A highly stable targeted linker-drug conjugate

Abstract

The application discloses a targeted linker-drug conjugate with high stability and excellent anti-tumor effect, and belongs to the field of drugs. The application specifically provides a drug link assembly unit with a specific linker structure, and a targeted linker-drug conjugate formed by connecting the drug link assembly unit with an antibody. The targeted linker-drug conjugate with the specific linker structure has low polymer content and bare antibody percentage, a suitable DAR value, excellent plasma stability, storage stability and anti-tumor effect, and has a wide application prospect in the preparation of drugs for preventing and / or treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a highly stable targeting linker-drug conjugate. Background Technology

[0002] Antibody-drug conjugates (ADCs) selectively deliver drugs to cancer cells and kill them, while having minimal impact on normal cells, ushering in a new era in cancer treatment. Several ADCs have already received FDA approval, such as Mylotarg (a CD33 antibody linked to chazimic acid), Adcetris (a CD30 antibody linked to auristatin E) for the treatment of Hodgkin's lymphoma and undifferentiated large cell lymphoma, DS-8201 (a Her2 antibody linked to a camptothecin derivative Dxd) for the treatment of HER2-positive breast cancer, and Sacituzumab govitecan (targeting TROP-2 antigen, also known as epithelial glycoprotein 1, EGP-1) for triple-negative breast cancer.

[0003] To date, FDA-approved antitumor drug (ADC) primarily targets DNA or tubulin. As small molecule compounds with antitumor effects, camptothecin derivatives (such as SN-38, Dxd, and Dx-8951), which inhibit DNA topoisomerase I to achieve antitumor activity, have been shown to kill various cancer cells both in vivo and in vitro, exhibiting strong antitumor effects. Compounds that inhibit tubulin to achieve antitumor activity, such as eryribulin, MMAE, MMAF, and maytansine, have also been shown to kill various cancer cells both in vivo and in vitro, exhibiting strong antitumor effects. The known structural formulas of compounds that can act on DNA or tubulin are as follows:

[0004]

[0005] According to the classic mechanism of action of antibody-drug conjugates (ADCs), antibody-drug conjugates can specifically bind to cell surface proteins, and the resulting conjugates are endocytosed by the cell, thereby achieving targeted delivery of drug molecules into the cell. Therefore, the intracellular drug concentration is directly related to the distribution density of target sites on the cell surface that can be specifically recognized by the antibody. Unfortunately, the density of target sites on the molecular surface that can be recognized by the antibody is usually low, resulting in a low drug concentration within the target cell. To address this issue, a commonly used method is to increase the antibody-antibody ratio (DAR) of the ADC, thereby increasing the amount of drug entering the cell. However, according to the study by Hamblett et al. (ClinCancer Res. 2004, 10, 7063), increasing the DAR value of the ADC often has the opposite effect in pharmacokinetic exposure. ADC molecules exhibit severe aggregation, reduced stability, and an increase in small molecule toxins in the blood, leading to toxic side effects.

[0006] Daiichi Sankyo Co., Ltd. has filed numerous patent applications since 2013 (CN201380053256.2, CN201910768778.X, CN201980061665.4, etc.), disclosing a series of antibody-drug conjugates with specific linker-toxin structures, and specifically disclosing ADCs with the following typical structures that exhibit superior effects. However, their stability and therapeutic efficacy still need further improvement.

[0007]

[0008] Therefore, it is of great significance to develop a drug conjugate that can significantly improve aggregation, enhance stability and therapeutic efficacy, and target the lesion site. Summary of the Invention

[0009] The purpose of this invention is to provide a highly stable drug linking assembly unit and a target connector-drug conjugate.

[0010] This invention provides a drug linker assembly unit, or its stereoisomer, optical isomer, salt, or deuterated derivative thereof, wherein the drug linker assembly unit comprises a connector and a drug, the connector end being capable of connecting to a target connector, the target connector being a substance capable of targeting and binding to a lesion site; the connector includes the structure shown in Formula A, as shown in Formula A-1:

[0011]

[0012] Among them, W1, W3, and W4 are each independently selected from N and N + E -R1, CR1; R1 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy; N + It is a monovalent nitrogen cation, E - It is a monovalent negative ion;

[0013] W2 is selected from N, N + E - R2, CR2; R2 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy;

