Compounds and Uses

Abstract

Conjugate compounds capable of targeting cells and degrading abnormal or undesirable proteins and uses thereof, as well as linker compounds useful in the preparation of the conjugate compounds.

Description

[Technical Field]

[0001] The present application relates to the field of biomedical chemistry. More specifically, the present application relates to conjugate compounds capable of target binding to cells and degrading target proteins, and uses thereof, as well as linker compounds useful for preparing the conjugate compounds. [Background technology]

[0002] Proteolysis Targeting Chimeras ("PROTACs"), proposed and validated by Raymond Deshaies and Craig Crews in 2001, are a novel drug discovery technology for degrading target proteins ("POIs" or "Protein of Interest") using the ubiquitin-proteasome system. This is an improved, versatile platform technology for inducing protein degradation. The chimeric bifunctional small molecules formed by this technology platform have a target protein ligand at one end that binds to the target protein and an E3 ligase ligand at the other end that binds to the E3 ligase, with the two ligands linked via a single linking moiety. After entering cells, the chimera attracts the target protein and E3 ligase to form a target protein-PROTAC-E3 ligase ternary complex. At the same time, ubiquitin is attached to the target protein via the proximity of the ubiquitin-conjugating enzymes E2 and E3 ligase, and further via the E3 ligase (target protein ubiquitination). The ubiquitin-attached target protein is recognized and degraded by the proteasome, thereby selectively reducing the target protein level. During this process, the target protein ligand in the PROTAC does not need to occupy the binding site for a long period of time; the transient formation of the ternary complex is sufficient to instantly complete the ubiquitination of the target protein, and the PROTAC can function multiple times within the cell. In theory, catalytic amounts of a PROTAC drug can eliminate any overexpressed or mutated pathogenic proteins, thereby treating the disease.

[0003] PROTAC technology has many advantages, including: 1) PROTACs can overcome drug resistance to tumor drugs. While drug resistance exhibited by cancer treatments such as chemotherapy, kinase inhibitors, and immunotherapy hinders their clinical application and increases the difficulty of drug development, PROTACs can degrade the entire target protein at relatively low doses and eliminate all of its functions, thereby potentially solving the current problem of drug resistance; and 2) PROTACs can degrade undruggable targets. Currently, only 20-25% of protein targets have been studied for drug discovery. Most small molecule drugs and large molecule antibodies require enzyme or receptor active sites to act; however, 80% of proteins in human cells lack such sites. PROTACs do not require such sites; they simply provide binding activity, triggering the proximity of the target protein to an E3 enzyme, thereby mediating ubiquitination and degradation of the target protein.

[0004] In 2013, Arvinas developed the first PROTAC drug (ARV-110) and advanced it to clinical research in 2019. Pharmaceutical companies around the world are competing to utilize PROTAC technology to develop targeted protein degradation drugs. By the end of 2021, 16 PROTAC drugs had already entered or were about to enter clinical research. However, PROTACs also have limitations. For example, PROTACs have low druggability, and their molecular weights generally range from 700 to 1200. This relatively large size typically results in poor membrane permeability, poor physicochemical properties (e.g., poor water solubility), and poor pharmacokinetics, potentially complicating their in vivo application. Furthermore, PROTACs have poor targeting properties. While the efficacy of PROTACs has been demonstrated against a wide variety of targets, the majority of reported degradation products to date have shown limited tissue selectivity and relatively poor selectivity for different cell types.

[0005] The present invention combines the advantages of ligand-drug conjugate and PROTAC technologies, coupling a PROTAC to a ligand with targeting activity, thereby utilizing the PROTAC drug itself to overcome drug resistance of tumor drugs, target targets that are traditionally difficult to drug, and have low toxicity, while improving the targeting and druggability of PROTACs. Summary of the Invention [Means for solving the problem]

[0006] One aspect of the present application provides a conjugate compound or a pharmaceutically acceptable salt thereof, the conjugate compound or the pharmaceutically acceptable salt thereof comprising targeting molecules of at least two ligands that specifically bind to cell surface proteins, and a payload linked to the targeting molecules, wherein the payload is a proteolytic targeting chimera.

[0007] In some embodiments, the targeting molecule binds to the cell surface proteins FOLR1, TRPV6, PSMA, LHRH, EGFR, Her2, Trop2, Her3, Claudin18.2, c-Met, or any combination thereof. In some embodiments, the targeting molecule binds to the cell surface proteins FOLR1, TRPV6, PSMA, c-Met, or any combination thereof.

[0008] In some embodiments, the targeting molecule binds to the following combinations of cell surface proteins: FOLR1 and TRPV6, FOLR1 and PSMA, TRPV6 and PSMA, TRPV6 and c-Met, FOLR1 and c-Met, PSMA and c-Met, FOLR1, TRPV6 and PSMA, FOLR1, TRPV6 and c-Met, FOLR1, PSMA and c-Met, TRPV6, PSMA and c-Met, or FOLR1, PSMA, TRPV6 and c-Met. In some embodiments, the targeting molecule comprises two ligands and binds to FOLR1 and TRPV6, FOLR1 and PSMA, TRPV6 and PSMA, FOLR1 and c-Met, PSMA and c-Met, or TRPV6 and c-Met, respectively.

[0009] In some embodiments, the ligand that binds to FOLR1 comprises folic acid or an analog thereof and pteroic acid, and preferably the folic acid analog is selected from 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 10-formyltetrahydrofolic acid, methotrexate, 5,10-formyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, aminopterin, and raltitrexed.

[0010] In some embodiments, the ligand that binds to TRPV6 comprises the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

[0011] In some embodiments, the ligand that binds to PSMA comprises a polypeptide, an antibody, or a small molecule. In some embodiments, the ligand that binds to PSMA is selected from the following structures: [ka]

[0012] In some embodiments, the ligand that binds to c-MET is Cys a -X1-Cys c -X2-Gly-Pro-Pro-X3-Phe-Glu-Cys d -Trp-Cys b X1 is Asn, His or Tyr, X2 is Gly, Ser, Thr or Asn, X3 is Thr or Arg, X4 is Ala, Asp, Glu, Gly or Ser, X5 is Ser or Thr, X6 is Asp or Glu, and Cys a-d are each a cysteine ​​residue, preferably the residue Cys a and Cys b , and Cys c and Cys d each cyclizes to form two independent disulfide bonds. In some embodiments, the carboxyl group of the terminal lysine of the c-MET ligand may be amidated. In some embodiments, the N-terminus of the c-MET ligand may be acetylated. In some embodiments, the ligand that binds to c-MET is Ala-Gly-Ser-Cys a -Tyr-Cys c -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys d -Trp-Cys b -Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys, the amino acid sequence of which is set forth in SEQ ID NO: 4, a-d are each a cysteine ​​residue, preferably the residue Cys a and Cys b , and Cys c and Cys dare each cyclized to form two independent disulfide bonds, and preferably the amino group of the terminal Ala is acetylated. In some embodiments, the ligand that binds to c-MET comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4, and wherein Cys a-d are each a cysteine ​​residue, preferably the residue Cys a and Cys b , and Cys c and Cys d Each cyclizes to form two independent disulfide bonds.

[0013] In some embodiments, the ligands in the targeting molecule are linked directly or via a spacer region, preferably by dehydration condensation. In some embodiments, the spacer region comprises an amino acid or combination of amino acids selected from the group consisting of Lys, Cys, Lys-Cys, Cys-Cys, Lys-Cys-Lys, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg, Asp-Asp-Lys-Cys, and Pro-Leu-Gly, and optionally, the amino acids may be amidated. In some embodiments, Lys is amidated, for example, to 2,6-diaminohexanamide. In some embodiments, the spacer region comprises one or more homologous or dissimilar compounds formed by dehydration condensation, selected from the group consisting of: [ka] In this case, n is 0 or 1.

[0014] In some embodiments, a targeting molecule having the following structural moiety is linked to a payload to form a conjugate compound: [ka] [ka] [ka] wherein Q is an activating group. In some embodiments, the targeting molecule is linked to the payload via an activating group Q, and preferably, the activating group Q is an alkynyl group or a mercapto group.

[0015] In some embodiments, a targeting molecule having the following structure is linked to a payload to form a conjugate compound: [ka] [ka] [ka]

[0016] In some embodiments, a proteolysis targeting chimera having the structure shown in Formula (I) is linked to a targeting molecule to form a conjugate compound, (T P ) n -LT E3 (I), Among them, Above T p is a target protein ligand moiety that binds to a target protein, wherein n is 1, 2, or 3; The above L is T P and T E3 and a connecting portion that connects the Above T E3 is the E3 ligase ligand moiety that binds to the E3 ligase.

[0017] In some embodiments, the E3 ligase ligand is bound to a VHL (von Hippel-Lindau) protein, a CRBN (cereblon) protein, an MDM2 protein, or an XIAP protein, and preferably, the E3 ligase ligand is a VHL ligand or a CRBN ligand.

[0018] In some embodiments, the VHL ligand is VHL-L or an analog thereof, wherein VHL-L has a structure selected from the group consisting of: [ka] (wherein R is hydrogen or a methyl group) and [ka]

[0019] In some embodiments, the CRBN ligand is lenalidomide, pomalidomide, thalidomide, or an analog thereof.

[0020] In some embodiments, the proteolysis targeted chimera has a structure selected from the group consisting of: [ka] Among them, Ra, Rb and Rc are each independently hydrogen, an alkyl group, NH, OH or a halogen, and preferably, the alkyl group is a methyl group and the halogen is fluorine; Rd is CH2, C=O or C=S; p is 0 or 1; the target protein ligand is capable of binding to a specific target protein; The target protein ligand is linked to the E3 ligase group via the linking moiety.

[0021] In some embodiments, the target protein is a nuclear receptor (e.g., AR and ER), a kinase protein (e.g., RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, and FAK), AKT, a PAN-BET protein, a BET protein (e.g., BRD2, BRD3, BRD4, and BRD6), BLK, BRAF1, BCL-x1, ERRα, FKBP12, FRS2α, JAK1, JAK3, IKZF1, IRAK4, MDM2, MetAP2, PLK1, PSK-J3, RAS, TACC3, Tau, TrkB, MDM2, or any combination thereof. In some embodiments, the target protein is an AR protein, an ER protein, a RAS protein, a BRD4 protein, a PLK1 protein, a FAK protein, an MDM2 protein, or any combination thereof.

[0022] In some embodiments, the target protein ligand moiety has a structure selected from the following: [ka]

[0023] In some embodiments, the linking moiety in the proteolysis targeting chimera has a structure selected from the group consisting of: [ka] Among them, p1 is an integer from 0 to 4, p2 is an integer from 0 to 6, p3, p4, p5, and p6 are each independently 0 or 1; G 1 is a 4- to 14-membered linear alkylene group, and optionally, 2 to 5 carbon atoms in the 4- to 14-membered linear alkylene group are selected from the group consisting of -O-, -NH-, -N(C 1-6 alkyl)- or -C≡C-; G 2is an amino acid residue, and preferably, G2 is a Lys residue; G 3 teeth, [ka] selected from the group consisting of G 4 is a 5- to 9-membered linear alkylene group, and optionally, 2 to 4 carbon atoms in the 5- to 9-membered linear alkylene group are selected from the group consisting of -O-, -NH-, -N(C 1-6 It may be substituted with -C≡C- or -C≡C-.

[0024] In some embodiments, the linking moiety in the proteolysis targeting chimera has a structure selected from the group consisting of: [ka]

[0025] In some embodiments, the proteolysis targeting chimera has the structure shown below: [ka] [ka] [ka]

[0026] In some embodiments, the payload is directly linked to the targeting molecule, or the payload is linked to the targeting molecule via a linker. In some embodiments, the link between the payload and the targeting molecule, between the payload and the linker, or between the linker and the targeting molecule is completed by a chemical reaction. In some preferred embodiments, the link between the targeting molecule and the linker is completed by a click reaction.

[0027] In some embodiments, the linker comprises link module 1 and link module 2.

[0028] In some embodiments, the linker comprises linking module 2.

[0029] In some embodiments, the linker comprises a linking module 1 and a PEG chain attached to the linking module 1; [ka] In some preferred embodiments, the PEG chain comprises a C1-C8 alkyl chain, -NH-, -O-, -C(O)-, or any combination thereof. [ka] In some preferred embodiments, the linker binds to link module 1. [ka] As will be appreciated by those skilled in the art, these groups may be attached to a linking moiety on Link Module 1 through a left attachment point and to a payload through a right attachment point, or may be attached to a linking moiety on Link Module 1 through a right attachment point and to a payload through a left attachment point.

[0030] In some embodiments, Link Module 1 is linked to Link Module 2 via a bond or a chemical group. In some embodiments, the chemical group is a PEG chain, [ka] or any combination thereof. In some preferred embodiments, the PEG chain is PEG1, PEG2, or PEG3. In some preferred embodiments, the chemical group is [ka] As will be appreciated by those skilled in the art, these chemical groups may be attached to Link Module 1 via a left-hand attachment point and to Link Module 2 via a right-hand attachment point, or to Link Module 1 via a right-hand attachment point and to Link Module 2 via a left-hand attachment point.

[0031] In some embodiments, the linking module 1 comprises: [ka] Includes:

[0032] In some embodiments, linkage module 2 comprises a cathepsin enzyme cleavage group, a plasmin enzyme cleavage group, an asparagine endonuclease enzyme cleavage group, a β-glucuronidase enzyme cleavage group, a sulfatase enzyme cleavage group, a phosphatase enzyme cleavage group, a β-galactosidase enzyme cleavage group, a glutathione enzyme cleavage group, or an acid labile group.

[0033] In some embodiments, the cathepsin enzyme-cleavable group is a CB enzyme-cleavable group.

[0034] In some preferred embodiments, the cathepsin enzyme cleavage group comprises an amino acid or a combination of an amino acid and an autolytic fragment, hi some preferred embodiments, the amino acid is Val-Cit, Gly-Gly-Phe-Gly, CycloBut-Cit, Phe-Cit, Val-Ala, Glu-Val-Cit, Ala-Cit, Val-Cit-Pro, or Val-Cit-Pro-Gly.

[0035] In some preferred embodiments, the plasmin enzyme-cleavable group comprises an amino acid or a combination of an amino acid and an autolytic fragment, hi some preferred embodiments, the amino acid is Val-Leu-Lys.

[0036] In some preferred embodiments, the asparagine endonuclease group comprises an amino acid or a combination of an amino acid and an autolytic fragment, hi some preferred embodiments, the amino acids are selected from Gly-Asn-Asn or Ala-Ala-Asn.

[0037] In some preferred embodiments, the autolytic fragments include PAB and its derivatives, [ka] In some preferred embodiments, the PAB is [ka] In some preferred embodiments, the PAB derivative is [ka] is.

[0038] In some preferred embodiments, the β-glucuronidase enzyme-cleavable group is [ka] In some preferred embodiments, the sulfatase enzyme-cleavable group is [ka] In some preferred embodiments, the phosphatase enzyme-cleavable group is [ka] In some preferred embodiments, the β-galactosidase enzyme-cleavable group is [ka] In some preferred embodiments, the glutathione enzyme-cleavable group is [ka] In some preferred embodiments, the acid labile group is [ka] is.

[0039] As will be appreciated by those skilled in the art, the active site of the enzyme-cleavage group may optionally be linked to Linking Module 1, a chemical group, a targeting molecule, or a payload after appropriate reaction. For example, the β-glucuronidase enzyme-cleavage group, after being effectively linked to Linking Module 1, a chemical group, a targeting molecule, or a payload, may be [ka] wherein the sulfatase enzyme cleavage group, after being effectively linked to a linking module 1, a chemical group, a targeting molecule, or a payload, [ka] wherein the phosphatase enzyme cleavage group, after being effectively linked to a linking module 1, a chemical group, a targeting molecule, or a payload, [ka] wherein the β-galactosidase enzyme cleavage group, after being operatively linked to a linking module 1, a chemical group, a targeting molecule, or a payload, [ka] wherein the glutathione enzyme cleavage group, after being effectively linked to linkage module 1, chemical group, targeting molecule, or payload, [ka] wherein the acid labile group, after operatively linking to a linking module 1, chemical group, targeting molecule, or payload, [ka] It has.

[0040] In some preferred embodiments, when the linker comprises Linking Module 2, Linking Module 2 is a glutathione enzyme cleavage group. In some preferred embodiments, the glutathione enzyme cleavage group is [ka] is.

[0041] In some embodiments, the conjugate compound has the structure: W 1 -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W 2 , Among them, L 1 teeth, [ka] -SS- or bond, EG is -CH2CH2O- or -OCH2CH2-, n 1 is an integer from 0 to 6, n 2 is an integer from 0 to 8, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, B, in each occurrence, is independently -C(=O)-, [ka] -NH-CH2CH2-C(=O)-, -C(=O)-NH-, -CH(CH3)-CH2-O-, -CH2CH2O-, -NH-C(=O)- or -NH-, n6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 5, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] -NH-CH2-O- or a bond, wherein X is a bond, -NH-, -C(=O)-, oxygen, or [ka] and R2 is a bond, —O—CH2— or —NH—CH2CH2—NH—C(═O)—; R3 is hydrogen or [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, [ka] and R4 is hydrogen or C 1-8 is an alkyl group, R5 is hydrogen, amino group, nitro group, phosphate group, -OR a or [ka] Among them, R a is a methyl group, an ethyl group, a propyl group, or an isopropyl group, W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0042] In some embodiments, EG is -CH2CH2O-. 1 is 2. In some embodiments, n 1 is 0. In some embodiments, n 2 is 1. In some embodiments, n 2 is 4. In some embodiments, n 2 is 5. In some embodiments, n 5 is 0. In some embodiments, n 6 is 1. In some embodiments, n 6 is 3. In some embodiments, n 1 is 2 and n 2 is 1 and n 3 and n 4 is 0 and L 2 is a bond.

[0043] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro, or Asn. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and -Ala-Ala-Asn-.

[0044] In some embodiments, n 4 is 1, Q is -C(=O)-, and n 4 is 2, Q is —O— and —C(═O)—, respectively, and n 4 When n is 3, Q is -O-, -O-, and a bond, respectively. 4 is 0.

[0045] In some embodiments, L 2 is a bond, [ka] is.

[0046] In some embodiments, the conjugate compound has the structure: W 1 -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W 2 Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, n 2 is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH2CH2O-, -NH-CH2CH2-C(=O)- or -NH-; n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] and wherein X is a bond, -NH-, -C(=O)- or [ka] and R2 is a bond, R3 is [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, or [ka] and R4 is hydrogen; R5 is hydrogen, -OCH3 or [ka] and W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0047] In some embodiments, L 2 teeth, [ka] is.

[0048] In some embodiments, L 1 teeth [ka] and n 1 , n 5 , and n 4 are both 0.

[0049] In some embodiments, L 1 teeth [ka] and n 2 is 1 or 2.

[0050] In some embodiments, n 5 If is 1, then n 2 and n 4 are both 0.

[0051] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, or Ala. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-.

[0052] In some embodiments, the conjugate compound has the structure: W 1 -L 1 -(EG)n 1 -(CH2)n 2 -(C=O)-(AA)n 3 -L 2 -W 2 Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, n 2 is an integer from 0 to 5, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 1 to 4, L 2 teeth, [ka] or a bond, wherein X is -NH-; R2 is a bond, R4 is hydrogen; R5 is hydrogen or -OCH3; W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0053] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 teeth [ka] is.

[0054] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 is a bond.

[0055] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 teeth [ka] is.

[0056] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 is a bond.

[0057] In some embodiments, each occurrence of AA is independently a Val, Cit, Glu, or Ala amino acid residue. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Leu-Lys- and -Gly-Asn-Asn-.

[0058] In some embodiments, the conjugate compound has the structure: [ka] [ka] [ka] [ka] [ka] [ka] Among them, W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0059] In some embodiments, the conjugate compound has the structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0060] Another aspect of the present application provides a pharmaceutical composition comprising a conjugate compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used for intravenous, subcutaneous, oral, intramuscular, or intraventricular administration.

[0061] Another aspect of the present application provides methods for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. In some embodiments, the disease is selected from the group consisting of cancer, immune disease, cardiovascular disease, metabolic disease, and neurological disease. In some embodiments, the disease is characterized in the subject by expression of a target protein or aberrant expression of a target protein. In some embodiments, the method further comprises administering one or more therapeutic agents in combination with the conjugate compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0062] In some embodiments, the cancer is characterized by high expression of FOLR1, TRPV6, PSMA, or c-MET in cancer cells. In some embodiments, the cancer is characterized by high expression of FOLR1 and TRPV6, FOLR1 and c-MET, c-MET and TRPV6, c-MET and PSMA, PSMA and TRPV6, or FOLR1 and PSMA in cancer cells. In some embodiments, the cancer is selected from the group consisting of human breast ductal carcinoma, human brain astrocytoma, bladder transitional cell papilloma, prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, gastric cancer, cervical cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, lymphoma, and multiple myeloma. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0063] In some embodiments, the immune disease is an autoimmune disease, hi some embodiments, the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0064] In some embodiments, the cardiovascular disease is selected from the group consisting of angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.

[0065] In some embodiments, the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.

[0066] In some embodiments, the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, dizziness, stupor, and epilepsy.

[0067] Another aspect of the present application provides the use of a conjugate compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, in the preparation of a medicament for treating a disease in a subject.

[0068] Another aspect of the present application provides a compound or a salt thereof, said compound having the structure: L a -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L b , Among them, L a teeth, [ka] and EG is -CH2CH2O- or -OCH2CH2-, n 1 is an integer from 0 to 6, n 2 is an integer from 0 to 8, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, B is -C(=O)-, [ka] -NH-CH2CH2-C(=O)-, -C(=O)-NH-, -CH(CH3)-CH2-O-, -CH2CH2O-, -NH-C(=O)- or -NH-, n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 5, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L b teeth, [ka] -OH or -NH-CH2-OH, wherein X is a bond, -NH-, -C(=O)-, oxygen, or [ka] and R2 is a bond, —O—CH2— or —NH—CH2CH2—NH—C(═O)—; R3 is hydrogen or [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, [ka] and R4 is hydrogen or C 1-8 is an alkyl group, R5 is hydrogen, amino group, nitro group, phosphate group, -OR a or [ka] Among them, R a is a methyl group, an ethyl group, a propyl group, or an isopropyl group, R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0069] In some embodiments, EG is -CH2CH2O-. 1 is 2. In some embodiments, n 1 is 0. In some embodiments, n 2 is 1. In some embodiments, n 2 is 4. In some embodiments, n 2 is 5. In some embodiments, n 5 is 0. In some embodiments, n 6 is 1. In some embodiments, n 6 is 3. In some embodiments, n 1 is 2 and n 2 is 1 and n 3 ~n 4 is 0 and L b is -OH.

[0070] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro, or Asn. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and -Ala-Ala-Asn-. 4 is 1, Q is -C(=O)-, and n 4 is 2, Q is —O— and —C(═O)—, respectively, and n4 When n is 3, Q is -O-, -O-, and a bond, respectively. 4 is 0.

[0071] In some embodiments, L b teeth, [ka] is.

[0072] In some embodiments, the compound has the structure: L a -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L b Among them, L a teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer between 2 and 4, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, n 2 is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH2CH2O-, -NH-CH2CH2-C(=O)- or -NH-; n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L b is -OH, [ka] and wherein X is a bond, -NH-, -C(=O)- or [ka] and R2 is a bond, R3 is [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, or [ka] and R4 is hydrogen; R5 is hydrogen, -OCH3 or [ka] and R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0073] In some embodiments, L b is -OH, [ka] is.

[0074] In some embodiments, n 5 If is 1, then n 2 and n 4 are both 0.

[0075] In some embodiments, L a teeth [ka] and n 1 , n 5 , and n 4 are both 0.

[0076] In some embodiments, L a teeth [ka] and n 2 is 1 or 2.

[0077] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, or Ala. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-.

[0078] In some embodiments, the compound has the structure: L a -(EG)n 1 -(CH2)n 2 -(C=O)-(AA)n 3 -L b Among them, L a teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, n 2 is an integer from 0 to 5, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, L b teeth, [ka] or -OH, wherein X is -NH-; R2 is a bond, R4 is hydrogen; R5 is hydrogen or -OCH3; R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0079] In some embodiments, L a teeth [ka] and n 1 is 2 and n 2 is 1 and L b teeth [ka] is.

[0080] In some embodiments, L a teeth [ka] and n 1 is 2 and n 2 is 1 and L b is -OH.

[0081] In some embodiments, L a teeth [ka] and n 1 is 0, and n 2 is 5 and L b teeth [ka] is.

[0082] In some embodiments, L a teeth [ka] and n 1 is 0, and n 2 is 5 and L b is -OH.

[0083] In some embodiments, each occurrence of AA is independently a Val, Cit, Glu, or Ala amino acid residue. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Leu-Lys- and -Gly-Asn-Asn-.

[0084] In some embodiments, the compound has a structure selected from the group consisting of: [ka] [ka] [ka] [ka] wherein R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0085] Another aspect of the present application is [ka] or a salt thereof.

[0086] Another aspect of the present application provides a conjugate compound or a pharmaceutically acceptable salt thereof, said conjugate compound having the structure: W a -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W b , Among them, L 1 teeth, [ka] -SS- or bond, EG is -CH2CH2O- or -OCH2CH2-, n 1 is an integer from 0 to 6, n 2 is an integer from 0 to 8, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, B, in each occurrence, is independently -C(=O)-, [ka] -NH-CH2CH2-C(=O)-, -C(=O)-NH-, -CH(CH3)-CH2-O-, -CH2CH2O-, -NH-C(=O)- or -NH-, n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 5, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] -NH-CH2-OH or a bond, wherein X is a bond, -NH-, -C(=O)-, oxygen, or [ka] and R2 is a bond, —O—CH2— or —NH—CH2CH2—NH—C(═O)—; R3 is hydrogen or [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, [ka] and R4 is hydrogen or C 1-8 is an alkyl group, R5 is hydrogen, amino group, nitro group, phosphate group, -OR a or [ka] Among them, R a is a methyl group, an ethyl group, a propyl group, or an isopropyl group, W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0087] In some embodiments, EG is -CH2CH2O-. 1 is 2. In some embodiments, n 1 is 0. In some embodiments, n 2 is 1. In some embodiments, n 2 is 4. In some embodiments, n 2is 5. In some embodiments, n 5 is 0. In some embodiments, n 6 is 1. In some embodiments, n 6 is 3. In some embodiments, n 1 is 2 and n 2 is 1 and n 3 and n 4 is 0 and L 2 is a bond.

[0088] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro, or Asn. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and Ala-Ala-Asn-.

[0089] In some embodiments, n 4 is 1, Q is -C(=O)-, and n 4 is 2, Q is —O— and —C(═O)—, respectively, and n 4 When n is 3, Q is -O-, -O-, and a bond, respectively. 4 is 0.

[0090] In some embodiments, L 2 is a bond, [ka] is.

[0091] In some embodiments, the compound has the structure: Wa -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W b Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer between 2 and 4, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, n 2 is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH2CH2O-, -NH-CH2CH2-C(=O)- or -NH-; n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] and wherein X is a bond, -NH-, -C(=O)- or [ka] and R2 is a bond, R3 is [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, or [ka] and R4 is hydrogen; R5 is hydrogen, -OCH3 or [ka] and W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0092] In some embodiments, L 2 teeth, [ka] is.

[0093] In some embodiments, n 5 If is 1, then n 2 and n 4 are both 0.

[0094] In some embodiments, L 1 teeth [ka] and n 1 , n 5 , and n 4 are both 0.

[0095] In some embodiments, L 1 teeth [ka] and n 2 is 1 or 2.

[0096] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, or Ala. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-.

[0097] In some embodiments, the compound has the structure: W a -L 1 -(EG)n 1 -(CH2)n 2 -(C=O)-(AA)n 3 -L 2 -W b Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, n 2 is an integer from 0 to 5, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 1 to 4, L 2 teeth, [ka] or a bond, wherein X is -NH-; R2 is a bond, R4 is hydrogen; R5 is hydrogen or —OCH3; W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0098] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 teeth [ka] is.

[0099] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 is a bond.

[0100] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 teeth [ka] is.

[0101] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 is a bond.

[0102] In some embodiments, the conjugate compound has a structure selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] Among them, W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0103] Another aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound is [ka] Among them, W a and W b are each independently a targeting molecule capable of binding to a payload or a cell surface protein.

[0104] Another aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound is [ka] Among them, W a and W b are each independently a targeting molecule capable of binding to a payload or a cell surface protein.

[0105] Another aspect of the present application provides a proteolysis targeting chimera or a pharmaceutically acceptable salt thereof, wherein the proteolysis targeting chimera compound has the following structure: [ka] Among them, Ra and Rb are each independently hydrogen, an alkyl group, NH, OH, or halogen; A, at each occurrence, is independently an amino acid residue; a is 0 or 1, b is an integer from 0 to 5, X is [ka] or a bond, The target protein ligand is [ka] is.

[0106] In some embodiments, Ra is hydrogen. In some embodiments, Rb is hydrogen. In some embodiments, A is an amino acid residue formed by Lys. In some embodiments, a is 1. In some embodiments, a is 0. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5.

[0107] In some embodiments, the proteolysis targeted chimera has a structure selected from the group consisting of: [ka] [ka] [Brief explanation of the drawings]

[0108] [Figure 1] 1 shows an exemplary conjugate compound of the present application.

[0109] [Figure 2]The compound CR20214 degrades the target protein. Western Blot experiment results show that the compound CR20214 degrades the target protein.

[0110] [Figure 3] The compound CR20295 degrades the target protein. Western Blot experiment results show that the compound CR20295 degrades the target protein.

[0111] [Figure 4] The results of Western blot experiments show that compounds CR202V6 and CR202AL degrade the target protein.

[0112] [Figure 5] FIG. 5A shows the changes in tumor volume in tumor-bearing mice of the human liver cancer model HepG2 after administration of a control, CR20274, and the conjugate compound of the present application, and FIG. 5B shows the changes over time of administration in tumor-bearing mice of the human liver cancer model HepG2 compared to their initial body weights.

[0113] [Figure 6] FIG. 6A shows the changes in tumor volume in mice bearing a human breast cancer model MDA-MB-231 tumor after administration of a control, CR20274, and the conjugate compound of the present application, and FIG. 6B shows the changes over time of administration in mice bearing a human breast cancer model MDA-MB-231 tumor compared to their initial body weight.

[0114] [Figure 7] FIG. 7A shows the changes over time in body weight of tumor-bearing mice of human prostate cancer model 22RV1 compared to the initial body weight after administration of the control, CR20274, and the conjugate compound of the present application, and FIG. 7B shows the changes in tumor volume in tumor-bearing mice of human prostate cancer model 22RV1.

[0115] [Figure 8]FIG. 8A shows the changes over time in body weight of mice bearing a human prostate cancer model C4-2 tumor compared to the initial body weight after administration of a control, CR20274, and the conjugate compound of the present application, and FIG. 8B shows the changes in tumor volume in mice bearing a human prostate cancer model C4-2 tumor.

[0116] [Figure 9] FIG. 9A shows the changes in tumor volume in HT-29 tumor-bearing mice, a human colon cancer model, after administration of a control, CR20274, and the conjugate compound of the present application, and FIG. 9B shows the changes over time of administration in HT-29 tumor-bearing mice, a human colon cancer model, compared with the initial body weight.

[0117] [Figure 10] The changes in tumor volume in mice bearing the human prostate cancer model 22RV1 tumor after administration of the control, CR20274, and the conjugate compound of the present application (FIG. 10A) and the changes over time of administration of the human prostate cancer model 22RV1 compared to the initial body weight (FIG. 10B) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0118] The following description is only for illustrating various embodiments of the present application. Therefore, the detailed description of the present application should not be interpreted as limiting the scope of the application. Those skilled in the art can easily obtain various equivalent methods and modifications based on the spirit of the present invention and the description of the present specification, and it should be understood that such equivalent embodiments are included in the scope of the present invention. All documents cited in this application, including published publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0119] As used herein, the singular forms "a," "one," "an," and "the," or "the above" include plural references unless the context clearly dictates otherwise.

[0120] As used herein, terms such as "comprises," "comprising," "containing," "containing," and "having" are intended to be exhaustive or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting of" is closed-ended.

[0121] One aspect of the present specification discloses a conjugate compound or a pharmaceutically acceptable salt thereof, the conjugate compound or the pharmaceutically acceptable salt thereof comprising targeting molecules of at least two ligands that specifically bind to cell surface proteins, and a payload linked to the targeting molecules, wherein the payload is a proteolytic targeting chimera.

[0122] As used herein, the term "targeting molecule" refers to any molecule or moiety that can be targeted to a target site, target tissue, target organ, target cell, or target intracellular region. In some embodiments, the targeting molecule causes a moiety linked to the target molecule to distribute more, e.g., at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more, to the target site, target tissue, target organ, target cell, or target intracellular region compared to a non-target site, non-target tissue, non-target organ, non-target cell, or non-target intracellular region. In some embodiments, a targeting molecule causes a conjugated compound or reagent bearing the targeting molecule to distribute to a target site, tissue, organ, cell, or intracellular region to a greater extent, e.g., at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more, compared to one lacking the targeting molecule. In some embodiments, a targeting molecule can induce or promote specific binding of a conjugated compound containing such a targeting molecule to a target molecule, induce or promote endocytosis of the conjugated compound by the target cell, induce or promote concentration of the conjugated compound around and / or entry into the target cell.

[0123] The term "ligand" as used in this application can include a variety of chemical molecules or polypeptides that have specific binding affinity to a selected target, which can be a cell surface protein (e.g., a cell surface receptor or a cell surface antigen), a specific protein, a cell, a tissue, an organ, etc. In some embodiments, a ligand can specifically bind to a cell surface receptor. In some embodiments, a ligand can specifically bind to a cell surface antigen. In some embodiments, a ligand can specifically bind to a specific protein that can cause a disease. In some embodiments, overexpression of the specific protein causes the disease, or the specific protein is a mutant protein that causes a disease. In some embodiments, the ligand of the present application can be 10 -6 ~10 -11 M(K d In some embodiments, the ligands of the present application bind to the target with an affinity of at least 10 -6 , at least 10 -7 , at least 10 -8 , or at least 10 -9 M(K d In some embodiments, the ligands of the present application bind to a target with an affinity of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more than the affinity of the same non-target (e.g., another cell surface receptor, cell surface antigen, or specific protein). In some embodiments, the cell surface receptors, cell surface antigens, and specific proteins of the present application are expressed on the surface of or within target cells (e.g., cancer cells or cells with physiological dysfunction) at a significantly higher level than in normal cells. As used in this application, the term "significant" refers to a statistically significant difference or a significant difference that can be recognized by one of ordinary skill in the art.

[0124] In some embodiments, the expression level of a cell surface receptor, cell surface antigen, or specific protein of the present application on the surface of or in a target cell (e.g., a cancer cell or a cell with a physiological dysfunction) is 2-fold to 1,000,000-fold higher than the expression level in a normal cell, e.g., the expression level in a target cell (e.g., a cancer cell or a cell with a physiological dysfunction) is 2-fold to 10-fold, 2-fold to 100-fold, 2-fold to 1,000-fold, 2-fold to 10,000-fold, 2-fold to 100,000-fold, or 2-fold to 1,000,000-fold higher than the expression level in a normal cell (which may be equal to any value within the above numerical range, including the endpoints of the range). In some embodiments, the expression level of a cell surface receptor in a target cell (e.g., a cancer cell or a cell with a physiological dysfunction) is at least 10-fold, at least 100-fold, at least 1,000-fold, at least 10,000-fold, or at least 100,000-fold higher than the expression level in a normal cell. In some embodiments, the level of a cell surface receptor, cell surface antigen, or specific protein on a normal cell is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% when compared to the level of the cell surface receptor, cell surface antigen, or specific protein expressed by a target cell (e.g., a cancer cell or a physiologically dysfunctional cell). In some embodiments, the cell surface receptor, cell surface antigen, or specific protein described herein is undetectable on a normal cell.

[0125] In some embodiments, the cell surface receptor is selected from FOLR1, TRPV6, or c-Met. In some embodiments, the cell surface antigen is selected from PSMA.

[0126] In some embodiments, the specific protein is selected from nuclear receptors (e.g., AR and ER), kinase proteins (e.g., RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, and FAK), AKT, PAN-BET proteins, BET proteins (e.g., BRD2, BRD3, BRD4, and BRD6), BLK, BRAF1, BCL-x1, ERRα, FKBP12, FRS2α, JAK1, JAK3, IKZF1, IRAK4, MDM2, MetAP2, PLK1, PSK-J3, RAS, TACC3, Tau, TrkB, MDM2, or any combination thereof. In some embodiments, the specific protein is selected from an AR protein, an ER protein, a RAS protein, a BRD4 protein, a PLK1 protein, a FAK protein, an MDM2 protein, or any combination thereof. In some embodiments, the target protein ligand moiety in the proteolysis targeting chimera targets the specific protein.

