H-pyrrolo[3,4-d]imidazolyl, benzimidazolyl, octahydro-benzo[d]imidazolyl, decahydroquinolinyl, hexahydrothiophenezimidazolyl, hexahydrofuranzimidazolyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, octahydrocyclopenten[c]pyrroleyl, dihydroindolyl, dihydroisoindolyl, benzoxazolyl, benzothiazolyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl, 4H-1,3-benzoxazinyl, etc.

[0317] In this document, the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon group, such as 6-20 aryl, 6-10 aryl, 5-8 aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracene, phenanthrene, etc. The "6-20 aryl" refers to an aryl group containing 6-20 ring atoms.

[0318] In this document, the term "heteroaryl" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Optionally, the ring atom (e.g., a carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may be oxidized. Specific examples include, but are not limited to, 5-10-membered heteroaryl, 5-10-membered nitrogen-containing heteroaryl, 6-10-membered oxygen-containing heteroaryl, 6-8-membered nitrogen-containing heteroaryl, 5-8-membered oxygen-containing heteroaryl, etc., such as furanyl, thiophene, pyrrole, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazole, 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, pyridinyl, 2-pyridone, 4 -Pyridinone, pyrimidin, 1,4-dioxazinyl, 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-tetraazinyl, aziridine-heptanetrienyl, 1,3-diazacycloheptanetrienyl, aziridine-octatetraenyl, etc.

[0319] Beneficial effects of the invention

[0320] This disclosure provides a novel class of bioactive molecular conjugates by improving the conjugation method between the drug and the target moiety in ADCs or SMDCs. In some embodiments of this disclosure, the bioactive molecular conjugates are obtained by nucleophilic substitution of a heteroaromatic ring on an ADC drug linker with a free thiol group in an antibody molecule. The conjugates obtained using the above conjugation method can achieve at least one of the following technical effects:

[0321] (1) It has high stability;

[0322] (2) It has a high drug loading capacity. In some embodiments, the DAR value of the conjugate can reach 5-8.

[0323] (3) It has extremely high coupling efficiency; in some embodiments, the coupling efficiency can reach 90%.

[0324] (4) The conjugates obtained by the above conjugation method can effectively improve the stability of drug molecules in circulation and reduce the shedding of non-target drugs from non-target cells.

[0325] (5) The conjugate can also increase the effective release of bioactive molecules into cells, thereby achieving the purpose of enhancing efficacy and reducing toxicity;

[0326] (6) The conjugate has good tumor tissue targeting properties; and

[0327] (7) The conjugate has good therapeutic effects in tumor animal models.

[0328] In addition, the conjugation method described in this 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. Attached Figure Description

[0329] Figure 1 shows the TIC (total ion chromatogram) of BT001002.

[0330] Figure 2 shows the deconvolution plot of the BT001002 coupled light chain.

[0331] Figure 3 shows the deconvolution plot of the BT001002 coupled heavy chain.

[0332] Figure 4 shows the TIC (total ion chromatogram) of BT001004.

[0333] Figure 5 shows the deconvolution plot of the BT001004 coupled light chain.

[0334] Figure 6 shows the deconvolution plot of the BT001004 coupled heavy chain.

[0335] Figure 7 shows the SEC chromatogram of BT001002.

[0336] Figure 8 shows the molecular weight marker SEC chromatogram of BT001002.

[0337] Figure 9 shows the SEC chromatogram of BT001004.

[0338] Figure 10 shows the deconvolution plot of the BT001012 coupled light chain.

[0339] Figure 11 is a deconvolution diagram of the BT001012 coupled heavy chain.

[0340] Figure 12 is a deconvolution diagram of the BT001013 coupled light chain.

[0341] Figure 13 is a deconvolution diagram of the BT001013 coupled heavy chain.

[0342] Figure 14 is a deconvolution diagram of the BT001018 coupled light chain.

[0343] Figure 15 shows the deconvolution plot of the BT001018 coupled heavy chain.

[0344] Figure 16 shows the deconvolution diagram of the BT001021 coupled light chain.

[0345] Figure 17 shows the deconvolution diagram of the BT001021 coupled heavy chain.

[0346] Figure 18 shows the deconvolution plot of the BT001023 coupled light chain.

[0347] Figure 19 shows the deconvolution plot of the BT001023 coupled heavy chain.

[0348] Figure 20 shows the deconvolution plot of the BT001040 coupled light chain.

[0349] Figure 21 shows the deconvolution plot of the BT001040 coupled heavy chain.

[0350] Figure 22 shows the deconvolution plot of the BT001041 coupled light chain.

[0351] Figure 23 is a deconvolution diagram of the BT001041 coupled heavy chain.

[0352] Figure 24 shows the deconvolution plot of the BT001042 coupled light chain.

[0353] Figure 25 shows the deconvolution plot of the BT001042 coupled heavy chain.

[0354] Figure 26 shows the deconvolution plot of the BT001043 coupled light chain.

[0355] Figure 27 shows the deconvolution plot of the BT001043 coupled heavy chain.

[0356] Figure 28 shows the deconvolution plot of the BT001044 coupled light chain.

[0357] Figure 29 shows the deconvolution plot of the BT001044 coupled heavy chain.

[0358] Figure 30 shows the deconvolution plot of the BT001046 coupled light chain.

[0359] Figure 31 shows the deconvolution plot of the BT001046 coupled heavy chain.

[0360] Figure 32 shows the deconvolution plot of the BT001047 coupled light chain.

[0361] Figure 33 shows the deconvolution plot of the BT001047 coupled heavy chain.

[0362] Figure 34 shows the SEC chromatogram of BT001012.

[0363] Figure 35 shows the SEC chromatogram of BT001013.

[0364] Figure 36 shows the SEC chromatogram of BT001018.

[0365] Figure 37 shows the SEC chromatogram of BT001021.

[0366] Figure 38 shows the SEC chromatogram of BT001023.

[0367] Figure 39 shows the SEC chromatogram of BT001042.

[0368] Figure 40 shows the SEC chromatogram of BT001043.

[0369] Figure 41 shows the SEC chromatogram of BT001044.

[0370] Figure 42 shows the SEC chromatogram of BT001046.

[0371] Figure 43 shows the SEC chromatogram of BT001047.

[0372] Figure 44 shows the growth changes of tumor volume in each group of mice in the NCI-N87 human gastric cancer model.

[0373] Figure 45 shows the changes in body weight of mice in each group in the NCI-N87 human gastric cancer model.

[0374] Figure 46 shows the growth changes in tumor volume in each group of mice in the HCC1806 human breast cancer model.

[0375] Figure 47A shows the growth changes in tumor volume in each group of mice in the HCC827 human non-small cell lung cancer xenograft model.

[0376] Figure 47B shows the changes in body weight of mice in each group in the HCC827 human non-small cell lung cancer xenograft model.

[0377] Figure 48A shows the growth changes in tumor volume in each group of mice in the NCI-N87 human gastric cancer xenograft model.

[0378] Figure 48B shows the changes in body weight of mice in each group in the NCI-N87 human gastric cancer xenograft model.

[0379] Figure 49A. Growth changes in tumor volume in different groups of mice in the MDA-MB-231 human breast cancer tumor-bearing mouse model.

[0380] Figure 49B. Changes in body weight of mice in different groups in the MDA-MB-231 human breast cancer tumor-bearing mouse model. Detailed Implementation

[0381] The present disclosure is further illustrated below through a description of specific embodiments, but this is not intended to limit the scope of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the present disclosure without departing from its basic ideas and scope.

[0382] The abbreviations used in this invention have the following meanings:

[0383]

[0384] Preparation scheme

[0385] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0386] Nuclear magnetic resonance (NMR) 1 The H NMR (H2N) assay was performed using a Bruker 400MHz NMR spectrometer; the assay solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0387] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0388] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0389] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0390] The preparative liquid chromatography method is as follows:

[0391] Method A:

[0392] Column: Daisogel C18 10μm 100x250mm

[0393] Mobile phase A: Water; Mobile phase B: Acetonitrile

[0394]

[0395] Method B:

[0396] Column: Daisogel C18 10μm 50x250mm

[0397] Mobile phase A: Water; Mobile phase B: Acetonitrile

[0398]

[0399] Method C:

[0400] Column: Daisogel C18 10μm 50x250mm

[0401] Mobile phase A: Water containing 0.1% trifluoroacetic acid; Mobile phase B: Acetonitrile

[0402]

[0403] Method D: Column: Waters SunFire C18 5μm 19x250mm

[0404] Mobile phase A: Acetonitrile; Mobile phase B: Water containing 0.05% formic acid

[0405] Time: 0 min - 16 min; Mobile phase A: 10% - 90%; Flow rate: 28 mL / min

[0406] I. Synthesis of Bioactive Molecules

[0407] Example 1: Synthesis of (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-propanoyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyramido)-N,3-dimethylbutyramide (T001)

[0408]

[0409] Step 1: Synthesis of tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)carbamate

[0410] At room temperature, 1-hydroxybenzotriazole (2.0 mg, 14.74 μmol) was dissolved in N,N-dimethylformamide (4 mL), cooled to 0 °C, and then (4-methylaminobenzyl)-tert-butyl carbamate (4.0 mg, 16.1 μmol), N,N-diisopropylethylamine (8.5 mg, 66.8 μmol), and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-( (S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine (10.0 mg, 13.5 μmol, commercially available), stirred for 5 min, then 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (10.0 mg, 20.1 μmol) was added, and the mixture was stirred at 0 °C for 1 h. The reaction was monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS) to ensure complete reaction. The reaction solution was purified by preparative liquid chromatography (Method D) to give the title compound as a white solid, 9.0 mg. ESI-MS (m / z): 950.5 [M+H] + .

[0411] Step 2: Synthesis of (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-propionyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamid

[0412] At room temperature, tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-3-phenylpropionamido)methyl)phenyl)carbamate (9.0 mg, 0.02 mmol) was dissolved in 1,4-dioxane (0.5 mL), cooled to 0 °C, and dioxane hydrochloride solution (1 mL, 4.0 M) was added. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction of the reactants was monitored by high performance liquid chromatography-mass spectrometry (HPLC-MS) until complete. The solvent was removed under reduced pressure, and the crude product was purified by preparative liquid chromatography (Method C) to give 5.0 mg of the trifluoroacetate of the title compound as a white solid. ESI-MS (m / z): 850.5 [M+H] + .

[0413] Example 2: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino)1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide (T011)

[0414]

[0415] Step 1: Synthesis of tert-butyl (S)-(4-((2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido)methyl)phenyl)carbamate

[0416] At 0 °C, 222 mg (1.0 mmol) of 4-aminobenzylamine and 306 mg (1.5 mmol) of N-methylmorpholine were added to a solution of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-phenylpropionic acid (387 mg, 1.0 mmol) in N,N-dimethylformamide (5 mL). Then, 203 mg (1.5 mmol) of 1-hydroxybenzotriazole and 288 mg (1.5 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added sequentially. The reaction was carried out overnight at 0 °C. The reaction solution was poured into water (50 mL), and a white solid precipitated. The mixture was filtered, and the filter cake was washed with water (20 mL × 3). The solid was purified by silica gel column chromatography to give the title compound as a white solid, 380 mg. ESI-MS (m / z): 592.3 [M+H] + .

[0417] Step 2: Synthesis of (S)-(4-((2-amino-3-phenylpropamido)methyl)phenyl)carbamate tert-butyl ester

[0418] Lithium hydroxide monohydrate (21 mg, 0.51 mmol) was dissolved in water (1 mL) and added to a solution of (S)-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropamido)methyl)phenyl)carbamate tert-butyl ester (102 mg, 0.17 mmol) in tetrahydrofuran (2 mL). The reaction mixture was reacted at room temperature for 2 h. Water (20 mL) was added to the reaction mixture, 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 drying agent was filtered off, the solvent was removed under reduced pressure, and the residue was purified by preparative liquid chromatography (Method D) to give the title compound as a white solid, 65 mg. ESI-MS (m / z): 370.2 [M+H] + .

[0419] Step 3: Synthesis of (4-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-(methylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)carbamate

[0420] At 0 °C, (S)-(4-((2-amino-3-phenylpropamido)methyl)phenyl)carbamate tert-butyl ester (15 mg, 0.04 mmol) and N-methylmorpholine (12 mg, 0.12 mmol) were added to (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl- In a solution of 3-methoxy-2-methylpropionic 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 sequentially. The reaction was carried out overnight at 0 °C. The reaction solution was purified by preparative liquid chromatography (Method D) to give the title compound as a white solid, 24 mg. ESI-MS (m / z): 950.6 [M+H] + .

[0421] Step 4: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino)-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide

[0422] Trifluoroacetic acid (0.5 mL) was added to a solution of (4-((S)-2-((2R,3R)-3-((S)-1-(3R,4S,5S)-4-((S)-3-(methylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)carbamate (14.0 mg, 0.015 mmol) in dichloromethane (1.5 mL), and 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 give the trifluoroacetate of the title compound as a white solid, 4.2 mg. ESI-MS (m / z): 850.6 [M+H] + .

[0423] The following molecules can be synthesized using similar synthetic methods:

[0424]

[0425] Example 3: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)-N-isopropylacetamide

[0426]

[0427] Bellotrin hydrochloride (1.0 g, 2.13 mmol) and triethylamine (0.65 g, 0.9 mL) were dissolved in dichloromethane (50 mL) at room temperature. Acetic anhydride (0.22 g, 2.13 mmol) was slowly added dropwise, and 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. Insoluble matter was filtered off, solvent was evaporated, and the mixture was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give 1 g of the title compound. ESI-MS (m / z): 476.2 [M+H] + .

[0428] Example 4: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)-N-isopropylmethanesulfonamide

[0429]

[0430] Methanesulfonyl chloride (462 mg, 12.77 mmol, approximately 70% purity) was added dropwise to a solution of beloteccan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in 40 mL of dichloromethane and reacted at room temperature for 2 h. The mixture was filtered, and the filter cake was washed three times with 3 mL of dichloromethane to give 2.2 g of the title compound.

[0431] The structural characterization data are as follows:

[0432] 1 H NMR (400MHz, DMSO-d6) δ8.32 (d, J=8.4Hz, 1H), 8.20 (dd, J=8.4, 1.2Hz, 1H), 7.93-7. 84 (m, 1H), 7.79 (t, J = 7.6Hz, 1H), 7.35 (s, 1H), 6.56 (s, 1H), 5.44 (d, J = 9.2Hz, 4H), 3 .98 (p, J=6.7Hz, 1H), 3.50 (t, J=8.0Hz, 2H), 3.42-3.35 (m, 2H), 3.00 (s, 3H), 1.93-1 .82 (m, 2H), 1.15 (d, J=6.7Hz, 6H), 0.88 (t, J=7.3Hz, 3H).ESI-MS (m / z): 512.2[M+H] + [α] D 20 The temperature is +28.19° (c = 0.101 g / 100 mL, CH3CN).

[0433] The remaining bioactive molecules, whose synthesis methods are not specified, are commercially available or can be prepared using methods disclosed in the prior art.

[0434] II. Synthesis of compounds containing cellular bioactive molecules and linkers

[0435] Example 5: Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butamido)-3-methylbutamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-5-ureidopentanamide

[0436]

[0437] Step 1: Synthesis of tert-butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyrate (compounds 1-2)

[0438] Compound 1-1 (500 mg, 3.27 mmol) was dissolved in N,N-dimethylformamide (10 mL) at room temperature. Sodium hydride (130 mg, 3.27 mmol) was slowly added in portions, and the mixture was stirred at room temperature for 10 min. Tert-butyl 4-bromobutyrate (725 mg, 3.27 mmol) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 h. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The mixture was washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure to give the title compound, 500 mg. ESI-MS (m / z): 296.1 [M+H] + .

[0439] Step 2: Synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (compounds 1-3)

[0440] Compounds 1-2 (500 mg, 1.69 mmol) were dissolved in dichloromethane (6 mL) at room temperature, followed by the addition of trifluoroacetic acid (3 mL). After the addition was complete, the mixture was reacted at room temperature for 4 h. The solvent was then removed by vacuum evaporation to give the title compound, 400 mg. ESI-MS (m / z): 240.1 [M+H] + .

[0441] Step 3: Synthesis of (9H-fluorene-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 (compounds 1-5)

[0442] At room temperature, 4-(N-Boc-aminomethyl)-aniline (6.0 g, 27 mmol), compounds 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 mixture was heated to 45 °C and maintained at this temperature for 8.0 h. Upon cooling to room temperature, a large amount of solid precipitated. Filtering yielded the title compound, 3.65 g. ESI-MS (m / z): 701.4 [M+H] + .