[0014] P1, P2, P3, P4, P5, P6, P7, and P8 are each independently selected from none, unsubstituted, or replaced. Replacement C 1～10 Alkylene; n' is an integer from 1 to 50, R' is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 5’ )2, R 5’ Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0015] G1 and G2 are each independently selected from N and N + B - R7; N + B is a monovalent nitrogen cation. - R7 is a monovalent negative ion, selected from C. 1～10 Alkyl, C 1～10 Alkoxy, n1 is an integer from 1 to 50, and R8 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R9)2, R8 selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0016] R3 and R4 are each independently selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, n is an integer from 1 to 50, and R is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R5)2, where R5 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl; or, R3 and R4 linked together to form j is an integer selected from 0 to 10;

[0017] t1 is selected from 0 or 1;

[0018] It is either non-covalent or covalent; when When t2 is zero, t2 is 0; when When the bond is covalent, t2 is 1;

[0019] t1 and t2 are not both 1;

[0020] k1 and k2 are each independently selected from integers from 0 to 10;

[0021] a1, a2, a3, a4, a5, and a6 are each independently selected from integers from 0 to 10;

[0022] a7 is selected from integers from 1 to 50;

[0023] R i Selected from hydrogen, C 1～10 alkyl;

[0024] b1 and b2 are each independently selected from integers from 0 to 10.

[0025] The present invention also provides a drug linker assembly unit, or a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated thereof, wherein the drug linker assembly unit comprises a connector and a drug, the connector end being capable of connecting to a target connector, the target connector being a substance capable of targeting and binding to a lesion site; the connector includes the structure shown in Formula A, the structure shown in Formula A being as shown in Formula A-1:

[0026]

[0027] Among them, W1, W3, and W4 are each independently selected from N and N + E - R1, CR1; R1 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy; N + It is a monovalent nitrogen cation, E- It is a monovalent negative ion;

[0028] W2 is selected from N, N + E - R2, CR2; R2 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy;

[0029] P1, P2, P3, P4, P5, P6, P7, and P8 are each independently selected from none, unsubstituted, or replaced. Replacement C 1～10 Alkylene; n' is an integer from 1 to 50, R' is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 5’ )2, R 5’ Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0030] G1 and G2 are each independently selected from N and N + B - R7; N + B is a monovalent nitrogen cation. - R7 is a monovalent negative ion, selected from C. 1～10 Alkyl, C 1～10 Alkoxy, n1 is an integer from 1 to 50, and R8 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R9)2, R8 selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0031] R3 and R4 are each independently selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, n is an integer from 1 to 50, and R is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R5)2, where R5 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl; or, R3 and R4 linked together to form j is an integer selected from 0 to 10;

[0032] t1 is selected from 0 or 1;

[0033] It is either non-covalent or covalent; when When t2 is zero, t2 is 0; when When the bond is covalent, t2 is 1;

[0034] t1 and t2 are not both 1;

[0035] k1 and k2 are each independently selected from integers from 0 to 10;

[0036] a1, a2, a3, a4, a5, and a6 are each independently selected from integers from 0 to 10;

[0037] a7 is selected from integers from 1 to 50;

[0038] R i Selected from hydrogen, C 1～10 alkyl;

[0039] b1 and b2 are each independently selected from integers from 0 to 10;

[0040] And when W1 is N, W2 is N, and a1, a2, a3, a4, b1, b2, t1, and t2 are all 0, If P1 is not C and P2 and P4 are not C, then P1 is not C. 1～2 Alkylene, P3 is not C 1～2 Alkylene;

[0041] And when W1 is N, W2 is N, W3 is N, and a1, a2, a3, a4, b1, b2, a5, a6, and t2 are all 0, If P1 is not C and P2 and P4 are not C, then P1 is not C. 1～2 Alkylene, P3 is not C 1～2 Alkylene, P5 is not C 1～2 Alkylene, P6 is not C 1～2 Alkylene.

[0042] Furthermore, the structure shown in equation A is as shown in equation B-1:

[0043]

[0044] Wherein, W1 is selected from N, N + E-R1, CR1; R1 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy; N + E- is a monovalent nitrogen cation, and E- is a monovalent anion;

[0045] W2 is selected from N and CR2; R2 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy;

[0046] P1, P2, P3, and P4 are each independently selected from none, unsubstituted, or replaced. Replacement C 1～10 Alkylene; n' is an integer from 1 to 50, R' is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 5’ )2, R 5’ Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0047] R3 and R4 are each independently selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, n is an integer from 1 to 50, and R is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R5)2, where R5 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl; or, R3 and R4 linked together to form j is an integer selected from 0 to 10;

[0048] k1 and k2 are each independently selected from integers from 0 to 10;

[0049] a1, a2, a3, and a4 are each independently selected from integers from 0 to 10;

[0050] b1 and b2 are each independently selected from integers from 0 to 10;

[0051] And when W1 is N, W2 is N, a1, a2, a3, a4, b1, b2 are all 0, P2 and P4 are zero, P1 is not zero, and C 1～2 Alkylene, P3 not absent, C 1～2 Alkylene.