[0127] Folate receptor 1 (FOLR1) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) that binds folate with nanomolar affinity, thereby facilitating receptor-mediated endocytosis. Rapidly growing solid malignant tumors, including ovarian and lung cancer, all depend on folate for metabolism and nucleic acid synthesis. In some embodiments, the targeting molecules described herein comprise ligands that target FOLR1.

[0128] A ligand that specifically binds to FOLR1, or a "FOLR1 ligand," refers to an antibody, polypeptide, nucleic acid aptamer, small molecule, etc., that can specifically recognize and bind to FOLR1. The FOLR1 ligand of the present application includes currently existing or subsequently generated FOLR1 ligands. In some embodiments, the FOLR1 ligand includes folic acid or an analog thereof and pteroic acid. As used herein, the term "analog" includes structural analogs and functional analogs. Structural analogs refer to a class of compounds that have similar chemical structures, which may contain one or more different atoms or one or more different functional groups. Functional analogs refer to a class of compounds that have the same or similar chemical, biological, or pharmacological effects. In some embodiments, the folic acid analog is selected from the group consisting of 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 10-formyltetrahydrofolic acid, methotrexate, 5,10-formyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, aminopterin, and raltitrexed.

[0129] Transient receptor potential cation channel subfamily V member 6 (TRPV6) is a highly selective transmembrane calcium ion transporter that mediates the active transport of calcium ions from extracellular to intracellular spaces. TRPV6 is expressed in normal human tissues, including the kidney, gastrointestinal tract, pancreas, mammary gland, and salivary gland, but is primarily expressed in intestinal epithelial cells. Because TRPV6 is involved in intracellular calcium ion transport, alterations in the number or function of TRPV6 channels can lead to altered calcium ion regulation and, in turn, structural or functional abnormalities in related tissues and organs. Compared with normal tissues, TRPV6 expression is significantly elevated in malignant tumors, such as breast cancer, cholangiocarcinoma, ovarian cancer, lung squamous cell carcinoma, and prostate cancer, and its aberrant expression may be involved in tumor formation and progression.

[0130] A ligand that specifically binds to TRPV6, or a "TRPV6 ligand," refers to an antibody, polypeptide, nucleic acid aptamer, small molecule, etc., that can specifically recognize and bind to TRPV6. The TRPV6 ligand of the present application includes currently existing or subsequently generated TRPV6 ligands. In some embodiments, the TRPV6 ligand comprises the amino acid sequence set forth in SEQ ID NO: 1 (KEFLHPSKVDLPR), the amino acid sequence set forth in SEQ ID NO: 2 (EGKLSSNDTEGGLCKEFLHPSKVDLPR), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0131] Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein consisting of 750 amino acids, with 19 amino acids in the intracellular domain, 24 amino acids in the transmembrane domain, and 707 amino acids in the extracellular domain, present in the membrane of prostate epithelial cells. Prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but its expression level in prostate cancer cells is much higher. Compared with prostate-specific antigens conventionally used in clinical trials, prostate-specific membrane antigen is a more sensitive and specific tumor marker for prostate cancer, and is particularly highly expressed in hormone-refractory prostate cancer and prostate cancer metastasis, and has high sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors. At the same time, prostate-specific membrane antigen is also highly and specifically expressed on tumor vascular endothelial cells in several types of non-prostate-derived solid tumors (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer, and colorectal cancer).

[0132] A ligand that specifically binds to prostate-specific membrane antigen or a "prostate-specific membrane antigen ligand" refers to an antibody, polypeptide, nucleic acid aptamer, small molecule, etc., that can specifically recognize and bind to prostate-specific membrane antigen. The prostate-specific membrane antigen ligand of the present application includes currently existing or subsequently generated prostate-specific membrane antigen ligands, and further includes fragments of the aforementioned ligands, so long as the fragments retain the ability to bind to prostate-specific membrane antigen. Antibody-type ligands are the most common prostate-specific membrane antigen ligands, including, but not limited to, monoclonal antibodies J591, J533, J415, and E99 (see, for example, Liu H, Rajasekaran AK, Moy P et al., Constitutive and antibody-induced internalization of prostate-specific member antigen [J]. Cancer Res., 1998, 58(18):4055-4060). Nucleic acid aptamers are single-stranded DNA or RNA that can bind to prostate-specific membrane antigen (PSMA) with high affinity and specificity, and are obtained by technical screening using an exponential enrichment ligand system. Examples of such PSA ligands include, but are not limited to, xPSM-A10 and xPSM-A9 nucleic acid aptamers (e.g., Lupoid SE et al., "Identification and Characterization of Nuclease-Stabilized RNA Molecules that Bind Human Prostate Cancer Cells via the Prostate-Specific Membrane Antigen," Cancer Res., 2002, 62(14):4029-4033). Compared with antibody and nucleic acid aptamer ligands, PSA small molecule ligands have advantages such as small molecular weight, high permeability, low immunogenicity, and ease of synthesis, and include, but are not limited to, glutamylurea and phosphoramidate small molecule ligands.

[0133] As used herein, the terms "polypeptide," "protein," and "peptide" may refer to a single amino acid or a polymer of amino acids. The polypeptides, proteins, or peptides described herein can contain naturally occurring and non-naturally occurring amino acids, or amino acid analogs and mimetics. Polypeptides, proteins, or peptides can be obtained by any method known in the art, including, but not limited to, isolation and purification from natural sources, recombinant expression, and chemical synthesis. In some embodiments, a polypeptide contains 2 to 50, 2 to 40, 2 to 30, 2 to 25, 2 to 22, 2 to 20, 2 to 18, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 4 to 50, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 22, 5 to 20, 5 to 18, 5 to 15, 5 to 12, 5 to 10, 6, 7, 8, or 9 amino acids.

[0134] As used herein, "small molecule ligand" refers to a compound having a molecular weight of about 3 kDa or less. In some embodiments, a small molecule compound has a molecular weight of about 2 kDa or less. In some embodiments, a small molecule compound has a molecular weight of about 1.5 kDa or less. In some preferred embodiments, a small molecule compound has a molecular weight of about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da or less.

[0135] In some embodiments, the prostate specific membrane antigen small molecule ligand of the present application may be selected from the group consisting of 2-[[methylhydroxyphosphinyl]methyl]glutaric acid, 2-[[ethylhydroxyphosphinyl]methyl]glutaric acid, 2-[[propylhydroxyphosphinyl]methyl]glutaric acid, 2-[[butylhydroxyphosphinyl]methyl]glutaric acid, 2-[[cyclohexylhydroxyphosphinyl]methyl]glutaric acid, 2-[[phenylhydroxyphosphinyl]methyl]glutaric acid, 2-[[2-(tetrahydrofuranyl) )hydroxyphosphinyl]methyl]glutaric acid, 2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl ]methyl]glutaric acid, 2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]glutaric acid, and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]glutaric acid, 2-[[methylhydroxyphosphinyl]oxy]glutaric acid, 2-[[ethylhydroxyphosphinyl]oxy]glutaric acid, 2-[[propylhydroxyphosphinyl]oxy] Glutaric acid, 2-[[butylhydroxyphosphinyl]oxy]glutaric acid, 2-[[phenylhydroxyphosphinyl]oxy]glutaric acid, 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]oxy]glutaric acid, 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]oxy]glutaric acid, 2-[[(phenylmethyl)hydroxyphosphinyl]oxy]glutaric acid, and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]glutaric acid, 2-[[(N-hydroxy)carbamoyl]methyl]glutaric acid,2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]glutaric acid, 2-[[(N-butyl-N-hydroxy)carbamoyl]methyl]glutaric acid, 2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]glutaric acid, 2-[[(N-hydroxy-N-phenyl)carbamoyl]methyl]glutaric acid, 2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]glutaric acid, 2-[[(N-ethyl-N-hydroxy)carbamoyl]methyl]glutaric acid, 2-[[(N-hydroxy-N-propyl)carbamoyl 2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]glutaric acid, 2-[[(N-hydroxy-N-4-pyridyl)carbamoyl]methyl]glutaric acid, 2-[[(N-hydroxy)amido]methyl]glutaric acid, 2-[[N-hydroxy(methyl)amido]methyl]glutaric acid, 2-[[N-hydroxy(benzyl)amido]methyl]glutaric acid, 2-[[N-hydroxy(phenyl)amido]methyl]glutaric acid, 2-[[N-hydroxy(2-phenylethyl)amido]methyl ]glutaric acid, 2-[[N-hydroxy(ethyl)amido]methyl]glutaric acid, 2-[[N-hydroxy(propyl)amido]methyl]glutaric acid, 2-[[N-hydroxy(3-phenylpropyl)amido]methyl]glutaric acid, and 2-[[N-hydroxy(4-pyridyl)amido]methyl]glutaric acid, 2-[(sulfinyl)methyl]glutaric acid, 2-[(methylsulfinyl)methyl]glutaric acid, 2-[(ethylsulfinyl)methyl]glutaric acid, 2-[(propylsulfinyl)methyl]glutaric acid, 2-[(butylsulfinyl) (phenyl)methyl]glutaric acid, 2-[(phenylsulfinyl)methyl]glutaric acid, 2-[[(2-phenylethyl)sulfinyl]methyl]glutaric acid, 2-[[(3-phenylpropyl)sulfinyl]methyl]glutaric acid, 2-[[(4-pyridyl)sulfinyl]methyl]glutaric acid, 2-[(benzylsulfinyl)methyl]glutaric acid, 2-[(sulfonyl)methyl]glutaric acid, 2-[(methylsulfonyl)methyl]glutaric acid, 2-[(ethylsulfonyl)methyl]glutaric acid, 2-[(propylsulfonyl)methyl]glutaric acid2-[(butylsulfonyl)methyl]glutaric acid, 2-[(phenylsulfonyl)methyl]glutaric acid, 2-[[(2-phenylethyl)sulfonyl]methyl]glutaric acid, 2-[[(3-phenylpropyl)sulfonyl]methyl]glutaric acid, 2-[[(4-pyridyl)sulfonyl]methyl]glutaric acid, 2-[(benzylsulfonyl)methyl]glutaric acid, 2-[(sulfoximinyl)methyl]glutaric acid, 2- [(Methylsulfoximinyl)methyl]glutaric acid, 2-[(ethylsulfoximinyl)methyl]glutaric acid, 2-[(propylsulfoximinyl)methyl]glutaric acid, 2-[(butylsulfoximinyl)methyl]glutaric acid, 2-[(phenylsulfoximinyl]methyl]glutaric acid, 2-[[(2-phenylethyl)sulfoximinyl]methyl]glutaric acid, 2-[[(3-phenylpropyl)sulfoximinyl]methyl]glutaric acid, 2- [[(4-pyridyl)sulfoximinyl]methyl]glutaric acid, and 2-[(benzylsulfoximinyl)methyl]glutaric acid, N-[methylhydroxyphosphinyl]glutamic acid, N-[ethylhydroxyphosphinyl]glutamic acid, N-[propylhydroxyphosphinyl]glutamic acid, N-[butylhydroxyphosphinyl]glutamic acid, N-[phenylhydroxyphosphinyl]glutamic acid, N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid, N-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid, and N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid. The prostate-specific membrane antigen ligands of the present application further include all of the prostate-specific membrane antigen small molecule ligands disclosed in PCT applications WO2010 / 108125 and WO2006 / 093991, the two above-mentioned patent applications being incorporated herein in their entireties.

[0136] In some embodiments, the prostate specific membrane antigen ligand of the present application is a glutaric acid derivative. In some embodiments, the prostate specific membrane antigen ligand of the present application is an aminocarbonyl group derivative of glutaric acid.

[0137] In some embodiments, the prostate specific membrane antigen ligand of the present application comprises the following structure: [ka]

[0138] In some embodiments, the prostate specific membrane antigen ligand included in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure: [ka]

[0139] In some embodiments, the prostate specific membrane antigen ligand included in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure: [ka]

[0140] In some embodiments, the prostate specific membrane antigen ligand included in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure: [ka]

[0141] In some embodiments, the prostate specific membrane antigen ligand included in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure: [ka]

[0142] In some embodiments, the prostate specific membrane antigen ligand included in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure: [ka]

[0143] In some embodiments, the prostate specific membrane antigen ligand included in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure: [ka]

[0144] C-MET is a protein product encoded by the C-MET proto-oncogene and a receptor for hepatocyte growth factor (HGF). It has tyrosine kinase activity, associates with multiple oncogene products and regulatory proteins, and is involved in the control of cell signaling and cytoskeletal reorganization, playing an important role in cell proliferation, differentiation, and motility. C-MET is closely associated with the development and metastasis of multiple cancers. Studies have shown that many tumor patients have overexpression and gene amplification of C-MET during both tumor development and metastasis. Because C-MET acts on different substrates in different cells and at different differentiation stages, it exhibits multiple functions under certain conditions, such as promoting the division of hepatocytes, endothelial cells, and melanocytes, inducing epithelial cell dispersion, and inducing cell morphological changes. In some embodiments, the targeting molecule described herein comprises a ligand that targets C-MET.

[0145] A ligand that specifically binds to C-MET or a "C-MET ligand" refers to an antibody, polypeptide, nucleic acid aptamer, small molecule, and the like that can specifically recognize and bind to C-MET. C-MET ligands of the present application include C-MET ligands that already exist today or that are subsequently generated. In some embodiments, a C-MET ligand is a C-MET ligand that is a Cys a -X1-Cys c -X2-Gly-Pro-Pro-X3-Phe-Glu-Cys d -Trp-Cys bX1 is Asn, His or Tyr, X2 is Gly, Ser, Thr or Asn, X3 is Thr or Arg, X4 is Ala, Asp, Glu, Gly or Ser, X5 is Ser or Thr, X6 is Asp or Glu, and Cys a-d are each a cysteine ​​residue. In some preferred embodiments, the residues Cys a and Cys b , and Cys c and Cys d each cyclizes to form two independent disulfide bonds. In some embodiments, the ligand of c-MET is Ala-Gly-Ser-Cys a -Tyr-Cys c -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys d -Trp-Cys b -Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys, the amino acid sequence of which is set forth in SEQ ID NO: 4, a-d are each a cysteine ​​residue. In some preferred embodiments, the residues Cys a and Cys b , and Cys c and Cys d each cyclizes to form two independent disulfide bonds. In some preferred embodiments, the carboxyl group of the terminal lysine may be amidated. The C-MET ligands of the present application further include all of the C-MET ligands disclosed in PCT application WO2008139207A3, the disclosure of which is incorporated herein by reference in its entirety.

[0146] The corresponding Japanese names and abbreviations of amino acids used herein are shown in the table below. The term "amino acid" or the Japanese name or abbreviation of a specific amino acid used in this application is intended to include all stereoisomers thereof, for example, L-configuration and D-configuration. For example, "Arg" used herein may be L-Arg or D-Arg. For example, amidated Lys may be L-2,6-diaminohexanamide or D-2,6-diaminohexanamide. [Table 1]

[0147] In some embodiments, the ligands in the targeting molecule are linked directly or via a spacer region. In some preferred embodiments, the linkage between the ligands or the spacer region is achieved by dehydration condensation. As used herein, "dehydration condensation" refers to a process in which two groups react to eliminate one molecule of water and form a new chemical bond. For example, the amino group (-NH2) in one amino acid reacts with the carboxyl group (-COOH) in another amino acid to eliminate one molecule of water and form a new peptide bond.

[0148] In some embodiments, the spacer region comprises an amino acid or combination of amino acids selected from the group consisting of Lys, Cys, Lys-Cys, Cys-Cys, Lys-Cys-Lys, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg, Asp-Asp-Lys-Cys, and Pro-Leu-Gly. Optionally, the amino acids in the spacer region may be amidated. In some preferred embodiments, lysine (Lys) is amidated, for example, to 2,6-diaminohexanamide.

[0149] In some embodiments, the spacer region comprises one or more homologous or dissimilar compounds formed by dehydration condensation selected from the group consisting of: [ka] wherein n is 0 or 1. In some embodiments, the spacer region comprises: [ka] may be.

[0150] In some embodiments, a targeting molecule having the following structural moiety is linked to a payload to form a conjugate compound: [ka] [ka] Among them, Q is an active group. In some embodiments, the targeting molecule is linked to the payload via an active group Q, wherein the active group Q is an alkynyl group or a mercapto group.

[0151] In some embodiments, a targeting molecule having the following structure is linked to a payload to form a conjugate compound: [ka] [ka]

[0152] The term "payload" as used herein refers to a molecule or substance intended to be delivered to a target cell or tissue. In some embodiments, the payload may be a molecule or substance for the diagnosis, treatment, or prevention of a disease in a subject. The payload may be, but is not limited to, a proteolytic targeting chimera intended for use in the diagnosis, treatment, or prevention of a disease in a subject.

[0153] In some embodiments, the proteolytic target chimera comprises a target protein ligand portion that binds to a target protein (specific protein), an E3 ligase ligand portion that binds to an E3 ligase, and a linking portion that links the target protein ligand portion and the E3 ligase ligand portion. The proteolytic target chimera of the present application includes proteolytic target chimeras that already exist or that are subsequently generated. The proteolytic target chimera of the present application further includes all proteolytic target chimeras disclosed in PCT Application WO2021205391A1, U.S. Application US20190151457A1, and Chinese Applications CN113563414, CN104736569A, and CN108366992A, the disclosures of which are incorporated herein by reference in their entireties.

[0154] In some embodiments, a proteolysis targeting chimera having the structure shown in Formula (I) is linked to a targeting molecule to form a conjugate compound, (T P ) n -LT E3 (I), Among them, Above T p is a target protein ligand moiety that binds to a target protein, wherein n is 1, 2, or 3; The above L is T P and T E3 and a connecting portion that connects the Above T E3 is the E3 ligase ligand moiety that binds to the E3 ligase.

[0155] In some embodiments, the proteolysis targeted chimera has a structure selected from the group consisting of: [ka] Among them, Ra, Rb and Rc are each independently hydrogen, an alkyl group, NH, OH or a halogen, and preferably, the alkyl group is a methyl group and the halogen is fluorine; Rd is CH2, C=O or C=S; p is 0 or 1; the target protein ligand is capable of binding to a specific target protein; The target protein ligand is linked to the E3 ligase group via the linking moiety.

[0156] In some embodiments, the target protein is a specific protein described herein. In some embodiments, the target protein is selected from nuclear receptors (e.g., AR and ER), kinase proteins (e.g., RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, and FAK), AKT, PAN-BET proteins, BET proteins (e.g., BRD2, BRD3, BRD4, and BRD6), BLK, BRAF1, BCL-x1, ERRα, FKBP12, FRS2α, JAK1, JAK3, IKZF1, IRAK4, MDM2, MetAP2, PLK1, PSK-J3, RAS, TACC3, Tau, TrkB, MDM2, or any combination thereof. In some preferred embodiments, the target protein is selected from an AR protein, an ER protein, a RAS protein, a BRD4 protein, a PLK1 protein, a FAK protein, an MDM2 protein, or any combination thereof.

[0157] In some embodiments, a target protein ligand moiety disclosed herein has a structure selected from the following: [ka]

[0158] In some embodiments, the linking moiety in the proteolysis targeting chimera has a structure selected from the group consisting of: [ka] Among them, p1 is an integer from 0 to 4, p2 is an integer from 0 to 6, p3, p4, p5, and p6 are each independently 0 or 1; G 1 is a 4- to 14-membered linear alkylene group, and optionally, 2 to 5 carbon atoms in the 4- to 14-membered linear alkylene group are selected from the group consisting of -O-, -NH-, -N(C 1-6 alkyl)- or -C≡C-; G 2 is an amino acid residue, and preferably, G2 is a Lys residue; G 3 teeth, [ka] selected from the group consisting of G 4 is a 5- to 9-membered linear alkylene group, and optionally, 2 to 4 carbon atoms in the 5- to 9-membered linear alkylene group are selected from the group consisting of -O-, -NH-, -N(C 1-6 It may be substituted with -C≡C- or -C≡C-.

[0159] In some embodiments, the linking moiety in the proteolysis targeting chimeras disclosed herein has a structure selected from the group consisting of: [ka]

[0160] In some embodiments, the proteolysis targeted chimeras disclosed herein have the structure shown below: [ka] [ka] [ka] [ka]

[0161] In some embodiments, the conjugate compound, or a pharmaceutically acceptable salt thereof, comprises one, two, three, four, or more payloads.

[0162] In some embodiments, the payload of the present application has a free amino or carboxyl group prior to linking to the targeting molecule or linker of the present application (i.e., prior to forming the conjugate compound), and the payload is coupled to the conjugate compound by an acylation reaction between the amino or carboxyl group and a group of a corresponding moiety (e.g., linker) of the conjugate compound.

[0163] In some embodiments, the payload is directly linked to the targeting molecule, or the payload is linked to the targeting molecule via a linker. In some embodiments, the link between the payload and the targeting molecule, between the payload and the linker, or between the linker and the targeting molecule is completed by a chemical reaction. In some preferred embodiments, the link between the targeting molecule and the linker is completed by a click reaction.

[0164] The "click reaction" described in this application is a type of chemical synthesis method with high efficiency and selectivity. The click reaction splices multiple molecular fragments together to form an efficient linking unit, and the entire reaction process is simple, rapid, and produces few by-products. Some examples of click reactions include, but are not limited to, the addition reaction of a thiol with an olefin and the cyclization reaction of an azide with an alkyne.

[0165] The term "linker," as used herein, refers to a molecule or moiety that covalently attaches a payload to a targeting molecule. The linker comprises a functional group for linking the payload to at least one targeting molecule. In some embodiments, the functional group may comprise two reactive moieties, one for linking to the payload and the other for linking to the targeting molecule. In some embodiments, the functional groups are different from each other. In some embodiments, the functional group comprises a group containing a sulfhydryl-reactive moiety and an amine-reactive moiety. In some embodiments, the functional groups are the same as each other. In some embodiments, the carboxylic acid in the amino acid contained in the linker is amidated. In some embodiments, the linker comprises a short-chain polyethylene glycol (e.g., comprising 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeating units). In some embodiments, the linker comprises an azide group. In some embodiments, the linker comprises an alkynyl group. In some embodiments, the linker is sufficiently stable to avoid accidental release of the payload into the blood circulation, thereby increasing the effective amount of the payload delivered to the target cell or tissue and avoiding toxicity. In some embodiments, the linker can release the payload around or inside the target cell to effectively kill or block the function of the target cell.

[0166] In some embodiments, the linker comprises link module 1 and link module 2.

[0167] In some embodiments, the linker comprises linking module 2.

[0168] In some embodiments, the linker comprises a linking module 1 and a PEG chain attached to the linking module 1; [ka] In some preferred embodiments, the PEG chain comprises a C1-C8 alkyl chain, -NH-, -O-, -C(O)-, or any combination thereof. [ka] In some preferred embodiments, the linker binds to link module 1. [ka] As will be appreciated by those skilled in the art, these groups may be attached to a linking moiety on Link Module 1 through a left attachment point and to a payload through a right attachment point, or may be attached to a linking moiety on Link Module 1 through a right attachment point and to a payload through a left attachment point.

[0169] In some embodiments, Link Module 1 is linked to Link Module 2 via a bond or a chemical group. In some embodiments, the chemical group is a PEG chain, [ka] C1-C8 alkyl chain, -NH-, -O-, -C(O)-, [ka] or any combination thereof. In some preferred embodiments, the PEG chain is PEG1, PEG2, or PEG3. In some preferred embodiments, the chemical group is [ka] As will be appreciated by those skilled in the art, these chemical groups may be attached to Link Module 1 via a left-hand attachment point and to Link Module 2 via a right-hand attachment point, or to Link Module 1 via a right-hand attachment point and to Link Module 2 via a left-hand attachment point.

[0170] In some embodiments, the linking module 1 comprises: [ka] Includes:

[0171] In some embodiments, linkage module 2 comprises a CB enzyme-cleavable group, a plasmin enzyme-cleavable group, an asparagine endonuclease enzyme-cleavable group, a β-glucuronidase enzyme-cleavable group, a sulfatase enzyme-cleavable group, a phosphatase enzyme-cleavable group, a β-galactosidase enzyme-cleavable group, a glutathione enzyme-cleavable group, or an acid-labile group.

[0172] In some preferred embodiments, the CB enzyme-cleavable group comprises an amino acid or a combination of an amino acid and an autolytic fragment, hi some preferred embodiments, the amino acid is Val-Cit, Gly-Gly-Phe-Gly, CycloBut-Cit, Phe-Cit, Val-Ala, Glu-Val-Cit, Ala-Cit, Val-Cit-Pro, or Val-Cit-Pro-Gly.

[0173] In some preferred embodiments, the plasmin enzyme-cleavable group comprises an amino acid or a combination of an amino acid and an autolytic fragment, hi some preferred embodiments, the amino acid is Val-Leu-Lys.

[0174] In some preferred embodiments, the asparagine endonuclease group comprises an amino acid or a combination of an amino acid and an autolytic fragment, hi some preferred embodiments, the amino acids are selected from Gly-Asn-Asn or Ala-Ala-Asn.

[0175] In some preferred embodiments, the autolytic fragments include PAB and its derivatives, [ka] In some preferred embodiments, the PAB is [ka] In some preferred embodiments, the PAB derivative is [ka] is.

[0176] In some preferred embodiments, the β-glucuronidase enzyme-cleavable group is [ka] In some preferred embodiments, the sulfatase enzyme-cleavable group is [ka] In some preferred embodiments, the phosphatase enzyme-cleavable group is [ka] In some preferred embodiments, the β-galactosidase enzyme-cleavable group is [ka] In some preferred embodiments, the glutathione enzyme-cleavable group is [ka] In some preferred embodiments, the acid labile group is [ka] is.

[0177] As will be appreciated by those skilled in the art, the active site of the enzyme-cleavage group may optionally be linked to Linking Module 1, a chemical group, a targeting molecule, or a payload after appropriate reaction. For example, the β-glucuronidase enzyme-cleavage group, after being effectively linked to Linking Module 1, a chemical group, a targeting molecule, or a payload, may be [ka] wherein the sulfatase enzyme cleavage group, after being effectively linked to a linking module 1, a chemical group, a targeting molecule, or a payload, [ka] wherein the phosphatase enzyme cleavage group, after being effectively linked to a linking module 1, a chemical group, a targeting molecule, or a payload, [ka] wherein the β-galactosidase enzyme cleavage group, after being operatively linked to a linking module 1, a chemical group, a targeting molecule, or a payload, [ka] wherein the glutathione enzyme cleavage group, after being effectively linked to linkage module 1, chemical group, targeting molecule, or payload, [ka] wherein the acid labile group, after operatively linking to a linking module 1, chemical group, targeting molecule, or payload, [ka] It has.

[0178] In some preferred embodiments, when the linker comprises Linking Module 2, Linking Module 2 is a glutathione enzyme cleavage group. In some preferred embodiments, the glutathione enzyme cleavage group is [ka] is.

[0179] The CB enzyme refers to cathepsin B, a cysteine ​​protease common in lysosomes. Plasmin refers to a serine protease common in plasma. Asparagine endonuclease (Legumain), also known as human legumain, is a peptidase in lysosomes that can target and cleave peptide fragments containing asparagine residues (-Asn-).

[0180] In some embodiments, the conjugate compound has the structure: W 1 -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W 2 , Among them, L 1 teeth, [ka] -SS- or bond, EG is -CH2CH2O- or -OCH2CH2-, n 1 is an integer from 0 to 6, n 2 is an integer from 0 to 8, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, B, in each occurrence, is independently -C(=O)-, [ka] -NH-CH2CH2-C(=O)-, -C(=O)-NH-, -CH(CH3)-CH2-O-, -CH2CH2O-, -NH-C(=O)- or -NH-, n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 5, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] -NH-CH2-O- or a bond, wherein X is a bond, -NH-, -C(=O)-, oxygen, or [ka] and R2 is a bond, —O—CH2— or —NH—CH2CH2—NH—C(═O)—; R3 is hydrogen or [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, [ka] and R4 is hydrogen or C 1-8 is an alkyl group, R5 is hydrogen, amino group, nitro group, phosphate group, -OR a or [ka] Among them, R a is a methyl group, an ethyl group, a propyl group, or an isopropyl group, W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0181] In some embodiments, EG is -CH2CH2O-. 1 is 2. In some embodiments, n 1 is 0. In some embodiments, n 2 is 1. In some embodiments, n 2 is 4. In some embodiments, n 2 is 5. In some embodiments, n 5 is 0. In some embodiments, n 6 is 1. In some embodiments, n 6 is 3. In some embodiments, n 1 is 2 and n 2 is 1 and n 3 and n 4 is 0 and L 2 is a bond.

[0182] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro, or Asn. 3- is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and -Ala-Ala-Asn-.

[0183] In some embodiments, n 4 is 1, Q is -C(=O)-, and n 4 is 2, Q is —O— and —C(═O)—, respectively, and n 4 When n is 3, Q is -O-, -O-, and a bond, respectively. 4 is 0.

[0184] In some embodiments, L 2 is a bond, [ka] is.

[0185] In some embodiments, the conjugate compound has the structure: W 1 -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W 2 Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, D is -C(=O)-NH-S(=O)2-NH-, and n5 is 0 or 1, n 2 is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH2CH2O-, -NH-CH2CH2-C(=O)- or -NH-; n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] and wherein X is a bond, -NH-, -C(=O)- or [ka] and R2 is a bond, R3 is [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, or [ka] and R4 is hydrogen; R5 is hydrogen, -OCH3 or [ka] and W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0186] In some embodiments, L 2 teeth, [ka] is.

[0187] In some embodiments, L 1 teeth [ka] and n 1 , n 5 , and n 4 are both 0.

[0188] In some embodiments, L 1 teeth [ka] and n 2 is 1 or 2.

[0189] In some embodiments, n 5 If is 1, then n 2 and n 4 are both 0.

[0190] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, or Ala. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-.

[0191] In some embodiments, the conjugate compound has the structure: W 1 -L 1 -(EG)n 1 -(CH2)n 2 -(C=O)-(AA)n 3 -L 2 -W 2 Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, n 2 is an integer from 0 to 5, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 1 to 4, L 2 teeth, [ka] or a bond, wherein X is -NH-; R2 is a bond, R4 is hydrogen; R5 is hydrogen or -OCH3; W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0192] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 teeth [ka] is.

[0193] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n2 is 1 and L 2 is a bond.

[0194] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 teeth [ka] is.

[0195] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 is a bond.

[0196] In some embodiments, each occurrence of AA is independently a Val, Cit, Glu, or Ala amino acid residue. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Leu-Lys- and -Gly-Asn-Asn-.

[0197] In some embodiments, the conjugate compounds disclosed herein have the structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] Among them, W 1 is a targeting molecule capable of binding to a cell surface protein, and W 2 is the payload.

[0198] In some embodiments, the present specification discloses the following conjugate compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0199] The term "complex compound" or "compound" as used in this application is intended to include all stereoisomers (e.g., enantiomers and diastereomers), geometric isomers, tautomers, and isotopes of the structures depicted.

[0200] The complex compounds or compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, e.g., enantiomers and diastereomers, are intended to be included. Complex compounds or compounds described herein containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials are well known in the art, for example, by resolution of racemic mixtures or by stereoselective synthesis. The complex compounds or compounds described herein may contain multiple geometric isomers of olefins, carbon-carbon double bonds, and the like, and all of these stable isomers are contemplated in this application. The present application describes cis and trans geometric isomers of the complex compounds or compounds, which can be isolated as a mixture of isomers or as a single isomer.

[0201] Racemic mixtures of compounds can be resolved by any of several methods known in the art. An exemplary method involves fractional crystallization using a chiral resolving acid, which is an optically active, salt-forming organic acid. Suitable resolving agents used in fractional crystallization methods include, for example, optically active acids (e.g., the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, and lactic acid) or various optically active camphorsulfonic acids. Other resolving agents suitable for fractional crystallization methods include stereoisomeric forms of N-methylbenzylamine, 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

[0202] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Appropriate elution solvent compositions may be determined by one skilled in the art.

[0203] The conjugate compounds or compounds of the present application further include tautomeric forms. Tautomeric forms are caused by the exchange of a single bond with an adjacent double bond accompanied by protolysis. Tautomeric forms include proton tautomers of isomeric protonation states with the same chemical formula and total charge. Examples of proton tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which the proton can occupy more than one position in the heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be equilibrated or sterically locked into a single form by appropriate substitution.

[0204] The complex compounds or compounds of the present application may also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include protium, deuterium, and tritium.

[0205] In some embodiments, the conjugate compound or compounds of the present application can be obtained by organic synthesis. The conjugate compound or compounds of the present application can be prepared using any known organic synthesis technique and can be synthesized based on multiple possible synthetic routes, including salts, esters, hydrates, or solvates thereof.

[0206] The reaction for preparing the conjugate compound or compounds of the present application can be carried out in a suitable solvent, and those skilled in the art of organic synthesis can easily select the solvent. A suitable solvent can be essentially unreactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction proceeds (which may range, for example, from the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of one or more solvents. Based on the specific reaction step, those skilled in the art can select an appropriate solvent for the particular reaction step.

[0207] The preparation of the conjugate compounds or compounds of the present application may involve the protection and deprotection of various chemical groups. Those skilled in the art can easily determine whether protection and deprotection are necessary and the selection of appropriate protecting groups. Protecting group chemistry can be found, for example, in T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), the entire contents of which are incorporated herein by reference.

[0208] The reaction can be monitored by any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13The purity of the compound can be monitored using spectroscopic techniques such as spectroscopy (e.g., infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or chromatography (e.g., high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin-layer chromatography (TLC). Those skilled in the art can purify the compound using several methods, including high performance liquid chromatography (HPLC) (see, e.g., "Preparative LC-MS Purification: Improved Compound Specific Method Optimization," Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, J. Combi. Chem. 2004, 6(6), 874-883, which is incorporated herein by reference in its entirety) and normal-phase silica gel column chromatography.

[0209] One aspect of the present application discloses a pharmaceutical composition comprising a conjugate compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition described herein is used for intravenous, subcutaneous, oral, intramuscular, or intraventricular administration.

[0210] As used herein, the term "pharmaceutically acceptable" refers to something that is, within the scope of reasonable medical judgment, suitable for contact with the cells of humans and other animals without undue toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit / hazard ratio.

[0211] As used herein, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of the conjugate compounds of the present application. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthoate, gentisinate, isethionate, di-p-toluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinic acid laurylsulfonate, etc. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. In some embodiments, sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases including, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide.Suitable amine base addition salts are prepared from amines which are sufficiently basic to form stable salts, and preferably include the following amines commonly used in pharmaceutical chemistry because of their relatively low toxicity and acceptability for medical use: ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenylethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabietic amine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids (e.g., lysine and arginine), and dicyclohexylamine.

[0212] The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert carrier agent for delivering the conjugate compound provided herein to a subject, which does not interfere with the structure and properties of the conjugate compound. Some of such carriers allow the conjugate compound to be formulated into, for example, tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and lozenges for oral ingestion by a subject. Some of such carriers allow the conjugate compound to be formulated for injection, infusion, or topical administration.

[0213] Pharmaceutically acceptable carriers used in the pharmaceutical compositions provided by the present application include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous carriers (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), non-aqueous carriers (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antibacterial agents, isotonicity agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., PEG-10 ... , sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof. Suitable ingredients can include, for example, fillers, binders, buffers, preservatives, lubricants, flavorings, thickeners, colorants, or emulsifiers.

[0214] In some embodiments, the pharmaceutical composition is an injectable preparation. Injectable preparations include sterile aqueous solutions or dispersions, suspensions, or emulsions. In all circumstances, injectable preparations should be sterile and fluid for easy injection. Injectable preparations should be stable under the conditions of manufacture and storage and must be protected against the contamination of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof, and / or vegetable oils. Injectable preparations should maintain suitable fluidity. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by using surfactants, and the like. Prevention of microorganisms can be achieved by various antibacterial and antifungal agents, such as parahydroxybenzoates, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0215] In some embodiments, the pharmaceutical composition is an oral formulation, including, but not limited to, capsules, cachets, pills, tablets, lozenges (usually using a flavoring base such as sucrose and gum arabic or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, etc.

[0216] In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), the complex compound is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium hydrogen phosphate, and / or any one of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) saccharides, such as glycerin; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as acetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.