[0443] Step 4: Synthesis of (9H-fluorene-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 (compounds 1-6)

[0444] At room temperature, trifluoroacetic acid (15 mL) was added to compounds 1-5 (3.0 g, 4.29 mmol), and the mixture was stirred at room temperature for 1.0 h. The solvent was removed by vacuum distillation to give a yellow oily substance. Anhydrous diethyl ether (20 mL) was added, resulting in the precipitation of a large amount of solid. The mixture was stirred vigorously for 0.5 h, filtered, and 3.06 g of the trifluoroacetate of the title compound was obtained. ESI-MS (m / z): 601.3 [M+H] + .

[0445] Step 5: Synthesis of (9H-fluorene-9-yl)-methyl-((S)-1-(((S)-1-((4-((((R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropamido)methyl)phenyl)amino-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (compounds 1-7)

[0446] At room temperature, Boc-D-phenylalanine (1.1 g, 4.2 mmol) and trifluoroacetates of compounds 1-6 (3.0 g, 4.2 mmol) were dissolved in N,N-dimethylformamide (40 mL), and the solution was cooled to 0 °C. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 g, 6.3 mmol), 1-hydroxybenzotriazole (0.9 g, 6.3 mmol), and N-methylmorpholine (1.7 g, 16.8 mmol) were added sequentially, and the reaction mixture was stirred at this temperature for 1.0 h. The reaction solution was then added dropwise to ice water (400 mL), and the mixture was stirred vigorously for 0.5 h. A large amount of solid precipitated out. The solid was filtered to give 3.3 g of the title compound. ESI-MS (m / z): 848.4 [M+H] + .

[0447] Step Six: Synthesis of (9H-fluorene-9-yl)-methyl-((S)-1-(((S)-1-((4-((((R)-2-amino-3-phenylpropamido)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (compounds 1-8)

[0448] Compounds 1-7 (3.0 g, 3.3 mmol) were dissolved in trifluoroacetic acid (30 mL) at room temperature and stirred for 1.0 h. The solvent was removed by vacuum distillation to give a yellow oily substance. Anhydrous diethyl ether (100 mL) was added, and the mixture was stirred vigorously for 0.5 h. A large amount of solid precipitated out. The solid was filtered to give 2.1 g of the trifluoroacetate of the title compound. ESI-MS (m / z): 748.4 [M+H] + .

[0449] Step 7: Synthesis of (9H-fluorene-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-butamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrole-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (compounds 1-9)

[0450] At room temperature, (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-(dimethylamino)-3-butamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrole-2-yl)-3-methoxy-2-methylpropionic acid (1.3 g, 2.17 mmol) and trifluoroacetate of compounds 1-8 (1.8 g, 2.17 mmol) were dissolved in N,N-dimethylformamide (20 mL). The mixture was cooled to 0 °C, and 1-hydroxybenzotriazole (440 mg, 3.26 mmol) and N-methylmorpholine (658 mg, 6.51 mmol) were added sequentially. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (624 mg, 1.38 mmol) was added. After the addition was complete, the reaction mixture was stirred at 0 °C for 5 h. Preparative liquid chromatography purification (Method D) yielded the title compound, 1.8 g. ESI-MS (m / z): 1329.2 [M+H] + .

[0451] Step 8: Synthesis of (S)-2-((S)-2-amino-3-butamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolo-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-5-ureidopentanamide (compounds 1-10)

[0452] Compounds 1-9 (500 mg, 0.38 mmol) were dissolved in N,N-dimethylformamide (5 mL) at room temperature, and piperidine (324 mg, 3.8 mmol) was added. The mixture was stirred at room temperature for 3 h. Preparative liquid chromatography (Method D) was performed to obtain the title compound, 350 mg. ESI-MS (m / z): 1107.2 [M+H] + .

[0453] Step Nine: Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butamido)-3-methylbutamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-5-ureidopentanamide (compound TL001)

[0454] Compound 1-10 (60 mg, 0.054 mmol) and 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (26 mg, 0.066 mmol) were dissolved in N,N-dimethylformamide (3 mL) at room temperature. The mixture was cooled to 0 °C, and N,N-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazol-1-oxotripyrrolinium hexafluorophosphate (281 mg, 0.54 mmol) were added sequentially. After the addition was complete, the reaction mixture was stirred at room temperature for 3 h. The mixture was purified by preparative liquid chromatography (Method D) to give the title compound, 30 mg. ESI-MS (m / z): 664.5 [M / 2+H] + .

[0455] Example 6 (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methanesulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butamido)-butamido)-5-ureidopentanamide

[0456]

[0457] Step 1: Synthesis of 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (compound 2-2)

[0458] At room temperature, 300 mg (1.25 mmol) of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid was dissolved in 8 mL of methanol, and sodium methanethiol (351 mg, 5.02 mmol) was added in a single batch. The mixture was heated to 50 °C and reacted overnight. The solution was purified by preparative liquid chromatography (Method D) to give the title compound, 120 mg. ESI-MS (m / z): 252.1 [M+H] + .

[0459] Step 2: Synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butamido)-butamido)-5-ureidopentanamide (compounds 2-3)

[0460] Following a similar procedure as described in step nine of Example 5, 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid was used instead of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid, and preparative liquid chromatography purification (Method D) was performed to obtain the title compound, 20 mg. ESI-MS (m / z): 670.5 [M / 2+H] + .

[0461] Step 3: Synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methanesulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butamido)-butamido)-5-ureidopentanamide (compound TL002)

[0462] Compound 2-3 (20 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL) at room temperature, and m-chloroperoxybenzoic acid (4.0 mg, 0.022 mmol) was added. After addition, the mixture was reacted at room temperature for 2 h. The solution was purified by preparative liquid chromatography (Method D) to give the title compound, 5.0 mg. ESI-MS (m / z): 686.5 [M / 2+H] + .

[0463] 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-methylbutamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide

[0464]

[0465] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (compound 3-2)

[0466] At room temperature, methyl 5-hexynate (500 mg, 3.97 mmol) and 5-bromo-2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 mL). Triethylamine (3 mL), cuprous iodide (75 mg, 0.4 mmol), and palladium dichloride dichloride (279 mg, 0.4 mmol) were added sequentially. The mixture was heated to 95 °C and stirred for 6 h under nitrogen protection. The reaction was quenched with water, extracted with ethyl acetate (20 mL × 3), and the organic phases were combined. The mixture was washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure. The mixture was purified by preparative liquid chromatography (Method D) to give the title compound, 300 mg. ESI-MS (m / z): 251.3 [M+H] + .

[0467] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (compound 3-3)

[0468] At room temperature, compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixture of tetrahydrofuran and water (4 mL / 4 mL), and lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added. The mixture was stirred at room temperature for 4 h, diluted with water, and extracted with ethyl acetate (20 mL × 2). The aqueous phase was adjusted to pH 3 with 1 N hydrochloric acid and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed by vacuum evaporation to give 120 mg of the title compound.

[0469] Step 3: Synthesis of 6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexynoic acid (compounds 3-4)

[0470] Compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL) at room temperature, and m-chloroperoxybenzoic acid (22 mg, 0.127 mmol) was added. After the addition was complete, the mixture was stirred overnight at room temperature. The solution was purified by preparative liquid chromatography (Method D) to give the title compound, 20 mg. ESI-MS (m / z): 269.1 [M+H] + .

[0471] Step 4: Synthesis of N-((S)-1-(((S)-1-((4-((((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutane-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide (compound TL003)

[0472] Following a similar procedure as described in step nine of Example 5, 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid was substituted for 6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexynic acid, and preparative liquid chromatography purification (Method D) was performed to obtain the title compound, 14 mg. ESI-MS (m / z): 679.0 [M / 2+H] + .

[0473] Example 8 (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)-ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]-indolazino[1,2-b]-quinoline-4-yl(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9,36-azatetracosane-41-amido)benzyl)carbonate

[0474]

[0475] Step 1: Synthesis of (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)-(9H-fluorenyl)-carbamate (compound 19-2)

[0476] Fmoc-L-citrulline (5.0 g, 12.58 mmol), p-aminobenzyl alcohol (6.20 g, 50.32 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (6.22 g, 25.16 mmol) were dissolved in dichloromethane (100 mL) at room temperature and reacted at 45 °C for 6 h. The reaction solution was concentrated under reduced pressure and slurried in anhydrous diethyl ether (100 mL) to give the title compound, 6.0 g. ESI-MS (m / z): 503.3 [M+H] + .

[0477] Step 2: Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (compound 19-3)

[0478] Compound 19-2 (1.0 g, 1.99 mmol) was dissolved in N,N-dimethylformamide (8 mL) at room temperature, and piperidine (339 mg, 3.98 mmol) was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 30 min, and then dichloromethane (10 mL) was added, followed by stirring for another 10 min. The reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography to give the title compound, 400 mg. ESI-MS (m / z): 281.2 [M+H] + .

[0479] Step 3: Synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (compound 19-4)

[0480] Compound 19-3 (150 mg, 0.54 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatricycloundecanoic acid (296 mg, 0.54 mmol) were dissolved in dichloromethane (10 mL), cooled to 0 °C, and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (145 mg, 0.58 mmol) were added. The mixture was then allowed to react overnight at room temperature. The reaction solution was concentrated under reduced pressure and purified by rapid silica gel column chromatography to give the title compound, 200 mg. ESI-MS (m / z): 817.5 [M+H] + .

[0481] Step 4: Synthesis of 4-((S)-35-azido-4,8-dioxo-2-(3-ureopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9-azatetracosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-4-yl) carbonate (compound 19-5)

[0482] (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-11-yl)ethyl)-N-isopropylmethanesulfonamide (200 mg, 0.39 mmol) was dissolved in dichloromethane (10 mL) at room temperature. The solution was cooled to 0 °C, and a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL) was added. Then, a solution of triphosgene (116 mg, 0.39 mmol) in dichloromethane (1.0 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at 0 °C for 1 h. A solution of compound 19-4 (159 mg, 0.18 mmol) in dichloromethane (2.0 mL) was added to the reaction mixture. After the addition was complete, the mixture was stirred at room temperature for 1 h. The title compound, 160 mg, was obtained by preparative high-performance liquid chromatography (method D). ESI-MS (m / z): 678.0 [M / 2+H] + .

[0483] Step 5: Synthesis of 4-((S)-35-amino-4,8-dioxo-2-(3-ureopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9-azatetracosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7] indo[1,2-b]quinoline-4-yl) carbonate (compound 19-6)

[0484] Compound 19-5 (80 mg, 0.059 mmol) was dissolved in tetrahydrofuran (1.0 mL) at room temperature. The solution was cooled to 0 °C, and a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 mL) was added. Platinum dioxide (15 mg, 0.059 mmol) was added in a single addition under nitrogen protection. After the addition was complete, the air was purged three times with hydrogen, and the reaction was allowed to proceed at room temperature for 6 h. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. Preparative high-performance liquid chromatography (Method D) was used to purify the crude product to obtain the title compound, 40 mg. ESI-MS (m / z): 665.0 [M / 2+H] + .

[0485] Step Six: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-4-yl(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9,36-azatetracosane-41-amido)benzyl)carbonate (compound TL019)

[0486] Compound 19-6 (30 mg, 0.016 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-5-hexynic acid (6.4 mg, 0.024 mmol) were dissolved in N,N-dimethylformamide (1 mL), cooled to 0 °C, and then benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate (16.5 mg, 0.032 mmol) and N,N-diisopropylethylamine (6.2 mg, 0.047 mmol) were added sequentially. After addition, the mixture was reacted at room temperature for 2 h. The mixture was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 10 mg. ESI-MS (m / z): 790.0 [M / 2+H] + .

[0487] Example 9 (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-4-yl-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl))-5-hexynamido)butamido)-5-ureopentamido)benzyl)carbonate

[0488]

[0489] Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentanamido-2-yl)amino)-3-methyl-butamido-2-yl)-(9H-fluorenyl)methyl-carbamate

[0490] Following a procedure similar to step one of Example 8, compound 19-1 was replaced with compound 28-1 to obtain the title compound, 310 mg. ESI-MS (m / z): 602.3 [M+H] + .

[0491] Step 2: Synthesis of (S)-2-((S)-2-amino-3-methylbutyramide)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (compound 28-2)

[0492] Following a procedure similar to step two of Example 8, compound 19-2 was replaced with compound 28-2 to obtain the title compound, 150 mg. ESI-MS (m / z): 380.3 [M+H] + .

[0493] 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-hexyneamide (compound 28-4)

[0494] Benzotriazol-1-yl-oxytripyrrolidinyl 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-hexynic acid (135 mg, 0.5 mmol) and (2S)-2-(((2S)-2-amino-3-methyl-butyryl)amino)-N-(4-(hydroxymethyl)phenyl)-5-ureoyl-pentanamide (190 mg, 0.5 mmol) in N,N-dimethylformamide (10 mL) at room temperature and stirred for 3 h at room temperature. Preparative high-performance liquid chromatography (method D) purification yielded the title compound, 78 mg. ESI-MS (m / z): 630.3 [M+H] + .

[0495] Step 4: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-4-yl-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methanesulfonyl)pyrimidin-5-yl))-5-hexynamido)butamido)-5-ureidopentamido)benzyl)carbonate (compound TL028)

[0496] Following a procedure similar to step four of Example 8, compound 19-4 was replaced with compound 28-4 to obtain the title compound, 1.76 mg. ESI-MS (m / z): 1167.4 [M+H] + .

[0497] Example 10 (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-4-yl-(4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatritriatrisamamido)benzyl)carbonate

[0498]

[0499] Step 1: Synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-azatetracosane)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (compound 29-1)

[0500] Following a procedure similar to step three of Example 8, compound 19-3 was replaced with compound 28-3 to obtain the title compound, 180 mg. ESI-MS (m / z): 916.5 [M+H] + .

[0501] Step 2: Synthesis of 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatritriatrisaccharamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]inzizo[1,2-b]quinoline-4-yl) carbonate (compound 29-2)

[0502] Following a procedure similar to step four of Example 8, compound 19-4 was replaced with compound 29-1 to obtain the title compound, 30 mg. ESI-MS (m / z): 727.5 [M / 2+H] + .

[0503] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolazino[1,2-b]quinoline-4-yl-(4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatritriatrisamamido)benzyl)carbonate (compound TL029)

[0504] Compound 29-2 (20 mg, 0.014 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(2-propynyl-1-yl)-5-hexynamide (4.3 mg, 0.014 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (1 mL / 0.25 mL) at room temperature. Cuprous bromide (3.95 mg, 0.027 mmol) was added, and the mixture was stirred for 1 h. Preparative high-performance liquid chromatography (method D) purification yielded the title compound, 15 mg. ESI-MS (m / z): 880.0 [M / 2+H] + .

[0505] Example 11 (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl(4-((2S,5S)-5-isopropyl-45-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,40-tetraoxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12,39-tetraazatriapentadecan-44-carbamoyl)benzyl) carbonate

[0506]

[0507] Step 1: Synthesis of (S)-2-((S)-35-amino-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)-N-(4-(hydroxymethyl)phenyl)-5-ureopentanamide

[0508] Compound 29-1 (400 mg, 0.44 mmol) was dissolved in methanol and tetrahydrofuran (2.0 mL: 4.0 mL) at 20 °C. After complete dissolution, platinum dioxide (40 mg) was added in a single step under nitrogen protection, followed by three purgings with hydrogen. The hydrogenation reaction was carried out at 20 °C for 2 h. The mixture was filtered, the filter cake was washed with methanol, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 200 mg. ESI-MS (m / z): 890.4 [M+H] + .

[0509] Step 2: Synthesis of N-((6S,9S)-1-amino-6-((4-(hydroxymethyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonoxy-2,7,10,16-tetraaza-tetradodecane-42-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide

[0510] Compound 22-1 (250 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (1.0 mL) at 20 °C. HATU (160 mg, 0.42 mmol) and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were added sequentially, and the mixture was stirred overnight at room temperature. The title compound, 250 mg, was obtained by preparative high-performance liquid chromatography (Method D).