[0052] Furthermore, the structure shown in Equation A is as shown in Equation B-2:

[0053]

[0054] Among them, W1 is selected from N and CR1, W2 is selected from N and CR2; R1 is hydrogen and C. 1～3 Alkyl group, R2 is hydrogen, C 1～3 alkyl;

[0055] P1, P2, P3, and P4 are each independently selected from none and C. 1～2 Alkylene;

[0056] a1 and a2 are each independently selected from integers from 1 to 6;

[0057] Preferably, W1 is selected from N and CR1, and W2 is selected from N and CR2; R1 is hydrogen and R2 is hydrogen.

[0058] P1, P2, P3, and P4 are each independently selected from the absence of methylene groups;

[0059] a1 and a2 are each independently selected from integers from 1 to 4.

[0060] Furthermore, the structure shown in Equation A is as shown in Equation B-3:

[0061]

[0062] Among them, W1 is selected from N and CR1, W2 is selected from N and CR2; R1 is hydrogen and C. 1～3 Alkyl group, R2 is hydrogen, C 1～3 alkyl;

[0063] P1, P2, P3, and P4 are each independently selected from C. 1～3 Alkylene;

[0064] R3 and R4 are each independently selected from hydrogen and C. 1～3 Alkyl, C 1～3 Alkoxy, n is an integer from 1 to 30, and R is selected from hydrogen and methyl; or, R3 and R4 are connected to form j is an integer selected from 0 to 5;

[0065] Preferably, W1 is selected from N, and W2 is selected from N;

[0066] P1, P2, P3, and P4 are each independently selected from C2 alkylene groups;

[0067] R3 and R4 are each selected independently. n is an integer from 1 to 30, and R is selected from hydrogen and methyl; or, R3 and R4 are connected to form j is 1.

[0068] Furthermore, the structure shown in Equation A is as shown in Equation B-4:

[0069]

[0070] Wherein, W1 is selected from N, N + E - R1 and W2 are selected from N and N. + E - R2; R1 is selected from C 1～3 Alkyl group, R2 is selected from C 1～3 Alkyl, N + It is a monovalent nitrogen cation, E - It is a monovalent negative ion;

[0071] r3, r4, r5, and r6 are each independently selected from integers from 1 to 3;

[0072] r1 and r2 are each independently selected from 0 or 1;

[0073] Furthermore, when W1 is N and W2 is N, r1 and r2 are not both 0 at the same time;

[0074] n' is an integer from 1 to 40, and R' is selected from hydrogen and methyl.

[0075] Preferably, W1 is selected from N, N + E - R1 and W2 are selected from N and N. + E - R2; R1 is methyl, R2 is methyl, N + It is a monovalent nitrogen cation, E - It is a monovalent negative ion;

[0076] r3, r4, r5, and r6 are each independently selected from 1;

[0077] r1 and r2 are each independently selected from 0 or 1;

[0078] Furthermore, when W1 is N and W2 is N, r1 and r2 are not both 0 at the same time;

[0079] n' is an integer from 1 to 30, and R' is selected from hydrogen and methyl.

[0080] Furthermore, the structure shown in equation A is as shown in equation C-1:

[0081]

[0082] Among them, W1 and W3 are each independently selected from N and N + E - R1, CR1; R1 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy; N + It is a monovalent nitrogen cation, E - It is a monovalent negative ion;

[0083] W2 is selected from N and CR2; R2 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy;

[0084] P1, P2, P3, P4, P5, and P6 are each independently selected from none, unsubstituted, or replaced. Replacement C 1～10 Alkylene; n' is an integer from 1 to 50, R' is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 5’ )2, R 5’ Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0085] G1 and G2 are each independently selected from N and N + B - R7; N + B is a monovalent nitrogen cation. - R7 is a monovalent negative ion, selected from C. 1～10 Alkyl, C 1～10 Alkoxy, n1 is an integer from 1 to 50, and R8 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R9)2, R8 selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0086] R3 and R4 are each independently selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, n is an integer from 1 to 50, and R is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R5)2, where R5 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl; or, R3 and R4 linked together to form j is an integer selected from 0 to 10;

[0087] k1 and k2 are each independently selected from integers from 0 to 10;

[0088] a1, a2, a3, a4, and a5 are each independently selected from integers from 0 to 10;

[0089] b1 and b2 are each independently selected from integers from 0 to 10.