[0217] In liquid dosage forms for oral administration, the conjugate compound is mixed with any one of pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the conjugate compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

[0218] In some embodiments, the pharmaceutical composition is an oral spray formulation or a nasal spray formulation. Spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposome formulations, or solid particle formulations. Aqueous aerosols are prepared by mixing an aqueous solution or suspension of the drug with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary based on the requirements of the specific compound, but typically include non-ionic surfactants (Tween or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solutions, salts, sugars, or sugar alcohols. Aerosols are usually prepared from isotonic solutions and can be delivered by spraying.

[0219] In some embodiments, the pharmaceutical composition can be used in combination with one or more other drugs. In some embodiments, the pharmaceutical composition includes at least one other drug. In some embodiments, the other drug is an anti-tumor drug, a cardiovascular drug, an anti-inflammatory drug, an antiviral drug, a gastrointestinal drug, a nervous system drug, a respiratory drug, an immune system drug, a dermatological drug, a metabolic drug, or the like.

[0220] In some embodiments, the pharmaceutical composition can be administered to a subject in need thereof by a suitable route, including, but not limited to, oral, injection (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal injection, etc.), mucosal (e.g., intranasal, buccal administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration. In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly, or intraventricularly.

[0221] Due to the properties of some payloads, such as high toxicity and hydrophilicity, it is desirable to deliver the payload more specifically and efficiently to a subject in need thereof. For example, in cancer treatment, it is desirable to deliver a chemotherapeutic agent specifically to cancer cells without causing toxicity to normal cells. Accordingly, another aspect of the present application discloses a method of delivering a payload to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein. The payload described herein may be any drug sought by researchers, veterinarians, physicians, or other practitioners to induce a biological or medical response in a tissue, system, individual animal, or human to prevent, inhibit, ameliorate, or treat a disease.

[0222] One aspect of the present application discloses a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

[0223] The term "therapeutically effective amount," as used herein, refers to an amount of a conjugate compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, that relieves to some extent one or more symptoms of a disease or condition in a subject, partially or completely restores to normal one or more physiological or biochemical parameters associated with or leading to the disease or condition, and / or reduces the likelihood of the disease or condition occurring. This amount typically varies based on several factors and can be determined and accounted for by one of ordinary skill in the art based on the scope of the specification provided by this application. These include, but are not limited to, the particular subject and their age, weight, height, general physical condition, and medical history, the particular compound used, as well as its formulation carrier and chosen route of administration, and the nature and severity of the condition being treated.

[0224] In some embodiments, the amount of the conjugate compound or pharmaceutically acceptable salt thereof, or pharmaceutical composition is sufficient to inhibit a disease or condition in a subject, or to prophylactically inhibit or prevent the onset of a disease or condition. The therapeutically effective amount can vary for different subjects, but is typically in the range of 0.01 to 100 mg / kg, e.g., 0.01 to 90 mg / kg, 0.01 to 80 mg / kg, 0.01 to 70 mg / kg, 0.01 to 60 mg / kg, 0.01 to 50 mg / kg, 0.01 to 40 mg / kg, 0.01 to 30 mg / kg, 0.01 to 20 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, 0.01 to 4 mg / kg, 0.01 to 3 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, or 0.01 to 0.1 mg / kg. The therapeutically effective amount described in this application can be equal to any value within the numerical ranges set forth above, including the endpoints of the ranges.

[0225] Another aspect of the present application discloses a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. In some embodiments, the method further comprises administering one or more therapeutic agents in combination with the conjugate compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0226] Another aspect of the present application discloses the use of a conjugate compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, in the preparation of a medicament for treating a disease in a subject.

[0227] The term "subject" as used in this application refers to humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals. A subject may also be a livestock animal, such as a cow, pig, sheep, poultry, and horse, or a domestic animal, such as a dog or cat. A subject may be male (e.g., man) or female (e.g., woman), and may be elderly, an adult, an adolescent, a child, or an infant. A human may be of Caucasian, African, Asian, Semitic, or other ethnic background, or half of these ethnic backgrounds.

[0228] In some embodiments, the disease described herein is selected from the group consisting of cancer, immune disorders, metabolic disorders, and neurological disorders. In some embodiments, the disease is characterized by cells in an organ or tissue in a subject overexpressing or abnormally expressing a cell surface receptor, cell surface antigen, or specific protein. In some embodiments, the cell surface receptor is selected from FOLR1, TRPV6, and c-Met. In some embodiments, the cell surface antigen is selected from PSMA. In some embodiments, the specific protein is selected from nuclear receptors (e.g., AR and ER), kinase proteins (e.g., RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, and FAK), AKT, PAN-BET proteins, BET proteins (e.g., BRD2, BRD3, BRD4, and BRD6), BLK, BRAF1, BCL-x1, ERRα, FKBP12, FRS2α, JAK1, JAK3, IKZF1, IRAK4, MDM2, MetAP2, PLK1, PSK-J3, RAS, TACC3, Tau, TrkB, MDM2, or any combination thereof.

[0229] In some embodiments, cancer cells or other physiologically dysfunctional cells have expression of a cell surface receptor or antigen referred to in the present application. In some embodiments, cancer cells or other physiologically dysfunctional cells have high expression of a cell surface receptor or antigen referred to in the present application (e.g., based on Depmap data (see https: / / depmap.org / portal / ), the corresponding gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more). In some embodiments, cancer cells or other physiologically dysfunctional cells have high expression of FOLR1, TRPV6, PSMA, c-Met, or any combination thereof.

[0230] Another aspect of the present application discloses a compound or a salt thereof, said compound having the following structure: L a -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L b , Among them, L a teeth, [ka] and EG is -CH2CH2O- or -OCH2CH2-, n 1 is an integer from 0 to 6, n 2 is an integer from 0 to 8, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, B is -C(=O)-, [ka] -NH-CH2CH2-C(=O)-, -C(=O)-NH-, -CH(CH3)-CH2-O-, -CH2CH2O-, -NH-C(=O)- or -NH-, n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 5, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L b teeth, [ka] -OH or -NH-CH2-OH, wherein X is a bond, -NH-, -C(=O)-, oxygen, or [ka] and R2 is a bond, —O—CH2— or —NH—CH2CH2—NH—C(═O)—; R3 is hydrogen or [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, [ka] and R4 is hydrogen or C 1-8 is an alkyl group, R5 is hydrogen, amino group, nitro group, phosphate group, -OR a or [ka] Among them, R a is a methyl group, an ethyl group, a propyl group, or an isopropyl group, R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0231] In some embodiments, EG is -CH2CH2O-. 1 is 2. In some embodiments, n 1 is 0. In some embodiments, n 2 is 1. In some embodiments, n 2 is 4. In some embodiments, n 2 is 5. In some embodiments, n 5 is 0. In some embodiments, n 6 is 1. In some embodiments, n 6 is 3. In some embodiments, n 1 is 2 and n 2 is 1 and n 3 ~n 4 is 0 and L b is -OH.

[0232] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro, or Asn. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and -Ala-Ala-Asn-. 4 is 1, Q is -C(=O)-, and n 4 is 2, Q is —O— and —C(═O)—, respectively, and n4 When n is 3, Q is -O-, -O-, and a bond, respectively. 4 is 0.

[0233] In some embodiments, L b teeth, [ka] is.

[0234] In some embodiments, the compound has the structure: L a -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L b Among them, L a teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer between 2 and 4, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, n 2 is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH2CH2O-, -NH-CH2CH2-C(=O)- or -NH-; n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L b is -OH, [ka] and wherein X is a bond, -NH-, -C(=O)- or [ka] and R2 is a bond, R3 is [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, or [ka] and R4 is hydrogen; R5 is hydrogen, -OCH3 or [ka] and R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0235] In some embodiments, L b is -OH, [ka] is.

[0236] In some embodiments, n 5 If is 1, then n 2 and n 4 are both 0.

[0237] In some embodiments, L a teeth [ka] and n 1 , n 5 , and n 4 are both 0.

[0238] In some embodiments, L a teeth [ka] and n 2 is 1 or 2.

[0239] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, or Ala. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-.

[0240] In some embodiments, the compound has the structure: L a -(EG)n 1 -(CH2)n 2 -(C=O)-(AA)n 3 -L b Among them, L a teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, n 2 is an integer from 0 to 5, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, L b teeth, [ka] or -OH, wherein X is -NH-; R2 is a bond, R4 is hydrogen; R5 is hydrogen or -OCH3; R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine), [ka] is.

[0241] In some embodiments, L a teeth [ka] and n 1 is 2 and n 2 is 1 and L b teeth [ka] is.

[0242] In some embodiments, L a teeth [ka] and n 1 is 2 and n 2 is 1 and L b is -OH.

[0243] In some embodiments, L a teeth [ka] and n 1 is 0, and n 2 is 5 and L b teeth [ka] is.

[0244] In some embodiments, L a teeth [ka] and n 1 is 0, and n 2 is 5 and L b is -OH.

[0245] In some embodiments, each occurrence of AA is independently a Val, Cit, Glu, or Ala amino acid residue. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Leu-Lys- and -Gly-Asn-Asn-.

[0246] In some embodiments, the compounds disclosed herein have a structure selected from the group consisting of: [ka] [ka] [ka] [ka] wherein R1 is a leaving group, preferably R1 is a halogen (e.g., chlorine) or [ka] and its derivatives.

[0247] Another aspect of the present application is [ka] or a salt thereof.

[0248] In some embodiments, the leaving group is a halogen, such as fluorine, chlorine, bromine, or iodine. In some preferred embodiments, the leaving group is chlorine. In some preferred embodiments, the leaving group is p-nitrophenol. In some preferred embodiments, the leaving group is pentafluorophenol.

[0249] Another aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, said conjugate compound having the following structure: W a -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W b , Among them, L 1 teeth, [ka] -SS- or bond, EG is -CH2CH2O- or -OCH2CH2-, n 1 is an integer from 0 to 6, n 2 is an integer from 0 to 8, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, B, in each occurrence, is independently -C(=O)-, [ka] -NH-CH2CH2-C(=O)-, -C(=O)-NH-, -CH(CH3)-CH2-O-, -CH2CH2O-, -NH-C(=O)- or -NH-, n 6is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 5, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] -NH-CH2-OH or a bond, wherein X is a bond, -NH-, -C(=O)-, oxygen, or [ka] and R2 is a bond, —O—CH2— or —NH—CH2CH2—NH—C(═O)—; R3 is hydrogen or [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, [ka] and R4 is hydrogen or C 1-8 is an alkyl group, R5 is hydrogen, amino group, nitro group, phosphate group, -OR a or [ka] Among them, R a is a methyl group, an ethyl group, a propyl group, or an isopropyl group, W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0250] In some embodiments, EG is -CH2CH2O-. 1 is 2. In some embodiments, n 1 is 0. In some embodiments, n 2 is 1. In some embodiments, n 2 is 4. In some embodiments, n 2 is 5. In some embodiments, n 5 is 0. In some embodiments, n 6 is 1. In some embodiments, n 6 is 3. In some embodiments, n 1 is 2 and n 2 is 1 and n 3 and n 4 is 0 and L 2 is a bond.

[0251] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro, or Asn. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, -Val-Cit-Pro-Gly, -Gly-Asn-Asn- and -Ala-Ala-Asn-.

[0252] In some embodiments, n 4 is 1, Q is -C(=O)-, and n 4 is 2, Q is —O— and —C(═O)—, respectively, and n 4 When n is 3, Q is -O-, -O-, and a bond, respectively. 4 is 0.

[0253] In some embodiments, L 2 is a bond, [ka] is.

[0254] In some embodiments, the compound has the structure: W a -L 1 -(EG)n 1 -(D)n 5 -(CH2)n 2 -(B)n 6 -(AA)n 3 -(CH2CH2-Q)n 4 -L 2 -W b Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer between 2 and 4, D is -C(=O)-NH-S(=O)2-NH-, and n 5 is 0 or 1, n 2 is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH2CH2O-, -NH-CH2CH2-C(=O)- or -NH-; n 6 is an integer from 0 to 3, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 0 to 3, Q is independently in each occurrence -C(=O)-, -O-, or a bond; n 4 is an integer from 0 to 3, L 2 teeth, [ka] and wherein X is a bond, -NH-, -C(=O)- or [ka] and R2 is a bond, R3 is [ka] Among them, R b and R c are each independently a hydrogen atom, a nitro group, or [ka] and R4 is hydrogen; R5 is hydrogen, -OCH3 or [ka] and W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0255] In some embodiments, L 2 teeth, [ka] is.

[0256] In some embodiments, n 5 If is 1, then n 2 and n 4 are both 0.

[0257] In some embodiments, L 1 teeth [ka] and n 1 , n 5 , and n 4are both 0.

[0258] In some embodiments, L 1 teeth [ka] and n 2 is 1 or 2.

[0259] In some embodiments, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu, or Ala. 3 - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-.

[0260] In some embodiments, the compound has the structure: W a -L 1 -(EG)n 1 -(CH2)n 2 -(C=O)-(AA)n 3 -L 2 -W b Among them, L 1 teeth, [ka] and EG is -CH2CH2O-, n 1 is an integer from 0 to 4, n 2 is an integer from 0 to 5, AA, each occurrence independently, is an amino acid residue; 3 is an integer from 1 to 4, L 2 teeth, [ka] or a bond, wherein X is -NH-; R2 is a bond, R4 is hydrogen; R5 is hydrogen or -OCH3; W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0261] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 teeth [ka] is.

[0262] In some embodiments, L 1 teeth [ka] and n 1 is 2 and n 2 is 1 and L 2 is a bond.

[0263] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 teeth [ka] is.

[0264] In some embodiments, L 1 teeth [ka] and n 1 is 0, and n 2 is 5 and L 2 is a bond.

[0265] In some embodiments, the present specification discloses a conjugate compound having a structure selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] Among them, W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0266] Another aspect of the present application provides a conjugate compound or a pharmaceutically acceptable salt thereof, said conjugate compound being [ka] Among them, W a and W b are each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0267] Another aspect of the present application provides a conjugate compound or a pharmaceutically acceptable salt thereof, said conjugate compound being [ka] Among them, W a and W bare each independently targeting molecules capable of binding to a payload or a cell surface protein.

[0268] Another aspect of the present application provides a proteolysis targeting chimera or a pharmaceutically acceptable salt thereof, wherein the proteolysis targeting chimera compound has the following structure: [ka] Among them, Ra and Rb are each independently hydrogen, an alkyl group, NH, OH, or halogen; A, at each occurrence, is independently an amino acid residue; a is 0 or 1, b is an integer from 0 to 5, X is [ka] or a bond, The target protein ligand is [ka] is.

[0269] In some embodiments, Ra is hydrogen. In some embodiments, Rb is hydrogen. In some embodiments, A is an amino acid residue formed by Lys. In some embodiments, a is 1. In some embodiments, a is 0. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5.

[0270] In some embodiments, the proteolysis targeted chimera has a structure selected from the group consisting of: [ka]

[0271] [ka]

[0272] The present application will now be described in more detail with reference to specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize that multiple non-critical parameters can be changed or modified to produce essentially the same results. [Example]

[0273] The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the specific compositions, materials, and methods described below, in whole or in part, are within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the present invention, but are merely intended to illustrate specific embodiments and are within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials, and methods without any additional creativity, without departing from the scope of the present invention, and without adding to the present invention. It should be understood that multiple modifications to the methods of the present invention may still be included within the scope of the present invention. The inventors intend such variations to be included within the scope of the present invention. Example 1 Preparation of Conjugate Compound

[0274] 1. Preparation of proteolysis-targeting chimeras

[0275] 1.1 Preparation of CR202CC, CR202CF, CR202CI, CR202CL, and CR202CP [ka]

[0276] CR202EC (240.0 mg, 0.44 mmol; for the synthetic route, see J. Med. Chem. 2020, 63, 17, 9977-9989, the disclosure of which is incorporated herein by reference) was added to DMF (5 mL), and HATU (175.0 mg, 0.46 mmol) and DIPEA (166.0 μL, 0.50 mmol) were added at 0-5°C. After stirring for half an hour, CR202EB (342.0 mg, 0.42 mmol; for the synthetic route, see paragraph

[0227] of WO2015 / 184349A2, the disclosure of which is incorporated herein by reference) was added. The mixture was kept warm and reacted for 2 hours to complete the reaction. The reaction mixture was then diluted with water (25 mL), ethyl acetate (25 The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to yield 14.0 mg of CR202ED as a pale yellow solid. CR202ED (14.0 mg, 0.01 mmol) was added to methanol (200 μL) and the mixture was heated to 0-5°C. 3 M HCl / methanol solution (50 μL) was added and the mixture was reacted at 20-25°C for 1 hour to complete the reaction. The mixture was then dried to yield 13.0 mg of CR202CC.

[0277] The proteolysis-targeted chimeras CR202CF, CR202CI, CR202CL and CR202CP can be obtained by steps similar to those described above.

[0278] 1.2 Preparation of CR20274 [ka]

[0279] Synthesis of UB-180961h

[0280] Patent document WO2021205391A1 discloses a method for preparing compound UB-180961h, the disclosure content of which is incorporated herein by reference.

[0281] UB-180961a (20 g, 71.1 mmol) was dissolved in dry DMF (80 mL) and cooled to 0 °C. NaH (16.8 g, 107 mmol) was added to the reaction mixture. After half an hour, UB-180961b (21.1 g, 107 mmol) was dissolved in dry DMF (20 mL) and added dropwise to the reaction mixture. The mixture was allowed to react at room temperature overnight. Ice water (100 mL) was added to the reaction mixture, and the mixture was extracted three times with EtOAc (60 mL). The combined organic phases were separated by column chromatography (PE / EtOAc = 0-100%) to obtain UB-180961c (25 g, 47% yield) as a colorless oil. Compound UB-180961c (15 g, 285.4 mmol) was dissolved in water (40 mL), concentrated HCl (10 mL) was added, and the mixture was allowed to react at room temperature overnight. The reaction mixture was extracted three times with DCM (50 mL), and the organic phase was dried over anhydrous sodium sulfate and then concentrated to give UB-180961d (7.6 g) as a colorless oil.

[0282] Ub-180961d (7.8 g, 68 mmol) and UB-180961e (7.6 g, 68 mmol) were dissolved in DCE (100 mL) and reacted at room temperature for 1 h. NaBH(OAc) (29.6 g, 136 mmol) was added and the reaction mixture was allowed to react overnight at room temperature. TEA (5 mL, sat) and BocO (6 g, 23.8 mmol) were added to the reaction mixture and the mixture was allowed to react at room temperature for 18 h. The reaction mixture was extracted twice with EtOAc (15 mL). The organic phase was dried over anhydrous NaSO and separated by column chromatography (PE / EtOAc = 0-100%) to give UB-180961f (4.6 g, 57% yield) as a yellow oil. UB-180961f (4.6 g, 15 mmol), A1-I (3.7 g, 10 mmol), Pd(PPh3)2Cl2 (701 mg, 1 mmol), CuI (190 mg, 1 mmol), and TEA (4.2 mL, 30 mmol) were dissolved in dry DMF (120 mL) and reacted overnight at 80 °C. The reaction mixture was extracted twice with EtOAc (15 mL). The organic phase was dried over anhydrous Na2SO4 and then separated by column chromatography (PE / EtOAc = 0-100%) to give UB-180961g (3.6 g, 57% yield) as a yellow solid.

[0283] UB-180961g (3.6 g, 33.8 mmol), TEA (10.3 g, 10.2 mmol), and DMAP (12.4 g, 10.2 mmol) were dissolved in dry DMF (140 mL) and TsCl (14.6 g, 7.7 mmol) was added at 0 °C. The reaction mixture was heated to 30 °C and reacted for 15 h. The reaction mixture was extracted twice with DCM (50 mL). The organic phase was concentrated and then separated by column chromatography (PE / EtOAc = 0-100%) to afford UB-180961h (3.6 g, 86% yield) as a white solid. [ka] Synthesis of CR20274b

[0284] UB-180961h (48.4 g, 1.0 eq) was dissolved in DMF (484 mL, 10 V), the mixture was purged with N2, CsF (31.2 g, 3 eq) and TMSN3 (azidotrimethylsilane, 23.7 g, 3 eq) were added, the internal temperature was raised to 80 °C, and the mixture was stirred for 4.5 h. The reaction mixture was added dropwise to saturated aqueous NaHCO3 (4.8 L) to precipitate a solid. The solid was dissolved in EA (ethyl acetate, 968 mL) and further washed with saturated brine (484 mL). The organic phase was dried and concentrated to give the crude product. The crude product was purified by column chromatography (DCM:MeOH = 45:1) to give CR20274b (31.1 g, purity: 95.6%, yield: 78.7%) as a white solid. LCMS: [M+H] + =579.1.

[0285] Synthesis of CR20274d

[0286] CR20274b (30.8 g, 1.0 eq) and CR20274c (21.7 g, 1.0 eq) were dissolved in tert-butanol (900 mL, 30 V). Water (450 mL, 15 V), CuSO4 (8.49 g, 1.0 eq), and sodium ascorbate (21.1 g, 2.0 eq) were added. The mixture was purged with nitrogen three times and incubated at 25–30°C for 3.1 h. DCM (900 mL) and saturated brine (300 mL) were added to the reaction mixture, which was then separated and extracted once more with DCM (900 mL). The organic phase was collected, dried, concentrated, and purified by column chromatography (eluent: DCM / MeOH = 200 / 1 to 50 / 1) and concentrated under reduced pressure to give product CR20274d (41.5 g, 78.8% yield).

[0287] Synthesis of CR20274

[0288] CR20274d was dissolved in DCM (3 mL), 0.5 mL of HCl / dioxane (4 M) was added, and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was concentrated, washed three times with diethyl ether (5 mL), and filtered to obtain CR20274 (purity 98.1%, MS: [M+H]) as a white solid. +=884.6).

[0289] 1.3 Preparation of CR202EE [ka]

[0290] Synthesis of CR202EM

[0291] CR206EP was added to DMF (5 mL), and HATU and DIPEA were added at 0-5°C. After stirring for half an hour, CR202EK was added and the mixture was incubated for 2 hours to complete the reaction. The reaction mixture was extracted with water (25 mL) and ethyl acetate (25 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain CR202EL. CR202EL was added to methanol (200 μL), and 3 M HCl / methanol solution (50 μL) was added at 0-5°C. The mixture was incubated for 1 hour at 20-25°C to complete the reaction. The mixture was then concentrated to obtain CR202EM.

[0292] Synthesis of CR202EN

[0293] CR206GT was added to DMF (5 mL), and HATU and DIPEA were added at 0-5°C. After stirring for half an hour, CR202EM was added and the mixture was allowed to react for 2 hours while keeping the temperature high until the reaction was complete. The reaction mixture was extracted with water (25 mL) and ethyl acetate (25 mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain CR202EN.

[0294] Synthesis of CR202EE

[0295] CR205EN (320.0 mg, 0.23 mmol) was added to a 20% piperidine / DMF solution (3.0 mL) and reacted at room temperature for 1 hour until the reaction was complete. Water (15 mL) was added, and the mixture was extracted with dichloromethane (15 mL), concentrated, and purified by column chromatography to obtain CR202EE181.0 mg.

[0296] 1.4 Synthesis of CR202Z5, CR202ES, CR202Z7, CR202Z8, CR202Z9, CR202Z1, CR202Z2, CR202Z3, CR202Z4, CR202Y2, and CR202Y4 (Source: MCE, Catalog Number: HY-114312)

[0297] The synthetic route for CR202Z5 can be found in the synthesis of ARCC-4 in the literature (Communications Biology, (2018) 1:100). The synthetic route for CR202ES can be found in the synthesis of ARV-771 in the literature (PNAS, 113(26) 7124-7129). The synthetic route for CR202Z7 can be found in the synthesis of ERD-308 in the literature (J. Med. Chem. 2019, 62, 3, 1420-1442). The synthetic route for CR202Z8 can be found in the synthesis of GNE-987 in the literature (ChemMedChem, 2020, 15, 17-25). The synthetic route for CR202Z9 can be found in the synthesis of ARD-69 in the literature (J. Med. Chem. 2019, 62, 2, 941-964). The synthetic route of CR202Z1 can be referenced to the synthesis of ARV-110 in the patent (CN 111892577 A). The synthetic route of CR202Z2 can be referenced to the synthesis of ARV-471 in the patent (US 2021 / 0139458 A1). The synthetic route of CR202Z3 can be referenced to the synthesis of ARV-825 in the literature (Chemistry & Biology, 2015, 22, 755-763). The synthetic route of CR202Z4 can be referenced to the synthesis of FC-11 in the literature (ACS Med Chem Lett. 2019, 11, 1855-1862). The synthetic route of CR202Y2 can be referenced to the synthetic route of MD-224 in the literature (Cancer Res. 2019, 79, 251-262). CR202Y4 was obtained commercially (MCE, catalog number: HY-114312). The disclosures of these documents are incorporated herein by reference in their entirety.

[0298] 2. Preparation of targeting molecules

[0299] 2.1 Synthesis of L-01, L-02, and L-04 [ka]

[0300] Solid-phase synthesis of target L-01, targeting TRPV6 and folate receptors, was performed using Fmoc chemistry amino resin. The resin was loaded into a solid-phase reaction column, DMF was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes. Fmoc-Arg-OH, DCC (dicyclohexylcarbodiimide), and DMAP were added and reacted at 25°C for 3 hours. The resin was then capped with acetic anhydride and pyridine for 1 hour, washed three times with DMF, and the Fmoc protecting group on the resin was removed using DBLK. The Fmoc-Pro-OH and HOBt were weighed and dissolved in DMF. The solution was added to the reaction column in an ice-water bath at 0°C, mixed, and activated for 5 minutes. The solution was added to the reaction column and reacted for 3 hours. The solvent was then removed by vacuum evaporation. The resin in the reaction column was then washed three times, and the Fmoc protecting group was then removed using DBLK. The above procedure was repeated to obtain peptide resins, sequentially coupled with Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu-OtBu, and pteroic acid according to the structure. The peptide resin was dissolved in the solvent, filtered, and the collected solution was poured into MTBE to precipitate the solid. The solid was washed with MTBE to obtain the crude product, which was purified by pre-HPLC to obtain L-01.

[0301] L-02 and L-04 can be obtained by steps similar to those described above. [ka]

[0302] 2.2 Synthesis of L-03 CR211D6 structure: [ka] Preparation process: [ka]

[0303] For the synthesis of CR211D6, see Nature Medicine, 2015, 21, 955-961(), the disclosure of which is incorporated herein by reference in its entirety.

[0304] CR194PF (150.0 mg, 145.9 μmol) was added to DMF (3.0 mL), and the mixture was dissolved under nitrogen gas protection and protected from light. The mixture was stirred at room temperature (25.5 °C). HOSu (25.2 mg, 219.0 μmol) and EDCI (42.0 mg, 219.0 μmol) were added sequentially to the reaction mixture. DIPEA was added dropwise to adjust the pH to 8. The mixture was then reacted at room temperature for 1 hour. EDCI (42.0 mg, 219.0 μmol) was then added and the reaction was continued for 1 hour. The reaction mixture was then added dropwise to MTBE (30.0 mL). The clear liquid was discarded, and the residue was washed with MTBE (5.0 mL × 2). The mixture was dried under vacuum at 20 °C to give a yellow oil. DMF (1.0 mL) was added to dissolve the yellow oil (i.e., a CR194PF activated ester solution) and the mixture was ready for use.

[0305] CR211D6 (284.0 mg, 102.13 μmol) was dissolved in DMF / water (2.0 mL / 1.0 mL), shielded from light, and stirred at room temperature (25.9 °C) under nitrogen gas protection. DIPEA was added dropwise to adjust the pH to 8, and the CR194PF activated ester solution was added dropwise. The reaction mixture was reacted at room temperature for 1 hour. The reaction mixture was purified by reverse-phase liquid separation and lyophilized to obtain CR194PV (50.0 mg).

[0306] CR194PV (56.2 mg, 14.8 μmmol) was added to TFA / DCM (1.4 mL / 1.4 mL), and the mixture was stirred at room temperature (22.7 °C) for 1 hour under nitrogen gas protection and shielded from light. MTBE (10.0 mL) was added for dilution and the mixture was dried by blowing with nitrogen gas. The residue was washed with MTBE (5.0 mL) and dried by blowing with nitrogen gas to obtain L-03.

[0307] 2.3 Synthesis of L-05 [ka] Preparation process: [ka]

[0308] SM1 (Fmoc-L-Pra-OH, 20.00 g, 60 mmol) was dissolved in DMF (150 mL). The atmosphere was purged with nitrogen gas three times, and the reaction mixture was cooled to -8 °C. HATU (27.40 g, 72 mmol, 1.2 eq) and SM2 (7.56 g, 72 mmol) were added. DIEA (15.50 g, 120 mmol) was weighed and dissolved in DMF (10 mL) and added dropwise to the mixture. The reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was monitored by HPLC using in-process control to confirm that the starting material SM1 was essentially completely reacted. The reaction mixture was added to ethyl acetate (250 mL) and water (800 mL), extracted, and separated. The aqueous phase was further extracted twice with an appropriate amount of EA. The combined organic phases were dried over an appropriate amount of anhydrous sodium sulfate for 1 h. After filtration, concentration under reduced pressure, and drying, the mixture was purified by column chromatography (DCM:MeOH=100:1) to give CR202HY (26.51 g, purity: 98.51%, yield: 100%) as a white solid, [M+H] + =423.4. 1H-NMR (400 MHz, CDCl3) δ 7.81 (d, J = 7.5 Hz, 2H), 7.64 (d, J = 7.3 Hz, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 6.94 (s, 1H), 5.76 (s, 1H), 4.49 (dd, J = 18.1, 11.0 Hz, 2H), 4.26 (t, J = 6.8 Hz, 1H), 3.86 - 3.69 (m,2H), 3.66 - 3.44 (m, 6H), 2.66 (dd, J = 71.1, 60.5 Hz, 4H).

[0309] Under nitrogen gas protection, SM3 (13.58 g, 96 mmol, 1.6 eq) was added to anhydrous DCM (600 mL). The reaction mixture was cooled to 0 °C, and a solution of CR202HY (25.80 g, 60 mmol) in DCM (200 mL) was slowly added dropwise. The reaction mixture was allowed to react for 30 min at 10 °C. The mixture was quenched by sampling with aqueous ammonia and acetonitrile. The reaction was monitored by HPLC until complete. Triethylamine (18.22 g, 180 mmol) was added and the pH was adjusted to 8. Finally, a solution of SM2 (9.47 g, 90 mmol) in DCM (10 mL) was added dropwise at 5 °C. After the addition was complete, the reaction mixture was stirred at 10 °C for 30 min. The reaction was monitored by HPLC to confirm that the reaction was essentially complete. The reaction mixture was added to dichloromethane (500 mL) and water (200 mL), extracted, and separated. The aqueous phase was extracted twice with 150 mL of dichloromethane. The combined organic phases were dried over an appropriate amount of anhydrous sodium sulfate for 1 hour. After filtration, concentration under reduced pressure, and drying, the mixture was purified by column chromatography (DCM:EA=1:1, 1% methanol and 0.5% acetic acid added) and concentrated to give CR202HZ (24.00 g, purity: 95.82%, yield: 63.3%) as a foamy solid. [M+H] + =633.14. [ka]

[0310] CR202JA was prepared in a manner similar to that used to prepare CR202HZ from CR202HY. [ka]

[0311] CR202JA (6.15 g, 7.29 mmol) was dissolved in DMF (100 mL). The atmosphere was purged with nitrogen gas three times, the reaction temperature was lowered to -5°C, and DBU (2.10 g, 13.68 mmol) was added dropwise. After the addition was complete, the reaction was stirred at 15°C for 1-4 hours and monitored by HPLC for essentially complete reaction. A DMSO solution of CR21532 (3.63 g, 7.29 mmol, predissolved in 80 mL of DMSO to clear) was added, followed by the addition of HATU (4.99 g, 13.12 mmol). The reaction was continued at 15°C for 30 minutes and monitored by HPLC for essentially complete reaction. The reaction was stopped, and the resulting crude product was purified (acetonitrile / 0.1% TFA-H2O) and lyophilized to give CR202JF (2.55 g, purity: 83.16%, yield: 31.87%) as a tan solid, [M+H] + =1100.9. [ka]

[0312] CR202JF (1.26 g, 1.14 mmol) was dissolved in anhydrous DMF (15 mL), spherical molecular sieves were added, and the mixture was dried for 30 min. The mixture was then filtered. The filtrate was transferred to a 250 mL jacketed bottle, and anhydrous TEA (0.92 g, 9.12 mmol, pH = 8-9) was added. The mixture was purged with nitrogen gas three times. The reaction was quenched by adding bis(4-nitrophenyl)carbonate (NPC, 0.6859 g, 2.3 mmol) at 25 °C and magnetically stirring for 8-10 h. 200 mL of MTBE was added to precipitate the solid, which was then centrifuged to give CR202JL (1.28 g, purity: 60.86%, yield: 87.50%) as a yellow solid. [M+H] +=1265.52. [ka]

[0313] CR202JL (600.0 mg, 0.474 mmol) was dissolved in DMF (40 mL), CR211D6 (1039.5 mg, 0.474 mmol), and HOBT (115.5 mg, 0.711 mmol) were added, and the mixture was purged with nitrogen gas three times. DIEA (125.9 mg, 0.948 mmol) was added, and the reaction mixture was maintained at 25 °C with magnetic stirring for 3-5 hours. The reaction was monitored for completeness by HPLC. The reaction was quenched, and 300 mL of MTBE precipitated solid was added. The mixture was stirred for 10 minutes and centrifuged to obtain a yellow solid, CR202JM (1.26 g, purity: 60.41%, yield: 68.11%). [M4+H] + =978.09. [ka]

[0314] A 100 mL jacketed bottle was charged with 40% TFA / DCM (40 mL) and cooled to -10 °C. CR202JM (1.26 g, 0.317 mmol) was added under nitrogen gas protection, gradually warmed to 18 °C, and stirred for 2-3 h. The reaction was monitored for completeness by HPLC. The reaction was quenched by adding the reaction mixture to 300 mL of cold MTBE to precipitate a solid. The mixture was stirred for 10 min and centrifuged to obtain the crude product, which was purified and lyophilized to give CR202JN (i.e., L-05, 160.9 mg, purity: 90.87%, yield: 26.67%) as a yellow solid with [M / 3+H]+ = 1285.5.

[0315] 3. Preparation of linkers [ka]

[0316] For the synthesis route of the linker CR20501, see J. Med. Chem. 2018, 61, 989-1000. For the synthesis route of CR19206, see Int. J. Mol. Sci. 2017, 18, 1860. The disclosures of these documents are incorporated herein by reference in their entirety. Other linker compounds herein can be synthesized by methods similar to those described above.

[0317] The polypeptide portion in the linker of the present invention can be prepared with reference to the solid phase synthesis method for the targeting molecule.

[0318] 4. Preparation of Complex Compounds

[0319] 4.1 Preparation of CR202F0 [ka]

[0320] Under nitrogen gas protection, the temperature was controlled at 0-10 °C. CR202F4 (13.5 mg, 0.077 mmol), HATU (29.3 mg, 0.077 mmol), and DIPEA (10.0 mg, 0.077 mmol) were added to DCM (1.0 mL) in this order and stirred magnetically for 0.5 h. Free CR20274 (45.5 mg, 0.05 mmol) was added and dissolved with magnetic stirring. The reaction mixture was then reacted under nitrogen gas protection at 20-25 °C for 4 h. The reaction mixture was analyzed for complete completion by HPLC. The reaction mixture was extracted with ice water (10.0 mL) and DCM (10.0 mL), and the layers were separated. The organic phase was washed sequentially with saturated citric acid (10.0 mL), saturated sodium bicarbonate (10.0 mL), and saturated brine (10.0 mL). The mixture was then dried over anhydrous sodium sulfate to yield 56.0 mg of a yellow solid (containing CR202F5). The crude product was used directly in the next step reaction.

[0321] The crude product (56.0 mg, 0.0054 mmol) containing CR202F5 was added to DCM (2.0 mL) and dissolved with magnetic stirring. Under nitrogen gas protection, the temperature was controlled at 0-10 °C, and TFA (0.5 mL) was added dropwise. The reaction was then incubated for 1 hour and monitored for complete completion by HPLC. The reaction mixture was concentrated, and DCM (10.0 mL) and saturated sodium bicarbonate (2.0 mL) were added. The layers were extracted and separated. The aqueous phase was further extracted with DCM (5.0 mL x 2). The organic phases were combined and dried over anhydrous sodium sulfate to yield 49.7 mg of a dusty gray solid (containing CR202F6).