[0511] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl(4-((2S,5S)-5-isopropyl-45-(2-(methanesulfonyl)pyrimidin-5-yl)-4,7,11,40-tetraoxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12,39-tetraazatrapentadecane-44-carbamoyl)benzyl) carbonate (compound TL022)

[0512] At 20°C, (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-11-yl)ethyl)-N-isopropylmethanesulfonamide (70 mg, 0.14 mmol) was dissolved in dichloromethane (4.0 mL), cooled to 0°C, and a dichloromethane (1.0 mL) solution of p-dimethylaminopyridine (200 mg, 1.64 mmol) was added. Then, a dichloromethane (1.0 mL) solution of triphosgene (40.6 mg, 0.14 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at 0°C for 1 h. Unreacted phosgene was removed by purging with nitrogen. A solution of compound 22-2 (139 mg, 0.12 mmol) in dichloromethane (2.0 mL) was added to the reaction mixture, and the mixture was stirred at 0 °C for 1 h. The title compound, 1.5 mg, was obtained by preparative high-performance liquid chromatography (Method D). ESI-MS (m / z): 839.5 [M / 2+H] + .

[0513] Example XII 4-((S)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9,36-triazatetradodecyl-41-acetylamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl)carbonate

[0514]

[0515] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazolyl-41-acetylamido)benzyl carbonate

[0516] At room temperature, 6-(-2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl acetic acid (12 mg, 0.045 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (21.2 mg, 0.056 mmol) and N,N-diisopropylethylamine (8.6 mg, 0.067 mmol). The mixture was stirred for 10 min, and then compound 24-1 (35 mg, 0.022 mmol) was added. The mixture was stirred for 1 h. The solution was purified by preparative high-performance liquid chromatography (Method B) to obtain the title compound, 20 mg. ESI-MS (m / z): 1821.8 [M+H] + .

[0517] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazolyl-41-acetylamidoyl)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolazin[1,2-b]quinoline-4-yl)carbonate (compound TL024)

[0518] 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, with stirring for 20 min. The solution was purified by preparative high-performance liquid chromatography (Method C) to obtain 12 mg of the trifluoroacetate of the title compound. ESI-MS (m / z): 1549.6 [M+H] + .

[0519] Example 13 Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl-4-((2S,5S)-5-isopropyl-2-methyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)yl)hex-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriaconamido)benzyl carbonate

[0520]

[0521] Step 1: Preparation of (S)-(9H-fluorene-9-yl)-methyl(1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)carbamate

[0522] At room temperature, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.31 g, 5.30 mmol) and p-aminobenzyl alcohol (593 mg, 4.82 mmol) were added to a solution of compound 30-1 (1.5 g, 4.82 mmol) in dichloromethane (35 mL), and the mixture was stirred for 3 h. The mixture was purified by silica gel column chromatography to give the title compound, 1.8 g. ESI-MS (m / z): 417.2 [M+H] +

[0523] Step 2: Preparation of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)propionamide

[0524] At room temperature, ethylenediamine (5 mL) was added to a solution of compound 30-2 (1.8 g, 4.32 mmol) in dichloromethane (20 mL), and the reaction was allowed to proceed for 2 h. Purification was achieved by silica gel column chromatography to give the title compound, 820 mg. ESI-MS (m / z): 195.1 [M+H]+

[0525] Step 3: Preparation of (9H-fluorene-9-yl)-methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobut-2-yl)-carbamate

[0526] At room temperature, (2S)-2-(9H-fluorene-9-ylmethoxycarbonylamino)-3-methyl-butyric acid (875 mg, 2.58 mmol), O-benzotriazole-tetramethylurea 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 sequentially added to a 2 mL solution of compound 30-3 (503 mg, 2.58 mmol), and the mixture was stirred for 4 h. The mixture was purified by silica gel column chromatography to give the title compound, 1.1 g. ESI-MS (m / z): 516.2 [M+H]+

[0527] Step 4: Preparation of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide

[0528] At room temperature, ethylenediamine (2 mL) was added to a solution of compound 30-4 (1.1 g, 2.13 mmol) in dichloromethane (8 mL). The reaction was stirred for 1 h. Purification was performed by silica gel column chromatography to give the title compound, 610 mg. ESI-MS (m / z): 294.2 [M+H] +

[0529] Step 5: Preparation of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-diazapentadecanoamido)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide

[0530] At room temperature, O-benzotriazole-tetramethylurea 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-nonoxy-6-azatricyclodecane-1-acid (156 mg, 0.28 mmol) were added to dichloromethane (3 mL) containing compound 30-5 (84 mg, 0.28 mmol), and the mixture was stirred for 4 h. The mixture was purified by silica gel column chromatography to give the title compound, 163 mg. ESI-MS (m / z): 830.4 [M+H] +

[0531] Step Six: Preparation of 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl) carbonate

[0532] 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] indo[1,2-b]quinoline-11-yl)ethyl)-N-isopropylmethanesulfonamide (45 mg, 0.09 mmol) in dichloromethane (0.7 mL) at 0 °C. The reaction was carried out for 1 h at 0 °C. Compound 30-6 (73 mg, 0.09 mmol) in dichloromethane (1 mL) was added dropwise to the reaction solution, and the reaction was carried out for 1 h at 0 °C. The mixture was purified by silica gel column chromatography to give the title compound, 33 mg. ESI-MS (m / z): 1367.6 [M+H] +

[0533] Step 7: Preparation of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl-4-((2S,5S)-5-isopropyl-2-methyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriaconamido)benzyl carbonate (compound TL030)

[0534] At room temperature, cuprous bromide (5 mg, 0.04 mmol) and compound 30-7 (20 mg, 15 μmol) were added dropwise to a mixture of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynylamide (9 mg, 0.007 mmol) in water and N,N-dimethylformamide (0.2 mL: 0.8 mL), and the mixture was stirred for 4 h. The solution was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 4.15 mg. ESI-MS (m / z): 1672.7 [M+H] +

[0535] Example Fourteen: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0536]

[0537] Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-yneamide

[0538] Proprynne-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL) at 25 °C. N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (1.63 g, 4.25 mmol) were added sequentially, and the mixture was stirred for 2 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by rapid silica gel column chromatography (ethyl acetate / petroleum ether = 3 / 1) to give the title compound, 700 mg. ESI-MS (m / z): 306.1 [M+H] + .

[0539] 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-nonazo-3,9-diazapentadecanoylamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3′,4′:6,7]inzizo[1,2-b]quinoline-4-yl) carbonate

[0540] Under nitrogen protection at 25°C, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, and a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added. Then, a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at 0°C for 20 min, and the reaction solution was purged with nitrogen for 20 min. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatriacetamyl)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added. The mixture was stirred at 0 °C for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Method A) to give the title compound, 500 mg. ESI-MS (m / z): 1597.5 [M+H] + .

[0541] Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]inzazido[1,2-b]quinoline-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide)benzyl)carbonate

[0542] Compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL) at room temperature, and cuprous bromide (11 mg, 0.08 mmol) was added. The mixture was stirred for 1 h. The solution was purified by preparative high-performance liquid chromatography (Method B) to give the title compound, 30 mg. ESI-MS (m / z): 815.9 [(M-273) / 2+H] + .

[0543] Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate (compound TL033)

[0544] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and reacted with trifluoroacetic acid (0.2 mL) at room temperature for 30 min. The solution was purified by preparative high-performance liquid chromatography (Method C) to give 20.0 mg of the trifluoroacetate salt of the title compound. Its structure is characterized as follows:

[0545] 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 It is -19.55° (c = 1.000 g / 100 mL, CH3CN).

[0546] Example 15 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]inzazido[1,2-b]quinoline-4-carbonate

[0547]

[0548] Step 1: Synthesis of 4-((S)-35-azido-2-(4-((((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo6,12,15,18,21,24,27-nonoxy-((S)-9-((tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1,2,3,4-tetrahydroquinoline-1H-pyrano[3′,4′:6,7]inzazo[1,2-b]quinoline-4-yl) carbonate

[0549] Compound 34-1 (100 mg, 0.2 mmol) was dissolved in dry dichloromethane (2 mL) under nitrogen protection at room temperature. The mixture was cooled to 0 °C, and a solution of 4-dimethylaminopyridine (144 mg, 1.18 mmol) in dry dichloromethane (0.5 mL) was added. Then, a solution of triphosgene (41 mg, 0.14 mmol) in dry dichloromethane (0.5 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at 0 °C for 1 h. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatriacetamyl)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (160 mg, 0.15 μmol) in dry dichloromethane (0.5 mL) was added to the reaction solution. After addition, the reaction was allowed to proceed at room temperature for 1 h. The compound was purified by preparative high-performance liquid chromatography (Method B) to obtain 60 mg of the title compound. ESI-MS (m / z): 1592.7 [M+H] + .

[0550] 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]indazine[1,2-b]quinoline-4-yl-4-((S)-2-(4-(((6-2-(methanesulfonyl)pyrimidin-5-yl)-35-(4-((6-2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-dioxo6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide) carbonate

[0551] Compound 34-2 (40 mg, 0.03 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (11.50 mg, 0.04 mmol) were dissolved in dimethyl sulfoxide and water (0.5 mL: 0.1 mL) at room temperature, and cuprous bromide (9.01 mg, 0.06 mmol) was added and the mixture was stirred for 1 h. The mixture was purified by preparative high-performance liquid chromatography (Method B) to give the title compound, 20 mg. ESI-MS (m / z): 1897.5 [M+H].

[0552] Step 3: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-((methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]inzazido[1,2-b]quinoline-4-carbonate (compound TL034)

[0553] Compound 34-3 (30 mg, 0.018 mmol) was dissolved in acetonitrile and water (0.4 mL: 0.1 mL) at room temperature. A mixed solution of trifluoroacetic acid and acetonitrile (0.5 mL: 0.5 mL) was added dropwise, and the mixture was stirred at room temperature for 2 h. The solution was purified by preparative high-performance liquid chromatography (Method C) to give 12 mg of the trifluoroacetate of the title compound. ESI-MS (m / z): 1511.5 [M+H] + .

[0554] Example XVI. Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-yneamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0555]

[0556] 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-nonoxy-3,9-diazapentadecanoamide)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]inzizo[1,2-b]quinoline-4-yl) carbonate

[0557] Using a synthetic method similar to step one of Example 15, but replacing compound 34-1 with compound 35-1, the title compound, 60 mg, was obtained. ESI-MS (m / z): 1561.5 [M+H] + .

[0558] Step 2: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indo[1,2-b]quinoline-4-yl)-4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide] carbonate

[0559] Using a synthetic method similar to step two of Example 15, but replacing compound 34-2 with compound 35-2, the title compound was obtained, 20 mg. ESI-MS (m / z): 1866.5 [M+H].

[0560] Step 3: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate (compound TL035)

[0561] Using a synthetic method similar to step three of Example XV, but replacing compound 34-3 with compound 35-3, 4.9 mg of the trifluoroacetate of the title compound was obtained. ESI-MS (m / z): 1594.5 [M+H] + .

[0562] Example XVII. Synthesis of 4-((S,Z)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazolyl-41-enamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl) carbonate

[0563]

[0564] Step 1: Synthesis of (Z)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-enoic acid

[0565] Compound 3-4 (200 mg, 0.67 mmol) was dissolved in methanol (8.0 mL) at 20 °C. Lindela catalyst (20 mg) was added under nitrogen protection, and the mixture was purged three times with hydrogen. The hydrogenation reaction was carried out at 20 °C for 3 h. The mixture was filtered, and the filtrate was evaporated to dryness to give 150 mg of the title compound. ESI-MS (m / z): 271.1 [M+H] + .

[0566] Step 2: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl(4-((S,Z)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9,36-triazatetrazol-41-enamido)benzyl carbonate

[0567] Compound 45-2 (8 mg, 0.030 mmol) was dissolved in dichloromethane (2 mL) at room temperature. 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (14.9 mg, 0.039 mmol) and N,N-diisopropylethylamine (8.8 mg, 0.068 mmol) were added. The reaction mixture was stirred at room temperature for 10 min. Compound 48-1 (30 mg, 0.020 mmol) was then added, and the reaction mixture was stirred at room temperature for 1 h. The compound was purified by preparative high-performance liquid chromatography (Method B) to give the title compound, 30 mg. ESI-MS (m / z): 1787.8 [M+H] + .

[0568] Step 3: Synthesis of 4-((S,Z)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazolyl-41-enamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolezino[1,2-b]quinoline-4-yl) carbonate (compound TL045)

[0569] Compound 45-3 (30 mg, 0.017 mmol) was dissolved in acetonitrile (1 mL) at room temperature, and a solution of trifluoroacetic acid (0.5 mL) in acetonitrile (0.5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 min. Preparative high-performance liquid chromatography (method C) purification yielded the title compound trifluoroacetate, 9 mg. ESI-MS (m / z): 1515.6 [M+H] + .

[0570] Example 18 4-((S)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9,36-triazatetrazolyl-41-acetylamidoyl)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3′,4′:6,7]indolazin[1,2-b]quinoline-4-yl)carbonate

[0571]

[0572] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazol-41-acetylamido)benzyl carbonate

[0573] The synthetic method was similar to step one of Example XII, except that compound 24-1 was replaced with compound 48-1. The title compound was obtained, 15 mg. ESI-MS (m / z): 1785.8 [M+H] + .

[0574] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-42-(2-(methanesulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9,36-triazatetrazolyl-41-acetylamidoyl)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl)carbonate (compound TL048)

[0575] The synthesis was performed using a method similar to step two of Example XII, except that compound 24-2 was replaced with compound 48-2. The trifluoroacetate of the title compound was obtained, 11.35 mg. ESI-MS (m / z): 1513.7 [M+H] + .

[0576] Example 19 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-((methanesulfonyl)pyrimidin-5-yl)thiazolyl-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecylamino)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3′,4′:6,7]indolazin[1,2-b]quinoline-4-yl)carbonate

[0577]

[0578] Step 1: Synthesis of 2-(2-(methylthio)pyrimidin-5-yl)thiazolyl-4-carboxylic acid

[0579] Compound 49-1 (100 mg, 0.40 mmol), 2-bromo-4-thiazolic acid (99.01 mg, 0.48 mmol), potassium carbonate (137.03 mg, 0.99 mmol), and [1,1′-bis(diphenylphosphine)ferrocene]palladium dichloride (29.02 mg, 0.04 mmol) were added to N,N-dimethylformamide (4 mL) and water (1 mL) under nitrogen protection. The reaction mixture was heated to 100 °C and stirred for 4 hours. The reaction solution was cooled to room temperature, added dropwise to water, filtered, and the filtrate was collected. The filtrate was extracted with ethyl acetate (10 mL × 3), and the aqueous phase was collected. The pH was adjusted to 3 with dilute hydrochloric acid, and a solid precipitated. The solid was filtered, and the filter cake was collected to give 70 mg of the title compound. ESI-MS (m / z): 254.0 [M+H] + .

[0580] Step 2: Synthesis of 2-(2-(methanesulfonyl)pyrimidin-5-yl)thiazolyl-4-carboxylic acid

[0581] Compound 49-2 (73 mg, 0.29 mmol) was dissolved in dichloromethane (15 mL), and m-chloroperoxybenzoic acid (175.53 mg, 0.87 mmol, 85%) was added. The reaction mixture was stirred overnight at room temperature. The solvent was concentrated under reduced pressure, and purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 20 mg. ESI-MS (m / z): 286.0 [M+H] + .

[0582] Step 3: Synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)thiazolyl-4-carboxamide

[0583] Compound 49-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL), and O-(7-azabenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (39.98 mg, 0.11 mmol) was added. The reaction mixture was cooled to 0 °C, and N,N-diisopropylethylamine (22.65 mg, 0.18 mmol) and propyneamine (4.63 mg, 0.09 mmol) were added. The reaction mixture was stirred at room temperature for 3 h. Preparative high-performance liquid chromatography (PHPLC) purification (Method D) yielded the title compound, 10 mg. ESI-MS (m / z): 323.0 [M+H] + .

[0584] Step 4: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone [3′,4′:6,7]indolazino[1,2-b]quinoline-4-yl-(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((2-(2-(2-(methanesulfonyl)pyrimidin-5-yl)thiazolyl-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecylamino)benzyl)carbonate

[0585] Compound 33-1 (30 mg, 0.02 mmol) and compound 49-4 (9.08 mg, 0.03 mmol) were dissolved in dimethyl sulfoxide and water (2 mL / 0.5 mL) at room temperature. Cuprous bromide (5.39 mg, 0.04 mmol) was added, and the mixture was stirred for 2 h. The mixture was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method B) to give the title compound, 20 mg. ESI-MS (m / z): 1647.3 [M+H-273] + .

[0586] Step 5: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-((methanesulfonyl)pyrimidin-5-yl)thiazolyl-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecylamino)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranone[3′,4′:6,7]indolazin[1,2-b]quinoline-4-yl) carbonate

[0587] Compound 49-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2 mL) at room temperature, and trifluoroacetic acid (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 min. The reaction mixture was concentrated, and the residue was purified by preparative high-performance liquid chromatography (Method C) to give 8 mg of the trifluoroacetate of the title compound. ESI-MS (m / z): 1647.9 [M+H] + .

[0588] Example 20: 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-((methanesulfonyl)pyrimidin-5-yl)-oxazol-4-formamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0589]

[0590] Step 1: Synthesis of ethyl 2-(2-(methylthio)pyrimidin-5-yl)oxazol-4-carboxylate

[0591] At 25 °C, ethyl 2-bromooxazol-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(diphenylphosphine)ferrocene]palladium dichloride (33 mg, 0.05 mmol) were added sequentially. Under N2 protection, the mixture was heated to 90 °C and reacted for 3 h. The reaction solution was filtered through diatomaceous earth, and the filtrate was diluted with water (50 mL). Extraction was performed with ethyl acetate (30 mL × 3). The organic phases were combined and dried. The desiccant was removed, 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] + .

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

[0593] Compound 50-1 (50 mg, 0.19 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (4 mL / 2 mL) at 25 °C. After complete dissolution, lithium hydroxide monohydrate (40 mg, 0.94 mmol) was added, and 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), and the aqueous phase was adjusted to pH 2-3 with 1 N dilute hydrochloric acid. Extraction was carried out with a dichloromethane / methanol mixed solvent (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 drying agent was filtered off, and the filtrate was concentrated to give the title compound, 40 mg. It was used directly in the next reaction without purification. ESI-MS (m / z): 238.1 [M+H] + .

[0594] Step 3: Synthesis of 2-(2-(methanesulfonyl)pyrimidin-5-yl)oxazol-4-carboxylic acid

[0595] Compound 50-2 (40 mg, 0.17 mmol) was dissolved in dichloromethane (6 mL) at 25 °C. After complete dissolution, m-chloroperoxybenzoic acid (29 mg, 0.17 mmol) was added. The mixture was stirred at 25 °C for 14 h. The reaction solution was concentrated, and the residue was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 20 mg. ESI-MS (m / z): 269.9 [M+H] + .

[0596] Step 4: Synthesis of 2-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazol-4-carboxamide

[0597] At 25°C, compound 50-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (4 mL), followed by the sequential addition of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (42 mg, 0.11 mmol) and N,N-diisopropylethylamine (19 mg, 0.15 mmol). The mixture was stirred for 5 min, then propyneamine (5.0 mg, 0.09 mmol) was added. After the addition was complete, the mixture 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 give the title compound, 5.0 mg. ESI-MS (m / z): 306.9 [M+H] + .

[0598] Step 5: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indo[1,2-b]quinoline-4-yl 4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((2-(2-(2-(methanesulfonyl)pyrimidin-5-yl)oxazol-4-formamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide)benzyl)carbonate

[0599] 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). Cuprous bromide (5.0 mg, 0.04 mmol) was added in a single addition, and the mixture was allowed to react 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)] + .

[0600] Step Six: Synthesis of 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-((methanesulfonyl)pyrimidin-5-yl)oxazol-4-formamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0601] 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 the reaction was carried out at 25 °C for 10 min. The reaction solution was concentrated, and the residue was purified by preparative high-performance liquid chromatography (Method C) to give 3.0 mg of the trifluoroacetate of the title compound. ESI-MS (m / z): 816.5 [M / 2+H] + .

[0602] 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-methylbutyramide)-N,3-dimethylbutyramide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2- (methylpropionamide)-3-phenylpropionamide)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxy-13,19,22,25,28,31,34,37,40-nonazo-2,7,10,16-tetraazaanthra-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide

[0603]

[0604] 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-butamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrole-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)amino)-1-oxo-5-pentylurea-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino-(9H-fluorene-9-yl)methyl-carboxylate

[0605] At room temperature, compound 51-1 (100 mg, 0.17 mmol) and ((S)-1-(((S)-1-((4-((((S)-2-amino-3-phenylpropamido)methyl)phenyl)amino)-1-oxo-5-pentylurea-2-yl)amino)-3-methyl-1-oxobut-2-yl)amino-(9H-fluorene-9-yl)methyl-carboxylic acid ester trifluoroacetate (144 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (2 mL). The mixture was cooled to 0 °C, and 1-hydroxybenzotriazole (34 mg, 0.25 mmol), N-methylmorpholine (51 mg, 0.51 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (48 mg, 0.25 mmol) were added sequentially. After the addition was complete, the reaction mixture was stirred at 0 °C for 5 h. The reaction solution was poured into water (20 mL), and a white solid precipitated. The solid was filtered, the filter cake was washed with water, and dried to give 200 mg of the title compound. ESI-MS (m / z): 1329.2 [M+H] + .

[0606] Step 2: Synthesis of (S)-2-((S)-2-amino-3-butamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butamido)-N,3-dimethylbutamido)-3-methoxy-5-methylheptanoyl)pyrrole-2-yl)-3-methoxy-2-methylpropamido)-3-phenylpropamido)methyl)phenyl)-5-ureidopentanamide

[0607] Compound 51-2 (200 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (5 mL) at room temperature, and piperidine (0.5 mL) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 65 mg. ESI-MS (m / z): 1107.2 [M+H] + .

[0608] Step 3: Synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diaza-3-pentadecylamino)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramide)-N,3-dimethylbutyramide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamide)-3-phenylpropionamide)methyl)phenyl)-5-ureidopentanamide

[0609] 32-Azide-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatricarboxylic acid (33.1 mg, 0.06 mmol) was added to N,N-dimethylformamide (5 mL), followed by O-(7-azabenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (38 mg, 0.10 mmol) and N,N-diisopropylethylamine (26 mg, 0.20 mmol). The reaction mixture was stirred at room temperature for 10 min, cooled to 0 °C, and compound 51-3 (55 mg, 0.05 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 56 mg. ESI-MS (m / z): 821.8 [M / 2+H] + .

[0610] Step 4: N-((1-((6S,9S)-1-amino-6-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyramide)-N,3-dimethylbutyramide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropane Synthesis of amide (-3-phenylpropionamide)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxy-13,19,22,25,28,31,34,37,40-nonazo-2,7,10,16-tetraazaanthra-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide

[0611] Compound 51-4 (56 mg, 0.04 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)-5-hexyneamide (16 mg, 0.05 mmol) were dissolved in a mixture of dimethyl sulfoxide and water (2 mL / 0.5 mL) at room temperature. Cuprous bromide (10 mg, 68.17 μmol) was added, and the mixture was stirred for 2 h. The mixture was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 50 mg. ESI-MS (m / z): 974.3 [M / 2+H] + .

[0612] Example 22 4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonazo-3,6,12-triazatetradodecyl)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)pyrrolidone-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0613]

[0614] Step 1: 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonazo-3,6,12-triazatetradodecyl)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R) Synthesis of 2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0615] Compound 53-1 (100 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (3 mL) at room temperature, followed by the addition of 1-hydroxybenzotriazole (13 mg, 0.09 mmol) and N,N-diisopropylethylamine (36 mg, 0.28 mmol), and then compound 52-1 (67 mg, 0.09 mol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 120 mg. ESI-MS (m / z): 830.1 [M / 2+H] + .

[0616] Step 2: 4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonazo-3,6,12-triazatetradodecyl)benzyl-((S)-1-(((S)-1-(( Synthesis of (3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0617] Compound 52-2 (22 mg, 0.07 mmol) was dissolved in a mixture of dimethyl sulfoxide and water (3 mL / 0.3 mL) at room temperature, and cuprous bromide (18 mg, 0.13 mmol) was added. The mixture was stirred for 1 h. The solution was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 92 mg. ESI-MS (m / z): 982.8 [M / 2+H] + .

[0618] Example 23 (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-carbamoyl)methyl)-1H- Synthesis of 1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriacontamido)butyrylamino)-5-ureidopentanoylamino)benzyl)oxy)carbonyl)amino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0619]

[0620] Step 1: Synthesis of 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazaoctadecanoamido)benzyl-(4-nitrophenyl)-carbonate

[0621] Compound 29-1 (500 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (10 mL) at 25 °C. N,N-diisopropylethylamine (141 mg, 1.09 mmol) was added, followed by dropwise addition of a dichloromethane solution of di(p-nitrobenzene) carbonate (332 mg, 1.09 mmol) in 1 mL. After addition, the mixture was stirred at 25 °C for 3 h. The reaction solution was purified by reverse-phase column chromatography (C18) (acetonitrile / water = 1:2) to give the title compound, 400 mg. ESI-MS (m / z): 1081.9 [M+H] + .

[0622] 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-nonoxy-6-azatriaconitamido)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0623] Compound 53-1 (60 mg, 0.06 mmol) and ((2R)-3-((2S)-1-((3R,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine (41 mg, 0.06 mmol) were dissolved in N,N-dimethylformamide (2 mL) at 25 °C. After complete dissolution, 1-hydroxybenzotriazole (8 mg, 0.06 mmol) was added. The mixture was stirred at 25 °C for 16 h. The reaction solution was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 38 mg. ESI-MS (m / z): 837.2 [M / 2+H] + .

[0624] Step 3: (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-carbamoyl)methyl)-1H-1 Synthesis of 2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriaconamido)butyrylamino)-5-ureidopentanoylamino)benzyl)oxy)carbonyl)amino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0625] At 25 °C, 2-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazol-4-carboxamide (9 mg, 0.03 mmol) and compound 53-2 (50 mg, 0.03 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (1 mL / 0.25 mL). After complete dissolution, cuprous bromide (11 mg, 0.08 mmol) was added. The mixture was stirred for 1 h under N2 protection. The mixture was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 25 mg. ESI-MS (m / z): 989.9 [M / 2+H] + .

[0626] Example 24 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecylamino)benzyl-((S)-1-(((S)-1-(((3R, 4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0627]

[0628] Step 1: Synthesis of (S)-4-(35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyryl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecylamino)benzyl-(4-nitrophenyl)-carbonate

[0629] Compound 54-1 (1 g, 0.95 mmol) was dissolved in dichloromethane (20 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (488 mg, 3.77 mmol), and then dropwise added to a solution of dichloromethane (10 mL) containing di-(p-nitrobenzene)-carbonate (860 mg, 2.83 mmol). The reaction mixture was stirred at room temperature for 6 h. Purification was achieved by silica gel column chromatography (dichloromethane / methanol = 40 / 1) to give the title compound, 900 mg. ESI-MS (m / z): 953.0 [M+H-273] + .

[0630] Step 2: 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyryl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecylamino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1 Synthesis of R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0631] At room temperature, 1-hydroxybenzotriazole (33 mg, 0.25 mmol) and N,N-diisopropylethylamine (48 mg, 0.37 mmol) were added to compound 54-2 (2 mL), followed by compound 52-1 (88 mg, 0.12 mmol). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was purified by preparative high-performance liquid chromatography (Method B) to give the title compound, 150 mg. ESI-MS (m / z): 1803.6 [M+H] + .

[0632] Step 3: 4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyryl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33- Synthesis of nonazo-3,9-diaza-3-pentadecylamino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0633] Compound 54-3 (100 mg, 0.06 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(propyl-2-ynyl-1-yl)-5-hexyneamide (26 mg, 0.08 mmol) were dissolved in dimethyl sulfoxide (2 mL) and water (0.5 mL) at room temperature. Cuprous bromide (16 mg, 0.11 mmol) was added, and the mixture was stirred for 2 h. The mixture was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method B) to give the title compound, 70 mg. ESI-MS (m / z): 1936.6 [M+H-273] + .

[0634] Step 4: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)-5-hexyneamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentadecylamino)benzyl-((S)-1-(((S)-1-(((3R,4S, Synthesis of 5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate

[0635] Compound 54-4 (70 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL) at room temperature, and trifluoroacetic acid (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 20 min. The reaction mixture was concentrated, and the residue was purified by preparative high-performance liquid chromatography (Method C) to give 55 mg of the trifluoroacetate of the title compound. ESI-MS (m / z): 918.8 [M / 2+H] + .

[0636] Example 25 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0637]

[0638] Step 1: Synthesis of methyl 4-(2-(methylthio)pyrimidin-5-yl)benzoate

[0639] Compound 49-1 (252 mg, 1.0 mmol), water (3 mL), Pd(dppf)Cl2 (40 mg, 0.05 mmol), and potassium carbonate (277 mg, 2.0 mmol) were added sequentially to a 1,4-dioxane (5 mL) solution of methyl p-bromobenzoate (215 mg, 1.0 mmol). The mixture was stirred at 80 °C for 4 h. The mixture was extracted with ethyl acetate (30 mL × 3), the organic phases were combined, dried, and the insoluble matter was filtered off. The residue was purified by silica gel column chromatography to give the title compound, 220 mg. ESI-MS (m / z): 261.0 [M+H] + .

[0640] Step 2: Synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic acid

[0641] At 25°C, lithium hydroxide monohydrate (322 mg, 7.68 mmol) and water (3 mL) were added separately to a tetrahydrofuran (3 mL) solution of compound 55-1 (500 mg, 1.92 mmol), and the mixture was stirred for 4 h. The pH of the reaction solution was adjusted to 3-4 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, dried, and the insoluble matter was filtered off. The residue was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 430 mg. ESI-MS (m / z): 246.9 [M+H] + .

[0642] Step 3: Synthesis of 4-(2-(methanesulfonyl)pyrimidin-5-yl)benzoic acid

[0643] 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), and the mixture was stirred for 5 h. The title compound, 180 mg, was purified by silica gel column chromatography. ESI-MS (m / z): 279.0 [M+H] + .

[0644] Step 4: Synthesis of 4-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)benzamide

[0645] At 25 °C, benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate (100 mg, 0.26 mmol) was added to a solution of compound 55-3 (50 mg, 0.18 mmol) in dichloromethane (10 mL), and the mixture was stirred for 30 min. Then, propyneamine (10 mg, 0.2 mmol) and N,N-diisopropylethylamine (70 mg, 0.5 mmol) were added to the reaction mixture, and the mixture was stirred for 2.5 h. The title compound, 20 mg, was purified by silica gel column chromatography. ESI-MS (m / z): 316.0 [M+H] + .

[0646] Step 5: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indo[1,2-b]quinoline-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((4-(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide)benzyl)carbonate

[0647] At 25°C, under N2 protection, cuprous iodide (10 mg, 0.05 mmol) and water (2 mL) were added sequentially to a dimethyl sulfoxide (2 mL) solution of compound 55-4 (16 mg, 0.05 mmol) and compound 33-1 (80 mg, 0.05 mmol), and the reaction was stirred for 1 h. Purification (Method B) yielded the title compound, 79 mg. ESI-MS (m / z): 1641.5 [M-273+H] + .

[0648] Step Six: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methanesulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7] indo[1,2-b]quinoline-4-yl) carbonate

[0649] Compound 55-5 (55 mg, 0.029 mmol) was added to a mixed solvent of trifluoroacetic acid (0.5 mL) and water / acetonitrile (0.1 mL / 0.5 mL) at 25 °C. The reaction mixture was stirred for 15 min, and the reaction solution was purified by preparative high-performance liquid chromatography (Method C) to give 42 mg of the trifluoroacetate of the title compound. ESI-MS (m / z): 821.0 [M / 2+H] + .

[0650] Example 26 N-((1-((6S,9S)-1-amino-6-((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3- Methoxy-2-methylpropamido)propyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonoxy-2,7,10,16-tetraaza-tetradodecane-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide

[0651]

[0652] Step 1: Synthesis of 32-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatridodecanoic acid

[0653] Compound 56-1 (750 mg, 1.28 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (496 mg, 1.54 mmol) were dissolved in dimethyl sulfoxide (10 mL) at 20 °C. Cuprous bromide (465 mg, 3.21 mmol) was added in a single batch, and the mixture was stirred for 12 h after the addition was complete. The reaction mixture was filtered, and the filtrate was purified by preparative high-performance liquid chromatography (Method D) to give 500 mg of the title compound. ESI-MS (m / z): 860.4 [M+H] + .

[0654] Step 2: Synthesis of (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropamido)propyl)phenyl)amino)-1-oxo-5-ureidopentanoylamino-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate

[0655] At 25°C, (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(4-aminophenyl)-3-azidopropyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3 - Dimethylbutyrylamino (185 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), and HATU (137 mg, 0.36 mmol) was added. The mixture was stirred for 5 min, then (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidovalerate (131 mg, 0.26 mmol) was added, and the mixture was stirred at room temperature for 30 min. The reaction solution was used directly for the next step. ESI-MS (m / z): 626.0 [M / 2+H] + .