[0322] Under nitrogen gas protection, CR19424 (29.9 mg, 0.063 mmol), HATU (24.1 mg, 0.063 mmol), and DIPEA (8.2 mg, 0.063 mmol) were sequentially added to DMF (2.0 mL) and magnetically stirred for 0.5 h. Free CR202F6 (39.7 mg, 0.042 mmol) was added and dissolved with magnetic stirring. The reaction was continued at 20-25 °C for 1 h under nitrogen gas protection. The reaction was then in-process and controlled by HPLC until complete. The reaction mixture was purified using a preparative liquid phase separation. Lyophilization yielded 21.1 mg of a white solid (containing CR202E9).

[0323] CR202E9 (17.0 mg, 0.012 mmol) and CR19208 (27.8 mg, 0.018 mmol) were added to DMF (2.5 mL) under nitrogen gas protection and temperature control at 20-25°C, protected from light, and magnetically stirred. DIPEA was added dropwise to adjust the pH to 8. The mixture was then incubated for 1 hour. HPLC was used to control the reaction until complete completion. The reaction mixture was purified using a preparative liquid phase. Lyophilization afforded 15.1 mg of a white solid (CR202F0). MS: [M+4H] 4+ / 4=730.58.

[0324] 4.2 Preparation of CR202F2 [ka]

[0325] Under nitrogen gas protection and temperature control at 0-10°C, CR194H8 (167.9 mg, 0.068 mmol), HATU (25.8 mg, 0.068 mmol), and DIPEA (13.2 mg, 0.10 mmol) were added to DMF (0.6 mL) in order and stirred magnetically for 0.5 hours. Free CR20274 (30.0 mg, 0.034 mmol) was added and dissolved with magnetic stirring. The reaction was carried out at 25°C under nitrogen gas protection for 2 hours, and the reaction was monitored by HPLC until complete. DMF (2 mL) was added to the reaction mixture, and CR19208 (155.2 mg, 0.10 mmol) was added to the reaction mixture, protected from light, at 15-25°C. The mixture was dissolved with stirring. DIPEA was added dropwise until the pH reached 8. The reaction was carried out for 1.5 hours and the reaction was monitored by HPLC until complete. The reaction mixture was purified using a preparative liquid phase. Lyophilization gave CR202F2 (25.5 mg). MS: [M+4H] 4+ / 4=658.78

[0326] 4.3 Preparation of CR20293, CR202CE, CR202CH, CR202CK, CR202CN, CR202CR, CR20214, CR20282, CR20285, CR20287, CR20295, and CR20297 [ka]

[0327] CR20502 was prepared using a method similar to that for CR19206.

[0328] CR20274 (151.3 mg, 0.171 mmol), CR20502 (277.3 mg, 0.353 mmol), and HOBt (5.7 mg, 0.042 mmol) were added sequentially to DMF (3.0 mL) and dissolved by magnetic stirring. The reaction was carried out under nitrogen gas protection at 25 °C for 24 h. The reaction was monitored by HPLC until complete. DMF (6 mL) was added to the reaction mixture, protected from light. CR19208 (775.0 mg, 0.509 mmol) was added at 15-25 °C and dissolved by stirring. DIPEA was added dropwise until the pH reached 8. The reaction was carried out for 2 h. A 20 μL sample was taken and diluted with acetonitrile / water (400 μL). The reaction mixture was monitored by HPLC until complete. The reaction mixture was purified using a preparative liquid phase. CR20293 (175.2 mg, purity: 95.85%, yield: 33.7%) was obtained.

[0329] Other conjugate compounds CR202CE, CR202CH, CR202CK, CR202CN, CR202CR, CR20214, CR20282, CR20285, CR20287, CR20295 and CR20297 were prepared using a method similar to that for the preparation of CR20293.

[0330] 4.4 Synthesis of CR202EZ [ka]

[0331] The crude product of L-03 (CR194PW) was dissolved in DMF / water (2.0 mL / 0.7 mL), and CR202X7 (21.8 mg, 14.8 μmol) was further added to dissolve. The mixture was shielded from light, and under nitrogen gas protection, the mixture was stirred at room temperature (25.9 °C). DIPEA was added dropwise to adjust the pH to 8. Cuprous bromide (2.1 mg, 14.8 μmol) was added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was purified by reverse-phase preparative liquid chromatography and lyophilized to obtain CR202EZ (5.0 mg, yield: 6.7%). [M+5H] 5+ / 5=1040.06.

[0332] 4.5 Synthesis of CR202W5, CR202W6, and CR202W9 [ka]

[0333] CR202Z5 (100.0 mg, 97.7 μmol), DCM (2 mL), and 4A molecular sieves (200.0 mg) were added to a 25 mL single-necked jacketed bottle R1 and cooled to 0 °C. Triethylamine (34.5 mg, 342.0 μmol) was added and stirred for 2 h. At 0 °C, a solution of triphosgene (14.5 mg, 48.9 μmol) in DCM (0.1 mL) was added dropwise and allowed to react for 5 min while maintaining the temperature. Simultaneously, DMF (0.5 mL), CR202FG (54 mg, 97.7 μmol), DMAP (12 mg, 97.7 μmol), and TEA (10 mg, 97.7 μmol) were added to a 25 mL single-necked jacketed bottle R2 and dissolved by stirring. 4A molecular sieves (0.1 g) were added and cooled to 0 °C. Under nitrogen gas protection, the mixture in R1 was added dropwise to R2, and the reaction was stirred for 0.5 hours while keeping the temperature at 40°C until the reaction was complete. Water (1 mL) was added to R2 to quench the reaction, and the reaction mixture was purified by reverse phase chromatography to obtain CR202GN (white powder, m = 90 mg, purity: 91.4%, yield: 58.4%). LCMS: [M+2] 2+ / 2=800.9. [ka]

[0334] CR202GN (75 mg, 46.9 μmol) and L-04 (CR202W4) (106.5 mg, 70.3 μmol) were dissolved in DMF (4.5 mL) and water (1.5 mL) under light-protected conditions. Under nitrogen gas protection, DIPEA was added to adjust the system to pH = 8, and CuBr (6.7 mg, 46.9 μmol) was added. The reaction mixture was then purified by reverse phase chromatography to obtain CR202W5 (white powder, 70.0 mg, purity: 100%, yield: 47.9%). LCMS: [M-3H] 3- / 3=1037.0.

[0335] Similarly, a similar method was employed to synthesize CR202W6 and CR202W9.

[0336] 4.6 Synthesis of CR202X1 and CR202W7 [ka]

[0337] CR202Z2 (100.0 mg, 138.0 μmol) and DMAP (20.0 mg, 166.0 μmol) were added to DMF (3.6 mL) and stirred at -10°C under nitrogen gas protection. Scandium trifluoromethanesulfonate (18.0 mg, 41.4 μmol), DIPEA (18.0 mg, 138.0 μmol), and CR202X6 (129.0 mg, 179.2 μmol) were added sequentially and dissolved in DMF (1.0 mL). The mixture was incubated and reacted for 48 hours. The reaction mixture was purified by reverse-phase preparative liquid separation to give CR202EQ (140.0 mg, yield: 78.2%) as a white solid. [M+H] + =1300.5.

[0338] For the synthesis of CR202X1, see the synthesis of CR202W5.

[0339] Similarly, a similar method was employed to synthesize CR202W7.

[0340] 4.7 Synthesis of CR202W8 [ka]

[0341] CR202Z8 (30 mg, 27.4 μmol) and NPC (67 mg, 220 μmol) were dissolved in DMF (0.3 mL) and stirred magnetically under nitrogen gas protection. DIEA (7.1 mg, 54.9 μmol) was added and stirred at 25°C for 15 hours. CR202GF (45 mg, 81.7 μmol), DMAP (4 mg, 32.7 μmol), and DIEA (7.1 mg, 54.9 μmol) were added and stirred at 25°C for 16 hours. Sampling was performed to control the process until the reaction was complete. Preparative purification afforded CR202ER (29 mg, purity: 98.24%, yield: 63%) as a white solid. LCMS: [M+2H] 2+ / 2=837.5 [ka]

[0342] CR202ER (50 mg, 29.9 μmol) and CR202W4 (54 mg, 35.6 μmol) were dissolved in DMF (2.5 mL) and water (0.8 mL) under light-shielded conditions, magnetically stirred, and nitrogen gas protection was maintained. DIEA (130 μL) was added to adjust the pH to 8, and CuBr (4.3 mg, 29.9 μmol) was added. The reaction was carried out at 25°C for 1 hour, and sampling was carried out to control the process until the reaction was complete. The reaction solution was purified by preparative liquid phase separation. CR202W8 (33 mg, yield: 41%) was obtained. LCMS: [M+3H] 3+ / 3=1063.1.

[0343] 4.8 Synthesis of CR202V4 [ka]

[0344] CR202FT (6.73 g, 15.81 mmol) was dissolved in DMF (50 mL) and stirred magnetically. The reaction mixture was cooled to 0 °C, HATU (6.61 g, 17.39 mmol) was added, and the mixture was stirred at 0 °C for 10 min. CR19154 (6.00 g, 15.8 mmol) was added, and DIEA (2.25 g, 17.39 mmol) was weighed and dissolved in DMF (10 mL) and added dropwise to the mixture. The reaction mixture was stirred at 0 °C for 1 h. The starting material was monitored for complete reaction. The reaction mixture was added to ethyl acetate (180 mL) and stirred for 0.5 h. The mixture was centrifuged, washed three times with ethyl acetate, centrifuged, and left in a dry box overnight (35 °C). CR202FQ (11.93 g, purity: 80%, yield: 76.7%) was obtained as a yellow solid. [M+H] + =787.47. [ka]

[0345] CR202FQ (6.5 g, 8.26 mmol) was dissolved in methanol (70 mL) and stirred magnetically. A 20% solution of piperidine in DMF (12.3 g, 28.91 mmol) was added, and the reaction mixture was stirred at 50 °C overnight. LCMS confirmed the complete reaction of the starting material. The methanol was removed by rotary evaporation, and the mixture was washed twice with ethyl acetate (50 mL × 2). Rotary evaporation was continued until approximately 15 mL remained. Methyl tert-butyl ether (200 mL) was added to precipitate a white solid. The solid was filtered, and the filter cake was rinsed with methyl tert-butyl ether (20 mL × 2). The solid was dried under vacuum to give CR202FR (4.09 g, purity: 91%, yield: 79.8%) as a milky white solid. [ka]

[0346] CR194Z0 (1.51 g, 7.97 mmol) was dissolved in DMF (30 mL) and stirred magnetically. The reaction mixture was cooled to 0 °C, HATU (3.03 g, 7.97 mmol) was added, and the mixture was stirred at 0 °C for 10 minutes. CR202FR (4.09 g, 7.24 mmol) was added, and DIEA (1.03 g, 7.97 mmol) was weighed and dissolved in DMF (10 mL) and added dropwise to the mixture. The reaction mixture was stirred at 0 °C for 1 hour. LCMS confirmed the complete reaction of the starting material. The reaction mixture was added to ethyl acetate (150 mL) and stirred overnight. The solid was separated out, centrifuged, washed once with methyl tert-butyl ether, and dried to give CR202FS (3.7 g, purity: 79.6%, yield: 69.5%) as a pale yellow solid. [M+H] + =736.51. [ka]

[0347] CR202FS (1.7 g, 2.3103 mmol) was dissolved in DMF (20 mL) and dissolved with magnetic stirring. NPC (1.4 g, 4.6206 mmol) and DIEA (447.9 mg, 3.4655 mmol) were added, and the reaction was stirred at room temperature for 1 h. Purification by reverse-phase preparative separation afforded CR202X4 (1.77 g, purity: 99.28%, yield: 85.04%) as a white solid. 1H-NMR (400 MHz, DMSO) δ 10.16 (s, 1H), 8.38 - 8.30 (m, 2H), 8.25 (d, J = 7.2 Hz, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.64 - 7.55 (m, 2H), 7.44 (d, J = 8.6 Hz, 2H), 6.06 (s, 0H), 5.27 (s, 2H), 4.51 - 4.37 (m, 3H), 4.24 (dd, J = 8.4, 6.9 Hz, 1H), 3.96 (s, 2H), 3.70 - 3.58 (m, 6H), 3.49 - 3.39 (m, 2H), 3.03 (ddd, J = 38.8, 13.1, 6.5 Hz, 2H), 2.29 - 2.19 (m, 2H), 1.98 (ddd, J = 20.3, 12.7, 6.0 Hz, 2H), 1.85 - 1.67 (m, 2H), 1.55 (ddd, J = 21.1, 11.2, 5.0 Hz, 2H), 1.41 (s, 10H), 0.87 (dd, J = 14.8, 6.8 Hz, 6H), [M + H] + = 901.46。

Chem.

[0348] Free CR20274: CR20274 hydrochloride (902.8 mg, 0.94 mmol) was added to DCM (50.0 mL) and methanol (10.0 mL) and magnetically stirred until the solid was completely dissolved. At 5-10 °C, 10% sodium bicarbonate solution (20 mL) was added dropwise and stirred for 30 min. The reaction mixture was extracted with DCM (50 mL), methanol (5 mL), and water (20 mL) (a relatively thick emulsion formed). The aqueous phase was extracted twice with a DCM / methanol mixture, each time with DCM (50 mL) and methanol (5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum at 30 °C to dryness. Free CR20274 (910.4 mg, purity: 92.04%, yield: 100.0%) was obtained as a solid and used directly in the next step without further purification.

[0349] Free CR20274 (60.0 mg, 0.068 mmol), CR202X4 (150.1 mg, 0.170 mmol), and HOBt (1.8 mg, 0.014 mmol) were added sequentially to DMF (1.5 mL), dissolved by magnetic stirring, and reacted at 25 °C for 24 h under nitrogen gas protection. DIPEA (12 μL, 0.068 mmol) was added and the reaction continued for 20 h. The reaction was controlled by HPLC until complete completion, and the reaction mixture was directly purified to give CR202AW (64.9 mg, purity: 92.96%, yield: 43.6%). [M+2H] + / 2=823.8. [ka]

[0350] CR202AW (50.0 mg, 0.03 mmol) was added to DCM (2.0 mL), and under nitrogen gas protection, the external temperature was controlled at 15°C. TFA (0.2 mL) was added dropwise, and the reaction was allowed to proceed for 3 hours. The reaction was then monitored by HPLC until complete completion. The external temperature was controlled at 10°C, and saturated NaHCO3 was added dropwise to adjust the pH to 7-8. The mixture was concentrated to remove DCM, and a small amount of solid precipitated. The solid was dissolved in acetonitrile (4 mL), and purified by reverse-phase liquid separation to obtain CR202X5 (20.6 mg, purity: 84.20%, yield: 42.9%). [ka]

[0351] In a 25 mL jacket, CR202X5 (16.0 mg, 10.1 μmol) and CR202W4 (18.3 mg, 12.1 μmol) were dissolved in DMF (1 mL) and water (0.3 mL) under light-protected conditions, magnetically stirred, and protected with nitrogen gas. DIEA (250 μL) was added to adjust the pH to 8, and CuBr (1.5 mg, 1.0 eq.) was added and the reaction was allowed to proceed at 25 °C for 2 hours. The reaction mixture was purified by preparative liquid phase separation. CR202V4 (5.7 mg, purity: 92.26%, yield: 13.9%) was obtained, and [M+3H] 3+ / 3=1035.98.

[0352] 4.9 Synthesis of CR202V5, CR202V6, CR202Z6 and CR202V7 [ka]

[0353] CR202Z0 (3.00 g, 15.81 mmol), HATU (6.01 g, 15.81 mmol), DIPEA (2.73 g, 21.08 mmol), and CR19154 (4.00 g, 10.54 mmol) were added to DMF (40 mL) in sequence, and the reaction was carried out under nitrogen gas protection at a temperature of 25°C for 2 hours. The reaction was then controlled by HPLC until the reaction was complete. The reaction mixture was then directly purified by reversed-phase preparative liquid chromatography to give CR202FG (1.88 g, yield: 43.2%). [M+H] + =551.31. [ka]

[0354] CR202FG (1.48 g, 2.68 mmol) and NPC (1.63 g, 5.36 mmol) were added to DMF (7.5 mL), and DIEA (519.7 mg, 4.02 mmol) was added dropwise. The reaction was carried out under nitrogen gas protection with magnetic stirring for 2 hours. The reaction was controlled by HPLC until complete completion, and the reaction mixture was directly purified by reverse-phase preparative liquid separation to obtain CR202X6 (2.07 g, purity: 100.0%, yield: 100%). 1H-NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.40 (d, J = 7.3 Hz, 1H), 8.34 (d, J = 9.1 Hz, 3H), 7.68 (d, J = 8.5 Hz, 2H), 7.63-7.57 (m, 2H), 7.49 (d, J = 9.0 Hz, 1H), 7.44 (d, J = 8.5 Hz, 3H), 6.04 (s, 1H), 5.27 (s, 2H), 4.42 (d, J = 5.3 Hz, 1H), 4.35 (dd, J = 8.8, 6.6 Hz, 1H), 3.99 (s, 2H), 3.73-3.58 (m, 8H), 3.43 (t, J = 4.9 Hz, 2H), 3.02 (d, J = 21.0 Hz, 2H), 2.03 (dd, J = 13.4, 6.7 Hz, 1H), 1.79-1.54 (m, 2H), 1.42 (dd, J = 21.6, 14.8 Hz, 2H), 0.91 (d, J = 6.7 Hz, 3H), 0.85 (d, J = 6.7 Hz, 3H), [M+H] + =716.30. [ka]

[0355] CR20274 release: CR20274 hydrochloride (902.8 mg, 0.94 mmol) was added to DCM (50.0 mL) and methanol (10.0 mL) and magnetically stirred until the solid was completely dissolved. At 5-10 °C, 10% sodium bicarbonate solution (20 mL) was added dropwise and stirred for 30 min. The reaction mixture was extracted with DCM (50 mL), methanol (5 mL), and water (20 mL) (a relatively thick emulsion formed). The aqueous phase was extracted twice with a DCM / methanol mixture, each time with DCM (50 mL) and methanol (5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum at 30 °C to give CR20274 (910.4 mg, yield: 100.0%, purity: 92.04%) as a solid, which was used directly in the next step without further purification.

[0356] Free CR20274 (200.0 mg, 0.23 mmol), CR202X6 (404.7 mg, 0.57 mmol), and HOBt (6.1 mg, 0.045 mmol) were added sequentially to DMF (5.0 mL), dissolved by magnetic stirring, and reacted at 25 °C for 24 hours under nitrogen gas protection. The reaction was controlled by HPLC until complete completion, and the reaction mixture was directly purified by reverse-phase preparative liquid phase to give CR202X7 (134.0 mg, purity: 98.32%, yield: 40.6%). [M+2H] 2+ =730.99. [ka]

[0357] CR202X7 (130.0 mg, 0.089 mmol) and CR202W4 (161.8 mg, 0.107 mmol) were dissolved in DMF (7.8 mL) and water (2.6 mL) under light-protected conditions, magnetically stirred, and protected with nitrogen gas. DIEA (250 μL) was added to adjust the pH to 8, followed by CuBr (12.7 mg, 0.089 mmol). The reaction was allowed to proceed at 25°C for 2 hours, with in-process control until the reaction was complete. The reaction mixture was purified by reverse-phase preparative liquid chromatography. CR202V5 (76.6 mg, purity: 95.70%, yield: 28.9%) was obtained. MS: [M+4H] 4+ / 4=745.02.

[0358] Similarly, a similar method was adopted to synthesize CR202V6, CR202Z6 and CR202V7.

[0359] 4.10 Synthesis of CR202V8 [ka]

[0360] Synthesis of CR202AS Resin swelling: 2-CTC resin (50.00 g, 32.5 mmol) was weighed, anhydrous DCM (500 mL) was added, mechanical stirring was initiated (120 rpm), and the resin was allowed to swell for 30 min at 25 °C under nitrogen gas protection. Amino acid coupling: Fmoc-Lys(boc)-OH (45.68 g, 97.5 mmol) and DIEA (21.00 g, 162.5 mmol) were added and the reaction was continued for 3 hours. Resin capping: The reaction mixture was transferred to a solid-phase synthesis reactor, suction-dried, and the resin was washed three times with DMF (500 mL x 3). DMF (500 mL) was added, and nitrogen gas was sprayed over the resin. Finally, methanol (10.41 g, 325 mmol) and DIEA (12.60 g, 97.5 mmol) were added, and the reaction was carried out at 25°C for 1 hour. The reaction mixture was suction-dried, and the resin was washed five times with DMF (500 mL x 5). Deprotection: A 20% piperidine solution (500 mL) was added, nitrogen gas was blown in, and the reaction was carried out at 25°C for 20 minutes. A few particles of resin were removed and washed three times with ethanol. Ninhydrin developer was then added and the mixture was heated at 110°C for 2 minutes. The resin turned purple, indicating completion of the reaction. The reaction solution was dried by suction, and the resin was washed five times with DMF (500 mL x 5). Amino acid coupling: Fmoc-Leu-OH (17.33 g, 48.8 mmol) and HOBT (6.59 g, 48.8 mmol) were weighed and added to DMF (250 mL). After dissolving with magnetic stirring, DIC (6.15 g, 48.98 mmol) was added. After stirring for 3 minutes, the mixture was added to the reaction vessel and reacted at 25°C for 1 hour. The resin was sampled and heated to develop a white color, indicating completion of the reaction. The reaction solution was dried under vacuum, and the resin was washed three times with DMF (500 mL x 3). In this order, Fmoc-Val-OH and N3-PEG2-CH2-COOH were sequentially coupled to the resin. Resin shrinkage: The resin was washed twice with DCM (500 mL x 2), shrunk with MeOH, dried, and weighed to obtain 66.00 g of resin. Dissolution: 22.00 g of resin was added to 240 mL of prepared dissolution solution (72 mL of trifluoroethanol diluted to 240 mL with DCM), mechanical stirring was initiated, and the reaction was carried out at 25° C. for 3 hours under nitrogen gas protection. Work-up: Remove the resin by suction filtration, wash the resin once with a small amount of dichloromethane, and directly concentrate and dry the filtrate to obtain 8.08 g of oily liquid CR202AS. Store at -20°C to harden the product. HPLC: 91.08%, yield: 118.0%, analysis of the cause of excess weight: trifluoroethanol remained in the product.

[0361] Synthesis of CR202AY [ka]

[0362] CR202AS (3.51 g, 5.6 mmol) and HATU (2.97 g, 7.8 mmol) were added sequentially to a mixed solvent of DMF (33 mL), EA (ethyl acetate, 18 mL) and water (3.5 mL), and the mixture was stirred magnetically and cooled to 0°C under nitrogen gas protection. p-Aminobenzyl alcohol solution (1.03 g, 8.4 mmol, dissolved in EA (7 mL) and DMF (1.8 mL)) and DIEA solution (1.3 mL, 7.8 mmol, dissolved in EA (7 mL)) were added dropwise in sequence, and the reaction was carried out at 0°C for 1 hour. The reaction was controlled by HPLC until the reaction was complete. The reaction solution was added to purified water (200 mL), extracted with EA (100 mL × 3), and the organic phases were combined and washed with purified water (100 mL) and saturated brine (100 mL). The mixture was washed with 1 mL of 1,000 ml ...

[0363] Synthesis of CR202CU [ka]

[0364] CR202AY (2.0783 g, 2.8 mmol) and bis(4-nitrophenyl)carbonate (1.5486 g, 5.1 mmol) were added to DCM (30 mL) and stirred magnetically until dissolved. DIEA (0.93 mL, 5.7 mmol) was added and the mixture was reacted under nitrogen gas protection at 25°C for 3 hours. The reaction was controlled by HPLC until complete, and the mixture was concentrated at 25°C. The reaction mixture was purified by preparative liquid phase separation to give CR202Z1 (1.3924 g, HPLC: 95.00%, yield: 52.8%) as a white solid.

[0365] Synthesis of CR202AZ [ka]

[0366] CR20274 (150 mg, 0.17 mmol) and CR202CU (275.0 mg, 0.31 mmol) were added to DMF (3.0 mL) and magnetically stirred until dissolved. HOBT (4.6 mg, 0.034 mmol) and DIEA (40 mg, 0.26 mmol, 1.5 eq) were added, and the reaction was carried out at 25°C for 18 hours under nitrogen gas protection. The reaction was controlled by HPLC until complete, and the reaction mixture was purified by preparative liquid phase separation. CR202AZ (170.0 mg, HPLC: 98.89%, yield: 60.9%) was obtained as a white solid, [M+2H]. 2+ / 2=823.46.

[0367] Synthesis of CR202BA [ka]

[0368] CR202AZ (170.0 mg, 0.10 mmol) was added to DCM (4.0 mL) under nitrogen gas protection. The external temperature was controlled at 15 °C, and TFA (0.4 mL) was added dropwise. The reaction was allowed to proceed for 2.5 hours, and the process was monitored by HPLC until the reaction was complete. The external temperature was controlled at 0 °C, and saturated sodium bicarbonate solution was added dropwise to adjust the pH to 7-8. The mixture was concentrated at 15 °C to remove DCM, and the residue was dissolved in acetonitrile (10 mL). The reaction mixture was purified by preparative liquid phase chromatography. CR202BA (34.0 mg, HPLC: 89.36%, yield: 21.4%) was obtained as a white solid, [M+2H]. 2+ / 2=773.3.

[0369] Synthesis of CR202V8 [ka]

[0370] CR202BA (55.0 mg, 0.036 mmol) and CR202W4 (64.7 mg, 0.43 mmol) were dissolved in DMF (4.0 mL) and water (1.3 mL) under light-protected conditions, maintained under nitrogen gas protection, and magnetically stirred until dissolved. DIEA was added to adjust the pH to 8, and CuBr (5.1 mg, 0.036 mmol) was added. The reaction was allowed to proceed at 25 °C for 2 h. Samples were taken to ensure complete reaction for process control. The reaction solution was purified by preparative liquid phase separation. Salt removal afforded CR202V8 (22.5 mg, purity: 95.03%, yield: 20.8%), which was then purified using [M+3H]. 3+ / 3=1020.48.

[0371] 4.11 Synthesis of CR202V9 [ka]

[0372] Synthesis of CR202AU Resin swelling: 2-CTC resin (50.00 g, 32.5 mmol) was weighed, anhydrous DCM (500 mL) was added, mechanical stirring was initiated, and the resin was allowed to swell for 30 min at 25° C. under nitrogen gas protection. Amino acid coupling: Fmoc-Asn(trt)-OH (58.18 g, 97.5 mmol) and DIEA (21.00 g, 162.5 mmol) were added and the reaction was continued for 3 hours. Resin capping: The reaction mixture was transferred to a solid-phase synthesis reactor, suction-dried, and the resin was washed three times with DMF (500 mL x 3). DMF (500 mL) was added, and nitrogen gas was sprayed over the resin. Finally, methanol (10.41 g, 325 mmol) and DIEA (12.60 g, 97.5 mmol) were added, and the reaction was carried out at 25°C for 1 hour. The reaction mixture was suction-dried, and the resin was washed five times with DMF (500 mL x 5). Deprotection: A 20% piperidine solution (500 mL) was added, nitrogen gas was blown in, and the reaction was carried out at 25°C for 20 minutes. A few particles of resin were removed and washed three times with ethanol. Ninhydrin developer was then added and the mixture was heated at 110°C for 2 minutes. The resin turned purple, indicating completion of the reaction. The reaction solution was dried by suction, and the resin was washed five times with DMF (500 mL x 5). Amino acid coupling: Fmoc-Asn(trt)-OH (17.33 g, 48.8 mmol) and HOBT (6.59 g, 48.8 mmol) were weighed and added to DMF (250 mL). After dissolving with magnetic stirring, DIC (6.15 g, 48.98 mmol) was added. After stirring for 3 minutes, the mixture was added to the reaction vessel and reacted at 25°C for 1 hour. The resin was sampled and heated to develop a white color, indicating completion of the reaction. The reaction solution was dried under vacuum, and the resin was washed three times with DMF (500 mL x 3). In this order, Fmoc-Gly-OH and N3-PEG2-CH2-COOH were sequentially coupled to the resin. Resin shrinkage: The resin was washed twice with DCM (500 mL x 2), shrunk with MeOH, dried, and weighed to obtain 96.42 g of resin. Dissolution: 32.14 g of resin was added to the dissolution solution (300 mL, DCM:TFA:Tis=4.9:4.9:0.2) prepared at 5°C, mechanical stirring was started, and the reaction was carried out at 25°C for 3 hours under nitrogen gas protection. Work-up: The resin was removed by suction filtration, washed once with a small amount of dichloromethane, and the filtrate was directly concentrated to remove DCM. Then, it was added to 600 mL of pre-chilled MTBE to precipitate a white solid. The mixture was stirred for 10 minutes, filtered, and the solid was washed twice with MTBE (100 mL × 2). After drying, 5.21 g of white solid CR202AU (HPLC: 92.71%, yield: 101.6%) was obtained. The excess weight was presumably due to residual TFA in the product.

[0373] Synthesis of CR202AV [ka]

[0374] CR202AU (2.50 g, 5.3 mmol) and p-aminobenzyl alcohol (0.54 g, 4.4 mmol) were added to DMF (12 mL) and stirred magnetically until the mixture dissolved and became clear. HATU (2.51 g, 6.6 mmol) and DIEA (2.20 mL, 13.2 mmol) were added and the mixture was allowed to react at 25 °C for 1.5 h. The reaction was monitored by HPLC until complete. The reaction mixture was added to pre-cooled MTBE (60 mL) to precipitate a white solid. The solid was stirred magnetically for 10 min, filtered, and washed twice with MTBE (20 mL × 2) and dried to give CR202AV (1.3194 g, purity: 87.79%, yield: 51.9%) as a white solid.

[0375] Synthesis of CR202CV [ka]

[0376] CR202AV (1.28 g, 2.2 mmol) and bis(4-nitrophenyl)carbonate (1.21 g, 4.0 mmol) were added to DMF (10 mL) and dissolved with magnetic stirring. DIEA (0.73 mL, 4.4 mmol) was added and the mixture was reacted at 25 °C for 2 h under nitrogen gas protection. The reaction was monitored by HPLC until complete, and the reaction mixture was added to MTBE (60 mL) to precipitate a white solid. The solid was stirred for 10 min, filtered, washed twice with MTBE (20 mL × 2), and dried under vacuum at 30 °C to give CR202CV (1.19 g, HPLC: 87.40%, yield: 84.8%) as a white solid.

[0377] Synthesis of CR202BD [ka]

[0378] CR20274 (51.2 mg, 56.6 μmol) and CR202CV (75.8 mg, 101.8 μmol) were added to DMF (1 mL) and dissolved with magnetic stirring. HOBT (1.5 mg, 11.3 μmol) and DIEA (15 μL, 84.8 μmol) were added, and the reaction was carried out under N2 protection at 25 °C for 18 hours. The reaction was controlled by HPLC until complete, and the reaction mixture was purified by preparative liquid phase separation to give CR202BD (68.6 mg, purity: 91.5%, yield: 81.7%) as a white solid.

[0379] Synthesis of CR202V9 [ka]

[0380] CR202BD (58.0 mg, 39.0 μmol) and CR202W4 (70.8 mg, 46.7 μmol) were dissolved in DMF (3.6 mL) and water (1.2 mL) under light-protected conditions and magnetic stirring under nitrogen gas protection. DIEA was added to adjust the pH to 8, and CuBr (5.6 mg, 39.0 μmol) was added. The reaction was carried out at 25°C for 2 hours, and samples were taken to control the process until the reaction was complete. The reaction solution was purified by preparative liquid phase separation. CR202V9 (37.4 mg, purity: 94.90%, yield: 31.6%) was obtained as [M+4]. 4+ / 4=751.89.

[0381] 4.12 Synthesis of CR202AL [ka]

[0382] Synthesis of activated ester of CR202Z0: CR202Z0 (3.00 g, 15.9 mmol) was added to DCM (30 mL). Under nitrogen gas protection, the external temperature was controlled at 0°C, and EDCI (4.57 g, 23.8 mmol) and HOSu (2.74 g, 23.8 mmol) were added in portions. After the addition was completed, the mixture was allowed to warm to room temperature and react at room temperature for 2 hours. The reaction mixture was poured into ice water (100 mL) and extracted with DCM (50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 4.6 g of a yellow oil.

[0383] Synthesis of CR202BQ: CR202BP (5.90 g, 19.1 mmol) was added to DMF (30 mL) and dissolved with magnetic stirring. DBU (2.40 g, 15.9 mmol) was added and the mixture was allowed to react at room temperature for 3 hours. DIPEA (4.5 mL) was added, and CR202Z0 activated ester (4.6 g) was added dropwise to the mixture. The mixture was allowed to react at room temperature for 2 hours. The reaction mixture was poured into ice water (250 mL) and extracted with n-heptane (100 mL × 2). The aqueous phase was adjusted to pH = 2 with 6 N hydrochloric acid and further extracted with DCM (100 mL × 2). The organic phases were combined and dried over anhydrous sodium sulfate to give CR202BR (2.30 g, yield: 56.1%) as a yellow oil. 1 H-NMR (400 MHz, CDCl3) δ 7.36 (s, 1H), 4.06 (s, 2H), 3.82-3.67 (m, 6H), 3.62 (dd, J = 12.1, 6.0 Hz, 2H), 3.52-3.41 (m, 2H), 2.67 (t, J = 6.0 Hz, 2H), MS:[M+H] + =261.4. [ka]

[0384] CR202Q8 (3.30 g, 6.8 mmol) was added to isopropanol (15 mL) and chloroform (55 mL). Under nitrogen gas protection, the external temperature was controlled at 0°C, and sodium borohydride (389.1 mg, 10.2 mmol) was added in several portions. The reaction was continued for 3.5 hours while maintaining the temperature. Complete reaction was confirmed by TLC (n-heptane:EA=1:1, Rf=0.5). The reaction mixture was poured into ice water (150 mL), 6 N hydrochloric acid (2 mL) was added dropwise, and the mixture was extracted with DCM (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give 4.6 g of crude product. EA:n-heptane (7.5 mL:7.5 mL) was added, the mixture was slurried at room temperature overnight, filtered, rinsed with EA:n-heptane (1 mL:1 mL), and dried in vacuo to give an off-white solid of CR202Q9 (3.60 g, yield: 83.7%). 1H-NMR (400 MHz, CDCl3) δ 7.86 (d, J = 2.0 Hz, 1H), 7.58 (dd, J = 8.5, 2.1 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 5.43-5.30 (m, 3H), 5.23 (d, J = MS:[M+H] + =475.2. [ka]

[0385] CR202Q9 (2.85 g, 5.9 mmol) was added to methanol (100 mL), 10% Pd / C (0.30 g, water content: 50%) was added, the mixture was purged with hydrogen gas three times, and hydrogenated at room temperature under atmospheric pressure for 4 hours. Complete reaction was confirmed by TLC, filtered, concentrated to dryness, EA:n-heptane (2 mL:4 mL) was added, the mixture was slurried at room temperature for 16 hours, filtered, rinsed with EA:n-heptane (1:2, 2 mL × 2), and dried in vacuo to give CR202R0 (2.60 g, purity: 91.63%, yield: 87.8%) as an off-white solid. 1 H-NMR (400 MHz, CDCl3) δ 6.94 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 1.7 Hz, 1H), 6.70 (dd, J = 8.2, 1.7 Hz, 1H), 5.42-5.31 (m, 3H), 5.06 (d, J = 7.3 Hz, 1H), 4.59 (s, 2H), 4.20 (d, J = 9.3 Hz, 1H), 3.80 (s, 3H), 2.14-2.08 (m, 9H), MS:[M+H] + =456.3. [ka]

[0386] CR202BR (1.82 g, 6.9 mmol) was added to DMF (24 mL). Under nitrogen gas protection, the external temperature was controlled at 0 °C. DIPEA (1.38 g, 10.6 mmol), HATU (2.62 g, 6.9 mmol), and CR202R0 (2.40 g, 5.3 mmol) were added sequentially. After the addition was completed, the mixture was allowed to warm to room temperature (16.0 °C) and react at room temperature for 2.5 hours. The reaction was then monitored by HPLC until complete completion. The reaction mixture was poured into ice water (200 mL) and extracted with EA (100 mL × 3). The organic phases were combined, washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, and the resulting crude product (5.4 g) was concentrated and purified to give CR202AH (2.18 g, purity: 97.52%, yield: 54.8%) as a foamy solid. 1 H-NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 8.08 (s, 1H), 7.47 (s, 1H), 7.10 (d, J = 7.0 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 5.45 (t, J = 9.5 Hz, 1H), 5.35 (dt, J = 14.0, 7.7 Hz, 2H), 5.09 (d, J = 7.6 Hz, 1H), 4.68 (s, 2H), 4.23 (d, J = 9.7 Hz, 1H), 4.06 (s, 2H), 3.80 (s, 3H), 3.71 (d, J = 5.1 Hz, 8H), 3.44 (t, J = 4.9 Hz, 2H), 2.76 (t, J = 5.6 Hz, 2H), 2.18-2.04 (m, 9H), MS:[M+H] + =698.3. [ka]

[0387] CR202AH (1.10 g, 1.58 mmol) was dissolved in DMF (10 mL). Under nitrogen gas protection, bis(p-nitrophenyl)carbonate (960 mg, 3.15 mmol) and DIPEA (306 mg, 2.37 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 3 hours, and the reaction was monitored by HPLC until complete. The reaction mixture was purified to give CR202AI (1.10 g, purity: 99.85%, yield: 80.9%) as a white solid. 1 H-NMR (400 MHz, CDCl3) δ 8.56 (d, J = 1.9 Hz, 1H), 8.35-8.28 (m, 2H), 8.13 (s, 1H), 7.57 (s, 1H), 7.45-7.39 (m, 2H), 7.17 (dd, J = 8.4, 2.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 5.47 (t, J = 9.5 Hz, 1H), 5.39-5.31 (m, 2H), 5.27 (s, 2H), 5.13 (d, J = 7.6 Hz, 1H), 4.25 (d, J = 9.7 Hz, 1H), 4.10 (s, 2H), 3.80 (s, 3H), 3.75-3.68 (m, 8H), 3.47-3.41 (m, 2H), 2.79 (t, J = 6.1 Hz, 2H), 2.14-2.08 (m, 9H), MS:[M+H] + =863.3. [ka]

[0388] Release of CR20274: CR20274 hydrochloride (2.8 g, 2.93 mmol, 1.0 eq.) was dissolved in DCM (100 mL) and methanol (20 mL) and cooled to 5–10 °C. NaHCO (2.0 g, 23.8 mmol) was weighed and dissolved in water (35 mL). The solution was then added dropwise to the DCM / MeOH solution of CR20274. The mixture was stirred at 5–10 °C for 3 minutes and then separated. The aqueous phase was extracted with DCM / MeOH (10 / 1, 50 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under vacuum at 30 °C. 2.5 g (purity: 98.02%, yield: 97%) of free CR20274 was obtained as a pale yellow solid and used directly in the next step.