[0656] Step 3: Synthesis of (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-(4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidovalamid

[0657] At 25°C, diethylamine (0.5 mL) was added to the reaction solution from step two. After the addition was complete, the mixture was stirred for 30 min. The reaction solution was purified by preparative high-performance liquid chromatography (Method D) to obtain 70 mg of the title compound. ESI-MS (m / z): 515.0 [M / 2+H] + .

[0658] 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)pyrrolidine-2-yl)-3-methoxy Synthesis of 2-methylpropamidopropylphenylcarbamoyl-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonoxy-2,7,10,16-tetraaza-tetradodecane-42-yl)-1H-1,2,3-triazol-4-ylmethyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide

[0659] At 25℃, (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)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidovalamid (95m 32-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamide)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatridocoanoic acid (79 mg, 0.092 mmol) was dissolved in N,N-dimethylformamide (4 mL), and HATU (70 mg, 0.184 mmol) was added in a single batch. The mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative high-performance liquid chromatography (Method D) to give the title compound, 30 mg. ESI-MS (m / z): 935.8 [M / 2+H] + .

[0660] III. Conjugation of antibodies with compounds containing cellular bioactive molecules and linkers

[0661] Example 27 Preparation of BT001002

[0662] Take 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg / mL), dilute 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. Incubate at room temperature for 30 min. Add 15 times the amount of TL003 dissolved in dimethyl sulfoxide to the above solution system, mix well, and incubate at room temperature for 2 h. Finally, add 6.1 μL of 100 mM cysteine ​​to terminate the reaction. Finally, the buffer solution was replaced with 20mM PB buffer solution at pH 6.44 using a G-25 gel column to obtain the product conjugated with Sacituzumab antibody TL003, named BT001002.

[0663]

[0664] Example 28: Preparation of BT001004

[0665] Take 0.285 mL of Sacituzumab antibody (anti-Trop-2, 17.6 mg / mL) and 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, mix well, and incubate at room temperature for 30 min. Add 9 times the amount of TL019 dissolved in dimethyl sulfoxide to the above solution system, mix well, and incubate at room temperature for 2 h. Finally, replace the buffer solution with PBS buffer solution at pH 6.5 using a G-25 gel column to obtain the product conjugated with TL019 and Sacituzumab antibody, named BT001004.

[0666]

[0667] Example 29: Preparation of BT001012

[0668] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL024 to obtain the product conjugated with the Sacituzumab antibody, named BT001012.

[0669]

[0670] Example 30: Preparation of BT001013

[0671] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL048 to obtain the product conjugated with the Sacituzumab antibody, named BT001013.

[0672]

[0673] Example 31: Preparation of BT001018

[0674] Using a method similar to that in Example 27, TL003 was replaced with TL030 to obtain the product conjugated with Sacituzumab antibody, named BT001018.

[0675]

[0676] Example 32 Preparation of BT001021

[0677] Take 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg / mL), dilute 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. Incubate at room temperature for 30 min. Add 10 times the amount of TL033 trifluoroacetate dissolved in dimethyl sulfoxide to the above solution system, mix well, and incubate at room temperature for 2 h. Finally, add 6.1 μL of 100 mM cysteine ​​to terminate the reaction. Finally, the buffer solution was replaced with PBS buffer solution at pH 6.5 using a G-25 gel column to obtain the product conjugated with TL033 and Sacituzumab antibody, named BT001021.

[0678]

[0679] Example 33 Preparation of BT001022

[0680] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL034 to obtain the product conjugated with the Sacituzumab antibody, named BT001022.

[0681]

[0682] Example 34 Preparation of BT001023

[0683] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL035 to obtain the product conjugated with the Sacituzumab antibody, named BT001023.

[0684]

[0685] Example 35 Preparation of BT001032

[0686] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL045 to obtain the product conjugated with the Sacituzumab antibody, named BT001032.

[0687]

[0688] Example 36: Preparation of BT001033

[0689] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL033, and the Sacituzumab antibody was replaced with the M1 antibody, resulting in a product conjugated with the M1 antibody, named BT001033.

[0690]

[0691] Example 37 Preparation of BT001034

[0692] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL033, and the Sacituzumab antibody was replaced with the M2 antibody, resulting in a product conjugated with the M2 antibody, named BT001034.

[0693]

[0694] Example 38: Preparation of BT001035

[0695] Take 0.3 mL of M3 antibody (anti-Trop-2, 33.5 mg / mL), dilute 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. Incubate at room temperature for 30 min. Add 10 times the amount of TL033 trifluoroacetate dissolved in dimethyl sulfoxide to the above solution system, mix well, and incubate at room temperature for 2 h. Finally, 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 the product conjugated with TL033 and M3 antibody, named BT001035.

[0696]

[0697] Example 39: Preparation of BT001036

[0698] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL033, and the Sacituzumab antibody was replaced with the trastuzumab antibody, resulting in a product conjugated with TL033 and the trastuzumab antibody, named BT001036.

[0699]

[0700] Example 40: Preparation of BT001040

[0701] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL049 to obtain the product conjugated with the Sacituzumab antibody, named BT001040.

[0702]

[0703] Example 41: Preparation of BT001041

[0704] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL050 to obtain the product conjugated with the Sacituzumab antibody, named BT001041.

[0705]

[0706] Example 42 Preparation of BT001042

[0707] Using a method similar to that in Example 27, TL003 was replaced with TL051 to obtain the product conjugated with TL051 and Sacituzumab antibody, named BT001042.

[0708]

[0709] Example 43: Preparation of BT001043

[0710] Using a method similar to that in Example 27, TL003 was replaced with TL052 to obtain a product conjugated with TL052 and Sacituzumab antibody, named BT001043.

[0711]

[0712] Example 44 Preparation of BT001044

[0713] Using a method similar to that in Example 27, TL003 was replaced with TL053 to obtain a product conjugated with Sacituzumab antibody, named BT001044.

[0714]

[0715] Example 45: Preparation of BT001045

[0716] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL054 to obtain the product conjugated with the Sacituzumab antibody, named BT001045.

[0717]

[0718] Example 46: Preparation of BT001046

[0719] Using a method similar to that in Example 27, TL003 was replaced with the trifluoroacetate of TL055 to obtain the product conjugated with the Sacituzumab antibody, named BT001046.

[0720]

[0721] Example 47 Preparation of BT001047

[0722] Using a method similar to that in Example 27, TL003 was replaced with TL056 to obtain a product conjugated with TL056 and Sacituzumab antibody, named BT001047.

[0723]

[0724] Example 48: LC-MS determination of the molecular weight of BT001002

[0725] LCMS molecular weight analysis was performed on the coupled BT001002.

[0726] Chromatographic determination conditions:

[0727] Liquid chromatography column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0728] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0729] Flow rate: 0.25 mL / min; Sample chamber temperature: 8℃; Column temperature: 60℃; Injection volume: 1 μg;

[0730] Time (minutes) 178913 Mobile phase A (volume %) 90 20 20 90 90 Mobile phase B (volume %) 10 80 80 10 10

[0731] Mass spectrometry determination conditions:

[0732] Mass spectrometer model: Triple TOF 5600+;

[0733] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10m / z600-5000;

[0734] The results are shown in Figure 1-3.

[0735] Theoretical and measured molecular weight of BT001002

[0736]

[0737] 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 / heavy chain and one cellular bioactive molecule; DAR2 represents a conjugate containing one antibody light / heavy chain and two cellular bioactive molecules; DAR3 represents a conjugate containing one antibody light / heavy chain and three cellular bioactive molecules; DAR4 represents a conjugate containing one antibody light / heavy chain and four cellular bioactive molecules; glycoform indicates the glycan structure on the two heavy chains; G0F indicates fucosylated galactose-free. The terms mAb, LC, HC, DAR1, DAR2, DAR3, DAR4, and G0F will be explained as above in the following text.

[0738] As shown in Figures 1-3, the molecular weights of both the light and heavy chains change after the antibody is conjugated with TL003. The light chain conjugates one cellular bioactive molecule, while the heavy chain conjugates three cellular bioactive molecules. Therefore, the total conjugation ratio (DAR) of the antibody to the cellular bioactive molecule is 8.

[0739] Example 49: LC-MS determination of the molecular weight of BT001004

[0740] The molecular weight of the coupled BT001004 was analyzed by LC-MS.

[0741] Chromatographic determination conditions:

[0742] Liquid chromatography column: ACQUITU Protein BEH C18 1.7μm, 2.1mm×100mm;

[0743] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0744] Flow rate: 0.25 mL / min; Sample chamber temperature: 8℃; Column temperature: 60℃; Injection volume: 1 μg;

[0745] Time (minutes) 220 2225 2630 Mobile phase A (volume %) 80 60 10 10 80 80 Mobile phase B (volume %) 20 40 90 90 20 20

[0746] Mass spectrometry determination conditions:

[0747] Mass spectrometer model: Triple TOF 5600+;

[0748] GS1 60; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m / z 600-5000;

[0749] The results are shown in Figure 4-6.

[0750] Theoretical and measured molecular weight of BT001004

[0751]

[0752] LC represents antibody light chain, and HC represents antibody heavy chain.

[0753] As shown in Figures 4-6, the antibody light chain in BT001004 is conjugated to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 14% and 86%, respectively), and the heavy chain is conjugated to 1-3 cellular bioactive molecules (DAR1, DAR2, and DAR3 ratios are 13%, 19%, and 68%, respectively). Therefore, the total conjugation ratio (DAR) of the antibody to cellular bioactive molecules is calculated to be 7.0.

[0754] Example 50: LC-MS determination of the molecular weight of BT001012

[0755] Using a method similar to that of Example 48, the results are shown in Figures 10 and 11.

[0756] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001012 obtained after conjugation of TL024 with the antibody are shown in the table below:

[0757]

[0758] As shown in Figures 10 and 11, in BT001012, the antibody light chain is conjugated to 0-1 toxins (LC, DAR1 ratios are 12.9% and 87.1%, respectively), and the heavy chain is conjugated to 1-3 toxins (DAR1, DAR2, DAR3 ratios are 13.4%, 10.8%, and 75.8%, respectively). Therefore, the antibody-toxin conjugation ratio (DAR) is calculated to be 7.0.

[0759] Example 51: LC-MS determination of the molecular weight of BT001013

[0760] Using a method similar to that of Example 48, the results are shown in Figures 12 and 13.

[0761] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001013 obtained after conjugation of TL048 with the antibody are shown in the table below:

[0762]

[0763]

[0764] As shown in Figures 12 and 13, in BT001013, the antibody light chain is conjugated to 0-1 toxins (LC, DAR1 ratios are 6.8% and 93.2%, respectively), and the heavy chain is conjugated to 1-4 toxins (DAR1, DAR2, DAR3, DAR4 ratios are 12.8%, 12.8%, 64.9%, and 9.5%, respectively). Therefore, the antibody-toxin conjugation ratio (DAR) is calculated to be 7.3.

[0765] Example 52: LC-MS determination of the molecular weight of BT001018

[0766] Using a method similar to that in Example 48, the results are shown in Figures 14 and 15.

[0767] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001018 obtained after conjugation of TL030 with the antibody are shown in the table below:

[0768]

[0769] As shown in Figures 14 and 15, in BT001018, the antibody light chain is conjugated to 0-1 toxins (LC, DAR1 ratios are 55.3% and 44.7%, respectively), and the heavy chain is conjugated to 1-3 toxins (DAR1, DAR2, DAR3 ratios are 19.6%, 23.3%, and 49.6%, respectively). Therefore, the antibody-toxin conjugation ratio (DAR) is calculated to be 5.2.

[0770] Example 53: LC-MS determination of the molecular weight of BT001021

[0771] LCMS molecular weight analysis was performed on the coupled BT001021.

[0772] Chromatographic determination conditions:

[0773] Liquid chromatography column: ACQUITU Protein BEH C4 1.7μm, 2.1mm x 100mm;

[0774] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0775] Flow rate: 0.25 mL / min; Sample chamber temperature: 8℃; Column temperature: 60℃; Injection volume: 1 μg;

[0776] Time (minutes) 178913 Mobile phase A (volume %) 90 20 20 90 90 Mobile phase B (volume %) 10 80 80 10 10

[0777] Mass spectrometry determination conditions:

[0778] Mass spectrometer model: Triple TOF 5600+;

[0779] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10m / z600-5000;

[0780] The results are shown in Figures 16 and 17.

[0781] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001021 obtained after conjugation of TL033 with the antibody are shown in the table below:

[0782]

[0783] As shown in Figures 16 and 17, in BT001021, the antibody light chain is conjugated to 0-1 toxins (LC, DAR1 ratios are 4.5% and 95.5%, respectively), and the heavy chain is conjugated to 1-3 toxins (DAR1, DAR2, DAR3 ratios are 15.3%, 17.6%, and 67.1%, respectively). Therefore, the antibody-toxin conjugation ratio (DAR) is calculated to be 6.9.

[0784] Example 54: LC-MS determination of the molecular weight of BT001023

[0785] Using a method similar to that of Example 48, the results are shown in Figures 18 and 19.

[0786] The theoretical and measured molecular weights (calculated based on the major glycoform G0F) of BT001023 obtained after conjugation of TL035 with the antibody are shown in the table below:

[0787]

[0788] As shown in Figures 18 and 19, in BT001023, the antibody light chain is conjugated to 0-1 toxins (LC, DAR1 ratios are 15% and 85%, respectively), and the heavy chain is conjugated to 0-3 toxins (HC, DAR1, DAR2, DAR3 ratios are 6.7%, 16.7%, 12.7%, and 63.9%, respectively). Therefore, the antibody-toxin conjugation ratio (DAR) is calculated to be 6.4.

[0789] Example 55: LC-MS determination of the molecular weight of BT001040

[0790] LC-MS molecular weight analysis of the coupled BT001040

[0791] Liquid chromatography column: Thermo MabPac™ RP 4μm, 3.0mm * 100mm

[0792] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN; Mobile phase B: 0.1% FA / 2% H2O / 98% ACN

[0793] Flow rate: 0.25 mL / min; Sample chamber temperature: 8℃; Column temperature: 60℃; Injection volume: 1 μg

[0794] Time (minutes) 220 2225 2630

[0795] Mobile phase A (volume %) 80 60 10 10 80 80 Mobile phase B (volume %) 20 40 90 90 20 20

[0796] Mass spectrometry determination conditions:

[0797] Mass spectrometer model: Triple TOF 5600+

[0798] GS1 35; GS2 35; CUR30; TEM 350; ISVF5000; DP250; m / z 600-5000

[0799] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001040 obtained after conjugation of TL049 with the antibody are shown in the table below:

[0800]

[0801] As shown in Figures 20 and 21, in BT001040, the antibody light chain conjugates 0-1 cellular bioactive molecules (LC, DAR1 ratios are 4.9% and 95.1%, respectively), and the heavy chain conjugates 1-4 cellular bioactive molecules (DAR1, DAR2, DAR3, DAR4 ratios are 16.5%, 14.3%, 52.6%, and 16.6%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 7.3.

[0802] Example 56: LC-MS determination of the molecular weight of BT001041

[0803] Using a method similar to that in Example 55, the results are shown in Figures 22 and 23.

[0804] The theoretical and measured molecular weights (calculated based on the major glycoform G0F) of BT001041 obtained after conjugation of TL050 with the antibody are shown in the table below:

[0805]

[0806]

[0807] As shown in Figures 22 and 23, the antibody light chain in BT001041 is conjugated to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 10.5% and 89.5%, respectively), and the heavy chain is conjugated to 1-4 cellular bioactive molecules (DAR1, DAR2, DAR3, and DAR4 ratios are 21.3%, 14.8%, 57.9%, and 6.0%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 6.8.

[0808] Example 57: LC-MS determination of the molecular weight of BT001042

[0809] Using a method similar to that in Example 55, the results are shown in Figures 24 and 25.

[0810] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001042 obtained after conjugation of TL051 with the antibody are shown in the table below:

[0811]

[0812] As shown in Figures 24 and 25, the antibody light chain in BT001042 is conjugated to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 14.9% and 85.1%, respectively), and the heavy chain is conjugated to 1-3 cellular bioactive molecules (DAR1, DAR2, and DAR3 ratios are 19.7%, 9.4%, and 70.9%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 6.7.

[0813] Example 58: LC-MS determination of the molecular weight of BT001043

[0814] Using a method similar to that in Example 55, the results are shown in Figures 26 and 27.