[0389] Free CR20274 (150.0 mg, 0.17 mmol), CR202AI (292.8 mg, 0.34 mmol), HOBt (4.6 mg, 0.034 mmol), and DIPEA (32.9 mg, 0.25 mmol) were added sequentially to DMF (4.0 mL), dissolved by magnetic stirring, and reacted at 25 °C for 20 h under nitrogen gas protection. DIPEA (8 μL, 0.045 mmol) was added and the reaction was continued for 24 h. The reaction was controlled by HPLC until complete completion, and the reaction solution was purified by preparative liquid phase separation to give CR202AJ (218.1 mg, purity: 99.56%, yield: 54.5%). [ka]

[0390] CR202AJ (210.0 mg, 130.0 μmol) was dissolved in THF (21 mL). The external temperature was controlled at 0-5°C, and 0.1 M aqueous LiOH solution (7.2 mL, 718.0 μmol) was added dropwise. The reaction mixture was then incubated for 1 hour. 5% aqueous TFA solution was added dropwise to adjust the pH to 6-7. The mixture was concentrated under reduced pressure to remove THF, and the residue was purified by liquid-phase separation to obtain CR202AK (35.0 mg, purity: 94.84%, yield: 17.8%). [ka]

[0391] In a 25 mL jacket, CR202AK (85.0 mg, 57.8 μmol, 1.0 eq.) and CR202W4 (104.9 mg, 69.4 μmol, 1.2 eq.) were dissolved in DMF (6.4 mL) and water (2.5 mL) under light-protected conditions, magnetically stirred, and protected with nitrogen gas. DIEA was added to adjust the pH to 8, and CuBr (8.3 mg, 57.8 μmol, 1.0 eq.) was added. The reaction was allowed to proceed at 25 °C for 2 hours, and samples were taken to control the process until the reaction was complete. The reaction mixture was purified by preparative liquid phase separation. Salts were removed with purified water to give CR202AL (23.7 mg, purity: 94.48%, yield: 13.9%), which was then purified with [M+4H]. 4+ / 4=746.40.

[0392] 4.13 Synthesis of CR202W1 [ka]

[0393] In a 100 mL three-neck flask, sulfonyl chloride (9.65 g, 71.5 mmol, 1.0 eq.) was diluted with anhydrous THF (15 mL), magnetically stirred, and cooled to -70°C under nitrogen gas protection. Neopentyl alcohol (6.3 g, 71.5 mmol) and pyridine (5.66 g, 71.5 mmol) were dissolved in anhydrous THF (30 mL) and slowly added dropwise to the three-neck flask over 1 hour. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 2 hours. The solid was removed by filtration to obtain neopentyl sulfurochloridate. [ka]

[0394] In a 250 mL three-neck flask, CR202BE (6 g, 35.9 mmol), TEA (7.27 g, 71.8 mmol), and DMAP (4.4 g, 36.0 mmol) were dissolved in THF (120 mL) and cooled to 0 °C under nitrogen gas protection. A THF solution of neopentyl sulfurochloridate (71.5 mmol) was added dropwise. The mixture was stirred at 0 °C for 1 h and quenched by the addition of 1 M HCl (150 mL) and ethyl acetate (150 mL). The layers were separated, and the organic phase was washed successively with saturated NaHCO (100 mL) and 10% NaSO (100 mL), dried over anhydrous NaSO, and filtered. The filtrate was concentrated and applied to a silica gel column (n-heptane / ethyl acetate = 10 / 1 to 5 / 1) to give CR202BN (6.4 g, purity: 98.49%, yield: 50%) as a pale yellow solid. H-NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.76 (d, J = 2.0 Hz, 1H), 8.39 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 4.36 (s, 2H), 0.97 (s, 9H). [ka]

[0395] In a 100 mL jacket, CR202BN (3 g, 9.45 mmol) and bromopropyne (1.69 g, 14.2 mmol) were dissolved in THF (40 mL) and protected with nitrogen gas. The mixture was cooled to -10 °C and zinc powder (927 mg, 14.2 mmol) was added. The condensation was stopped, the mixture was allowed to warm to room temperature (10–20 °C), and the mixture was stirred overnight. The reaction was sampled and monitored for complete completion. The mixture was quenched by adding saturated NH4Cl (200 mL) and extracted with ethyl acetate (150 mL × 2). The combined organic phase was washed with water (150 mL) and 10% Na2SO4 (150 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated and purified by column chromatography (n-heptane / ethyl acetate = 6 / 1 to 4 / 1) to give CR202BG (5.0 g, purity: 94.86%, yield: 64%) as a yellow oil. H-NMR (400 MHz, CDCl) δ 8.09 (d, J = 2.0 Hz, 1H), 7.71 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 4.97 (t, J = 6.4 Hz, 1H), 4.22 (s, 2H), 2.73-2.60 (m, 2H), 2.13 (t, J = 6.4 Hz, 1H), 1.02 (s, 9H). [ka]

[0396] CR202BG (5 g, 14.0 mmol) and CR202BS (2.44 g, 14.0 mmol) were dissolved in DMF (50 mL) and water (15 mL) and protected with nitrogen gas. DIEA (2.3 mL) was added to adjust the pH to 8. CuBr (2.0 g, 13.9 mmol) was added and the mixture was stirred at 25 °C for 1 h. Samples were taken and the reaction was controlled in process until complete. The reaction mixture was purified by preparative liquid phase separation to give CR202BH (7.2 g, purity: 86.69%, yield: 59%) as a colorless oil. LCMS: [M+H]+ = 532.3. [ka]

[0397] CR202BH (7.2 g, 9.48 mmol), CR194Z0 (1.8 g, 9.52 mmol), and DIEA (3.65 g, 28.2 mmol) were dissolved in DMF (50 mL) and protected with nitrogen gas. HATU (3.6 g, 9.47 mmol) was added. The mixture was stirred at 14 °C for 2 h, and the reaction was sampled and in-process controlled until complete. Water (150 mL) and ethyl acetate (100 mL x 2) were added for extraction. The combined organic phase was washed with water (100 mL) and 10% Na2SO4 (100 mL) successively, dried over anhydrous Na2SO4, and then filtered. The filtrate was purified by column chromatography (DCM / MeOH = 30 / 1 to 20 / 1) to give CR202BJ (6.4 g, purity: 94.11%, yield: 90%) as a yellow oil. [M+H] + =703.4 (4.43 minutes). H-NMR (400 MHz, DMSO-d6) δ 8.09 (d, J = 2.0 Hz, 1H), 7.80 - 7.78 (m, 2H), 7.64 (d, J = 8.4 Hz, 1H), 7.61 (t, J = 5.6 Hz, 1H), 5.00 (t, J = 5.6 Hz, 1H), 4.46 (t, J = 5.6 Hz, 2H), 4.29 (s, 2H), 3.88 (s, 2H), 3.76 (t, J = 5.2 Hz, 2H), 3.62 - 3.59 (m, 6H), 3.51 - 3.39 (m, 9H), 3.26 (q, J = 6.0 Hz, 2H), 3.02 (d, J = 6.0 Hz, 2H), 0.96 (s, 9H). [ka]

[0398] CR202BJ (6.4 g, 9.11 mmol), NPC (5.5 g, 18.1 mmol), and DIEA (1.77 g, 13.7 mmol) were dissolved in DMF (30 mL) and protected with nitrogen gas. The mixture was stirred at 25°C for 1.5 hours, and sampling was performed to ensure complete reaction. The reaction mixture was purified by preparative liquid phase separation to give CR202Z4 (6.0 g, purity: 97.24%, yield: 72%) as a colorless gel. [M+H] + =868.3. H-NMR (400 MHz, DMSO-d6) δ 8.30 (d, J = 9.2 Hz, 2H), 8.25 (d, J = 2.0 Hz, 1H), 7.94 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.86 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.52 (d, J = 9.2 Hz, 2H), 6.17 (t, J = 6.4 Hz, 1H), 4.48 (t, J = 4.8 Hz, 2H), 4.31 (s, 2H), 3.87 (s, 2H), 3.76 (t, J = 4.8 Hz, 2H), 3.61 - 3.58 (m, 6H), 3.46 - 3.36 (m, 10H), 3.24 (q, J = 6.0 Hz, 2H), 0.95 (s, 9H). [ka]

[0399] CR20274 (100 mg, 113 μmol), CR202Z4 (196 mg, 226 μmol), HOBT (3 mg, 22.2 μmol), and DIEA (29.2 mg, 226 μmol) were dissolved in DMF (1.5 mL) and protected with nitrogen gas. The mixture was stirred at 25°C for 21 hours, and samples were taken to control the process until the reaction was complete. The reaction mixture was purified by preparative liquid phase separation to give CR202BK (82 mg, purity: 95.21%, yield: 53%). LCMS: [M / 2+H] + =806.9 (RT=5.45 min). [ka]

[0400] CR202BK (117 mg, 101 μmol) was dissolved in DMF (3 mL), and 5 M aqueous NH4OAc (3 mL) was added. The mixture was protected with nitrogen gas. The mixture was stirred at 45°C for 17 hours, and samples were taken to control the process until the reaction was complete. The reaction mixture was purified by preparative liquid phase separation to give CR202BL (78 mg, purity: 99.54%, yield: 70%). LCMS: [MH] - =1540.8. [ka]

[0401] CR202BL (70 mg, 45.4 μmol) and CR202W4 (83 mg, 54.8 μmol) were dissolved in DMF (6 mL) and water (2 mL) under light-protected conditions, magnetically stirred, and protected with nitrogen gas. DIEA (250 μL) was added to adjust the pH to 8, followed by CuBr (6.5 mg, 45.4 μmol) and reaction at 25°C for 1 hour. CR202W4 (18 mg, 11.9 μmol), DIEA (100 μL), and CuBr (3.2 mg, 22.7 μmol) were added and reaction was continued at 25°C for 1 hour. Sampling was performed to control the process until the reaction was complete. The reaction mixture was purified by preparative liquid phase separation. Salts were removed with purified water to give CR202W1 (33 mg, purity: 94.46%, yield: 19%). LCMS: [M+4H] 4+ / 4=764.94.

[0402] 4.14 Synthesis of CR202GM [ka]

[0403] SM1 (500 mg, 3.53 mmol, 1.0 eq.) was dissolved in DCM (50.0 mL) and cooled to 0 °C under nitrogen gas protection. SM2 (307 mg, 3.53 mmol, 1.0 eq.) was added and stirred at 0 °C for 30 min. TEA (714 mg, 7.06 mmol, 2.0 eq.) and SM3 (371 mg, 3.53 mmol, 1.0 eq.) were added and stirred at 5 °C for 1 h. The reaction was monitored for complete completion by HPLC. NPC (2.15 g, 7.07 mmol, 2.0 eq.) and TEA (358 mg, 3.54 mmol, 1.0 eq.) were added and stirred at 25 °C for 3 h. The reaction mixture was concentrated and subjected to silica gel column chromatography (n-heptane / ethyl acetate / acetic acid = 50 / 50 / 1) to give CR202HD (1.2 g, purity: 98.47%, yield: 74%) as a colorless viscous oil. LCMS: [M+H] + =463.4. 1 H-NMR (400 MHz, CDCl3) δ 8.30 - 8.26 (m, 2H), 8.02 (s, 1H), 7.43 - 7.40 (m, 2H), 5.72 (t, J = 6.0 Hz, 1H), 4.46 - 4.44 (m, 2H), 4.33 (t, J = 4.8 Hz, 2H), 3.79 - 3.77 (m, 2H), 3.67 (t, J = 4.8 Hz, 2H), 3.53 (t, J = 4.8 Hz, 2H), 3.35 (q, J = 4.8 Hz, 2H). [ka]

[0404] CR202HD (402 mg, 0.869 mmol, 1.1 eq.) was dissolved in DMF (3 mL), CR19154 (300 mg, 0.791 mmol, 1.0 eq.) and TEA (240 mg, 2.37 mmol, 3.0 eq.) were added, and the mixture was stirred at 25 °C for 17 h. The reaction was controlled by HPLC until complete. The reaction mixture was concentrated and purified by column chromatography (DCM / MeOH / AcOH = 100 / 10 / 1) to give CR202HE (560 mg, purity: 100%, yield: 90%) as a white foamy solid. LCMS: [M+H] + =703.4. 1 H-NMR (DMSO-d6, 400 MHz): δ 9.92 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.8 Hz, 2H), 5.98 (t, J = 5.6 Hz, 1H), 5.40 (s, 2H), 5.08 (s, 1H), 4.42 - 4.37 (m, 3H), 4.07 - 4.03 (m, 4H), 3.88 (dd, J = 8.4 Hz, 6.8 Hz, 1H), 3.56 - 3.54 (m, 2H), 3.48 - 3.46 (m, 4H), 3.01 - 2.93 (m, 4H), 2.01 - 1.92 (m, 1H), 1.74 - 1.30 (m, 4H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H). [ka]

[0405] CR202HE (460 mg, 1.21 mmol, 1.0 eq.) was dissolved in DMF (6 mL), NPC (405 mg, 1.33 mmol, 1.1 eq.) and DIEA (235 mg, 1.82 mmol, 1.5 eq.) were added, and the mixture was stirred at 25 °C for 17 h. The reaction was controlled by HPLC until complete. The reaction mixture was directly purified by reverse-phase preparative liquid separation to give CR202HF (230 mg, purity: 98.59%, yield: 37%) as a white solid. LCMS: [M+H] + =868.3. 1 H-NMR (DMSO-d6, 400 MHz): δ 11.37 (s, 1H), 10.14 (s, 1H), 8.36 - 8.32 (m, 2H), 8.13 (d, J = 7.6 Hz, 1H), 7.84 (t, J = 5.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.61 - 7.58 (m, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.8 Hz, 1H), 6.03 (s, 1H), 5.27 (s, 2H), 4.47 - 4.42 (m, 1H), 4.25 (t, J = 4.8 Hz, 2H), 4.14 - 4.05 (m, 2H), 3.95 - 3.91 (m, 1H), 3.61 - 3.59 (m, 4H), 3.52 - 3.51 (m, 2H), 3.13 - 2.96 (m, 4H), 2.04 - 1.96 (m, 1H), 1.78 - 1.58 (m, 2H), 1.54 - 1.34 (m, 2H), 0.90 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H). [ka]

[0406] CR202HF (200.0 mg, 0.23 mmol), CR20274 (free form, 220.0 mg, 0.23 mmol), HOBt (6.4 mg, 0.05 mmol), and DIPEA (89.0 mg, 0.69 mmol) were added sequentially to DMF (4.0 mL). The reaction was carried out under nitrogen gas protection at room temperature (26.0 °C) for 16 hours. The reaction was controlled by HPLC until complete completion. The reaction mixture was purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN) and lyophilized to give CR202HG (130.0 mg, purity: 97.81%, yield: 35.0%). LCMS: [M+H] + =1612.93. [ka]

[0407] Under nitrogen gas protection, CR202HG (120.0 mg, 0.074 mmol) and CR202W4 (134.0 mg, 0.088 mmol) were added to a mixture of DMF (6 mL) and water (2 mL) and stirred until dissolved, protected from light. DIPEA was added dropwise and the pH was adjusted to 8. CuBr (13.0 mg, 0.074 mmol) was added and the reaction mixture was allowed to react at room temperature (25.0 °C) for 2.5 h. The reaction was then monitored for complete completion by HPLC. The reaction mixture was directly purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN). The purified product was desalted with purified water and lyophilized to give CR202GM (52.1 mg, purity: 95.83%, yield: 22.4%). LCMS: [M+4H] 4+ =782.49.

[0408] 4.15 Synthesis of CR202HJ [ka]

[0409] CR202X6 (1.00 g, 1.39 mmol), Fmoc-ethylenediamine (0.39 g, 1.39 mmol), and DMF (10 mL) were added to a 100 mL single-neck flask and stirred until dissolved. DIEA (0.22 g, 1.67 mmol) was added and the mixture was allowed to react at 20.7 °C for 0.5 h. The reaction mixture was sampled and the process controlled until the reaction was complete. The reaction mixture was poured into pre-cooled (0-10 °C) MTBE (80 mL), kept warm, stirred for 5 min, and centrifuged. The precipitated solid was washed with cold MTBE (8 mL x 5) to obtain CR202MH (1.12 g, purity: 93.66%, yield: 93.8%) as a white solid. [ka]

[0410] CR202MH (1.08 g, 1.26 mmol) was added and dissolved in DMF (7 mL), and a solution of DBU (0.19 g, 1.26 mmol) in DMF (3 mL) was added dropwise. The reaction was carried out at 22.2 °C for 0.5 hours. Sampling was carried out during the reaction, and the process was controlled until the reaction was complete. The reaction solution was purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN) and lyophilized to obtain CR202MJ (913.7 mg, purity: 99.8%). [ka]

[0411] CR202MJ (0.83 g, 1.30 mmol) and NPC (0.79 g, 2.60 mmol) were added to DMF (8 mL) and completely dissolved. DIEA (0.25 g, 1.95 mmol) was added, and the mixture was allowed to react at room temperature for 0.5 hours. Sampling was performed during the reaction, and the process was controlled until the reaction was complete. The reaction mixture was purified by preparative HPLC and lyophilized to give CR202BX (254.9 mg, purity: 96.46%, yield: 24.5%). LCMS: [M+1H] + =802.4. 1H-NMR (400 MHz, DMSO) δ 10.06 (s, 1H), 8.36 (d, J = 7.4 Hz, 1H), 8.28 (d, J = 7.2 Hz, 2H), 8.10 (s, 1H), 7.61 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 9.0 Hz, 1H), 7.43 (d, J = 9.1 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 6.01 (s, 1H), 5.43 (s, 1H), 4.99 (s, 2H), 4.42 (dd, J = 13.4, 7.8 Hz, 1H), 4.38-4.32 (m, 1H), 3.99 (s, 2H), 3.70-3.61 (m, 6H), 3.45-3.42 (m, 3H), 3.17 (s, 4H), 3.01 (dd, J = 22.5, 9.5 Hz, 2H), 2.07-1.97 (m, 1H), 1.77-1.56 (m, 2H), 1.53-1.32 (m, 2H), 0.90 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H). [ka]

[0412] CR202BX (190.0 mg, 0.24 mmol), CR20274 (free form, 251.4 mg, 0.28 mmol), HOBt (6.4 mg, 0.05 mmol), and DIPEA (91.9 mg, 0.71 mmol) were added sequentially to DMF (3.8 mL). The mixture was reacted at room temperature under nitrogen gas protection for 8 hours. The reaction mixture was purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN) and lyophilized to give CR202HH (230.0 mg, purity: 98.27%, yield: 59.7%). LCMS: [M+H] + =1546.63. [ka]

[0413] Under nitrogen gas protection, CR202HH (210.0 mg, 0.14 mmol) and CR202W4 (240.0 mg, 0.16 mmol) were added to a mixture of DMF (12 mL) and water (4 mL) and stirred in the dark to dissolve. DIPEA was added dropwise to adjust the pH to 8, and CuBr (18.9 mg, 0.14 mmol) was added. The reaction was allowed to proceed at room temperature (25.0 °C) for 5 hours. Samples were taken during the reaction, and the reaction was controlled until complete. The reaction mixture was directly purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN). The product purified by preparative HPLC was desalted with purified water and lyophilized to give CR202HJ (215.7 mg, purity: 96.69%, yield: 47.3%). LCMS: [M+3H] 3+ =1021.20.

[0414] 4.16 Synthesis of CR202FU [ka]

[0415] Under nitrogen gas protection, lithium aluminum tetrahydride (0.47 g, 12.24 mmol) was added to THF (3 mL) and cooled to 0 °C. A solution of CR202MA (1.50 g, 8.28 mmol) in THF (15 mL) was added dropwise and kept at 0 °C for 2 hours. The reaction was sampled and monitored by TLC until complete. The reaction mixture was poured into ice water (5 mL), 15% sodium hydroxide solution (1 mL) was added dropwise, stirred at room temperature for 15 minutes, and extracted with ethyl acetate (15 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography (PE / EA = 1:1) to give CR202MB (1.15 g, purity: 95.03%, yield: 86.15%). LCMS: [M+H] + =154.6.

[0416] Under nitrogen gas protection, CR202MB (1.15 g, 7.50 mmol) was added to DCM (12 mL), and imidazole (1.53 g, 22.52 mmol) and TBSCl (1.69 g, 11.26 mmol) were added sequentially. The reaction was allowed to proceed at room temperature for 2 hours. The reaction was sampled and monitored by TLC until complete. The reaction mixture was quenched by pouring into ice water (5 mL) and extracted with DCM (15 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography (DCM / MeOH = 10:1) to give CR202MC (1.50 g, purity: 99.15%, yield: 74.07%). 1 H-NMR (400 MHz, DMSO) δ 6.73 (s, 1H), 6.62 (d, J = 7.9 Hz, 1H), 6.57 (d, J = 7.9 Hz, 1H), 4.60 (s, 2H), 4.54 (s, 2H), 3.74 (s, 3H), 0.88 (s, 9H), 0.05 (s, 6H).

[0417] CR202MD (1.66 g, 3.73 mmol) was added to DMF (15 mL), the temperature was controlled at 25 °C, HATU (1.99 g, 5.22 mmol) was added, and the mixture was stirred for 15 minutes. CR202MC (1.50 g, 5.59 mmol) and DIEA (0.67 g, 5.22 mmol) were added, and the reaction was sampled and monitored until complete. The reaction mixture was poured into ice water (75 mL), and extracted with DCM (30 mL x 3). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product CR202ME was used directly in the next step of the reaction. LCMS: [M+H] + =695.6. [ka]

[0418] CR202ME (2.78 g, 3.73 mmol) was added to THF (30 mL), TBAF (2.09 g, 7.46 mmol) was added, and the mixture was reacted at 25° C. for 1 hour. The reaction was sampled and monitored by TLC until the reaction was complete. The reaction mixture was concentrated to dryness, and the crude product CR202MG was used directly in the next step of the reaction. [ka]

[0419] The crude CR202MG was added to DMF (30 mL) and completely dissolved. Bis(4-nitrophenyl)carbonate (NPC, 2.26 g, 7.44 mmol) and DIPEA (0.72 g, 5.58 mmol) were added and the reaction was allowed to proceed at 20-25°C for 1 hour. Sampling was performed during the reaction and the reaction was monitored until complete. The reaction mixture was purified by preparative HPLC and lyophilized to give CR202BW (1.35 g, purity: 98.5%, yield: 48.7%) as a white solid. LCMS: [M+H] + =746.6, 1 H-NMR (400 MHz, DMSO) δ 9.17 (s, 1H), 8.46-8.48 (d, 1H), 8.31-8.33 (d, 1H), 8.05-8.07 (d, 1H), 7.57-7.59 (d, 1H), 7.46-7.49 (d, 1H), 7.17 (s, 1H), 7.02-7.07 (d, 1H), 6.00 (bs,1H), 5.26 (s, 2H), 4.49-4.54 (m, 2H), 4.33-4.37 (dd, 2H), 3.97 (s, 2H), 3.86 (s, 3H), 3.62-3.65 (m, 6H), 3.40-3.43(t, 2H), 2.97-3.00 (t, 2H),2.01-2.03(m, 1H), 1.59-1.61 (m, 2H), 1.42-1.45 (m, 2H), 0.88-0.90 (d, 3H), 0.82-0.84 (d, 3H). [ka]

[0420] Under nitrogen gas protection, CR202BW (250.1 mg, 0.34 mmol), CR20274 (free form, 326.0 mg, 0.37 mmol), HOBt (9.0 mg, 0.07 mmol), and DIPEA (140.0 mg, 1.02 mmol) were added sequentially to DMF (5.0 mL) and reacted at room temperature for 16 hours. The reaction mixture was purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN) and lyophilized to give CR202HP (236.2 mg, purity: 96.14%, yield: 47.3%). LCMS: [M+H] + =1490.96. [ka]

[0421] Under nitrogen gas protection, CR202HP (211.9 mg, 0.15 mmol) and CR202W4 (268.0 mg, 0.148 mmol) were added to a mixture of DMF (12 mL) and water (4 mL) and stirred until completely dissolved. The pH was adjusted to 8 by dropwise addition of DIPEA. CuBr (6.4 mg, 0.05 mmol) was added and the mixture was allowed to react at room temperature (23.7 °C) for 5 h. The reaction mixture was directly purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN). The purified product was desalted with pure water and lyophilized to give CR202FU (132.4 mg, purity: 98.67%, yield: 28.5%). LCMS: [M+4H] 4+ =751.89.

[0422] 4.17 Synthesis of CR202GQ [ka]

[0423] For the synthesis of CR202DS, refer to the preparation of Ligand-01: LCMS: [M+H] + =600.4, 1H-NMR (400 MHz, DMSO) δ 8.29 (d, J = 7.2 Hz, 1H), 8.10 (t, J = 5.8 Hz, 1H), 7.41 (d, J = 9.1 Hz, 1H), 4.49-4.40 (m, 1H), 4.35-4.23 (m, 2H), 3.94 (s, 2H), 3.78 (dd, J = 17.5, 6.0 Hz, 1H), 3.73-3.54 (m, 9H), 3.45-3.37 (m, 2H), 2.95 (s, 2H), 2.08-1.78 (m, 5H), 1.73-1.63 (m, 1H), 1.56-1.32 (m, 3H), 0.81 (dd, J = 19.2, 6.7 Hz, 6H)

[0424] Under nitrogen gas protection, CR202DS (350.0 mg, 0.58 mmol) was added to DMF (4.0 mL) and stirred until completely dissolved. The mixture was then cooled to 5 °C. HATU (220.5 mg, 0.58 mmol) and DIPEA (224.9 mg, 1.74 mmol) were added and the mixture was allowed to react for 0.5 hours. CR20274 (free form, 541.8 mg, 0.61 mmol) was added and the mixture was allowed to warm to room temperature (27.0 °C) and reacted for 16 hours. The reaction mixture was purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN) and lyophilized to give CR202GP (204.9 mg, purity: 91.88%, yield: 23.8%). LCMS: [M+H] + =1465.32. [ka]

[0425] Under nitrogen gas protection, CR202GP (200.0 mg, 0.14 mmol) and CR202W4 (249.5 mg, 0.16 mmol) were added to a mixture of DMF (12 mL) and water (4 mL) and stirred until completely dissolved. The mixture was allowed to warm to room temperature (23.0 °C). DIPEA was added dropwise to adjust the pH to 8. CuBr (20.0 mg, 0.14 mmol) was added and the mixture was allowed to react for 1.5 h. The reaction mixture was directly purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN). The purified product was desalted with pure water and lyophilized to give CR202GQ (156.7 mg, purity: 95.61%, yield: 36.6%). LCMS: [M+4H] 4+ =745.93.

[0426] 4.18 Synthesis of CR202LB [ka]

[0427] 5-Formylsalicylic acid (5.03 g, 2.96 mmol) was added to thionyl chloride (75 mL) and heated in an oil bath (82 °C) to react overnight. The reaction mixture was then cooled to 50 °C and concentrated under vacuum at 35 °C to dryness. Dry THF (30 mL × 3) was added, and the remaining thionyl chloride was removed by distillation three times to obtain 5-formylsalicylic acid chloride (yellow solid).

[0428] Azide-triethyleneglycol-amine (4.30 g, 1.97 mmol) was added to THF (80 mL), cooled to 2°C, and DIPEA (10 mL) was added. 5-Formylsalicylic acid chloride was dissolved in dry THF (20 mL) and cooled to 2-10°C under temperature control. The solution was added dropwise to the reaction mixture (pH = 5-6). 1 mL of DIPEA (pH = 8) was added, the mixture was heated to 20°C, and stirred overnight. Water (80 mL) was added to the reaction mixture, and the mixture was extracted with DCM (100 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a crude oil. The crude product was purified by silica gel column chromatography (DCM: EtOAc = 10:1 to 2:1) to give CR202KV (0.8 g, purity: ~90% (TLC), yield: 15%). LC-MS: [M+H] + :367.06. 1 H-NMR (400 MHz, DMSO) δ 9.71 (s, 1H), 8.37 (d, J = 2.1 Hz, 1H), 7.78 (dd, J = 8.6, 2.2 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H), 3.60 - 3.48 (m, 10H), 3.46 (dd, J = 7.3, 4.2 Hz, 4H), 3.38-3.26 (m, 2H).

[0429] CR194KV (0.80 g, 2.18 mmol) was dissolved in DCM (40 mL) and stirred. The mixture was cooled to 0 °C in an ice-salt bath. DIPEA (1.0 mL, 5.45 mmol) and CBr (2.79 g, 8.73 mmol) were added and stirred until completely dissolved. Dibenzyl phosphite (1.14 g, 4.36 mmol) was added dropwise. After the addition was complete, the ice-salt bath was removed and the reaction mixture was cooled to an internal temperature of 5 °C in an ice bath. 5% aqueous NaHPO (30 mL) was added dropwise and extracted with DCM (50 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated in vacuo at 35 °C to give an orange oil, which was purified by column chromatography (DCM: EtOAc = 10:1 to 2:1) to give CR202KW (1.3 g, 60% yield). [M+H] + :627.19, 1 H-NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 8.34 (s, 1H), 7.85 (dd, J = 8.5, 2.1 Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.30 (dt, J = 10.0, 5.1 Hz, 10H), 7.20 (s, 1H), 5.12 (d, J = 7.5 Hz, 4H), 3.63-3.51 (m, 14H), 3.30 (t, J = 5.0 Hz, 2H). [ka]

[0430] CR202KW (1.30 g, 2.07 mmol) was added to a DCM / MeOH mixture (50 mL, V / V = 1 / 1) and dissolved until clear. The internal temperature was cooled to 0 °C in an ice-salt bath. NaBH4 (0.16 g, 4.14 mmol) was added in portions and stirred at 0 °C for 30 min. Water (50 mL) and DCM (50 mL) were added to the reaction mixture, the organic phase was separated, and the aqueous phase was further extracted with DCM (50 mL × 2). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under vacuum at 35 °C to give crude CR202KX (1.29 g, purity: 85.30%, yield: 99.2%). The crude product was directly used in the next step without further purification. LC-MS: [M+H] + :629.14, 1 H-NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.37-7.26 (m, 13H), 5.10 (d, J = 8.5 Hz, 4H), 4.66 (s, 2H), 3.57 (d, J = 11.9 Hz, 4H), 3.30 (t, J = 5.0Hz, 2H). [ka]

[0431] CR202KX (1.09 g, 1.73 mmol) was added to acetonitrile (20 mL) and dissolved to a clear solution. The mixture was cooled in an ice bath, and TEA (0.25 mL, 1.73 mmol) was added. A solution of bis(pentafluorophenyl)carbonate (1.06 g, 2.6 mmol) in acetonitrile (2.5 mL) was then added dropwise to the reaction mixture. The ice bath was removed, and the mixture was stirred for 1.5 hours. After completion of the reaction, the reaction mixture was cooled in an ice bath and extracted with saturated aqueous sodium bicarbonate (30 mL) and ethyl acetate (70 mL). The organic phase was collected. The organic phase was washed with water (1 x 40 mL) and saturated sodium chloride (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo at 30 °C to give an oil. The oil was purified by column chromatography (n-heptane:ethyl acetate = 10:1 to 1:1) to give CR202KY (1.02 g, purity: 96.53%, yield: 70.3%). LC-MS: [M+H] + :839.12, H-NMR: 1 H-NMR (400 MHz, CDCl3) δ 7.91 (s, 1H), 7.43-7.26 (m, 12H), 5.28 (s, 2H), 5.11 (d, J = 8.9 Hz, 4H), 3.66-3.47 (m, 14H), 3.30 (t, J = 5.0 Hz, 2H). [ka]

[0432] CR20274 (0.57 g, 0.65 mmol) was added to DMF (10 mL) and dissolved to a clear solution, followed by cooling in an ice bath. HOBT (88.0 mg, 0.65 mmol) and DIPEA (140 μL, 0.78 mmol) were added sequentially. CR202KY (0.66 g, 0.78 mmol) was dissolved in DMF (2.5 mL) and added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed and the mixture was stirred for 1 hour. The reaction mixture was purified and lyophilized to give CR202KZ (0.79 g, purity: 93.01%, yield: 80.0%). LC-MS: [M+2H] 2+ :769.92. [ka]

[0433] A 50% solution of TFA in dichloromethane (14 mL, V / V = 1 / 1) was cooled to 0 °C, and CR202KZ (0.40 g, 0.26 mmol) was added and stirred until completely dissolved. The mixture was then heated to 24 °C and stirred for 2 hours. The reaction mixture was concentrated in vacuo at 27 °C to give CR202LA (0.36 g), which was directly used in the next step according to the theoretical yield. LC-MS: [M+2] 2+ :680.25. [ka]

[0434] CR202LA (0.36 g, 0.26 mmol) was added to a DMF:HO (12 mL, V / V = 1 / 1) mixed solvent and cooled to 4 °C. DIPEA (1.7 mL) was added dropwise to adjust the pH to 8. CR202W4 (0.39 g, 0.26 mmol) and CuBr (37.1 mg, 10.26 mmol) were added sequentially. The internal temperature was raised to 24.2 °C, and the mixture was stirred for 1.5 hours. The reaction mixture was sampled and monitored for completion by HPLC. The reaction mixture was purified by preparative HPLC and lyophilized to give CR202LB (0.29 g, purity: 98.34%, two-step yield: 38.8%).

[0435] 4.19 Synthesis of CR202HV [ka]

[0436] Under nitrogen gas protection, cesium carbonate (53.18 g, 179.48 mmol) was added to THF (290 mL), and thioacetic acid (13.66 g, 179.48 mmol) was added with stirring. The mixture was allowed to react at 25 °C for 30 minutes. After that, (s)-(-)-methyl 2-chloropropionate (20.00 g, 163.16 mmol) was added and the mixture was allowed to react for 1 hour. The reaction mixture was filtered, and the filtrate was added to water (1.2 L) and extracted twice with MTBE (1 L x 2). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness to give CR202KQ (24.34 g, purity: 90.09%, yield: 92.00%). [M+H] + =163.11. 1 H-NMR (400 MHz, CDCl3) δ 4.16 (q, J = 7.3 Hz, 1H), 3.66 (s, 4H), 2.28 (s, 3H), 1.43 (d, J = 7.3 Hz, 3H).

[0437] Under nitrogen gas protection, LiAlH4 (9.36 g, 246.64 mmol) was added to THF (150 mL) and stirred to dissolve, then the temperature was lowered to 0 °C. At the same time, CR202KQ (10.00 g, 61.65 mmol) was dissolved in THF (50 mL). This solution was slowly added dropwise to the LiAlH4 solution. After the addition was complete, the temperature was raised to room temperature and the reaction was allowed to proceed for 1.5 hours. The reaction was then monitored by HPLC until complete completion. The temperature was then lowered to 0 °C, and 2 M HCl solution was slowly added dropwise until no further bubbles were generated. Stirring was continued for 15 minutes, followed by filtration. The filter cake was washed once with THF (100 mL). The combined filtrate was discarded. The filter cake was soaked in methanol (300 mL) and then filtered. This procedure was repeated once. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give CR202KR (9.0104 g, purity: 92.21%, yield: 158.63%, note: the yield exceeded 100% due to the relatively large amount of residual methanol).

[0438] 2,2'-Dithiodipyridine (10.37 g, 47.07 mmol) was added to methanol (40 mL), and CR202KR (8.6763 g, 94.14 mmol) was added. The reaction was allowed to proceed at 25 °C for 2 hours. HPLC analysis confirmed the reaction was complete. The reaction mixture was concentrated to give a crude product, which was purified by column chromatography (n-Heptane / EA = 6 / 1) to give CR202KS (2.5298 g, purity: 94.35%, yield: 26.7%). LCMS: [M+H] + =201.97. 1 H-NMR (400 MHz, DMSO) δ 8.43 (m, 1H), 7.81 (m, 2H), 7.22 (td, J = 5.3, 2.8 Hz, 1H), 5.08 (t, J = 5.7 Hz, 1H), 3.46 (m, 2H), 3.05 (dd, J = 13.0, 6.5 Hz, 1H), 1.22 (t, J = 6.5 Hz, 3H). [ka]

[0439] CR202KS (1.20 g, 5.96 mmol) was added to DCM (12 mL), followed by p-nitrophenyl chloroformate (1.44 g, 7.14 mmol) and pyridine (0.49 g, 6.26 mmol). The reaction was allowed to proceed at 25°C for 1 hour, and the process was controlled by HPLC until the reaction was complete. The reaction mixture was concentrated to give the crude product, which was then purified by column chromatography (n-Heptane / EA = 10 / 1) to give CR202KT (2.01 g, purity: 87.42%, yield: 92.2%). LCMS: [M+H] + =366.98, 1H-NMR (400 MHz, DMSO) δ 8.44 (d, J = 4.5 Hz, 1H), 8.31 (m, 2H), 7.81 (m, 2H), 7.55 (m, 2H), 7.24 (ddd, J = 6.5, 4.8, 1.5 Hz, 1H), 4.34 (qd, J = 11.2, 6.0 Hz, 2H), 3.46 (dt, J = 13.1, 6.5 Hz, 1H), 1.33 (d, J = 7.0 Hz, 3H). [ka]

[0440] CR202KT (314.0 mg, 0.86 mmol) was added to DMF (5 mL), followed by CR20274 (500.0 mg, 0.57 mmol), HOBT (76.4 mg, 0.57 mmol), and DIPEA (146.0 mg, 1.13 mmol). The reaction was carried out at 25 °C for 16 h, with in-process control until the reaction was complete. After purification by preparative HPLC and lyophilization, CR202KU (0.4567 g, purity: 88.69%, yield: 72.69%) was obtained. [ka]

[0441] Under nitrogen gas protection, CR19208 (205.5 mg, 0.13 mmol) was added to a 50% DMF / HO solution (8 mL, V / V = 1 / 1), and 0.1 M NaHCO solution was added dropwise until the reaction solution reached pH 7. CR202KU (150.0 mg, 0.13 mmol) was dissolved in THF (3 mL) and added dropwise to the reaction solution (the reaction solution became cloudy). After adding 10 mL of 50% DMF / HO solution, 10 mL of DMF, and 5 mL of 0.1 M NaHCO solution (the reaction solution became clear), the reaction was allowed to proceed at 25 °C for 0.5 h, and the process was monitored by HPLC until the reaction was complete. The reaction mixture was purified by preparative HPLC (mobile phase: 0.1% TFA / HO and 0.1% TFA / ACN) and lyophilized to give CR202HV (99.5 mg, purity: 98.73%, yield: 29.26%). LCMS: [M+3H] 3+ =841.76.

[0442] 4.20 Synthesis of CR202GC [ka]

[0443] CR20274 (313.9 mg, 0.3551 mmol) was added to a 25 mL single-neck flask. Under nitrogen gas protection, THF (10.0 mL) was added and the mixture was cooled to 0 °C. Triphosgene (42.2 mg, 0.1420 mmol) was added, at which point a white solid appeared. DIEA was added dropwise until the solid completely dissolved, and the mixture was heated to 0 °C and reacted for 40 min. Another 25 mL single-neck flask was charged with CR202JC (191.6 mg, 0.3551 mmol) and THF (2.0 mL). The mixture was cooled to 0 °C, and then NaH (85.2 mg, 2.1306 mmol) was added and stirred until no more bubbles were generated. The CR20274 reaction solution was added to the CR202JC reaction solution. After the addition was complete, the mixture was gradually returned to room temperature and reacted for 30 min. The reaction was then monitored by HPLC until complete. The reaction mixture was purified by preparative HPLC to obtain 44.2 mg of CR202JD (purity: 80.42%, yield: 8.6%) as a white solid. [ka]

[0444] CR202JD (40.0 mg, 0.0276 mmol, 1.0 eq.) and CR202W4 (41.8 mg, 0.0276 mmol, 1.0 eq.) were weighed and placed in a 50 mL jacketed bottle. A DMF / HO solution (2.0 mL, V / V = 3 / 1) was added, and the temperature was controlled at 25 °C under light-protected conditions. DIEA was added dropwise to the reaction solution until pH = 8, and CuBr (4.5 mg, 0.0276 mmol, 1.0 eq.) was added. The mixture was kept at 25 °C and stirred for 10 minutes. 20 μL of the reaction solution was diluted to 300 μL with ACN / MeOH / HO, and the reaction was monitored for completion by HPLC. The reaction mixture was refrigerated at 2 to 8°C for 2 hours and purified by preparative HPLC to give CR202GC as a white solid (22.0 mg, purity: 98.53%).

[0445] 4.21 Synthesis of CR202JG [ka]

[0446] Compound 8 (20.0 g, 226 mmol, 24.4 mL, 1.40 eq.) and pyridine (12.8 g, 162 mmol, 13.1 mL, 1.00 eq.) were dissolved in DCM (200 mL). The temperature was controlled between -78 °C and -60 °C, and the solution was slowly added dropwise to a solution of sulfonyl chloride (21.8 g, 162 mmol, 16.2 mL, 1.00 eq.) in DCM (200 mL). The reaction mixture was allowed to react at room temperature for 10 hours. The reaction mixture was concentrated to give a yellow oil, which was then distilled to give compound 1a (10.5 g, crude product) as a colorless oil. 1 H NMR: (400 MHz, CDCl3) δ: 4.18 (s, 2H), 1.06 (s, 9H). [ka]

[0447] Compound 1a (5.00 g, 40.9 mmol, 1.00 eq.), DMAP (5.00 g, 40.9 mmol, 1.00 eq.), and EtN (8.29 g, 81.8 mmol, 11.4 mL, 2.00 eq.) were dissolved in THF (70.0 mL). A solution of compound 1 (9.55 g, 51.1 mmol, 1.25 eq.) in THF (35.0 mL) was added dropwise to the above solution and reacted at room temperature for 12 hours. The reaction was quenched by adding 10% citric acid (500 mL), extracted with ethyl acetate (200 mL × 2), and the combined organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give a yellow oil, which was purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to give compound 2 (9.00 g, 32.7 mmol, purity: 99.0%, yield: 79.9%) as a colorless oil. 1H NMR: (CDCl3, 400 MHz) δ 9.94 (s, 1H), 7.91 - 7.87 (m, 2H), 7.41 (dd, J = 10.8 Hz, 2.40 Hz, 2H), 4.04 (s, 2H), 0.90 (s, 9H). [ka]

[0448] Compound 2 (9.00 g, 32.7 mmol, 1.00 eq.) and 3-bromopropyne (7.30 g, 49.0 mmol, 5.29 mL, purity: 80%, 1.50 eq.) were dissolved in DMF (135 mL), Zn (3.44 g, 52.6 mmol, 1.61 eq.) was added at -10 °C, and the mixture was allowed to react for 12 hours after warming to room temperature. The reaction was quenched by adding aqueous ammonium chloride (400 mL), extracted with ethyl acetate (250 mL x 3), the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography (PE / EA = 6 / 1) to give compound 3 (10.0 g, crude product) as a yellow oil. LCMS: [M+H] + =313.1, 1 H NMR: (CDCl3, 400 MHz) δ 7.46 (d, J = 8.80 Hz, 2H), 7.32 - 7.28 (m, 2H), 4.90 (t, J = 6.40 Hz, 1H), 4.09 (s, 2H), 2.65 - 2.62 (m, 2H), 2.09 (t, J = 2.80 Hz, 1H), 1.01 (s, 9H). [ka]

[0449] Compound 3 (6.87 g, 19.5 mmol, 1.00 eq.) and compound 3a (3.41 g, 19.5 mmol, 1.00 eq.) were dissolved in acetonitrile (60 mL) and water (18.0 mL). DIEA (2.03 g, 15.6 mmol, 2.73 mL, 0.80 eq.) and CuBr (2.81 g, 19.6 mmol, 596 μL, 1.00 eq.) were added at room temperature and the mixture was allowed to react for 0.5 hours. The reaction mixture was acidified with acetic acid to give a blue solution. This was purified by preparative HPLC (0.05% TFA system) to give compound 4 (10.7 g, 17.5 mmol, purity: 98.6%, yield: 89.6%, TFA salt). LCMS: [M+H] + =487.2, 1 H NMR:(CDCl3, 400 MHz) δ 8.01 (br s, 3H), 7.46 (br s, 2H), 7.25 (s, 2H), 4.49 - 4.46 (m, 2H), 4.10 (s, 2H), 3.85 - 3.55 (m, 10H), 3.11 (s, 3H), 1.01 (s, 9H). [ka]

[0450] Compound 4 (6.30 g, 10.3 mmol, 1.00 eq., TFA salt), compound 4a (1.62 g, 8.58 mmol, 0.83 eq.), and DIPEA (4.01 g, 31.0 mmol, 5.40 mL, 3.00 eq.) were dissolved in DMF (30.0 mL), and HATU (3.93 g, 10.3 mmol, 1.00 eq.) was added at room temperature. The mixture was allowed to react for 2 hours. The reaction mixture was purified by preparative HPLC (0.01% TFA system) to give compound 5 (3.30 g, 4.95 mmol, purity: 98.6%, yield: 47.8%). LCMS: [M+H] + =658.4, 1H NMR:(CDCl3, 400 MHz) δ 7.51 (s, 1H), 7.39 (d, J = 8.40 Hz, 2H), 7.21 - 7.20 (m, 2H), 7.04 (s, 1H), 5.00 (dd, J = 8.80 Hz, 3.20 Hz, 1H), 4.46 - 4.43 (m, 2H), 4.01 (s, 2H), 3.89 (s, 2H), 3.78 - 3.59 (m, 2H), 3.58 - 3.32 (m, 16H), 3.04 - 2.99 (m, 2H), 0.94 (s, 9H). [ka]

[0451] Compound 5 (3.50 g, 5.32 mmol, 1.00 eq.) and DIPEA (2.75 g, 21.3 mmol, 3.71 mL, 4.00 eq.) were dissolved in THF (35.0 mL), and p-nitrophenyl chloroformate (2.25 g, 11.2 mmol, 2.10 eq.) was added at room temperature. The mixture was allowed to react at room temperature for 1 hour. N-tert-butoxycarbonyl-1,2-ethylenediamine (1.71 g, 10.6 mmol, 1.67 mL, 2.00 eq.) was then added and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was purified by preparative HPLC (0.01% TFA system) to give compound 6 (3.50 g, 4.00 mmol, purity: 96.4%, yield: 75.1%). LCMS: [M+H]+ = 844.4. 1H NMR:(CDCl3, 400 MHz) δ 7.43 (s, 1H), 7.35 (d, J = 8.80 Hz, 2H), 7.24 - 7.22 (m, 2H), 7.09 (s, 1H), 5.92 - 5.89 (m, 1H), 5.77 (s, 1H), 5.18 (s, 1H), 4.48 - 4.46 (m, 2H), 4.06 (s, 2H), 3.98 (s, 2H), 3.80 - 3.66 (m, 2H), 3.65 - 3.55 (m, 14H), 3.53 (d, J = 8.40 Hz, 2H), 3.37 - 3.19 (m, 6H), 1.40 (s, 9H), 0.98 (s, 9H). [ka]

[0452] Compound 6 (3.50 g, 4.00 mmol, 1.00 eq.) was dissolved in acetonitrile (44.0 mL), and TsOH (2.41 g, 13.9 mmol, 3.50 eq.) was added at room temperature. The mixture was allowed to react at room temperature for 12 hours. The reaction mixture was concentrated to give compound 7 (4.00 g, crude product) as a yellow oil. The crude product was used directly in the next step. [ka]

[0453] Compound 7 (2.00 g, 2.18 mmol, 1.00 eq., crude product) and DIEA (1.13 g, 8.73 mmol, 1.52 mL, 4.00 eq.) were dissolved in acetonitrile (20 mL), and bis(4-nitrophenyl)carbonate (531 mg, 1.75 mmol, 0.80 eq.) was added at 10°C. The reaction mixture was reacted at 10°C for 0.5 hours. The reaction mixture was purified by preparative HPLC (0.01% TFA system) to give CR202DR (1.05 g, 1.14 mmol, yield: 52.2%, purity: 98.7%) as a colorless oil. LCMS: [M+H] + =909.3, 1H NMR:(CDCl3, 400 MHz) δ 8.23 ​​(d, J = 8.80 Hz, 2H), 7.48(s, 1H), 7.38 (d, J = 8.40 Hz, 2H), 7.31 - 7.25 (m, 4H), 7.16 (s, 1H), 6.46 (s, 1H), 5.97 (s, 2H), 4.48 (t, J = 4.80 Hz, 2H), 4.10 (s, 2H), 4.01 (s, 2H), 3.82 - 3.67 (m, 2H), 3.58 - 3.26 (m, 22H), 1.01(s, 9H). [ka]

[0454] CR202DR (350 mg, 385 μmol) was dissolved in DMF (7 mL) and cooled in an ice bath. CR20274 (340 mg, 385 μmol), HOBT (10 mg, 77 μmol), and DIEA (100 mg, 770 μmol) were added under nitrogen gas protection, and the mixture was stirred at 25°C for 6 hours. The reaction mixture was poured into 70 mL of MTBE, and an oily substance was deposited at the bottom of the bottle. The supernatant was discarded. 50 mL of MTBE was then added to the oily substance and stirred. The oily substance partially hardened, and the resulting semi-solid crude product, CR202JQ, was used directly in the next step (purity: 78.22%). LCMS: [M+H] + =1654.2. [ka]

[0455] Under nitrogen gas protection, CR202JQ obtained in the previous step was added to a 100 mL recovery flask and dissolved in DMF (7.5 mL). 5 M aqueous NHOAc solution (7.5 mL) was added and the mixture was stirred at 55°C for 16 hours. Sampling was performed during the reaction process to control the reaction until it was completely completed. The reaction mixture was purified by preparative HPLC to obtain CR202JR (219.3 mg, purity: 85.83%, yield: 55.2%). [M+H] +=1504.07. [ka]

[0456] Under nitrogen gas protection, CR202JR (151.0 mg, 94.7 μmol) and CR202W4 (170.2 mg, 94.7 μmol) were added to a 25 mL recovery flask, and DMF (7.5 mL) and water (7.5 mL) were added. The mixture was stirred under light-protected conditions until completely dissolved. DIEA was added to adjust the pH to 8, and CuBr (13.6 mg, 94.7 μmol) was added. The mixture was allowed to react at 25°C for 2.5 hours. Samples were taken during the reaction process to control the reaction until it was complete. The reaction mixture was purified by preparative HPLC to give CR202JG (62.3 mg, purity: 96.78%, yield: 21.2%). LCMS: [M-2H] 2- =1547.85.

[0457] 4.22 Synthesis of CR202HT [ka]

[0458] Under nitrogen gas protection, TEA (2.54 g, 25.08 mmol) was added to DCM (20 mL) and cooled to 0 °C. CR202MQ (1.95 g, 10.53 mmol) was added dropwise, followed by DCM (4 mL) to rinse the syringe. The mixture was then incubated for 0.5 h. CR202MN / DCM (2.20 g / 8 mL, 10.03 mmol) was added dropwise via syringe pump. The addition was completed in 1 h. The mixture was then incubated for 1 h. CR202MP / DCM (1.35 g / 5 mL, 11.24 mmol) was added dropwise. The addition was completed in 10 min. The mixture was then incubated for 1 h at 0 °C. The mixture was concentrated to remove DCM, diluted with EA / n-heptane (V / V=1 / 4, 60 mL), washed three times with purified water (60 mL × 3), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under vacuum at 35 °C and purified by column chromatography (n-heptane / EA=100 / 0 to 8 / 1) to give CR202MK (1.59 g, purity: ∼80%, yield: 54.9%) as a pale yellow oil. 1 H-NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 4.02 (t, J = 5.2 Hz, 2H), 3.70 (m, 12H), 3.42 (m, 2H), 1.23 (m, 2H), 1.13 (dd, J = 7.1, 3.4 Hz, 12H). [ka]

[0459] CR202MK (112.8 mg, 0.25 mmol) was added to methanol (1.1 mL), and sodium borohydride (10.0 mg, 0.25 mmol) was added at an external temperature of -4.0°C. The reaction was sampled and monitored by TLC until the reaction was complete. Purified water (2.0 mL) was added dropwise to quench the reaction, and EA (3.0 mL) was added for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated under vacuum at 35°C to obtain CR202ML (181.5 mg) as a pale yellow oil. 1H-NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 4.65 (s, 2H), 4.00 (t, J = 5.4 Hz, 2H), 3.70 (m, 12H), 3.41 (m, 2H), 1.19 (m, 2H), 1.11 (dd, J = 6.9, 2.5 Hz, 12H). [ka]

[0460] CR202ML (170.0 mg, 0.373 mmol) was added to THF (3.0 mL) and stirred to form a suspension. Under nitrogen gas protection, DIPEA (120.5 mg, 0.933 mmol) and p-nitrophenol chloroformate (112.8 mg, 0.559 mmol) were added and the mixture was allowed to react at room temperature (26.2 °C) for 6 hours. DCM (1.2 mL) was added until the reaction mixture became clear, and the reaction was continued for 44 hours. The reaction was then essentially complete by HPLC. The mixture was extracted with EA (5.0 mL) and purified water (5.0 mL). The mixture was separated and washed twice with purified water (5 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by preparative HPLC (acetonitrile and water mobile phase) and lyophilized to give CR202KL (219.0 mg, purity 98.23%, yield: 61.8%) as an oil. [ka]

[0461] Under nitrogen gas protection, CR202KL (2170 mg, 0.35 mmol), CR20274 (370.8 mg, 0.42 mmol), and HOBt (10.1 mg, 0.07 mmol) were added to DMF (4.0 mL), and DIPEA (90.5 mg, 0.70 mmol) was added. The reaction was allowed to proceed at room temperature (24.9 °C) for 7 h. Samples were taken to confirm complete reaction. The reaction mixture was purified by preparative HPLC (acetonitrile and water mobile phase). Lyophilization afforded CR202KM (107.7 mg, purity: 95.0%, yield: 22.4%). [ka]

[0462] CR202KM (107.0 mg, 0.078 mmol) was dissolved in DMF (6.5 mL), purified water (3.5 mL) was added, and the reaction mixture was turbid. CR202W4 (142.5 mg, 0.095 mmol) was added under light-protected conditions. The mixture was purged with nitrogen gas three times and stirred at 25 °C until the mixture was clear. DIPEA (100 μL) was added dropwise to adjust the pH to 8. Cuprous bromide (11.2 mg, 0.078 mmol) was added, and the mixture was incubated for 2 hours. Samples were taken to confirm completion of the reaction. The reaction mixture was purified by preparative HPLC (15 nM sodium acetate / water-acetonitrile system, acetonitrile-water desalting mobile phase) and lyophilized to give CR202HT (26.1 mg, purity: 88.29%, 16.0 mg, purity: 73.74%). LCMS: [M+3H] 3+ =960.92.

[0463] 4.23 Synthesis of CR202HU [ka]

[0464] Zinc powder (9.03 g, 0.138 mol) was added to a 500 mL single-neck flask, followed by dry THF (150 mL). Propargyl bromide (16.41 g, 0.138 mol) was added dropwise. The mixture was stirred for 30 minutes. CR202BE (9.98 g, 0.060 mol) was dissolved in dry THF (150 mL) and added dropwise to the reaction mixture at -10°C to -5°C. After the addition was complete, the ice bath was removed and the mixture was allowed to react for 15 minutes. The reaction mixture became gel-like, and a sample was taken for in-process control by HPLC. The reaction mixture was cooled to 0°C, 100 mL of saturated ammonium chloride was added dropwise, and then 100 mL of water was added. The reaction mixture was transferred to a separatory funnel and extracted twice with 300 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum at 35°C to give a brown oil. This was purified by column chromatography (n-heptane:ethyl acetate = 20:1 to 2:1) to give CR202BF (12.00 g, purity: 91.14%, yield: 97.6%). 1 H-NMR (400 MHz, CDCl3) δ 10.53 (s, 1H), 8.13 (d, J = 1.9 Hz, 1H), 7.62 (dd, J = 8.7, 2.0 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 4.86 (dd, J = 9.5, 6.2 Hz, 1H), 2.71 - 2.55 (m, 2H), 2.48 (d, J = 3.6 Hz, 1H), 2.11 - 2.04 (m, 1H). [ka]

[0465] Silver carbonate (13.24 g, 48.03 mmol) was added to a 250 mL single-neck flask, acetonitrile (130 mL) was further added, the ice-salt bath was kept cooling, HMTTA (2.09 g, 9.09 mmol) was added, and the mixture was stirred for 7 minutes. CR202BF (2.70 g, 12.98 mmol) and CR202KC (6.94 g, 16.88 mmol) were added, the ice-salt bath was removed, the mixture was reacted for 1 hour, and samples were taken for in-process control by HPLC. The reaction mixture was filtered to remove the silver carbonate, and ethyl acetate (150 mL) and water (100 mL) were added to the filtrate. The organic phase was separated, and the aqueous phase was further extracted with ethyl acetate (100 mL). The organic phases were combined and washed with saturated sodium chloride (100 mL). The mixture was washed with 1 mL of HCl, dried over anhydrous sodium sulfate, and filtered (diatomaceous earth + silica gel + diatomaceous earth triple filtration). The filtrate was then concentrated under vacuum at 28 °C and purified by column chromatography (n-heptane:ethyl acetate = 4:1 to 1:2) to obtain CR202KD (10.00 g, yield: 120.9% (the product contains ethyl acetate)). 1 H-NMR (400 MHz, CDCl3) δ 7.83 (dd, J = 9.1, 2.1 Hz, 1H), 7.54 (td, J = 9.3, 2.2 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 5.51 (dd, J = 10.5, 7.9 Hz, 1H), 5.44 (d, J = 2.8 Hz, 1H), 5.06 (ddd, J = 9.2, 8.5, 1.9 Hz, 2H), 4.88 (t, J = 6.2 Hz, 1H), 4.30 - 4.19 (m, 1H), 4.18 - 3.98 (m, 2H), 2.71 - 2.54 (m, 2H), 2.16 (s, 3H), 2.13 - 2.07 (m,3H), 2.04 (s, 3H), 2.01 (s, 3H). [ka]

[0466] CR202KD (9.50 g, 0.018 mol) was dissolved in acetonitrile / HO (280 mL, acetonitrile:HO = 3:1). The mixture was cooled to 3 °C in an ice-salt bath. N3-(PEG)2-NH2 (3.08 g, 0.018 mol) was added, followed by CuBr (2.54 g, 0.018 mol). The mixture was allowed to react at 3 °C for 20 min, and then sampled and analyzed by HPLC for in-process control. The reaction mixture was adjusted to pH 5 by dropwise addition of 5% TFA (60 mL). The product was purified and lyophilized to give CR202KE (11.02 g, purity: 93.38%, yield: 115.7% (product contains reagents). LC-MS: [M+H] + :712.17. [ka]

[0467] CR202KE (10.00 g, 0.014 mol) was added to a 500 mL single-neck flask, dried DMAc (100 mL) was added, the ice bath was cooled to an internal temperature of 4°C, CR202Z0 (2.66 g, 0.014 mol) and HATU (5.34 g, 0.014 mol) were added, DIPEA (11 mL, 0.063 mol) was added dropwise, and the reaction was carried out in an ice bath for 25 minutes. Sampling was carried out for process control, and the reaction was terminated by adding 300 mL of water dropwise to the reaction solution, controlling the temperature at <20°C, and the mixture was extracted with ethyl acetate (200 mL × 2). The organic phases were combined and washed with saturated brine (50 The crude product was purified by column chromatography (n-heptane:ethyl acetate=4:1, DCM:MeOH=100:1 to 30:1) to give CR202KF (6.20 g, purity: 90.02%, yield: 61.4%). 1H-NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.73 (d, J = 4.3 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H), 7.13 (s, 1H), 5.51 (dd, J = 10.3, 8.1 Hz, 1H), 5.44 (d, J = 3.2 Hz, 1H), 5.14 - 5.00 (m, 3H), 4.61 - 4.50 (m, 2H), 4.22 (dd, J = 11.2, 7.0 Hz, 1H), 4.15 (dd, J = 11.6, 6.6 Hz, 1H), 3.94 (s, 2H), 3.84 (t, J = 4.8 Hz, 2H), 3.70 - 3.61 (m, 6H), 3.58 (s, 4H), 3.51 (t, J = 5.3 Hz, 2H), 3.48 - 3.41 (m, 2H), 3.41 - 3.35 (m, 2H), 3.16 (dd, J = 15.1, 3.2 Hz, 1H), 3.03 (dd, J = 15.0, 8.8 Hz, 3H), 2.16 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H). [ka]

[0468] CR202KF (2.00 g, 2.27 mmol) was dissolved in dry DMAc (60 mL), NPC (1.38 g, 4.53 mmol) was added, and DIPEA (1.2 mL, 6.81 mmol) was added dropwise. The mixture was stirred for 17 hours, and 300 mL of acetonitrile / 0.1% TFA / water (1 / 1) reagent was added dropwise. The temperature was controlled at <20°C, and the mixture was purified and lyophilized to give CR202KG (1.65 g, purity: 98.3%, yield: 67.5%). LC-MS: [M+H] + :1048.29. [ka]

[0469] CR202KG (0.82 g, 0.76 mmol) was dissolved in dry DMAc (13 mL) and cooled to an internal temperature of 10 °C in an ice bath. CR20274 (0.74 g, 0.84 mmol) and HOBT (0.1032 g, 0.76 mmol) were added, followed by DIPEA (0.16 mL). The ice bath was removed and the mixture was stirred for 3.5 hours. The reaction mixture was purified and lyophilized to give CR202KH (1.0781 g, purity: 98.23%, yield: 78.7%). LC-MS: [M+2] 2+ :897.21. [ka]

[0470] CR202KH (0.50 g, 0.28 mmol) was dissolved in methanol (50 mL), cooled to 10°C, and 0.1 M LiOH (25 mL) was added dropwise. The temperature was controlled at 10-15°C and the reaction was allowed to proceed for 25 minutes. Sampling was performed to confirm completion of the reaction. 5% AcOH was added dropwise to adjust the pH to 5. The mixture was purified and lyophilized to give CR202KJ (0.24 g, purity: 98.84%, yield: 45.3%). LC-MS: [M+2] 2+ :813.18. [ka]

[0471] CR202KJ (0.22 g, 0.135 mmol) and CR202W4 (0.25 g, 0.162 mmol) were dissolved in 6.6 mL of DMF:HO (1:1). The temperature was controlled at 20 °C, and DIPEA was added dropwise to adjust the pH to 8.0. CuBr (20.1 mg, 0.135 mmol) was added, and the mixture was stirred for 1.5 h. Samples were taken and the reaction was monitored by HPLC until complete completion. The reaction mixture was then cooled to 0 °C, and 2% TFA was added dropwise to adjust the pH to 3. CR202HU (0.11 g, purity: 95.3%, yield: 26.2%) was obtained by preparative purification.

[0472] 4.24 Synthesis of CR202HR [ka]

[0473] CR202JN (520.0 mg, 0.135 mmol) was dissolved in DMF / water (9 / 1, 40 mL), CR202HG (250.0 mg, 0.155 mmol) and CuBr (48.4 mg, 0.337 mmol) were added, and the mixture was purged with nitrogen gas three times, adjusted to pH 7, and the reaction mixture was kept at 25°C with magnetic stirring for 3 to 5 hours. The reaction was monitored by HPLC for complete reaction, and the reaction was terminated. The reaction mixture was purified and lyophilized to give yellow solid powder CR202HR (358.6 mg, purity 97.78%, yield: 40.75%). [M / 4+H] + =1367.50.

[0474] 4.25 Synthesis of CR202MT [ka]

[0475] A similar method to L-01 was employed to prepare a yellow solid, CR202MS.

[0476] CR20275 was prepared using a method similar to that for CR20282.

[0477] CR20275 (150.1 mg, 0.10 mmol) and CR202MS (81.2 mg, 0.12 mmol) were added to 50% ACN / HO (15 mL), and the reaction mixture was adjusted to pH 4-6 with 10 mmol / L NaHPO. The reaction mixture was then incubated at 25 °C for 1 hour, and the reaction was monitored by HPLC until complete. The reaction mixture was directly purified to give CR202MT (90.0 mg, purity: 93.46%, yield: 41.3%). Example 2 Solubility Experimental Study

[0478] General procedure for solubility experiments: Add 0.05 mL of PBS (pH 7.2-7.4) to the complex compound (1.0 mg) and the single proteolytic chimera in several portions, vortexing for 2 minutes until the solids are dissolved. The solubility is calculated as follows: C = m / V, where C is concentration, m is sample weight, and V is the total volume of PBS. [Table 2] Experimental Conclusion: As can be seen from the table above, the solubility of the conjugated compound was significantly higher than that of the proteolytic chimera alone (i.e., CR20274). Example 3 Experimental study of enzymatic cleavage

[0479] 1. Experimental study of cathepsin B enzymatic cleavage of CR20282, CR20293, CR20295, and CR20297

[0480] Sample information (enzyme cleavage experiment): CR20282, CR20293, CR20295, CR20297, cathepsin B-human liver (source: Sigma), cysteine ​​(source: Sigma), sodium acetate and EDTA (Shanghai Lingfeng).

[0481] Experimental method: Each conjugate compound was dissolved in acetonitrile-water (1:1, containing 0.1% TFA) to prepare a 1 mg / mL stock solution. 30 μL of the 1 mg / mL stock solution was added to 570 μL of 50 mM NaOAc (containing 1 mM EDTA and 10 mM cysteine) solution and mixed thoroughly. 7 μL of cathepsin B (0.05 U / μL) was then added and mixed gently to homogenize. The entire reaction mixture was placed in a 37°C incubator and incubated for 0, 1, 2, 4, and 8 hours. Samples were then taken for HPLC analysis at each time point.

[0482] Experimental Results and Analysis: As can be seen from Tables 2 and 3, under the enzymatic cleavage action of cathepsin B, each drug has a relatively good drug-loading release rate. [Table 3] [Table 4]

[0483] 2. Study of the cathepsin B enzyme cleavage rate of CR202V4, CR202V5, CR202V6, CR202V7, and CR202Z6

[0484] Sample information (enzyme cleavage experiment): CR202V4, CR202V5, CR202V6, CR202V7, CR202Z6, cathepsin B-human liver (source: Sigma), cysteine ​​(source: Sigma), sodium acetate and EDTA (source: Shanghai Lingfeng).

[0485] Experiment steps: 1) Prepare a 500 μg / mL mother solution of each conjugate compound using 0.1% TFA-acetonitrile water (1:1). 2) Take 60 μL of the 500 μg / mL compound stock solution and add it to 536 μL of 50 mM NaOAc (containing 1 mM EDTA + 10 mM Cys), add 4 μL of cathepsin B (CB enzyme), and shake gently to mix evenly. 3) The enzyme cleavage reaction product in 2) above was placed in a 37°C constant temperature incubator and incubated for 0, 1, 2, 4, and 24 hours, and samples were injected at each time point and sampled for LC-MS analysis.

[0486] Experimental Results and Analysis: As can be seen from Tables 4 and 5, under the action of CB enzyme, each drug has a relatively good drug-loading release rate. [Table 5] [Table 6]

[0487] 3. Study of the enzymatic cleavage rate of CR202V8 and CR202W1

[0488] Due to the enzymatic cleavage action of plasmin and sulfatase, the structural stability of the conjugate compounds CR202V8 (containing a plasmin enzyme-cleavable group) and CR202W1 (containing a sulfatase enzyme-cleavable group) was observed.

[0489] Experiment steps: 1) Each compound was prepared into a 500 μg / mL stock solution using PBS (pH = 7.4). 2) Take 60 μL of 500 μg / mL compound stock solution and add it to 420 μL of PBS (pH = 7.4), add 60 μL of 0.2 U / mL plasmin (or 60 μL of 0.5 U / mL sulfatase), and shake gently to mix evenly. 3) The enzyme cleavage reaction product in 2) above was placed in a 37°C constant temperature incubator and incubated for 0, 1, 2, 4, and 24 hours, and samples were injected at each time point and sampled for LC-MS analysis.

[0490] Experimental Results and Analysis: As can be seen from Tables 6 and 7, both CR202V8 and CR202W1 have relatively fast drug release rates, with the drug release rate of CR202V8 being faster than that of CR202W1. [Table 7] [Table 8]

[0491] 4. Study of β-glucosidase enzyme cleavage rate of CR202AL

[0492] The structural stability of the complex compound CR202AL was observed by the enzymatic cleavage action of β-glucosidase, and the presence or absence of other enzymatic cleavage fragments was observed to determine the enzymatic cleavage site.

[0493] Experiment steps: 1) Prepare CR202AL as a 500 μg / mL solution using PBS (pH 6.8). 2) Take 100 μL of the 500 μg / mL compound mother solution and add it to 775 μL of PBS (pH = 6.8), add 125 μL of 100 U / mL β-glucosidase, and shake gently to mix evenly. 3) The enzyme cleavage reaction product in 2) above was placed in a constant temperature incubator at 25°C and incubated for 0, 1, 2, 4, and 24 hours. At the appropriate times, samples were injected and sampled for LC-MS analysis.

[0494] Experimental results and analysis: As can be seen from Tables 8 and 9, under the enzymatic cleavage action of β-glucosidase, CR202AL has a relatively good drug-loading release rate. [Table 9] [Table 10] Example 4 Plasma Stability Studies

[0495] 1. Plasma stability study of CR20282, CR20285, CR20293, CR20295 and CR20297

[0496] Sample information: CR20282, CR20285, CR20293, CR20295, CR20297, methanol and mouse plasma.

[0497] Experimental method: Each conjugate compound was dissolved in acetonitrile-water (1:1) or acetonitrile-water (1:1, containing 0.1% TFA) to prepare a 1 mg / mL mother solution. 10 μL of the 1 mg / mL mother solution was added to 90 μL of plasma and gently mixed to homogenize. The plasma drug samples were placed in a 37°C incubator and incubated for 0, 0.5, 1, 2, 4, 6, and 24 hours. After reaching the reaction time points, the samples were removed and added with 300 μL of methanol solution (containing 10 ng / mL terfenadine) to precipitate proteins. The samples were then centrifuged (12 min, 4°C, 12,000 rpm). The supernatant was collected and stored in a refrigerator at -80°C. Samples from all time points were collected and analyzed by LC-MS.

[0498] Experimental Results and Analysis: As can be seen from Tables 10 and 11, each conjugate compound is relatively stable in plasma, and the payload released in plasma is relatively small and the rate is relatively slow. [Table 11] [Table 12]

[0499] 2. Plasma Stability Studies of CR202V4, CR202V5, CR202V6, CR202V7, CR202V8, CR202V9, CR202Z6, CR202W1, CR202AL, and CR20282

[0500] Sample information: CR202V4, CR202V5, CR202V6, CR202V7, CR202V8, CR202V9, CR202Z6, CR202W1, CR202AL, CR20282, methanol and mouse plasma.