[0815] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001043 obtained after conjugation of TL052 with the antibody are shown in the table below:

[0816]

[0817]

[0818] As shown in Figures 26 and 27, the antibody light chain in BT001043 is conjugated to 0-1 cellular bioactive molecules (LC and DAR1 ratios are 9.1% and 90.9%, respectively), and the heavy chain is conjugated to 1-3 cellular bioactive molecules (DAR1, DAR2, and DAR3 ratios are 20.1%, 11.4%, and 68.4%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 6.8.

[0819] Example 59: LC-MS determination of the molecular weight of BT001044

[0820] Using a method similar to that in Example 55, the results are shown in Figures 28 and 29.

[0821] The theoretical and measured molecular weights (calculated based on the major glycoform GOF) of BT001044 obtained after conjugation of TL053 with the antibody are shown in the table below:

[0822]

[0823] As shown in Figures 28 and 29, in BT001044, the antibody light chain conjugates 0-1 cellular bioactive molecules (LC and DAR1 ratios are 23.0% and 77.0%, respectively), and the heavy chain conjugates 1-3 cellular bioactive molecules (DAR1, DAR2, and DAR3 ratios are 19.4%, 11.4%, and 69.3%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 6.5.

[0824] Example 60: LC-MS determination of the molecular weight of BT001046

[0825] Using a method similar to that in Example 55, the results are shown in Figures 30 and 31.

[0826] The theoretical and measured molecular weights (calculated based on the major glycoform G0F) of BT001046 obtained after conjugation of TL055 with the antibody are shown in the table below:

[0827]

[0828]

[0829] As shown in Figures 30 and 31, in BT001046, the antibody light chain conjugates 0-1 cellular bioactive molecules (LC and DAR1 ratios are 33.8% and 66.2%, respectively), and the heavy chain conjugates 0-3 cellular bioactive molecules (DAR0, DAR1, DAR2, and DAR3 ratios are 21.9%, 6.1%, 9.6%, and 62.3%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 5.6.

[0830] Example 61: LC-MS determination of the molecular weight of BT001047

[0831] Using a method similar to that in Example 55, the results are shown in Figures 32 and 33.

[0832] The theoretical and measured molecular weights (calculated based on the major glycoform G0F) of BT001047 obtained after conjugation of TL056 with the antibody are shown in the table below:

[0833]

[0834] As shown in Figures 32 and 33, in BT001047, the antibody light chain conjugates 0-1 cellular bioactive molecules (LC, DAR1 ratios are 13.7% and 86.3%, respectively), and the heavy chain conjugates 1-3 cellular bioactive molecules (DAR1, DAR2, DAR3 ratios are 22.2%, 13.5%, and 64.3%, respectively). Therefore, the antibody-to-cellular bioactive molecule conjugation ratio (DAR) is calculated to be 6.6.

[0835] Example 62 Size Exclusion Chromatography Analysis

[0836] The coupling reaction was monitored by SEC-HPLC, and the coupling compound was detected by SEC.

[0837] Chromatographic conditions:

[0838] Liquid chromatography column: TOSOH TSKgel SuperSW mAb, 4μm, 7.8mm x 300mm;

[0839] Mobile phase: 100 mmol / L Na2HPO4, 100 mmol / L NaCl, 5% isopropanol, pH 7.0;

[0840] Flow rate: 0.5 ml / min; Detection wavelength: 280 nm; Column temperature: room temperature; Sample chamber temperature: 8 °C;

[0841] Injection volume: 30 μg; isocratic run: 30 min.

[0842] The SEC chromatogram and molecular weight marker SEC chromatogram of BT001002 obtained by conjugating antibody with TL003 are shown in Figures 7 and 8, respectively. According to the molecular weight marker, the molecular weight corresponding to the main peak of the conjugated product is approximately 150 kDa. That is, the light and heavy chains of BT001002 obtained by conjugating antibody with TL003 are not separated, and the antibody still maintains its overall structure.

[0843] The SEC chromatogram of BT001004 obtained by conjugating TL019 with the antibody is shown in Figure 9. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001004 obtained by conjugating TL019 with the antibody still maintains the complete structure of the antibody.

[0844] The SEC chromatogram of BT001012 obtained by conjugating TL024 with the antibody is shown in Figure 34. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001012 obtained by conjugating TL024 with the antibody still maintains the complete structure of the antibody.

[0845] The SEC chromatogram of BT001013 obtained by conjugating TL048 with the antibody is shown in Figure 35. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001013 obtained by conjugating TL048 with the antibody still maintains the complete structure of the antibody.

[0846] The SEC chromatogram of BT001018 obtained by conjugating TL030 with the antibody is shown in Figure 36. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001018 obtained by conjugating TL030 with the antibody still maintains the complete structure of the antibody.

[0847] The SEC chromatogram of BT001021 obtained by conjugating TL033 with the antibody is shown in Figure 37. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001021 obtained by conjugating TL033 with the antibody still maintains the complete structure of the antibody.

[0848] The SEC chromatogram of BT001023 obtained by conjugating TL035 with the antibody is shown in Figure 38. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001023 obtained by conjugating TL035 with the antibody still maintains the complete structure of the antibody.

[0849] The SEC chromatogram of BT001042 obtained by conjugating TL051 with the antibody is shown in Figure 39. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001042 obtained by conjugating TL051 with the antibody still maintains the complete structure of the antibody.

[0850] The SEC chromatogram of BT001043 obtained by conjugating TL052 with the antibody is shown in Figure 40. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001043 obtained by conjugating TL052 with the antibody still maintains the complete structure of the antibody.

[0851] The SEC chromatogram of BT001044 obtained by conjugating TL053 with the antibody is shown in Figure 41. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001044 obtained by conjugating TL053 with the antibody still maintains the complete structure of the antibody.

[0852] The SEC chromatogram of BT001046 obtained by conjugating TL055 with the antibody is shown in Figure 42. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001046 obtained by conjugating TL055 with the antibody still maintains the complete structure of the antibody.

[0853] The SEC chromatogram of BT001047 obtained by conjugating TL056 with the antibody is shown in Figure 43. Based on the SEC retention time and peak area ratio, the molecular weight of the main conjugation product was confirmed to be approximately 150 kDa, meaning that BT001047 obtained by conjugating TL056 with the antibody still maintains the complete structure of the antibody.

[0854] Example 63: Detection of the inhibitory effect of bioactive molecules and antibody-drug conjugates on in vitro cell activity.

[0855] First, tumor cells MDA-MB-468 (Trop-2 positive cell line) and HCC1806 (Trop-2 positive cell line) were cultured. The bioactive molecules and ADC molecules disclosed herein were co-cultured with the tumor cells, and then CCK8 reagent (Dongren Chemical Technology Co., Ltd., Cat: CK04, Lot: JJ744) was added. The activity of mitochondrial dehydrogenases was measured using a microplate reader (manufacturer: Molecular Devices, model: SpectraMax M2) at a detection wavelength of 450 nm to evaluate the inhibitory effect of ADC on cell proliferation. The tumor cell sources are shown in Table 1.

[0856] Table 1.

[0857] Cell Name, Tumor Type, Source: MDA-MB-468 (Breast Cancer), Conotech HCC1806 (Breast Cancer), Nanjing Kebai Biotechnology

[0858] In vitro cell viability assay: The bioactive molecules or ADCs (12 concentration gradients) were diluted with the corresponding detection medium (containing 2% FBS). Tumor cells were digested using trypsin according to standard methods, collected, and counted, then resuspended in the corresponding detection medium (containing 2% FBS). The diluted bioactive molecules or ADCs were added to 96-well plates, followed by the cells. Then, 20 μL of CCK8 reagent was added to each well, and the reaction was allowed to proceed for 4 hours. The results were then read using a microplate reader (detection wavelength: 450 nm). The experimental conditions and detection results are shown in Tables 2 and 3.

[0859] Table 2. Results of cell killing by bioactive molecules

[0860]

[0861]

[0862] Test results show that all bioactive molecules have tumor cell killing effects.

[0863] Table 3. Cell line killing results of conjugate (ADC)

[0864]

[0865] The test results show that the ADC molecules obtained using the new coupling method have tumor cell killing effects. This indicates that the ADCs formed using the new coupling method can kill tumor cells, and the new coupling method is effective when applied to ADC molecules.

[0866] Example 64: In vivo efficacy testing of antibody-drug conjugates and active biomolecules

[0867] test drug

[0868] Drug name, source, and preparation method:

[0869] BT001021, with a liquid concentration of 5.44 mg / ml, should be aliquoted and stored at -20℃. When using, it should be diluted with physiological saline according to the dosage to obtain the test solution.

[0870] Immu-132 (prepared according to Example 2 of WO2015 / 012904A2, DAR=5.4, also referred to as IMMU-132), with a liquid concentration of 13.158 mg / ml, was aliquoted and stored at -20°C. When used, it was diluted with physiological saline according to the dosage to obtain the test solution.

[0871] T-030, a solid powder, was prepared with 100% DMSO (Sigma) to a concentration of 5.2 mg / ml, aliquoted and stored at -20°C. When used, it was diluted with physiological saline according to the dosage to obtain a test solution.

[0872] SN-38 (also referred to as SN38) is a solid powder prepared with 100% DMSO (Sigma) to a concentration of 3.23 mg / ml. It is then aliquoted and stored at -20°C. When used, it is diluted with physiological saline according to the dosage to obtain a test solution.

[0873] Note: The dosage of the toxin is prepared according to the equimolar ratio of the ADC sample.

[0874] The structures of T-030, SN-38, and Immu-132 are as follows:

[0875]

[0876] Laboratory animals and cell lines

[0877] 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), breast cancer cell line HCC1806 (Nanjing Kebai).

[0878] Experimental grouping and evaluation method

[0879] Tumor-bearing mice with a tumor volume of 100-200 mm 3 were randomly selected (the number of groups was determined according to the number of samples), 6 mice per group. The administration volume was 10 mL / kg, and the administration route was intravenous injection via the tail vein. The tumor diameter was measured with a vernier caliper twice a week while administering the drug, and the tumor volume was 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 was observed and recorded daily.

[0880] The following formula was used to calculate the tumor growth inhibition rate TGI (%) to evaluate the antitumor efficacy of the antibody-drug conjugate:

[0881] TGI (%) = [1 - (V T末 - V T始 ) / (V C末 - V C始 )] * 100%

[0882] where V T末 : mean tumor volume at the end of the experiment in the treatment group

[0883] V T始 : mean tumor volume at the start of drug administration in the treatment group

[0884] V C末 : mean tumor volume at the end of the experiment in the solvent control group

[0885] V C始 : mean tumor volume at the start of drug administration in the solvent control group

[0886] The following Experimental Examples 1 and 2 evaluated 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 experiment, 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. After the tumor volume grew to about 100 mm 3 , they were randomly grouped, and after grouping, BT001021 was administered intravenously twice a week for a total of 6 times. The tumor volume and animal body weight changes were measured twice a week to evaluate the drug effect (antitumor efficacy) of the antibody-drug conjugate on tumor-bearing mice.

[0887] Experimental Example 1. Inhibition of NCI-N87 by Antibody-Drug Conjugates and Active Biomolecules

[0888] Experimental methods:

[0889] 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 subcutaneously inoculated into female Balb / c-nu mice to establish a gastric cancer model. The tumor volume was measured to be approximately 90 mm². 3 Mice were randomly assigned to groups based on tumor size: a saline group, a BT001021 group (3 mg / kg, intravenous injection, twice weekly for 3 weeks), a positive control group (Immu-132, 3 mg / kg, intravenous injection, twice weekly for 3 weeks), a T030 group, and a SN38 group. After grouping, the corresponding drug was administered via tail vein injection twice weekly for a total of 6 weeks. Tumor volume and body weight were observed and measured periodically after drug administration. Specific results are shown in Table 4, Figures 44 and 45.

[0890] Experimental conclusion:

[0891] This experiment used the human gastric cancer cell line NCI-N87 to construct a human gastric cancer subcutaneous xenograft model and evaluated the efficacy of BT001021 in the NCI-N87 human gastric cancer tumor-bearing mouse model.

[0892] The experimental results showed that BT001021 (3 mg / kg, intravenous injection, twice a week for 3 weeks) could significantly inhibit tumor growth in NCI-N87 gastric cancer xenograft mice, and tumor regression occurred at the end of the administration. Its anti-tumor activity was superior to that of positive Immu-132. No animals died or had significant weight loss in any of the treatment groups during the observation period, indicating that BT001021 had no obvious toxicity.

[0893] Table 4. Gastric cancer NCI-N87 model

[0894]

[0895]

[0896] Experimental Example 2. Inhibition of HCC1806 by Antibody-Drug Conjugates

[0897] Experimental methods:

[0898] HCC1806 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC1806 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female Balb / c-nu mice to establish a breast cancer model. The tumors were cultured until the average volume reached approximately 130 mmHg.3 Mice were randomly divided into three groups based on tumor size: a saline group, a BT001021 group (10 mg / kg, intravenous injection, twice weekly for 3 weeks), and a positive control group (Immu-132 group (10 mg / kg, intravenous injection, twice weekly for 3 weeks)). After grouping, the corresponding drug was administered via tail vein injection twice weekly for a total of 5 weeks. Tumor volume was observed and measured periodically after administration. Specific results are shown in Table 5 and Figure 46.

[0899] Experimental conclusion:

[0900] This experiment used the human breast cancer cell line HCC1806 to construct a human breast cancer subcutaneous xenograft model and evaluated the efficacy of BT001021 in the HCC1806 human breast cancer xenograft mouse model.

[0901] The experimental results show that BT001021 (10 mg / kg, intravenous injection, twice a week for 3 weeks) significantly inhibited tumor growth in HCC1806 breast cancer xenograft mice, and its anti-tumor activity was superior to that of positive Immu-132.

[0902] Table 5: HCC1806 model of breast cancer

[0903]

[0904] As shown in Tables 4 and 5 and Figures 44-46, the antibody drug BT001021 of this invention significantly inhibits tumor growth in the mouse NCI-N87 model, showing significantly better performance than Immu-132 at the same dose, without significant weight loss or drug toxicity. In the mouse HCC1806 model, due to the high malignancy of the tumor, Immu-132 did not show significant inhibitory activity even at a dose increased to 10 mg / kg, while BT001021 significantly inhibited tumor growth. These results indicate that BT001021 of this invention has good efficacy and excellent safety.

[0905] In the subcutaneous xenograft tumor models of Experiments 1 and 2, BT001021 showed significantly better antitumor activity than Immu-132 at the same dose, suggesting that BT001021 has the potential to treat solid tumors. Compared with Immu-132, BT001021 is expected to benefit more patients in clinical practice.

[0906] Experimental Example 3. Inhibition of HCC827 by Antibody-Drug Conjugates

[0907] Example 3 evaluates the inhibitory effects of BT001021 and BT001035 on the proliferation of a tumor-bearing mouse model constructed by subcutaneous transplantation of human tumor cells from HCC827 non-small cell lung cancer. Specifically, in this experiment, a tumor-bearing mouse model was constructed by subcutaneous transplantation of the human non-small cell lung cancer cell line HCC827. The tumor volume was increased to 100 mm². 3 Mice were randomly divided into left and right groups. After grouping, BT001021 and BT001035 were administered intravenously twice a week for a total of 6 weeks. Tumor volume and body weight changes were measured twice a week, and the efficacy (tumor inhibition effect) of BT001021 and BT001035 on tumor-bearing mice was calculated.

[0908] Experimental methods:

[0909] HCC827 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC827 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated 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 divided into four groups based on tumor size: a saline group, a positive control group treated with Immu-132 (10 mg / kg, IV, BIW×3W), a group treated with BT001021 (10 mg / kg, IV, BIW×3W), and a group treated with BT001035 (10 mg / kg, IV, BIW×3W). The corresponding drug was administered via tail vein injection after each grouping, twice weekly for a total of six weeks. Tumor volume and body weight were observed and measured periodically after drug administration. Specific results are shown in Table 6, Figures 47A and 47B.

[0910] Experimental conclusion:

[0911] The experimental results show that BT001021 and BT001035 can significantly inhibit tumor growth in HCC827 non-small cell lung cancer xenograft mice, and tumor regression occurs at the endpoint of administration. Their anti-tumor activity is superior to that of the positive group Immu-132. No animals died or lost significant weight during the observation period in any of the treatment groups, and no obvious drug toxicity was observed. The mice tolerated the evaluated drugs well during the treatment period.