[0501] Experiment steps: 1) Each conjugate compound was prepared into a 1 mg / mL mother solution using 0.1% TFA-acetonitrile water (1:1), 2) Take 10 μL of 1 mg / mL compound stock solution, add it to 90 μL of fresh plasma, gently shake to mix evenly, place it in a constant temperature incubator at 37°C and incubate for 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours. 3) After the reaction time point was reached, the sample was removed and 300 μL of methanol solution (containing 10 ng / mL terfenadine) was added to perform protein precipitation. 4) The mixture was centrifuged (13 minutes, 4°C, 12000 rpm), the supernatant was collected and stored in a refrigerator at -80°C, and samples were collected at all time points and analyzed by LC-MS.

[0502] Experimental Results and Analysis: As can be seen from Tables 12 and 13, each conjugate compound is relatively stable in plasma, and the payload released in plasma is relatively small and the rate is relatively slow. [Table 13] [Table 14] Example 5: Experimental study of the conjugate compound CR20214 inhibiting proliferation of different tumor cells

[0503] Cell line information [Table 15]

[0504] Key reagents: 1640 folate-free medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

[0505] Experimental steps 1) Seeding cells onto plates Cells were prepared in advance, digested with trypsin, collected, counted, and diluted to 2 × 10 cells using complete cell culture medium. 4The cells were diluted to 100 μL / mL and seeded into a 96-well plate. 100 μL of the diluted cell suspension was added to each well, and negative and blank control wells were also included. The 96-well plate containing the cells was placed in a 37°C, 5% CO2 incubator and incubated overnight. 2) Dilution and sample addition The samples were diluted with culture medium (initial concentration 10 μM, 5-fold gradient dilution) to set up a total of nine concentration gradients. The culture medium in the well plate was aspirated and discarded, and 100 μL of drug solution was added to each well of a 96-well plate, with three parallel wells, as well as a negative control and a blank control. The wells were then incubated at 37°C in a 5% CO2 incubator for 72 hours. 3) Color development and plate reading CCK-8 color development solution was taken and 10 μL (10% of the liquid volume in the well) was added to each well, and the plate was incubated at 37°C for an appropriate period of time (preferably to achieve an OD value in the range of 1.0 to 2.5). The culture plate cover was then removed from the 96-well plate, and the plate was placed in a microplate reader (Molecular Devices SpectraMax iD5) and the reading was taken at 450 nm. 4) Data processing Perform a four-parameter fitting curve using GraphPad Prism 8.0.2 to determine the IC 50 got the value. 5) Experimental results and analysis Experimental results: See Table 15. As can be seen from Table 15, CR20214 has killing effects on various types of tumor cells. [Table 16] Example 6: Experimental study of the inhibition of proliferation of different tumor cells by the conjugate compounds CR20282, CR20285, CR20287, CR20293, CR20295, CR20297 and CR202F0

[0506] Cell line information [Table 17]

[0507] Key reagents: 1640 folate-free medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

[0508] Experimental steps 1) Seeding cells onto plates Cells were prepared in advance, digested with trypsin, collected, counted, and diluted to 2 × 10 cells using complete cell culture medium. 4 The cells were diluted to 100 μL / mL and seeded into a 96-well plate. 100 μL of the diluted cell suspension was added to each well, and negative and blank control wells were also included. The 96-well plate containing the cells was placed in a 37°C, 5% CO2 incubator and incubated overnight. 2) Dilution and sample addition The samples were diluted with culture medium (initial concentration 10 μM, 5-fold gradient dilution) to set up a total of nine concentration gradients. The culture medium in the well plate was aspirated and discarded, and 100 μL of drug solution was added to each well of a 96-well plate, with three parallel wells, as well as a negative control and a blank control. The wells were then incubated at 37°C in a 5% CO2 incubator for 72 hours. 3) Color development and plate reading CCK-8 color development solution was taken and 10 μL (10% of the liquid volume in the well) was added to each well, and the plate was incubated at 37°C for an appropriate period of time (preferably to achieve an OD value in the range of 1.0 to 2.5). The culture plate cover was then removed from the 96-well plate, and the plate was placed in a microplate reader (Molecular Devices SpectraMax iD5) and the reading was taken at 450 nm. 4) Data processing Perform a four-parameter fitting curve using GraphPad Prism 8.0.2 to determine the IC 50 got the value. 5) Experimental results and analysis Experimental results: See Table 17. As can be seen from Table 17, CR20282, CR20285, CR20293, CR20295 and CR20297 have very strong killing effects on tumor cells expressing FOLR1 and PSMA. [Table 18] Example 7: Experimental study of compound CR20274 and conjugate compound CR20282 inhibiting proliferation of different tumor cells

[0509] Cell line information [Table 19]

[0510] Key reagents: 1640 folate-free medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

[0511] Experimental steps 1) Seeding cells onto plates Cells were prepared in advance, digested with trypsin, collected, counted, and diluted to 2 × 10 cells using complete cell culture medium. 4 The cells were diluted to 100 μL / mL and seeded into a 96-well plate. 100 μL of the diluted cell suspension was added to each well, and negative and blank control wells were also included. The 96-well plate containing the cells was placed in a 37°C, 5% CO2 incubator and incubated overnight. 2) Dilution and sample addition The samples were diluted with culture medium (initial concentration 10 μM, 5-fold gradient dilution) to set up a total of nine concentration gradients. The culture medium in the well plate was aspirated and discarded, and 100 μL of drug solution was added to each well of a 96-well plate, with three parallel wells, as well as a negative control and a blank control. The wells were then incubated at 37°C in a 5% CO2 incubator for 72 hours. 3) Color development and plate reading CCK-8 color development solution was taken and 10 μL (10% of the liquid volume in the well) was added to each well, and the plate was incubated at 37°C for an appropriate period of time (preferably to achieve an OD value in the range of 1.0 to 2.5). The culture plate cover was then removed from the 96-well plate, and the plate was placed in a microplate reader (Molecular Devices SpectraMax iD5) and the reading was taken at 450 nm. 4) Data processing Perform a four-parameter fitting curve using GraphPad Prism 8.0.2 to determine the IC 50 got the value. 5) Experimental results and analysis Experimental results: See Table 19. As can be seen from Table 19, CR20274 still had a relatively strong killing effect on cell lines that did not express PSMA or FOLR1, while CR20282 had almost no killing effect on cells that did not express PSMA or FOLR1, indicating that the lack of FOLR1 and PSMA on the cell membrane surface inhibited the targeted delivery of the dual-ligand PROTAC, indicating that the dual-ligand PROTAC has better targeting ability than the naked PROTAC. [Table 20] Example 8 Experimental study of the inhibition of proliferation of conjugate compounds CR202V4, CR202V5, CR202V6, CR202V7, CR202V9, CR202W1, CR202Z6, CR202AL, CR202GM, CR202GQ, CR202FU, CR202HJ, CR202JG, CR202HU, CR202HV and CR202V8 against different tumor cells

[0512] Cell line information: 22RV1 (human prostate cancer cells), C4-2 (human prostate cancer cells), and MDA-MB-231 (human breast cancer cells)

[0513] Key reagents: 1640 Folate-Free Medium, Fetal Bovine Serum, Penicillin-Streptomycin Solution, L-Glutamine, CellTiter-Glo® Luminescent Cell Viability Assay Reagent

[0514] Experiment steps: 1) Seeding cells onto plates Cells were prepared in advance, digested with trypsin, collected, counted, and diluted to 1.5 x 10 cells using complete cell culture medium. 4 The cells were diluted to 100 μL / mL and seeded into a 96-well plate. 100 μL of the diluted cell suspension was added to each well, and negative and blank control wells were also included. The 96-well plate containing the cells was placed in a 37°C, 5% CO2 incubator and incubated overnight. 2) Dilution and sample addition The samples were diluted with culture medium (initial concentration 10 μM, 3-fold gradient dilution) to set up a total of nine concentration gradients. The culture medium in the well plate was aspirated and discarded, and 100 μL of drug solution was added to each well of a 96-well plate, with three parallel wells, as well as a negative control and a blank control. The wells were then placed in an incubator at 37°C with 5% CO2 for 72 hours. 3) Detection of cell activity using CellTiter-Glo luminescence assay The CellTiter-Glo buffer solution was thawed and allowed to warm to room temperature. The CellTiter-Glo substrate was allowed to warm to room temperature. CellTiter-Glo buffer was added to one tube of CellTiter-Glo substrate to dissolve the substrate, thereby preparing the CellTiter-Glo working solution. The substrate was then gently vortexed and shaken to ensure complete dissolution. The cell culture plate was removed and allowed to equilibrate to room temperature for 30 minutes. 50 μL of CellTiter-Glo working solution (equivalent to half the volume of the cell culture medium in each well) was added to each well. The cell plate was covered with aluminum foil to protect from light. The culture plate was shaken on an orbital shaker for 2 minutes to promote cell lysis. The culture plate was then allowed to warm to room temperature for 10 minutes to allow the luminescence signal to stabilize. The luminescence signal was detected by placing it in a microplate reader (Molecular Devices SpectraMax iD5). 4) Data processing A four-parameter fitting curve was performed using GraphPad Prism 8.0.2 to obtain IC50 values. 5) Experimental results and analysis Experimental results: Referring to Table 20, as can be seen from Table 20, the conjugate compounds CR202V4, CR202V5, CR202V6, CR202V7, CR202V9, CR202W1, CR202Z6, CR202AL, CR202GM, CR202GQ, CR202FU, CR202HJ, CR202JG, CR202HU, CR202HV and CR202V8 all have relatively excellent tumor inhibitory effects against different tumor cells. [Table 21] Note: " / " indicates undetected. Example 9: Experimental study of the proliferation inhibition of different tumor cells by the conjugate compounds CR202W5, CR202W8, CR202W9 and CR202W6, and the single proteolytic chimeras CR202Z5, CR202Z8, CR202Z9 and CR202ES

[0515] Cell line information: LNCaP (human prostate cancer cells), VCaP (human prostate cancer cells)

[0516] Key reagents: 1640 Folate-Free Medium, Fetal Bovine Serum, Penicillin-Streptomycin Solution, L-Glutamine, CellTiter-Glo® Luminescent Cell Viability Assay Reagent

[0517] Experiment steps: 6) Seeding cells onto plates Cells were prepared in advance, digested with trypsin, collected, counted, and diluted to 1.5 x 10 cells using complete cell culture medium. 4 The cells were diluted to 100 μL / mL and seeded into a 96-well plate. 100 μL of the diluted cell suspension was added to each well, and negative and blank control wells were also included. The 96-well plate containing the cells was placed in a 37°C, 5% CO2 incubator and incubated overnight. 7) Dilution and sample addition The samples were diluted with culture medium (initial concentration 10 μM, 3-fold gradient dilution) to set up a total of nine concentration gradients. The culture medium in the well plate was aspirated and discarded, and 100 μL of drug solution was added to each well of a 96-well plate, with three parallel wells, as well as a negative control and a blank control. The wells were then placed in an incubator at 37°C with 5% CO2 for 72 hours. 8) Detection of cell activity using CellTiter-Glo luminescence assay The CellTiter-Glo buffer solution was thawed and allowed to warm to room temperature. The CellTiter-Glo substrate was allowed to warm to room temperature. CellTiter-Glo buffer was added to one tube of CellTiter-Glo substrate to dissolve the substrate, thereby preparing the CellTiter-Glo working solution. The substrate was then gently vortexed and shaken to ensure complete dissolution. The cell culture plate was removed and allowed to equilibrate to room temperature for 30 minutes. 50 μL of CellTiter-Glo working solution (equivalent to half the volume of the cell culture medium in each well) was added to each well. The cell plate was covered with aluminum foil to protect from light. The culture plate was shaken on an orbital shaker for 2 minutes to promote cell lysis. The culture plate was then allowed to warm to room temperature for 10 minutes to allow the luminescence signal to stabilize. The luminescence signal was detected by placing it in a microplate reader (Molecular Devices SpectraMax iD5). 9) Data processing A four-parameter fitting curve was performed using GraphPad Prism 8.0.2 to obtain IC50 values. 10) Experimental results and analysis Experimental Results: See Table 21. As can be seen from Table 21, the conjugate compounds CR202W5, CR202W8, CR202W9 and CR202W6 have comparable or even superior killing effects against different tumor cells compared to the single proteolytic chimeras CR202Z5, CR202Z8, CR202Z9 and CR202ES. [Table 22] Example 10: Experimental study of compounds CR202CC, CR202CF and CR202CI inhibiting proliferation of tumor cells

[0518] Cell line information [Table 23]

[0519] Key reagents: 1640 folate-free medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

[0520] Experimental steps 1) Seeding cells onto plates Cells were prepared in advance, digested with trypsin, collected, counted, and diluted to 2 × 10 cells using complete cell culture medium. 4 The cells were diluted to 100 μL / mL and seeded into a 96-well plate. 100 μL of the diluted cell suspension was added to each well, and negative and blank control wells were also included. The 96-well plate containing the cells was placed in a 37°C, 5% CO2 incubator and incubated overnight. 2) Dilution and sample addition The samples were diluted with culture medium (initial concentration 10 μM, 5-fold gradient dilution) to set up a total of nine concentration gradients. The culture medium in the well plate was aspirated and discarded, and 100 μL of drug solution was added to each well of a 96-well plate, with three parallel wells, as well as a negative control and a blank control. The wells were then incubated at 37°C in a 5% CO2 incubator for 72 hours. 3) Color development and plate reading CCK-8 color development solution was taken and 10 μL (10% of the liquid volume in the well) was added to each well, and the plate was incubated at 37°C for an appropriate period of time (preferably to achieve an OD value in the range of 1.0 to 2.5). The culture plate cover was then removed from the 96-well plate, and the plate was placed in a microplate reader (Molecular Devices SpectraMax iD5) and the reading was taken at 450 nm. 4) Data processing Perform a four-parameter fitting curve using GraphPad Prism 8.0.2 to determine the IC 50 got the value. 5) Experimental results and analysis Experimental results: see Table 23. As can be seen from Table 23, CR202CC, CR202CF and CR202CI have very strong killing effects on tumor cells expressing FOLR1 and PSMA. [Table 24] Example 11 Target protein degradation activity experiments of CR20214, CR20295, CR202V6, and CR202AL

[0521] Cell line information: MDA-MB-231 (human breast cancer cells), 22RV1 (human prostate cancer cells)

[0522] [Table 25]

[0523] Experiment steps:

[0524] Protein sample extraction: 1) An appropriate amount of RIPA lysis solution was taken and Protease Inhibitor Cocktail (1:100) was added a few minutes before use. 2) The medium was removed from the cells, scraped off with a cell scraper, centrifuged, and washed twice with pre-chilled PBS. 300 μL of lysis solution was added per dish. The centrifuge tube was placed on ice and pipetted with a pipette head for 5 minutes to ensure sufficient contact between the lysis solution and the cells. 3) After 30 minutes of lysis, the lysate was collected and transferred to a centrifuge tube, and the sample was centrifuged for 15 minutes at 12,000 xg to pellet the cell debris. 4) The supernatant can be transferred to a new centrifuge tube for the next Western procedure.

[0525] Concentration determination by BCA method: 1) 4× Laemmli sample buffer was prepared by adding 100 μL of mercaptoethanol to each 900 μL solution immediately before use. 2) Sample dilution: Based on the protein amount of 20 μg to 40 μg per well and the loading amount of 15 to 20 μL per well, the volume of the lysis solution, the volume of PBS, and the volume of 4x loading buffer were calculated, and the three were thoroughly mixed uniformly. 3) Denatured sample: heated at 90-95°C for 5 minutes.

[0526] ExpressPlus TM PAGE electrophoresis: An appropriate amount of protein sample solution is added to each well, and the protein solution to which a pre-stained marker has been added is selected as an electrophoresis marker. After loading, electrophoresis is performed to the bottom of the bromophenol blue latex under a constant voltage of 140 V.

[0527] Membrane transfer: 1) First, treat the nitrocellulose membrane with methanol for 5 to 10 seconds, then with pure water for 1 to 2 minutes. Then, equilibrate the nitrocellulose membrane and thick filter paper in membrane transfer buffer for 30 minutes. 2) The adhesive and nitrocellulose membrane were laid flat between two layers of thick filter paper to form a "sandwich," which was then transferred to a wet membrane transfer apparatus and subjected to a constant current of 300 mA for 2 hours. 3) After membrane transfer was complete, I removed the membrane with tweezers and marked it in a position that was easy for me to remember.

[0528] blocking: The nitrocellulose membrane washed with 1x TBST was immersed in 5% Nonfat Dry Milk blocking solution prepared with 1x TBST buffer and incubated at 20-25°C for 2 hours. The membrane was then washed twice with 1x TBST solution and once with 1x TBS solution, for 5 minutes each time.

[0529] Primary antibody incubation: After blocking was completed, the prepared primary antibody solution and nitrocellulose membrane were added to the hybridization bag overnight at 4°C, and the membrane was washed twice with 1x TBST solution and once with 1x TBS solution, each time for 5 minutes.

[0530] Secondary antibody incubation: After incubating the nitrocellulose membrane with diluted secondary antibody for 1.5 h, discard the excess antibody solution and wash the membrane twice with 1x TBST solution and once with 1x TBS solution, each time for 5 min.

[0531] Color development and exposure: After color development using an HRP-labeled antibody-conjugated enhanced chemiluminescence reagent, the membrane was exposed to X-ray film, developed, washed, and fixed, and the location of the antigen-antibody (primary antibody)-antibody (secondary antibody) complex on the nitrocellulose membrane was determined. The bands appearing on the X-ray film were scanned using a gel imaging system, and GAPDH was used as a reference.

[0532] Analysis of Results: As can be seen from the Western blot experiments (see Figures 2-4), CR20214, CR20295, CR202V6, and CR202AL were able to rapidly degrade their target proteins. Of these, CR20214 was able to almost completely degrade BRD4 and PLK1 at a concentration of 100 nM in 6 hours (Figure 2). CR20295 was able to almost completely degrade BRD4 at a concentration of 100 nM in 6 hours (Figure 3A). CR20295 was able to almost completely degrade BRD4 at a concentration of 10 nM in 24 hours (Figure 3B). CR202V6 and CR202AL were able to almost completely degrade BRD4 at a concentration of 3 nM in 6 hours (Figure 4A), demonstrating excellent target protein degradation activity, and still showed relatively strong degradation activity at 24 hours (Figure 4B). Example 12 Pharmacodynamic evaluation of compounds against subcutaneously transplanted human liver cancer HepG2 cells in a BALB / c nude mouse model

[0533] Purpose of the experiment: To preclinically evaluate the efficacy of CR202 series compounds against a subcutaneous HepG2 hepatocellular carcinoma tumor model lacking FOLR1 and PSMA receptor expression in BALB / c nude mice.

[0534] Experimental method: 0.1 mL (10 × 10 6 HepG2 cells (100 cells / 1000 cells) were inoculated subcutaneously into the right dorsal side of each mouse until the tumor volume reached an average of 100–150 mm. 3 When the animals reached 100 mg / kg, they were randomly divided into groups to set up a model control group and a treatment group, and treatment began on the first day of grouping. Before the start of treatment, all animals were weighed and tumor volume was measured with a vernier caliper. After tumor cell inoculation, conventional monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, specifically the activity of the experimental animals, feeding and drinking status, weight gain or loss, eyes, hair coat, and other abnormal conditions. After the start of treatment, the weight of the mice and tumor size were measured regularly. Formula for calculating tumor volume: tumor volume (mm 3 )V=1 / 2×(a×b 2 ) (wherein a represents the major axis and b represents the minor axis). The tumor growth inhibition rate, TGI (%), was calculated as follows: TGI% = (1 - T / C) x 100% (T and C are the average tumor volumes at a specific time point in the treatment group and the control group, respectively). The experimental grouping and design are shown in Table 24 below: [Table 26] Note: biw×3w refers to administration twice a week for 3 weeks (and so on), and qw×3w refers to administration once a week for 3 weeks (and so on).

[0535] Analysis of experimental results: The negative control group had a mean tumor volume of 1726.90 mm on the 36th day after group allocation. 3 The test substance CR20274 did not show a consistent tumor growth inhibitory effect at a 4 mg / kg, biw × 3 administration regimen, and the mean tumor volume was 1329.84 mm on the 36th day after group allocation. 3The TGI value was 22.99%, which was not significantly different from the negative control group (P=0.382). The test substance CR20282 did not show consistent tumor growth inhibitory effects at the two administration regimens of 12.5 mg / kg, biw x 3 and 12.5 mg / kg, qw x 3, and there was no significant difference compared to the negative control group (P=0.701). Specifically, see Figures 5A-5B.

[0536] Neither test compound CR20274 nor CR20282 demonstrated significant antitumor effects on tumor growth in mice bearing the human liver cancer model HepG2, under the established dosing frequency regimen. Meanwhile, the mouse human liver cancer model HepG2 does not express FOLR1 or PSMA, demonstrating that dual-ligand PROTAC compounds lack significant antitumor effects in animal models lacking expression of the corresponding target proteins. Example 13 Pharmacodynamic evaluation of compounds against a subcutaneous xenograft human breast cancer MDA-MB-231 tumor model

[0537] Purpose of the experiment: To preclinically evaluate the pharmacodynamics of the test substances CR20274, CR20282 and CR20285 in the subcutaneous human breast cancer model MDA-MB-231 in BALB / c nude mice.

[0538] Experimental method: MDA-MB-231 tumor masses were inoculated subcutaneously into the right armpit of each mouse until the average tumor volume reached 200 mm 3 Once the animals reached the target size, they were divided into groups and treatment was initiated. After treatment, the animals were monitored daily for health and mortality. Routine examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavioral manifestations, behavioral activity, food and water intake, body weight changes, external signs, or other abnormalities. Tumor growth was examined to determine whether it was inhibited, delayed, or cured. Tumor volume was measured periodically. The formula for calculating tumor volume was V = 1 / 2 × (a × b 2), where a and b represent the long and short diameters of the tumor, respectively. The tumor growth inhibition rate (TGI) (%) was calculated as follows: TGI% = (1 - T / C) x 100% (T and C are the average tumor volumes at a particular time point in the treatment group and control group, respectively). The experimental groupings and administration regimens are shown in Table 25 below. [Table 27]

[0539] Analysis of Experimental Results: See Figure 6A for the changes in tumor volume in mice bearing the human breast cancer model MDA-MB-231 tumor, and see Figure 6B for the changes in body weight over time in mice bearing the human breast cancer model MDA-MB-231 tumor compared to the initial body weight. As can be seen from the above, CR20282 at 12.5 mg / kg biw x 3 produced significant antitumor effects on tumor growth in the mouse xenograft human breast cancer model MDA-MB-231 and was relatively safe. CR20282 at 12.5 mg / kg qw x 3, CR20285 at 12.5 mg / kg qw x 3, CR20285 at 6 mg / kg biw x 3, CR20282 at 3 mg / kg biw x 3, and CR20285 at 3 mg / kg biw x 3 also had certain antitumor effects on tumors in the mouse xenograft human breast cancer model MDA-MB-231. CR20274 4 mg / kg biw × 3 did not have an antitumor effect on the mouse xenograft human breast cancer model MDA-MB-231 tumor. As can be seen from these results, the conjugate compound linked to the targeting molecule can significantly improve the antitumor effect compared to the proteolytic targeting chimera. Example 14 Pharmacodynamic evaluation of compounds against subcutaneously transplanted human prostate cancer 22RV1 cells in a BALB / c nude mouse model

[0540] Aim of the experiment: To conduct a preclinical pharmacodynamic study of the compound in the treatment of a subcutaneous 22RV1 cell-implanted tumor model (high PSMA expression) in BALB / c nude mice.

[0541] Experimental method: 22RV1 tumor masses were inoculated subcutaneously into the right dorsal side of each mouse until the average tumor volume reached 150 mm 3 Once the animals reached the target size, they were divided into groups and treatment was initiated. After treatment, the animals were monitored daily for health and mortality. Routine examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavioral manifestations, behavioral activity, food and water intake, body weight changes, external signs, or other abnormalities. Tumor growth was examined to determine whether it was inhibited, delayed, or cured. Tumor volume was measured periodically. The formula for calculating tumor volume was V = 1 / 2 × (a × b 2 ) where a and b represent the long and short diameters of the tumor, respectively. The experimental groupings and administration regimens are shown in Table 26 below. [Table 28]

[0542] Analysis of experimental results: The changes in body weight over time in mice bearing the human prostate cancer model 22RV1 tumor compared to the initial weight are shown in Figure 7A. In this experiment, BALB / c tumor-bearing nude mice tolerated the test compound at different doses well, showed a tendency to gain weight, and no mice died during the experiment.

[0543] See Figure 7B for the changes in tumor volume in mice bearing the human prostate cancer model 22RV1 tumor. The negative control group had a mean tumor volume of 1861.79 mm on day 31 after grouping. 3 The test substance CR20274 did not show a consistent tumor growth inhibitory effect at a 4 mg / kg, biw × 3 administration regimen, and the mean tumor volume was 1274.47 mm on the 31st day after group allocation. 3 The TGI value was 31.55%, which was not significantly different from the negative control group (P>0.05). The test substance CR20282 showed a highly significant tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, biw×3, and the mean tumor volume was 21.22 mm on the 31st day after grouping. 3The TGI value was 98.86%, which was significantly different from the negative control group (P<0.001). The test substance CR20293 showed a highly significant tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, biw×3, and the mean tumor volume was 46.32 mm on day 31 after group allocation. 3 The TGI value was 97.51%, which was significantly different from the negative control group (P<0.001). The test substance CR20295 showed a highly significant tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, biw×3. On the 31st day after grouping, the mean tumor volume was 86.32 mm 3 The TGI value reached 95.36%, which was significantly different from the negative control group (P<0.001). The test substance CR20297 showed a highly significant tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, biw x 3, and by day 31 after grouping, the tumors had completely regressed, with a TGI value of 100.00%, which was significantly different from the negative control group (P<0.001).

[0544] As can be seen from the above, compared with the control group and CR20274, the test compounds CR20282, CR20293, CR20295 and CR20297 all produced significant antitumor effects on tumor growth in mice bearing the human prostate cancer model 22RV1 tumor. Furthermore, the BALB / c tumor-bearing nude mice tolerated the test compounds well, showed a tendency to gain weight, and no mice died during the experimental period. Example 15: Pharmacological evaluation of compounds against subcutaneously transplanted human prostate cancer C4-2 cells in NCG mouse model

[0545] Purpose of the experiment: To preclinically evaluate the efficacy of the compound in treating a subcutaneous C4-2 cell tumor model (high PSMA expression) in NCG mice.

[0546] Experimental method: C4-2 cells were inoculated subcutaneously into the right dorsal side of each mouse until the average tumor volume reached 100–150 mm. 3Once the dose reached 100 mg / kg, the animals were divided into groups and administered the treatment. After administration, the animals were routinely monitored for tumor growth and the effects of treatment on their normal behavior, including their activity, feeding and drinking habits, weight gain or loss, and abnormalities in the eyes, hair, and other areas. Any clinical symptoms observed during the test were recorded in the raw data. After administration began, the mice's weights and tumor sizes were measured regularly. The formula for calculating tumor volume was V = 1 / 2 × (a × b 2 ) where a and b represent the long and short diameters of the tumor, respectively. The experimental groupings and administration regimens are shown in Table 27 below. [Table 29]

[0547] Analysis of experimental results: The changes in body weight over time in mice bearing the C4-2 tumor, a mouse model of human prostate cancer, compared with the initial body weight are shown in Figure 8A. In this experiment, BALB / c tumor-bearing nude mice tolerated the test compounds at different doses well, showed a tendency to gain weight, and essentially no mice died during the experiment.

[0548] See Figure 8B for the changes in body weight over time in mice bearing the C4-2 tumor, a mouse-human prostate cancer model, compared with the initial body weight. The negative control group had a mean tumor volume of 1748.61 mm on day 23 after grouping. 3 The test substance CR20274 did not show a consistent tumor growth inhibitory effect at a 4 mg / kg, biw × 3 administration regimen, and the mean tumor volume was 2198.07 mm on the 23rd day after group allocation. 3 The TGI value was -25.70%, which was not significantly different from the negative control group (P>0.05). The test substance CR20282 showed a certain tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, biw×3, and the mean tumor volume was 557.12 mm on the 23rd day after grouping. 3The TGI value was 68.14%, which was significantly different from the negative control group (P<0.01). The test substance CR20293 showed a certain tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, biw×3, and the mean tumor volume was 778.70 mm on the 23rd day after grouping. 3 The TGI value was 55.47%, which was significantly different from the negative control group (P<0.05). The test substance CR20295 showed a certain tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, BIW x 3. On the 23rd day after grouping, the mean tumor volume was 415.38 mm 3 The TGI value was 76.25%, which was significantly different from the negative control group (P<0.001). The test substance CR20297 showed excellent tumor growth inhibitory effects at a dose regimen of 12.5 mg / kg, biw×3, and the mean tumor volume was 300.72 mm on day 23 after group allocation. 3 The TGI value was 82.80%, which was significantly different from the negative control group (P<0.001).

[0549] In summary, compared with the control group and CR20274, the test compounds CR20282, CR20293, CR20295 and CR20297 all showed relatively good antitumor effects and safety against tumor growth in the mouse human prostate cancer model C4-2. Example 16: Pharmacodynamic evaluation of compounds against HT-29 cell subcutaneously transplanted tumors in a BALB / c nude mouse model

[0550] Experimental objective: To preclinically evaluate the efficacy of CR202 series compounds in treating subcutaneous HT-29 cell-implanted tumor models in BALB / c nude mice.

[0551] Experimental method: HT-29 cells were inoculated subcutaneously into the right dorsal side of each mouse until the tumor volume reached an average of 100–150 mm. 3Once the dose reached 100 mg / kg, the animals were divided into groups and administered the treatment. After administration, the animals were routinely monitored for tumor growth and the effects of treatment on their normal behavior, including their activity, feeding and drinking habits, weight gain or loss, and abnormalities in the eyes, hair, and other areas. Any clinical symptoms observed during the test were recorded in the raw data. After administration began, the mice's weights and tumor sizes were measured regularly. The formula for calculating tumor volume was V = 1 / 2 × (a × b 2 ) where a and b represent the long and short diameters of the tumor, respectively. The experimental groupings and administration regimens are shown in Table 28 below. [Table 30]

[0552] Experimental Results and Analysis: In this experiment, CR20282, CR202EZ, and CR202HR demonstrated favorable tumor inhibition rates and safety at both low and high doses compared with equimolar doses of CR20274 (see Figures 9A-9B). Mice in the treatment groups tolerated the treatment well, showed a tendency to gain weight, and no abnormal deaths occurred during the experiment. The negative control group had a mean tumor volume of 2079.64 mm on day 25 after grouping. 3 reached and was euthanized.

[0553] The test substance CR20274 showed a certain tumor growth inhibitory effect at a dose regimen of 2 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 1575.84 mm 3 The TGI value was 24.23%, which was not significantly different from the negative control group (P>0.05).

[0554] The test substance CR20274 showed a certain tumor growth inhibitory effect at a dose regimen of 4 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 1316.64 mm 3 The TGI value was 36.69%, which was not significantly different from the negative control group (P>0.01).

[0555] The test substance CR20282 showed a certain tumor growth inhibitory effect at a dose regimen of 6 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 971.53 mm 3 The TGI value was 53.28%, which was significantly different from the negative control group (P<0.001).

[0556] The test substance CR20282 showed a certain tumor growth inhibitory effect at a dose regimen of 12.5 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 670.36 mm 3 The TGI value was 67.77%, which was significantly different from the negative control group (P<0.001).

[0557] The test substance CR202EZ did not show any consistent tumor growth inhibitory effect at a dosing regimen of 10.4 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 1082.77 mm 3 The TGI value was 47.93%, which was significantly different from the negative control group (P<0.001).

[0558] The test substance CR202EZ showed a certain tumor growth inhibitory effect at a dose regimen of 21.6 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 691.438 mm 3 The TGI value was 66.75%, which was significantly different from the negative control group (P<0.001).

[0559] The test substance CR202HR showed excellent tumor growth inhibitory effects at a dose regimen of 10.9 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 946.05 mm 3 The TGI value was 54.51%, which was significantly different from the negative control group (P<0.001).

[0560] The test substance CR202HR showed excellent tumor growth inhibitory effects at a dosing regimen of 22.7 mg / kg, BIW x 3. On day 25 after group allocation, the mean tumor volume was 614.73 mm 3The TGI value was 70.44%, which was significantly different from the negative control group (P<0.001).

[0561] In summary, the test compounds CR20282, CR202EZ, and CR202HR all exhibited relatively good antitumor effects against tumor growth in the HT-29 human colorectal cancer model subcutaneously transplanted into mice, and showed a clear dose-relationship. Example 17: Pharmacodynamic evaluation of compounds in a BALB / c nude male mouse model with subcutaneous xenografts of the human prostate cancer 22Rv-1 cell line

[0562] Aim of the experiment: To preclinically evaluate the antitumor effects of CR202 series compounds in a BALB / c nude male mouse model subcutaneously xenografted with the human prostate cancer 22Rv-1 cell line.

[0563] Experimental Method: 22Rv-1 (CL-00458) cells were cultured in PRMI 1640 medium containing 10% fetal bovine serum. 22Rv-1 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation in nude mice. Experimental mice were subcutaneously inoculated with 1 x 107 22Rv-1 cells in the right dorsal region and resuspended in PBS (0.1 mL / mouse) mixed with Matrigel at a 1:1 ratio. Tumor growth was monitored periodically until tumors reached an average volume of 100-150 mm. 3 When the tumors reached a size of 100 mm, the animals were randomly assigned to groups according to tumor size and administered the treatment. After administration, the animals were routinely monitored for tumor growth and the effects of treatment on their normal behavior, including activity, feeding and drinking, weight gain or loss, and abnormalities in the eyes, hair coat, and other areas. Any clinical symptoms observed during the study were recorded in the raw data. After administration began, the mice were regularly weighed and their tumors were measured. The formula for tumor volume was V = 1 / 2 × (a × b2), where a and b represent the long and short diameters of the tumor, respectively. The experimental groupings and administration regimens are shown in Table 29 below. [Table 31]

[0564] Experimental Results and Analysis: In this experiment, the changes in tumor volume in mice bearing human prostate cancer 22Rv-1 tumors were shown in Figure 10A, and the changes in body weight over time in mice bearing human prostate cancer 22Rv-1 tumors compared to their initial weight were shown in Figure 10B. As can be seen, both CR202LB and CR202HT demonstrated good tumor inhibition rates and safety. Mice in the treatment groups tolerated the treatment well and showed a tendency to gain weight. No abnormal deaths occurred during the experiment. The negative control group was euthanized on day 29 after grouping, when the average tumor volume reached 1651.17 mm3.

[0565] The test substance CR202LB showed a consistent tumor growth inhibitory effect at a dose of 2.87 mg / kg BIW x 3. On day 29 after grouping, the mean tumor volume reached 969.46 mm3, and the TGI value was 41.29%, showing a significant difference compared to the negative control group (P<0.001).

[0566] The test substance CR202HT showed a consistent tumor growth inhibitory effect at a dose of 2.88 mg / kg, BIW x 3. On day 29 after group allocation, the mean tumor volume reached 690.37 mm3, and the TGI value was 58.19%, showing a significant difference compared to the negative control group (P<0.001).

[0567] In summary, both the CR202LB and CR202HT compounds exhibited relatively good safety and a certain antitumor effect against the growth of human prostate cancer 22Rv-1 subcutaneously transplanted into mice.

[0568] Similar to the above experimental method, the tumor inhibitory effects of other conjugate compounds of the present application were detected. The experimental results showed that all other conjugate compounds of the present application had certain tumor inhibitory effects, and the tumor inhibitory effects of the conjugate compounds were superior to those of the naked drug.

[0569] Example 18: Single-dose PK evaluation of compounds in a human colorectal cancer HT-29 cell line subcutaneous xenograft BALB / c nude male mouse model

[0570] Aim of the study: Single-dose PK evaluation of compounds CR20214 and CR20274 in a BALB / c nude male mouse model with subcutaneous xenografts of human colorectal cancer HT-29 cell line.

[0571] Experimental method: HT-29 cell lines were inoculated subcutaneously on the right dorsal side of each mouse (purchased from Jiangsu Jiaxing Pharmaceutical Co., Ltd., weighing 20-25 g, male, aged 45-60 days), and the average tumor volume was approximately 300 mm. 3 The mice were divided into groups when they reached the age of 18. HT-29 tumor-bearing mice were given a single dose of 6.25 mg / kg CR20214 or 2 mg / kg CR20274 via tail vein injection. At 0.833, 0.25, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours after administration, the tumors were harvested, crushed, and homogenized for liquid-liquid extraction. After centrifugation, the supernatant was collected and injected into a triple quadrupole tandem mass spectrometer to quantitatively measure the CR20274 content in the tumor. See Table 30. [Table 32]

[0572] Experimental Results and Analysis: Under equimolar dose administration conditions, CR20214 had a higher concentration than CR20274 in tumor tissue, indicating that CR20214 has better targeting ability.

Claims

1. A conjugate compound or a pharma- ceutically acceptable salt thereof, comprising a targeting molecule of at least two ligands that specifically bind to cell surface proteins and a payload linked to said targeting molecule, wherein said payload is a proteolytic targeting chimera. A conjugate compound or a pharma- ceutically acceptable salt thereof.

2. The targeting molecule binds to the following cell surface proteins: FOLR1, TRPV6, PSMA, LHRH, EGFR, Her2, Trop2, Her3, Claudin18.2, c-Met, or any combination thereof; The conjugate compound according to claim 1 or a pharma- ceutically acceptable salt thereof.

3. The targeting molecule binds to the cell surface proteins FOLR1, TRPV6, PSMA, c-Met, or any combination thereof.

3. The conjugate compound according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof.

4. the targeting molecule binds to the following combinations of cell surface proteins: FOLR1 and TRPV6, FOLR1 and PSMA, TRPV6 and PSMA, TRPV6 and c-Met, FOLR1 and c-Met, PSMA and c-Met, FOLR1, TRPV6 and PSMA, FOLR1, TRPV6 and c-Met, FOLR1, PSMA and c-Met, TRPV6, PSMA and c-Met, or FOLR1, PSMA, TRPV6 and c-Met; The conjugate compound according to any one of claims 1 to 3, or a pharma- ceutically acceptable salt thereof.

5. the targeting molecule comprises two ligands and binds to FOLR1 and TRPV6, FOLR1 and PSMA, TRPV6 and PSMA, FOLR1 and c-Met, PSMA and c-Met, or TRPV6 and c-Met, respectively; The conjugate compound according to any one of claims 1 to 4, or a pharma- ceutically acceptable salt thereof.

6. The ligand that binds to FOLR1 includes folic acid or an analog thereof and pteroic acid, and preferably, the folic acid analog is selected from 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 10-formyltetrahydrofolic acid, methotrexate, 5,10-formyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, aminopterin, and raltitrexed; The conjugate compound according to any one of claims 2 to 5, or a pharma- ceutically acceptable salt thereof.

7. The ligand that binds to TRPV6 comprises an amino acid sequence set forth in SEQ ID NO:1, an amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:

2. The conjugate compound according to any one of claims 2 to 6, or a pharma- ceutically acceptable salt thereof.

8. The ligand that binds to PSMA includes a polypeptide, an antibody, or a small molecule. The conjugate compound according to any one of claims 2 to 7, or a pharma- ceutically acceptable salt thereof.

9. The ligand that binds to PSMA is selected from the following structures: 【Chemistry 1】 The conjugate compound according to any one of claims 2 to 8, or a pharma- ceutically acceptable salt thereof.

10. The ligand that binds to c-MET is Cys ａ -X1-Cys ｃ -X2-Gly-Pro-Pro-X3-Phe-Glu-Cys ｄ -Trp-Cys ｂ X1 is Asn, His, or Tyr; X2 is Gly, Ser, Thr, or Asn; X3 is Thr or Arg; X4 is Ala, Asp, Glu, Gly, or Ser; X5 is Ser or Thr; X6 is Asp or Glu; and ａ－ｄ are each a cysteine ​​residue, preferably the residue Cys ａ and Cys ｂ , and Cys ｃ and Cys ｄ each cyclizes to form two independent disulfide bonds, The conjugate compound according to any one of claims 2 to 9, or a pharma- ceutically acceptable salt thereof.

11. The ligand that binds to c-MET is Ala-Gly-Ser-Cys ａ -Tyr-Cys ｃ -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys ｄ -Trp-Cys ｂ -Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys, an amino acid sequence as shown in SEQ ID NO: 4, in which Cys ａ－ｄ are each a cysteine ​​residue, preferably the residue Cys ａ and Cys ｂ , and Cys ｃ and Cys ｄ each cyclizes to form two independent disulfide bonds, optionally the carboxyl group of the terminal lysine may be amidated, and optionally the N-terminal alanine may be acetylated; The conjugate compound according to any one of claims 2 to 10, or a pharma- ceutically acceptable salt thereof.

12. The ligand that binds to c-MET comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4, and wherein the amino acid sequence is selected from the group consisting of Cys ａ－ｄ are each a cysteine ​​residue, preferably the residue Cys ａ and Cys ｂ , and Cys ｃ and Cys ｄ each cyclizes to form two independent disulfide bonds, The conjugate compound according to any one of claims 2 to 11, or a pharma- ceutically acceptable salt thereof.

13. The ligands are linked directly or via a spacer region, and preferably the linking is carried out by dehydration condensation. The conjugate compound according to any one of claims 1 to 12, or a pharma- ceutically acceptable salt thereof.

14. the spacer region comprises an amino acid or combination of amino acids selected from the group consisting of Lys, Cys, Lys-Cys, Cys-Cys, Lys-Cys-Lys, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg, Asp-Asp-Lys-Cys, and Pro-Leu-Gly, optionally wherein the amino acids are amidated; 14. The conjugate compound of claim 13 or a pharma- ceutically acceptable salt thereof.

15. Lys is amidated, for example to 2,6-diaminohexanamide; 15. The conjugate compound of claim 14 or a pharma- ceutically acceptable salt thereof.

16. The spacer region comprises one or more identical or different compounds formed by dehydration condensation, selected from the group consisting of: 【Chemistry 2】 wherein n is 0 or 1; 14. The conjugate compound of claim 13 or a pharma- ceutically acceptable salt thereof.

17. A targeting molecule having the following structure is linked to a payload to form a conjugate compound: 【Chemistry 3】 【Chemistry 4】 wherein Q is an active group, and the targeting molecule is linked to a payload via the active group Q; preferably, the active group Q is an alkynyl group or a mercapto group; and preferably, the targeting molecule has the following structure: 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 17. The conjugate compound according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof.

18. A proteolytic targeting chimera having the structure shown in formula (I) is linked to a targeting molecule to form a conjugate compound, (T Ｐ ) ｎ -L-T Ｅ３ (I)、 Among them, Said T ｐ is a target protein ligand moiety that binds to a target protein, wherein n is 1, 2, or 3; The L is T Ｐ and T Ｅ３ and Said T Ｅ３ is an E3 ligase ligand moiety that binds to E3 ligase; 18. The conjugate compound according to any one of claims 1 to 17, or a pharma- ceutically acceptable salt thereof.

19. The E3 ligase ligand is bound to a VHL (von Hippel-Lindau) protein, a CRBN (cerebolon) protein, an MDM2 protein, or a XIAP protein, and preferably, the E3 ligase ligand is a VHL ligand or a CRBN ligand.

19. The conjugate compound of claim 18, or a pharma- ceutically acceptable salt thereof.

20. The VHL ligand is VHL-L or an analog thereof, and the CRBN ligand is lenalidomide, pomalidomide, thalidomide, or an analog thereof.

20. The conjugate compound of claim 19, or a pharma- ceutically acceptable salt thereof.

21. The proteolysis targeting chimera has a structure selected from the group consisting of: 【Chemistry 8】 Among them, Ra, Rb and Rc each independently represent a hydrogen atom, an alkyl group, NH ２ , OH, or a halogen, preferably, the alkyl group is a methyl group and the halogen is fluorine; Rd is CH ２ , C=O or C=S; p is 0 or 1; the target protein ligand is capable of binding to a specific target protein; The target protein ligand is linked to an E3 ligase group via the linking moiety. The conjugate compound according to any one of claims 18 to 20, or a pharma- ceutically acceptable salt thereof.

22. The target protein is a nuclear receptor (e.g., AR and ER), a kinase protein (e.g., RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, and FAK), AKT, PAN-BET protein, BET protein (e.g., BRD2, BRD3, BRD4, and BRD6), BLK, BRAF1, BCL-xl, ERRα, FKBP12, FRS2α, JAK1, JAK3, IKZF1, IRAK4, MDM2, MetAP2, PLK1, PSK-J3, RAS, TACC3, Tau, TrkB, MDM2, or any combination thereof; 22. The conjugate compound according to any one of claims 18 to 21, or a pharma- ceutically acceptable salt thereof.

23. The target protein is an AR protein, an ER protein, a RAS protein, a BRD4 protein, a PLK1 protein, a FAK protein, an MDM2 protein, or any combination thereof; 23. The conjugate compound according to any one of claims 18 to 22, or a pharma- ceutically acceptable salt thereof.

24. The target protein ligand portion has a structure selected from the following: 【Chemistry 9】 The conjugate compound according to any one of claims 18 to 23, or a pharma- ceutically acceptable salt thereof.

25. The linking moiety has a structure selected from the group consisting of: 【Chemistry 10】 Among them, p １ is an integer from 0 to 4, p ２ is an integer from 0 to 6, p ３ , p ４ , p ５ and p ６ are each independently 0 or 1; G １ is a 4-14 membered linear alkylene group, optionally 2 to 5 carbon atoms in said 4-14 membered linear alkylene group are selected from -O-, -NH-, -N(C １－６ alkyl)- or -C≡C-, G ２ is an amino acid residue, among which, preferably, ２ is a Lys residue, G ３ teeth, 【Chemistry 11】 Selected from the group consisting of: G ４ is a 5- to 9-membered linear alkylene group, and optionally, 2 to 4 carbon atoms in said 5- to 9-membered linear alkylene group are selected from -O-, -NH-, -N(C １－６ alkyl)- or -C≡C-, The conjugate compound according to any one of claims 18 to 24, or a pharma- ceutically acceptable salt thereof.

26. The linking moiety has a structure selected from the group consisting of: 【Chemistry 12】 26. The conjugate compound according to any one of claims 18 to 25, or a pharma- ceutically acceptable salt thereof.

27. The proteolytic targeting chimera has the structure shown below: 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 27. The conjugate compound according to any one of claims 18 to 26, or a pharma- ceutically acceptable salt thereof.

28. The payload is directly linked to the targeting molecule, or the payload is linked to the targeting molecule via a linker.

28. The conjugate compound according to any one of claims 1 to 27, or a pharma- ceutically acceptable salt thereof.

29. The link between the payload and the targeting molecule, between the payload and the linker, or between the linker and the targeting molecule is completed by a chemical reaction, preferably, the link between the targeting molecule and the linker is completed by a click reaction; 29. The conjugate compound according to claim 1, or a pharma- ceutically acceptable salt thereof.

30. The complex compound has the following structure: W １ -L １ -(EG)n １ -(D)n ５ -(CH ２ )n ２ -(B)n ６ -(AA)n ３ -(CH ２ CH ２ -Q)n ４ -L ２ -W ２ 、 Among them, L １ teeth, 【Chemistry 16】 -S-S- or a bond, EG is -CH ２ CH ２ O- or -OCH ２ CH ２ - and n １ is an integer from 0 to 6, n ２ is an integer from 0 to 8, D is -C(=O)-NH-S(=O) ２ -NH-, n ５ is 0 or 1, B is independently at each occurrence -C(=O)-, 【Chemistry 17】 -NH-CH ２ CH ２ -C(=O)-, -C(=O)-NH-, -CH(CH ３ )-CH ２ -O-, -CH ２ CH ２ -O-, -NH-C(=O)- or -NH-, n ６ is an integer from 0 to 3, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 5, Q is independently at each occurrence -C(=O)-, -O-, or a bond; ４ is an integer from 0 to 3, L ２ teeth, 【Chemistry 18】 -NH-CH ２ -O- or a bond, In the above, X is a bond, -NH-, -C(=O)-, oxygen, or 【Chemistry 19】 and R ２ is a bond, —O—CH ２ -or-NH-CH ２ CH ２ -NH-C(=O)-, R ３ is hydrogen or 【Chemistry 20】 Of these, R ｂ and R ｃ each independently represents a hydrogen atom, a nitro group, 【Chemistry 21】 and R ４ is hydrogen or C １－８ is an alkyl group, R ５ is a hydrogen atom, an amino group, a nitro group, a phosphate group, -OR ａ or 【Chemistry 22】 Of these, R ａ is a methyl group, an ethyl group, a propyl group, or an isopropyl group, W １ is a targeting molecule capable of binding to a cell surface protein, and W ２ is the payload, Preferably, the L ２ is a bond, 【Chemistry 23】 and Preferably, EG is -CH ２ CH ２ O-, Preferably, n １ is 2 or 0, Preferably, n ２ is 1, 4 or 5, Preferably, n ５ is 0, Preferably, n ６ is 1 or 3, Preferably, n １ is 2, and n ２ is 1, and n ３ and n ４ is 0, and L ２ is a bond, Preferably, each occurrence of AA is an amino acid residue formed independently by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro or Asn; Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of: -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and Ala-Ala-Asn-; Preferably, n ４ is 1, Q is -C(=O)-, Preferably, n ４ is 2, Q is —O— and —C(═O)—, respectively; Preferably, n ４ is 3, Q is respectively -O-, -O- and a bond; or Preferably, n ４ is 0, 30. The conjugate compound according to any one of claims 1 to 29, or a pharma- ceutically acceptable salt thereof.

31. The complex compound has the following structure: W １ -L １ -(EG)n １ -(D)n ５ -(CH ２ )n ２ -(B)n ６ -(AA)n ３ -(CH ２ CH ２ -Q)n ４ -L ２ -W ２ Among them, L １ teeth, 【Chemistry 24】 and EG is -CH ２ CH ２ O-, n １ is an integer from 0 to 4, D is -C(=O)-NH-S(=O) ２ -NH-, n ５ is 0 or 1, n ２ is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH ２ CH ２ O-, -NH-CH ２ CH ２ -C(=O)- or -NH-, n ６ is an integer from 0 to 3, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 3, Q is independently at each occurrence -C(=O)-, -O-, or a bond; ４ is an integer from 0 to 3, L ２ teeth, 【Chemistry 25】 and In the above, X is a bond, -NH-, -C(=O)-, or 【Chemistry 26】 and R ２ is a bond, R ３ teeth 【Chemical 27】 Of these, R ｂ and R ｃ are each independently a hydrogen atom, a nitro group, or 【Chemistry 28】 and R ４ is hydrogen, R ５ is hydrogen, -OCH ３ or 【Chemical 29】 and W １ is a targeting molecule capable of binding to a cell surface protein, and W ２ is the payload, Preferably, the L ２ teeth, 【Chemistry 30】 and Preferably, L １ teeth 【Chemistry 31】 and n １ , n ５ and n ４ are both 0, Preferably, L １ teeth 【Chemistry 32】 and n ２ is 1 or 2, Preferably, n ５ If n is 1, ２ and n ４ are both 0, Preferably, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu or Ala; or Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-; 31. The conjugate compound according to any one of claims 1 to 30, or a pharma- ceutically acceptable salt thereof.

32. The complex compound has the following structure: W １ -L １ -(EG)n １ -(CH ２ )n ２ -(C=O)-(AA)n ３ -L ２ -W ２ Among them, L １ teeth, 【Chemical 33】 and EG is -CH ２ CH ２ O-, n １ is an integer from 0 to 4, n ２ is an integer from 0 to 5, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 1 to 4, L ２ teeth, 【Chemical 34】 or a bond, In this, X is -NH-, and R ２ is a bond, R ４ is hydrogen, R ５ is hydrogen or -OCH ３ and W １ is a targeting molecule capable of binding to a cell surface protein, and W ２ is the payload, Preferably, L １ teeth 【Chemistry 35】 and n １ is 2, and n ２ is 1, and L ２ teeth 【Chemical Formula 36】 and Preferably, L １ teeth 【Chemical 37】 and n １ is 2, and n ２ is 1, and L ２ is a bond, Preferably, L １ teeth 【Chemical 38】 and n １ is 0, n ２ is 5, and L ２ teeth 【Chemical 39】 and Preferably, L １ teeth 【Chemistry 40】 and n １ is 0, n ２ is 5, and L ２ is a bond, Preferably, each occurrence of AA is independently a Val, Cit, Glu, or Ala amino acid residue; or Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Leu-Lys- and -Gly-Asn-Asn-, 32. The conjugate compound according to any one of claims 1 to 31, or a pharma- ceutically acceptable salt thereof.

33. The linker comprises a link module 1 and a link module 2, optionally wherein the link module 1 and the link module 2 are linked via a bond, and optionally wherein the link module 1 and the link module 2 are linked via a chemical group; 33. The conjugate compound according to any one of claims 28 to 32, or a pharma- ceutically acceptable salt thereof.

34. The linker comprises a linking module 2, preferably the linking module 2 may be enzymatically cleavable or acid labile; 33. The conjugate compound according to any one of claims 28 to 32, or a pharma- ceutically acceptable salt thereof.

35. The linker comprises a link module 1 and a group that binds to the link module 1, optionally the group that binds to the link module 1 is a PEG chain, 【Chemistry 41】 C １ -C ８ an alkyl chain, -NH-, -O-, -C(O)-, or any combination thereof; Preferably, the PEG chain is 【Chemistry 42】 and Optionally, the group attached to the linking module 1 is 【Chemistry 43】 That is, 33. The conjugate compound according to any one of claims 28 to 32, or a pharma- ceutically acceptable salt thereof.

36. The connection module 1 is 【Chemistry 44】 That is, 36. The conjugate compound according to any one of claims 33 to 35, or a pharma- ceutically acceptable salt thereof.

37. The linking module 2 has a structure including a CB enzyme cleavage group, a plasmin enzyme cleavage group, an asparagine endonuclease enzyme cleavage group, a β-glucuronidase enzyme cleavage group, a sulfatase enzyme cleavage group, a phosphatase enzyme cleavage group, a β-galactosidase enzyme cleavage group, a glutathione enzyme cleavage group, or an acid labile group, before being linked to a targeting molecule, a linking module 1, a chemical group, or a payload.

37. The conjugate compound according to any one of claims 33 to 36, or a pharma- ceutically acceptable salt thereof.

38. The chemical group may be a PEG chain, 【Chemistry 45】 C １ -C ８ Alkyl chain, -NH-, -O-, -C(O)-, 【Chemistry 46】 or any combination thereof, Preferably, the PEG chain is PEG1, PEG2, or PEG3; Preferably, the chemical group is 【Chemistry 47】 That is, 34. The conjugate compound of claim 33, or a pharma- ceutically acceptable salt thereof.

39. the CB enzyme cleavage group comprises an amino acid which is Val-Cit, Gly-Gly-Phe-Gly, CycloBut-Cit, Phe-Cit, Val-Ala, Glu-Val-Cit, Ala-Cit, Val-Cit-Pro or Val-Cit-Pro-Gly, or a combination of an amino acid and an autolytic fragment; the plasmin enzyme cleavage group comprises the amino acids Val-Leu-Lys, or a combination of amino acids and autolytic fragments; the asparagine endonuclease group comprises amino acids selected from Gly-Asn-Asn or Ala-Ala-Asn, or a combination of amino acids and autolytic fragments; The β-glucuronidase enzyme cleavable group is 【Chemistry 48】 and The sulfatase enzyme cleavable group is 【Chemistry 49】 and The phosphatase enzyme cleavable group is 【Chemistry 50】 and The β-galactosidase enzyme cleavable group is 【Chemistry 51】 and The glutathione enzyme cleavable group is 【Chemistry 52】 and The acid labile group is 【Chemistry 53】 and Optionally, the autolytic fragment is PAB and its derivatives, 【Chemical 54】 and Preferably, the PAB is 【Chemistry 55】 and Preferably, the PAB derivative is 【Chemistry 56】 That is, 38. The conjugate compound of claim 37, or a pharma- ceutically acceptable salt thereof.

40. The linking module 2 portion, after operatively linking with linking module 1, a chemical group, targeting molecule, or payload, has the following structure: 【Chemistry 57】 having 40. The conjugate compound of claim 39, or a pharma- ceutically acceptable salt thereof.

41. The complex compound has the following structure: 【Chemistry 58】 【Chemistry 59】 【Chemistry 60】 【Chemistry 61】 【Chemistry 62】 【Chemistry 63】 Among them, W １ is a targeting molecule capable of binding to a cell surface protein, and W ２ is the payload, 41. The conjugate compound according to any one of claims 1 to 40, or a pharma- ceutically acceptable salt thereof.

42. The conjugate compound has the following structure: 【Chemistry 64】 【Chemistry 65】 【Chemistry 66】 【Chemistry 67】 【Chemistry 68】 【Chemistry 69】 【Chemistry 70】 【Chemistry 71】 【Chemical Formula 72】 【Chemical Formula 73】 【Chemical 74】 【Chemistry 75】 42. The conjugate compound according to claim 1, or a pharma- ceutically acceptable salt thereof.

43. A method for the preparation of a compound according to any one of claims 1 to 42, or a pharma- ceutically acceptable salt thereof, comprising administering to said patient a pharma- ceutically acceptable carrier. Pharmaceutical compositions.

44. The composition is used for intravenous, subcutaneous, oral, intramuscular or intraventricular administration.

44. The pharmaceutical composition of claim 43.

45. A method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate compound according to any one of claims 1 to 42 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to claim 43 or 44. method.

46. The disease is selected from the group consisting of cancer, immune diseases, cardiovascular diseases, metabolic diseases, and neurological diseases; 46. ​​The method of claim 45.

47. The cancer is characterized in that cancer cells highly express FOLR1, TRPV6, PSMA or c-MET.

47. The method of claim 46.

48. The cancer is characterized in that the cancer cells highly express FOLR1 and TRPV6, FOLR1 and c-MET, c-MET and TRPV6, c-MET and PSMA, PSMA and TRPV6, or FOLR1 and PSMA.

48. The method of claim 47.

49. The disease is characterized in that the target protein is expressed or the target protein is abnormally expressed in the subject.

49. The method according to any one of claims 45 to 48.

50. The cancer is selected from the group consisting of human breast ductal carcinoma, human brain astrocytoma, bladder transitional cell papilloma, prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, gastric cancer, cervical cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, lymphoma, and multiple myeloma; 47. The method of claim 46.

51. The immune disease can be selected from an autoimmune disease, the autoimmune disease being selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus; 47. The method of claim 46.

52. The cardiovascular disease is selected from the group consisting of angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease; 47. The method of claim 46.

53. The metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia; 47. The method of claim 46.

54. The neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, dizziness, stupor, and epilepsy; 47. The method of claim 46.

55. The method further comprises administering one or more therapeutic agents in combination with the conjugate compound or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition.

55. The method according to any one of claims 45 to 54.

56. 45. Use of a conjugate compound or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 42, or a pharmaceutical composition according to claim 43 or 44, in the preparation of a medicament for treating a disease in a subject.

57. A compound or a salt thereof, the compound having the structure: L ａ -(EG)n １ -(D)n ５ -(CH ２ )n ２ -(B)n ６ -(AA)n ３ -(CH ２ CH ２ -Q)n ４ -L ｂ 、 Among them, L ａ teeth, 【Chemical Formula 76】 and EG is -CH ２ CH ２ O- or -OCH ２ CH ２ - and n １ is an integer from 0 to 6, n ２ is an integer from 0 to 8, D is -C(=O)-NH-S(=O) ２ -NH-, n ５ is 0 or 1, B is -C(=O)-, 【Chemical 77】 -NH-CH ２ CH ２ -C(=O)-, -C(=O)-NH-, -CH(CH ３ )-CH ２ -O-, -CH ２ CH ２ -O-, -NH-C(=O)- or -NH-, n ６ is an integer from 0 to 3, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 5, Q is independently at each occurrence -C(=O)-, -O-, or a bond; ４ is an integer from 0 to 3, L ｂ teeth, 【Chemical 78】 -OH or -NH-CH ２ -OH, In the above, X is a bond, -NH-, -C(=O)-, oxygen, or 【Chemical 79】 and R ２ is a bond, —O—CH ２ -or-NH-CH ２ CH ２ -NH-C(=O)-, R ３ is hydrogen or 【Chemistry 80】 Of these, R ｂ and R ｃ each independently represents a hydrogen atom, a nitro group, 【Chemistry 81】 and R ４ is hydrogen or C １－８ is an alkyl group, R ５ is a hydrogen atom, an amino group, a nitro group, a phosphate group, -OR ａ or 【Chemistry 82】 Of these, R ａ is a methyl group, an ethyl group, a propyl group, or an isopropyl group, R １ is a leaving group, preferably R １ is a halogen (e.g., chlorine), 【Chemistry 83】 and Preferably, the L ｂ teeth, 【Chemistry 84】 and Preferably, EG is -CH ２ CH ２ O-, Preferably, n １ is 0 or 2, Preferably, n ２ is 1, 4 or 5, Preferably, n ５ is 0, Preferably, n ６ is 1 or 3, Preferably, n １ is 2, and n ２ is 1, and n ３ and n ４ is 0, and L ｂ is —OH, Preferably, each occurrence of AA is an amino acid residue formed independently by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro or Asn; Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and -Ala-Ala-Asn-; Preferably, n ４ is 1, Q is -C(=O)-, Preferably, n ４ is 2, Q is —O— and —C(═O)—, respectively; Preferably, n ４ is 3, Q is respectively -O-, -O- and a bond; or Preferably, n ４ is 0, A compound or a salt thereof.

58. The compound has the following structure: L ａ -(EG)n １ -(D)n ５ -(CH ２ )n ２ -(B)n ６ -(AA)n ３ -(CH ２ CH ２ -Q)n ４ -L ｂ Among them, L ａ teeth, 【Chemistry 85】 and EG is -CH ２ CH ２ O-, n １ is an integer from 2 to 4, D is -C(=O)-NH-S(=O) ２ -NH-, n ５ is 0 or 1, n ２ is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH ２ CH ２ O-, -NH-CH ２ CH ２ -C(=O)- or -NH-, n ６ is an integer from 0 to 3, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 3, Q is independently at each occurrence -C(=O)-, -O-, or a bond; ４ is an integer from 0 to 3, L ｂ is -OH, 【Chemistry 86】 and In the above, X is a bond, -NH-, -C(=O)-, or 【Chemistry 87】 and R ２ is a bond, R ３ teeth 【Chemistry 88】 Of these, R ｂ and R ｃ are each independently a hydrogen atom, a nitro group, or 【Chemistry 89】 and R ４ is hydrogen, R ５ is hydrogen, -OCH ３ or 【Chemistry 90】 and R １ is a leaving group, preferably R １ is a halogen (e.g., chlorine), 【Chemistry 91】 and Preferably, L ｂ is -OH, 【Chemistry 92】 and Preferably, n ５ If n is 1, ２ and n ４ are both 0, Preferably, L ａ teeth 【Chemistry 93】 and n １ , n ５ and n ４ are both 0, Preferably, L ａ teeth 【Chemistry 94】 and n ２ is 1 or 2, Preferably, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu or Ala; or Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-; 58. The compound according to claim 57, or a salt thereof.

59. The compound has the following structure: . ａ (!)n １ () ２ )) ２ ___________________________ ３ fi ｂ Among them, L ａ teeth, 【Chemistry 95】 and EG is -CH ２ CH ２ O-, n １ is an integer from 0 to 4, n ２ is an integer from 0 to 5, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 3, L ｂ teeth, 【Chemistry 96】 or -OH, In this, X is -NH-, and R ２ is a bond, R ４ is hydrogen, R ５ is hydrogen or -OCH ３ and R １ is a leaving group, preferably R １ is a halogen (e.g., chlorine), 【Chemistry 97】 and Preferably, L ａ teeth 【Chemistry 98】 and n １ is 2, and n ２ is 1, and L ｂ teeth 【Chemistry 99】 and Preferably, L ａ teeth 【Chemistry 100】 and n １ is 2, and n ２ is 1, and L ｂ is —OH, Preferably, L ａ teeth 【Chemistry 101】 and n １ is 0, n ２ is 5, and L ｂ teeth 【Chemistry 102】 and Preferably, L ａ teeth 【Chemistry 103】 and n １ is 0, n ２ is 5, and L ｂ is —OH, Preferably, each occurrence of AA is independently a Val, Cit, Glu, or Ala amino acid residue; or Preferably, the -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Leu-Lys- and -Gly-Asn-Asn-, 59. A compound or a salt thereof according to claim 57 or 58.

60. The compound has a structure selected from the group consisting of: 【Chemistry 104】 【Chemistry 105】 【Chemistry 106】 【Chemistry 107】 Among them, R １ is a leaving group, preferably R １ is a halogen (e.g., chlorine), 【Chemistry 108】 That is, 60. The compound or salt thereof according to any one of claims 57 to 59.

61. 【Catalogue 109】 or a salt thereof.

62. A conjugate compound or a pharma- ceutically acceptable salt thereof, said conjugate compound having the structure: W ａ -L １ -(EG)n １ -(D)n ５ -(CH ２ )n ２ -(B)n ６ -(AA)n ３ -(CH ２ CH ２ -Q)n ４ -L ２ -W ｂ 、 Among them, L １ teeth, 【Chemistry 110】 -S-S- or a bond, EG is -CH ２ CH ２ O- or -OCH ２ CH ２ - and n １ is an integer from 0 to 6, n ２ is an integer from 0 to 8, D is -C(=O)-NH-S(=O) ２ -NH-, n ５ is 0 or 1, B is independently at each occurrence -C(=O)-, 【Chemistry 111】 -NH-CH ２ CH ２ -C(=O)-, -C(=O)-NH-, -CH(CH ３ )-CH ２ -O-, -CH ２ CH ２ -O-, -NH-C(=O)- or -NH-, n ６ is an integer from 0 to 3, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 5, Q is independently at each occurrence -C(=O)-, -O-, or a bond; ４ is an integer from 0 to 3, L ２ teeth, 【Chemistry 112】 -NH-CH ２ -OH or a bond; In the above, X is a bond, -NH-, -C(=O)-, oxygen, or 【Chemistry 113】 and R ２ is a bond, —O—CH ２ -or-NH-CH ２ CH ２ -NH-C(=O)-, R ３ is hydrogen or 【Chemistry 114】 Of these, R ｂ and R ｃ each independently represents a hydrogen atom, a nitro group, 【Chemistry 115】 and R ４ is hydrogen or C １－８ is an alkyl group, R ５ is a hydrogen atom, an amino group, a nitro group, a phosphate group, -OR ａ or 【Chemistry 116】 Of these, R ａ is a methyl group, an ethyl group, a propyl group, or an isopropyl group, W ａ and W ｂ are each independently a targeting molecule capable of binding to a payload or a cell surface protein; Preferably, the L ２ is a bond, 【Chemistry 117】 and Preferably, EG is -CH ２ CH ２ O-, Preferably, n １ is 0 or 2, Preferably, n ２ is 1, 4 or 5, Preferably, n ５ is 0, Preferably, n ６ is 1 or 3, Preferably, n １ is 2, and n ２ is 1, and n ３ and n ４ is 0, and L ２ is a bond, Preferably, each occurrence of AA is an amino acid residue formed independently by Val, Cit, Glu, Leu, Lys, Ala, Gly, Phe, Gln, Pro or Asn; Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit-, -Phe-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Cit-, -Val-Leu-Lys-, -Val-Cit-Pro-Gly-, -Gly-Asn-Asn- and -Ala-Ala-Asn-; Preferably, n ４ is 1, Q is -C(=O)-, Preferably, n ４ is 2, Q is —O— and —C(═O)—, respectively; Preferably, n ４ is 3, Q is respectively -O-, -O- and a bond; or Preferably, n ４ is 0, A conjugate compound or a pharma- ceutically acceptable salt thereof.

63. The complex compound has the following structure: W ａ -L １ -(EG)n １ -(D)n ５ -(CH ２ )n ２ -(B)n ６ -(AA)n ３ -(CH ２ CH ２ -Q)n ４ -L ２ -W ｂ Among them, L １ teeth, 【Chemistry 118】 and EG is -CH ２ CH ２ O-, n １ is an integer from 2 to 4, D is -C(=O)-NH-S(=O) ２ -NH-, n ５ is 0 or 1, n ２ is an integer from 0 to 5, B is -C(=O)-, -C(=O)-NH-, -CH ２ CH ２ O-, -NH-CH ２ CH ２ -C(=O)- or -NH-, n ６ is an integer from 0 to 3, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 0 to 3, Q is independently at each occurrence -C(=O)-, -O-, or a bond; ４ is an integer from 0 to 3, L ２ teeth, 【Chemistry 119】 and In the above, X is a bond, -NH-, -C(=O)-, or 【Chemistry 120】 and R ２ is a bond, R ３ teeth 【Chemistry 121】 Of these, R ｂ and R ｃ are each independently a hydrogen atom, a nitro group, or 【Chemistry 122】 and R ４ is hydrogen, R ５ is hydrogen, -OCH ３ or 【Chemistry 123】 and W ａ and W ｂ are each independently a targeting molecule capable of binding to a payload or a cell surface protein; Preferably, L ２ teeth, 【Chemistry 124】 and Preferably, n ５ If n is 1, ２ and n ４ are both 0, Preferably, L １ teeth 【Chemistry 125】 and n １ , n ５ and n ４ are both 0, Preferably, L １ teeth 【Chemistry 126】 and n ２ is 1 or 2, Preferably, each occurrence of AA is independently an amino acid residue formed by Val, Cit, Glu or Ala; or Preferably, -(AA)n ３ - is a peptide residue selected from the group consisting of -Val-Cit-, -Glu-Val-Cit-, -Ala-Cit- and -Val-Ala-; 63. The conjugate compound of claim 62, or a pharma- ceutically acceptable salt thereof.

64. The complex compound has the following structure: W ａ -L １ -(EG)n １ -(CH ２ )n ２ -(C=O)-(AA)n ３ -L ２ -W ｂ Among them, L １ teeth, 【Chemistry 127】 and EG is -CH ２ CH ２ O-, n １ is an integer from 0 to 4, preferably n ２ is an integer from 1 to 5, AA, each occurrence, is independently an amino acid residue; ３ is an integer from 1 to 4, L ２ teeth, 【Chemistry 128】 or a bond, In this, X is -NH-, and R ２ is a bond, R ４ is hydrogen, R ５ is hydrogen or -OCH ３ and W ａ and W ｂ are each independently a targeting molecule capable of binding to a payload or a cell surface protein; Preferably, L １ teeth 【Chemistry 129】 and n １ is 2, and n ２ is 1, and L ２ teeth 【Chemistry 130】 and Preferably, L １ teeth 【Chemistry 131】 and n １ is 2, and n ２ is 1, and L ２ is a bond, Preferably, L １ teeth 【Chemistry 132】 and n １ is 0, n ２ is 5, and L ２ teeth 【Chemistry 133】 and Preferably, L １ teeth 【Chemistry 134】 and n １ is 0, n ２ is 5, and L ２ is a bond, 63. The conjugate compound of claim 61 or 62, or a pharma- ceutically acceptable salt thereof.

65. The compound has a structure selected from the group consisting of: 【Chemistry 135】 【Chemistry 136】 【Chemistry 137】 【Chemistry 138】 【Chemistry 139】 Among them, W ａ and W ｂ are each independently a payload or a targeting molecule capable of binding to a cell surface protein; 65. The conjugate compound according to any one of claims 52 to 64, or a pharma- ceutically acceptable salt thereof.

66. A conjugate compound or a pharma- ceutically acceptable salt thereof, wherein the conjugate compound is 【Chemistry 140】 Of these, W ａ and W ｂ are each independently a payload or a targeting molecule capable of binding to a cell surface protein; A conjugate compound or a pharma- ceutically acceptable salt thereof.

67. A conjugate compound or a pharma- ceutically acceptable salt thereof, wherein the conjugate compound is 【Chemistry 141】 Of these, W ａ and W ｂ are each independently a payload or a targeting molecule capable of binding to a cell surface protein; A conjugate compound or a pharma- ceutically acceptable salt thereof.

68. A proteolysis targeted chimera or a pharma- ceutically acceptable salt thereof, wherein the proteolysis targeted chimera compound has the structure: 【Chemistry 142】 Among them, Ra and Rb are each independently hydrogen, C １－４ Alkyl group, NH ２ , OH, or halogen; A is an amino acid residue, a is 0 or 1, b is an integer from 0 to 5; X is, 【Chemistry 143】 or a bond, The target protein ligand is 【Chemistry 144】 That is, A proteolysis targeted chimera or a pharma- ceutically acceptable salt thereof.

69. Ra is hydrogen; 69. The protein degradation targeting chimera of claim 68, or a pharma- ceutically acceptable salt thereof.

70. Rb is hydrogen; 69. The protein degradation targeting chimera of claim 68, or a pharma- ceutically acceptable salt thereof.

71. A is an amino acid residue formed by Lys, 69. The protein degradation targeting chimera of claim 68, or a pharma- ceutically acceptable salt thereof.

72. The proteolysis targeted chimeric compound has a structure selected from the group consisting of: 【Chemistry 145】 【Chemistry 146】 69. The protein degradation targeting chimera of claim 68, or a pharma- ceutically acceptable salt thereof.

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