[0912] Table 6. HCC827 lung cancer model

[0913]

[0914] As shown in Table 6, Figures 47A and 47B, both BT001021 and BT001035 exhibited significant tumor growth inhibitory activity during the evaluation period. At the same dosage, their tumor inhibitory activity was significantly superior to Immu-132. During the administration period, no significant weight loss or drug toxicity was observed in any group of animals. These results indicate that BT001021 and BT001035 possess excellent antitumor activity.

[0915] In this subcutaneous xenograft model, BT001021 and BT001035 showed significantly better antitumor activity than Immu-132 at the same dose, suggesting that BT001021 and BT001035 have the potential to treat solid tumors. Compared with Immu-132, BT001021 and BT001035 are expected to benefit more patients in clinical practice.

[0916] Experimental Example 4. Inhibition of NCI-N87 by Antibody-Drug Conjugates

[0917] Experiment 4 evaluated the inhibitory effect of antibody-drug conjugate BT001036 on tumor proliferation in tumor-bearing mice constructed from subcutaneously transplanted human tumor cells. Specifically, in this experiment, a tumor-bearing mouse model was constructed by subcutaneously transplanting the human gastric cancer cell line NCI-N87. The tumor volume was increased to 140 mm². 3 Mice were randomly divided into two groups and administered BT001036 intravenously twice a week for a total of six weeks. Tumor volume and body weight changes were measured twice a week to evaluate the efficacy (tumor-suppressive effect) of the antibody-drug conjugate in tumor-bearing mice.

[0918] Experimental methods:

[0919] 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 subcutaneously inoculated into female Balb / c-nu mice to establish a gastric cancer xenograft model. The tumors were cultured until the average volume reached approximately 140 mmHg. 3 Mice were randomly divided into three groups based on tumor size: a 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 drug was administered via tail vein injection twice a week for a total of six weeks. Tumor volume and body weight were observed and measured periodically after administration. Specific results are shown in Table 7, Figures 48A and 48B.

[0920] Table 7. Gastric cancer NCI-N87 model

[0921]

[0922] Experimental conclusion:

[0923] This experiment used subcutaneous transplantation of the human gastric cancer cell line NCI-N87 to construct a human gastric cancer subcutaneous xenograft model, and evaluated the efficacy of BT001036 in the NCI-N87 human gastric cancer tumor-bearing mouse model.

[0924] The experimental results show that both high and low doses of BT001036 (1.5 mg / kg and 3 mg / kg) can significantly inhibit tumor growth in NCI-N87 gastric cancer xenograft mice, and tumor regression occurs at the endpoint of administration, demonstrating excellent anti-tumor activity. No animal deaths or significant weight loss were observed in any of the treatment groups during the observation period, and no obvious drug toxicity was observed. The mice showed good tolerance to the evaluated drugs during the treatment period.

[0925] Experimental Example 5. Inhibition of MDA-MB-231 by Antibody-Drug Conjugates

[0926] Experiment 5 evaluated the inhibitory effect of BT001021 on the proliferation of a mouse model of MDA-MB-231 breast cancer cells transplanted subcutaneously. Specifically, in this experiment, a mouse model of tumor-bearing cancer was constructed by subcutaneously transplanting the human breast cancer cell line MDA-MB-231. The tumor volume was increased to 130 mm². 3 Mice were randomly divided into left and right groups, and then administered BT001021 intravenously twice a week for a total of 6 weeks. Tumor volume and body weight changes were measured simultaneously, and the efficacy (tumor-suppressing effect) of BT001021 on tumor-bearing mice was calculated.

[0927] Experimental methods:

[0928] NCI-MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. MDA-MB-231 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female Balb / c-nu mice to establish a lung cancer xenograft model. The tumors were cultured until the average volume reached approximately 130 mmHg. 3 Mice were randomly divided into two groups based on tumor size: a saline group and a BT001021 (3 mg / kg) group. After grouping, the corresponding drugs were administered via tail vein injection twice a week for a total of six weeks. Tumor volume and body weight were observed and measured periodically after administration. Specific results are shown in Table 8, Figures 49A and 49B.

[0929] Experimental conclusion:

[0930] The experimental results show that BT001021 can significantly inhibit tumor growth in MDA-MB-231 breast cancer xenograft mice, and tumor regression occurred at the endpoint of administration. No animals died or lost significant weight during the observation period in any of the treatment groups, and no obvious drug toxicity was observed. The mice tolerated the evaluated drugs well during the treatment period.

[0931] Table 8. Breast cancer MDA-MB-231 model

[0932]

[0933] In the subcutaneous xenograft tumor model, the anti-tumor activity of BT001021 was significant, and no animal deaths or significant reduction in animal body weight occurred in all treatment groups during the observation period. No obvious drug toxicity was shown, and the mice tolerated the evaluated drugs well during the treatment period.

[0934] Example 65 In vivo pharmacokinetic test of antibody-drug conjugate and active biomolecule

[0935] Experimental Example 6 Evaluated the in vivo pharmacokinetics of antibody-drug conjugate and active biomolecule. Specifically, in this experiment, human gastric cancer cell line NCI-N87 was subcutaneously transplanted into Balb / c-nu mice to construct a tumor-bearing mouse model. When the tumor volume grew to 100 - 200 mm 3 , they were randomly grouped, and after grouping, BT001021 and T-030 were administered intravenously once. The concentration of T-030 in tumor tissues and serum was measured to evaluate the in vivo pharmacokinetic behavior of antibody conjugate drug BT001021 and active biomolecule T-030 in tumor-bearing mice.

[0936] Tested drugs

[0937] Drug name, preparation method:

[0938] 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;

[0939] T-030 was formulated into 1 mg / ml with dimethyl sulfoxide and diluted with normal saline according to the dose to obtain the test solution.

[0940] Experimental animals and cell lines:

[0941] Balb / c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., Production License No.: SCXK(Beijing)2016 - 0011); gastric cancer cell line NCI-N87 (ATCC).

[0942] Experimental grouping and evaluation method:

[0943] Tumor-bearing mice with a tumor volume of 100 - 200 mm 3 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.

[0944] Experimental Example 6. In vivo pharmacokinetic tests of BT001021 and T-030 in tumor-bearing mice

[0945] Experimental methods:

[0946] 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 subcutaneously inoculated into Balb / c-nu mice to establish a lung cancer xenograft model. The tumors were cultured until the average volume reached approximately 100-200 mmHg. 3 Based on tumor size, patients were randomly assigned to three groups: a 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. The corresponding drug was administered via tail vein injection after grouping. Serum and tumor tissue samples were collected from the T-030 group at 1 h, 2 h, 4 h, 8 h, 24 h, and 72 h after administration (T-030 was undetectable in both serum and tumor tissue at 72 h, therefore no samples were collected at 168 h). Serum and tumor tissue samples were also collected from the BT001021 group at 1 h, 2 h, 4 h, 8 h, 24 h, 72 h, and 168 h after administration. The concentration of T-030 in serum and tumor tissue was detected using LC-MS / MS. Specific results are shown in Table 9. The T-030 dosage (0.23 mg / kg) was converted to an equimolar value based on the BT001021 dosage (10 mg / kg).

[0947] Table 9. Pharmacokinetic parameters of tumor and serum T-030 in tumor-bearing mice after intravenous administration of T-030 and BT001021.

[0948]

[0949]

[0950] Experimental conclusion:

[0951] AUC in tumors and serum of BT001021 (10 mg / kg) treatment group last The AUCs in the T-030 administration group were 850.1 h*ng / ml and 174.97 h*ng / ml, respectively, in tumor cells and serum. lastThe concentrations were 3.85 h*ng / ml and 5.58 h*ng / ml, respectively. A comparison of the three groups showed that the exposure of T-030 in the BT001021 group was significantly increased compared to the T-030 group. Furthermore, the exposure of the active biomolecule T-030 in the tumor of the BT001021 group was significantly higher than that in the serum, while the exposure of the active biomolecule in serum and tumor of the T-030 group was essentially the same, indicating that the antibody-drug conjugate (BT001021) has strong tumor tissue targeting.

[0952] The C-level activity of biomolecule T-030 in tumors and serum was observed in the BT001021 (10 mg / kg) administration group. max The concentrations were 7.82 ng / ml and 11.7 ng / ml, respectively. The C60 concentration of the active biomolecule T-030 in the T-030-treated group was [not specified in the original text]. max The concentrations were 1.20 ng / ml and 1.81 ng / ml, respectively, indicating that the antibody-drug conjugate (BT001021) had higher concentrations of the active biomolecule (T-030) in tumor tissue and serum.

[0953] The BT001021 (10 mg / kg) administration group showed increased activity of the biomolecule T-030 in tumors. 1 / 2 The duration of action of the active biomolecule T-030 in the tumor was 93.14 h. 1 / 2 The half-life was 2.55 h, indicating that the antibody-drug conjugate (BT001021) has a longer half-life in tumor tissue.

[0954] In summary, compared with the corresponding active biomolecule (T-030), BT001021 has significant tumor tissue targeting and good pharmacokinetic properties.

[0955] Experimental Example 7. In vivo pharmacokinetic assay of antibody-drug conjugates BT001021 and Immu-132.

[0956] In this experiment, a tumor-bearing mouse model was established by subcutaneously transplanting the human gastric cancer cell line NCI-N87 into Balb / c-nu mice. The tumor volume was allowed to grow to 100-200 mm². 3 Mice were then randomly assigned to groups, and each group received a single intravenous administration of BT001021 and Immu-132. The concentrations of the active biomolecules T-030 and SN-38 corresponding to BT001021 and Immu-132, respectively, in tumor tissue and serum were measured to evaluate the in vivo pharmacokinetic behavior of the antibody-drug conjugates BT001021 and Immu-132 in tumor-bearing mice.

[0957] test drug

[0958] Drug name and preparation method:

[0959] BT001021, with a liquid concentration of 20 mg / ml, was sub-packaged and stored at -20°C. When used, it was diluted with normal saline according to the dose to obtain a test solution.

[0960] Immu-132 was diluted with normal saline according to the dose to obtain a test solution.

[0961] Experimental animals and cell lines:

[0962] 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).

[0963] Experimental grouping and evaluation methods:

[0964] Tumor-bearing mice with randomly selected groups (the number of groups was determined according to the number of samples) and a tumor volume of 100 - 200 mm 3 were selected, with 4 mice per group. The administration route was single-dose intravenous injection via the tail vein.

[0965] Experimental methods:

[0966] NCI-N87 cells were cultured in 1640 medium containing 10% heat-inactivated fetal bovine serum under the conditions of 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 lung cancer xenograft model. When the average tumor volume was approximately 100 - 200 mm 3 , they 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 administration, and the concentrations of T-030 or SN-38 in the serum and tumor were detected by LC-MS / MS method. 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

[0967]

[0968] Experimental conclusions:

[0969] The AUCs of the toxin small molecules 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 molecules in the tumor and serum of the Immu-132 administration group lastThe concentrations were 116.8 h*ng / ml and 422.7 h*ng / ml, respectively. The C-values ​​of the small molecule toxin in the tumor were [not specified in the original text]. max The concentration of the toxin small molecule in the tumor was 6.8 ng / ml in the Immu-132 dosing group. max The concentration was 2.8 ng / ml. These results indicate that, compared to Immu-132, BT001021 has better tumor tissue targeting and better pharmacokinetic properties, and a better therapeutic window.

[0970] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]-G Formula (I) in, T represents a bioactive molecular fragment, preferably a molecular fragment with antitumor bioactivity; L1 is selected from amino acids, peptides composed of 2-10 amino acids, oligosaccharides, and -(CH2). t1 -、-(CH2CH2O) t1 -(CH2) t2 -、 In this context, each of R, R', R1, and R2 is independently H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, cyano group, or C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl groups (e.g., -CH2CN), C 1-6 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl group, C 3-6 Cycloalkyl, 6-10 aryl or 5-12 heteroaryl, each Z1 is independently an amino acid or a peptide composed of 2-10 amino acids, each t1 and t2 is independently 0, 1, 2, 3, 4, 5 or 6, each x1 and x2 is independently 0, 1, 2, 3, 4, 5 or 6, each x3 is independently 0, 1, 2, 3 or 4, and the 1 position of L1 is connected to T; L2 is selected from amino acids, peptides composed of 2-10 amino acids, oligosaccharides, and -(CH2). t1 -、-(CH2CH2O) t1 -(CH2) t2 -、 Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CN, and C. 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 alkenyl, C 2-10 alkynyl group and C 3-6 Cycloalkyl, or R3, R4 or R5, R6 or R3, R5 together with the carbon atom to which they are attached to form a 3-8 membered ring, t1 and t2 are each independently 0, 1, 2, 3, 4, 5 or 6, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L2 is connected to L1 at position 1; L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, 3-8 membered cycloalkyl, 3-8 membered alicyclic, 6-12 membered bridged heterocyclic, 6-12 membered spirocyclic, 6-12 membered fused heterocyclic, 6-10 membered aryl, 5-12 membered heteroaryl, and 3-8 membered cycloalkyl-W-; wherein W is oxygen or NR8, and R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CN, carboxyl, sulfonic acid, C 1-6 Alkyl, Halogenated C 1-6 alkyl and cyano substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 2-10 alkenyl and C 2-10 The alkynyl group, R8, is independently selected from H (hydrogen), D (deuterium), C (hydrogen), D (deuterium), and C (hydrogen). 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Alkoxy and cyano C 1-2 Alkyl group, and L3 is connected to L2 at position 1; L4 is selected from Among them, Z5 is preferred from C 2-6 Olefins, C 2-6 Alkynes, amides, sulfones, sulfoxides, 6-10 aryl groups, 5-6 heteroaryl groups; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene, 6-10 aryl and 5-14 heteroaryl; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene, Halogenated C 1-6 alkylene and alkoxy-substituted C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6; and L4 is attached to E at the 2 position; E is selected from one or more R. 12 The following groups are substituted: pyrimidine, quinazoline, and pyrrolo[2,3-d]pyrimidine; wherein, R 12 Independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl and Halogenated C 1-6 alkyl; G is the leaving group for nucleophilic substitution reactions; m1, m2 and m3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from peptides composed of 2-5 amino acids, including Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, and Asn. In this context, each of R, R', R1, and R2 is independently H (hydrogen), D (deuterium), and C (carbon). 1-6 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 3-6 Cycloalkyl, Z1 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x1 is 0, 1, 2 or 3, x3 is 0, 1, 2, 3 or 4.

3. The compound of any one of claims 1-2 or a pharmaceutically acceptable salt thereof, wherein, 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, Where R, R', and R1 are each independently H (hydrogen), D (deuterium), and C (carbon). 1-6 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 3-6 Cycloalkyl, Z1 is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x1 and x3 are each independently 0, 1, 2 or 3.

4. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, In this context, 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 0, 1 or 2 independently.

5. The compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, 6. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein, L1 is selected from 7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from peptides composed of 2-5 amino acids, including Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, and Asn. Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CF3, CN, CH2CN, and C. 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group and C 3-6 The cycloalkyl group, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is connected to L1 at position 1; m1 can be 0, 1, 2 or 3.

8. The compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein, 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, Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), halogen, carboxylic acid, sulfonic acid, CF3, CN, CH2CN, and C. 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl, C 2-6 alkynyl group and C 3-6 The cycloalkyl group, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is connected to L1 at position 1; m1 can be 0, 1, or 2.

9. The compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from Among them, R3, R4, R5, and R6 are each independently selected from H (hydrogen), D (deuterium), and C. 1-4 Alkyl groups, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is connected to L1 at position 1; m1 is 1.

10. The compound of any one of claims 1-9 or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from 11. The compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein, L2 is selected from 12. The compound according to any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, 3-8 membered cycloalkyl, 3-8 membered alicyclic, 6-12 membered bridged heterocyclic, 6-12 membered spirocyclic, 6-12 membered fused heterocyclic, 6-10 membered aryl, 5-12 membered heteroaryl, and 3-8 membered cycloalkyl-W-; wherein W is oxygen or NR8, and R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 Alkyne group; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spirocyclic group, or 6-12 membered fused heterocyclic group contains one or more nitrogen atoms; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spirocyclic group, or 6-12 membered fused heterocyclic group contains one or more quaternized nitrogen atoms; preferably, the 3-8 membered alicyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spirocyclic group, or 6-12 membered fused heterocyclic group contains one or more nitrogen atoms, wherein at least one nitrogen atom is substituted with =O; R8 is independently selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, C 2-6 alkenyl, C 3-6 alkynyl group, C 3-6 cycloalkyl, C 1-6 Alkoxy and cyano C 1-2 alkyl; m2 can be 0, 1, 2, or 3.

13. The compound according to any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, 3-6 membered alicyclic and 5-10 membered heteroaryl; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 Alkyne group; preferably, the 3-6 membered alicyclic group contains one or more nitrogen atoms; preferably, the 3-6 membered alicyclic group contains one or more quaternized nitrogen atoms; preferably, the 3-6 membered alicyclic group contains one or more nitrogen atoms, wherein at least one nitrogen atom is substituted with =O; m2 can be 0, 1, or 2.

14. The compound according to any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein, L3 is selected from 5-6 heteroaryl groups optionally substituted with one or more R7 groups; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 alkynyl group; m2 is 1.

15. The compound according to any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein, L3 is selected from the following groups optionally substituted with one or more R7 groups: amino, N-methylpiperidine, pyrazole, and triazole; wherein R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 alkenyl and C 2-6 alkynyl group; m2 is 0 or 1.

16. The compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein, L3 is selected from: triazole; m2 is 0 or 1.

17. The compound of any one of claims 1-16 or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from Z4 is a 6-10 aryl group or a 5-6 heteroaryl group; R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position; and L4 is attached to E at the 2 position; m3 is selected from 0, 1, 2 or 3.

18. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from Z4 is a benzene ring, R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene, or R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6, and L4 is attached to E at the 2 position; and L4 is attached to E at the 2 position. m3 is selected from 0, 1, 2 or 3.

19. The compound according to any one of claims 1-18 or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from Z4 is a 5-6 member heteroaryl group; R 10 Selected from H (hydrogen), C 1-6 Alkyl; Z2 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene; R9 is selected from H (hydrogen), C 1-6 Alkyl group; Z3 is absent or selected from C 1-6 Alkylene; or, R9 and Z3 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic group; α is independently 0, 1, 2, 3, 4, 5 or 6; and L4 is attached to E at the 2 position; m3 is selected from 0, 1, 2 or 3.

20. The compound of any one of claims 1-19 or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from m3 is 1.

21. The compound according to any one of claims 1-20, or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from m3 is 1.

22. The compound according to any one of claims 1-21, or a pharmaceutically acceptable salt thereof, wherein, L4 is selected from m3 is 1.

23. The compound of any one of claims 1-22 or a pharmaceutically acceptable salt thereof, wherein, E is selected from one or more R. 12 Substituted pyrimidines; wherein, R 12 It is independently selected from H (hydrogen) and D (deuterium).

24. The compound according to any one of claims 1-23, or a pharmaceutically acceptable salt thereof, wherein, G is selected from halogens, OMs, OTs, OTf, nitro, and optionally by one or more Rs. 13 The following groups are substituted: alkyl sulfide group, aryl sulfide group, heteroaryl sulfide group, alkyl sulfoxide group, aryl sulfoxide group, heteroaryl sulfoxide group, alkyl sulfonyl group, aryl sulfonyl group, or heteroaryl sulfonyl group; wherein, R 13 Independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Alkoxy, 6-10 aryl and 5-12 heteroaryl.

25. The compound according to any one of claims 1-24, or a pharmaceutically acceptable salt thereof, wherein, G is selected from F, Cl, Br, I, OMs, OTs, OTf, methanesulfonyl, ethanesulfonyl, p-toluenesulfonyl, and naphthalenesulfonyl.

26. The compound according to any one of claims 1-25, or a pharmaceutically acceptable salt thereof, wherein, G is selected from F, Cl, Br, OMs, OTs, methanesulfonyl, and p-toluenesulfonyl.

27. The compound according to any one of claims 1-26, or a pharmaceutically acceptable salt thereof, wherein, G is selected from Cl and methanesulfonyl.

28. The compound according to any one of claims 1-27, or a pharmaceutically acceptable salt thereof, wherein, In this context, G is preferably methanesulfonyl, E is preferably pyrimidine, and m3 is 1.

29. The compound of any one of claims 1-28 or a pharmaceutically acceptable salt thereof, wherein, for Where m4 is preferably an integer from 0 to 6, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

30. The compound according to any one of claims 1-29, or a pharmaceutically acceptable salt thereof, wherein, for Where m5 is preferably an integer from 0 to 6, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

31. The compound according to any one of claims 1-30, or a pharmaceutically acceptable salt thereof, wherein, for Where m6 is preferably an integer from 0 to 6, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

32. The compound according to any one of claims 1-31, or a pharmaceutically acceptable salt thereof, wherein, for Where m7 is selected from integers from 1 to 5, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

33. The compound according to any one of claims 1-32, or a pharmaceutically acceptable salt thereof, wherein, for Where m8 is selected from integers from 1 to 5, and methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

34. The compound according to any one of claims 1-33, or a pharmaceutically acceptable salt thereof, wherein, for Where m9 is selected from integers from 1 to 5, R 13 Selected from hydrogen, C 1-6 Alkyl and methanesulfonyl groups are substituents for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

35. The compound according to any one of claims 1-34, or a pharmaceutically acceptable salt thereof, wherein, for Where m 10 The integers are selected from 0 to 6, and Z4 is selected from 5-6 heteroaryl groups; methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

36. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-35, wherein, for Z4 is derived from pyridine, pyrimidine, pyrazole, thiazole, oxazole, and triazole; methanesulfonyl is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

37. The compound according to any one of claims 1-36, or a pharmaceutically acceptable salt thereof, wherein, for Z4 is selected from oxazole and thiazole, and the methanesulfonyl group is a substituent for the carbon atom adjacent to the nitrogen atom in the pyrimidine ring.

38. The compound according to any one of claims 1-37 or a pharmaceutically acceptable salt thereof, wherein, for 39. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, -[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]-G is selected from the following structural segments:

40. The compound according to any one of claims 1-39, or a pharmaceutically acceptable salt thereof, wherein, T represents a bioactive molecular fragment, wherein the bioactive molecule is selected from metal complexes, such as platinum complexes (e.g., oxaliplatin) and gold complexes; glycopeptide antibiotics, such as bleomycin or bleomycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan), topoisomerase II inhibitors (e.g., actinomycin D, doxorubicin, doxorubicin, docalimicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); and DNA synthesis interfering drugs, such as methotrexate, 5-fluorouracil, and cytarabine. Glycosides, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nerabine; drugs acting on structural proteins, such as microtubule inhibitors, vinblastine alkaloids, vincristine, paclitaxel, docetaxel, or cabazitaxel; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cyclin inhibitors; maytansine derivatives; calichiomycin derivatives; olrital statin derivatives; pyrrolobenzodiazepine dimers (PBD) derivatives; melphalan; mitomycin C; chlorambucil; and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis, or necrosis.

41. The compound according to any one of claims 1-40, or a pharmaceutically acceptable salt thereof, wherein, The bioactive molecules are selected from Where R 14 Selected from R 15 Substituted acyl or sulfonyl group, R 15 Selected from C 1-6 Alkyl, Halogenated C 1-6 Alkyl, 6-10 aryl and 5-12 heteroaryl; R 16 Selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, R 17 Replacement C 1-6 alkyl, R 17 Selected from aryl and heteroaryl groups, including but not limited to phenyl and pyridyl groups, m 11 Selected from 0, 1, 2.

42. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-41, wherein, The bioactive molecules are selected from Where R 14 Selected from R 15 Substituted acyl or sulfonyl group, R 15 Selected from C 1-6 Alkyl, Halogenated C 1-6 Alkyl, 6-10 aryl and 5-12 heteroaryl; R 16 Selected from H (hydrogen), D (deuterium), C 1-6 Alkyl, R 17 Replacement C 1-6 alkyl, R 17 Selected from aryl and heteroaryl groups, m 11 Selected from 0, 1, 2.

43. The compound according to any one of claims 1-42, or a pharmaceutically acceptable salt thereof, wherein, The bioactive molecules are selected from 44. The compound according to any one of claims 1-43, or a pharmaceutically acceptable salt thereof, wherein, The bioactive molecules are selected from 45. The compound according to any one of claims 1-44, or a pharmaceutically acceptable salt thereof, wherein, The bioactive molecules are selected from 46. ​​The compound according to any one of claims 1-45, or a pharmaceutically acceptable salt thereof, wherein, The bioactive molecules are selected from 47. The compound according to any one of claims 1-46, or a pharmaceutically acceptable salt thereof, wherein, The bioactive molecules are selected from 48. The compound of any one of claims 1-47 or a pharmaceutically acceptable salt thereof, wherein, T is selected from 49. The compound according to any one of claims 1-48, or a pharmaceutically acceptable salt thereof, wherein, T is selected from 50. The compound according to any one of claims 1-49, or a pharmaceutically acceptable salt thereof, wherein, T is selected from 51. The compound according to any one of claims 1-50, or a pharmaceutically acceptable salt thereof, wherein, T is selected from 52. The compound according to any one of claims 1-51, or a pharmaceutically acceptable salt thereof, wherein, T is selected from 53. The compound according to any one of claims 1-52, or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from 54. The compound according to any one of claims 1-53, or a pharmaceutically acceptable salt thereof, wherein, The compound is selected from 55. A conjugate comprising a bioactive molecule, a linker, and a targeting moiety, wherein the targeting moiety is connected to the linker via an active group (e.g., a thiol group) to form the conjugate.

56. The coupling of claim 55, having the structure shown in formula (II): {T-[L1-(L2) m1 -(L3) m2 -(L4) m3 -E]} γ -A Equation (II) in, A is the target fraction (e.g., small molecule ligand, protein, peptide, non-protein reagent (e.g., sugar, RNA or DNA)); γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8 (e.g., 5, 6, 7 or 8); The remaining groups are as defined in any one of claims 1-54.

57. The coupling according to any one of claims 55-56, wherein, The targets of A are selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylyl cyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, O772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, F cRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1lc, 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, ANGPTL4, 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, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA and TENB2.

58. The coupling according to any one of claims 55-57, wherein, A is a small molecule ligand such as folic acid derivatives, glutamate urea derivatives, somatostatin derivatives, aryl sulfonamide derivatives (e.g., carbonic anhydrase IX inhibitors), polyenes linking two aliphatic indoles, anthocyanin dyes, and IR-783 or its derivatives.

59. The coupling according to any one of claims 55-58, wherein, A is selected from 60. The coupling according to any one of claims 55-59, wherein, A represents an antibody, such as a monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment includes Fab, Fab′, F(ab′)2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies (e.g., scFv), non-human antibodies, humanized antibodies, chimeric antibodies, fully human antibodies, proantibodies, bispecific antibodies, or multispecific antibodies.

61. The coupling according to any one of claims 55-60, wherein, A is a monoclonal antibody against Her 2 or a monoclonal antibody against Trop-2. Preferably, the monoclonal antibody against Trop-2 is selected from Sacituzumab antibody, antibody M1, M2 or M3; preferably, the monoclonal antibody against Her 2 is selected from trastuzumab or pertuzumab. in, The heavy chain of the antibody Sacituzumab has the amino acid sequence shown in SEQ ID No.: 19; the light chain has the amino acid sequence shown in SEQ ID No.:

20. The heavy chain variable region of antibody M1 has the amino acid sequence shown in SEQ ID No.: 11; the light chain variable region has the amino acid sequence shown in SEQ ID No.: 12; The heavy chain variable region of the antibody M2 has the amino acid sequence shown in SEQ ID No.: 13; the light chain variable region has the amino acid sequence shown in SEQ ID No.: 14; The heavy chain variable region of the antibody M3 has the amino acid sequence shown in SEQ ID No.: 15; the light chain variable region has the amino acid sequence shown in SEQ ID No.:

16. The heavy chain constant regions of antibodies M1, M2, and M3 have the amino acid sequence shown in SEQ ID No.: 10; the light chain constant regions have the amino acid sequence shown in SEQ ID No.:

9.

62. The coupling according to any one of claims 55-61, wherein, A is a monoclonal antibody against Her 2 or a monoclonal antibody against Trop-2. Preferably, the monoclonal antibody against Trop-2 is selected from Sacituzumab antibody, and the monoclonal antibody against Her 2 is selected from trastuzumab or pertuzumab.

63. The coupling according to any one of claims 55-62, wherein, A is selected from RGD peptides that recognize cell surface integrin receptors; growth factors such as EGF, PDGF, or VEGF that recognize cell surface growth factor receptors; and peptides that can recognize functional cell surface plasminogen activator, dermalin, bradykinin, somatostatin, or prostate-specific membrane antigen receptors.

64. The coupling according to any one of claims 55-63, wherein, A is selected from CD40 ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2 ligand, TACSTD2 ligand, and DR5 ligand.

65. The coupling according to any one of claims 55-64, wherein, The coupling element is selected from: Wherein, γ is selected from an integer or 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 or M3 antibody, and the monoclonal antibody against Her 2 is selected from trastuzumab or pertuzumab; preferably, γ is selected from an integer or decimal between 5 and 8 (e.g. 5, 6, 7 or 8).

66. The coupling according to any one of claims 55-65, wherein, The coupling element is selected from: Wherein, γ is selected from an integer or 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 antibody, and the monoclonal antibody against Her 2 is selected from trastuzumab or pertuzumab; preferably, γ is selected from an integer or decimal between 5 and 8 (e.g., 5, 6, 7 or 8).

67. The coupling according to any one of claims 55-66, wherein, The coupling element is selected from: Wherein, A1 is the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8.

68. The coupling according to any one of claims 55-67, wherein, The coupling element is selected from: Wherein, A1 is the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

69. The coupling according to any one of claims 55-68, wherein, The coupling element is selected from: Wherein, A1 is the Sacituzumab antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

70. The coupling compound according to any one of claims 55-69, wherein, The coupling element is selected from: Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

71. The coupling according to any one of claims 55-70, wherein, The coupling element is selected from: Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

72. The coupling compound according to any one of claims 55-71, wherein, The coupling element is selected from: Wherein, A2 is trastuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

73. The coupling compound according to any one of claims 55-72, wherein, The coupling element is selected from: Wherein, A3 is pertuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

74. The coupling according to any one of claims 55-73, wherein, The coupling element is selected from: Wherein, A3 is pertuzumab, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

75. The coupling compound according to any one of claims 55-74, wherein, The coupling element is selected from: Wherein, A4 is the M1 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

76. The coupling according to any one of claims 55-75, wherein, The coupling element is selected from: Wherein, A4 is the M1 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

77. The coupling according to any one of claims 55-76, wherein, Couplets are selected from: Wherein, A5 is the M2 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

78. The coupling compound according to any one of claims 55-77, wherein, The coupling element is selected from: Wherein, A5 is the M2 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

79. The coupling compound according to any one of claims 55-78, wherein, The coupling element is selected from: Wherein, A6 is the M3 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

80. The coupling according to any one of claims 55-79, wherein, The coupling element is selected from: Wherein, A6 is the M3 antibody, and γ is selected from an integer or decimal between 1 and 10; preferably, γ is selected from an integer or decimal between 5 and 8, such as an integer or decimal between 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.

81. A method for preparing the conjugate according to any one of claims 55-80, comprising the step of coupling the linker of the compound of formula (I) to the active group of the target portion.

82. The method for preparing the conjugate of claim 81, comprising the step of forming a CS bond between the linker of the compound of formula (I) and the targeting portion.

83. The method for preparing the conjugate according to any one of claims 81-82, wherein the targeting portion of the conjugate is a monoclonal antibody against Her 2 or a monoclonal antibody against Trop-2, or an active fragment or variant thereof; preferably, the monoclonal antibody against Trop-2 is selected from Sacituzumab antibody, antibody M1, M2 or M3, and the monoclonal antibody against Her 2 is selected from trastuzumab or pertuzumab.

84. The method for preparing the conjugate according to any one of claims 81-83, wherein the targeting portion of the conjugate is a monoclonal antibody against Her 2 or a monoclonal antibody against Trop-2, or an active fragment or variant thereof; preferably, the monoclonal antibody against Trop-2 is selected from Sacituzumab antibody, and the monoclonal antibody against Her 2 is selected from trastuzumab or pertuzumab.

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

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

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

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

89. Use of the compound of any one of claims 1-54 or a pharmaceutically acceptable salt thereof, or a conjugate of any one of claims 55-80, or the pharmaceutical composition of claim 87, in the treatment of diseases (e.g., cancer) associated with abnormal cellular activity.

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

91. A method of treating a disease associated with abnormal cellular activity (e.g., cancer), comprising administering to an individual in need an effective dose of a compound of any one of claims 1-54 or a pharmaceutically acceptable salt thereof, or a conjugate of any one of claims 55-80, or a pharmaceutical composition of claim 87.