[0090] Furthermore, the structure shown in equation A is as shown in equation C-2:

[0091]

[0092] Where W1 is N, W2 is N, and W3 is N;

[0093] P1, P2, P3, P4, and P5 are each independently selected from C. 1～3 Alkylene;

[0094] a1 and a2 are each independently selected from integers from 1 to 6;

[0095] Preferably, W1 is N, W2 is N, and W3 is N;

[0096] P1, P2, P3, P4, and P5 are each independently selected from C. 1～2 Alkylene;

[0097] a1 and a2 are each independently selected from integers from 1 to 4.

[0098] Furthermore, the structure shown in equation A is as shown in equation C-3:

[0099]

[0100] Where W1 is N, W2 is N, and W3 is N;

[0101] P1, P2, P3, P4, and P5 are each independently selected from C. 1～3 Alkylene;

[0102] G1 and G2 are each independently selected from N and N + B - R7; N + B is a monovalent nitrogen cation. - R7 is a monovalent negative ion, selected from C. 1～10 Alkyl, C 1～10 Alkoxy, n1 is an integer from 1 to 50, and R8 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R9)2, where R9 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0103] R3 and R4 are each independently selected from hydrogen and C. 1～3 Alkyl, C 1～3 Alkoxy, n is an integer from 1 to 30, and R is selected from hydrogen and methyl; or, R3 and R4 are connected to form j is an integer selected from 0 to 5;

[0104] Preferably, W1 is N, W2 is N, and W3 is N;

[0105] P1, P2, P3, P4, and P5 are each independently selected from C2 alkylene groups;

[0106] R3 and R4 are each selected independently. n is an integer from 1 to 30, and R is selected from hydrogen and methyl; or, R3 and R4 are connected to form j is 1.

[0107] Furthermore, the structure shown in equation A is as shown in equation C-4:

[0108]

[0109] Where W1 is N, W2 is N, and W3 is N;

[0110] r3, r4, r5, and r6 are each independently selected from integers from 1 to 3;

[0111] P5 is selected from C 1～3Alkylene;

[0112] r1 and r2 are each independently selected from 0 or 1;

[0113] n' is an integer from 1 to 40, and R' is selected from hydrogen and methyl.

[0114] Preferably, the structure shown in formula A is as shown in formula C-5:

[0115]

[0116] W1 is N, W2 is N, W3 is N;

[0117] r4 and r6 are each independently selected from integers from 1 to 3;

[0118] P5 is selected from C 1～3 Alkylene.

[0119] Furthermore, the structure shown in equation A is as shown in equation D-1:

[0120]

[0121] Among them, W1 and W4 are each independently selected from N and N + E - R1, CR1; R1 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy; N + It is a monovalent nitrogen cation, E - It is a monovalent negative ion;

[0122] W2 is selected from N and CR2; R2 is selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy;

[0123] P1, P2, P3, P4, P7, and P8 are each independently selected from unsubstituted or replaced samples. Replacement C 1～10 Alkylene; n' is an integer from 1 to 50, R' is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 5’ )2, R 5’ Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0124] R3 and R4 are each independently selected from hydrogen and C. 1～10 Alkyl, C 1～10 Alkoxy, n is an integer from 1 to 50, and R is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, N(R5)2, where R5 is selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkyne, 3-10 aryl, 3-10 heteroaryl, or R3 and R4 linked together

[0125] Preferably, W1 is N, W2 is N, and W4 is N;

[0126] P1, P2, P3, P4, P6, and P7 are each independently selected from C. 1～2 Alkylene;

[0127] R3 and R4 are each selected independently. n is an integer from 1 to 30, and R is selected from hydrogen and methyl.

[0128] Furthermore, the structure shown in equation A is as follows:

[0129]

[0130]

[0131] Where n is an integer selected from 1 to 24, R is hydrogen or methyl, n' is an integer selected from 1 to 24, R' is hydrogen or methyl, a7 is an integer selected from 1 to 24, R i R8 is hydrogen or methyl, n1 is an integer selected from 1 to 24, R8 is hydrogen or methyl, and E is halogen.

[0132] The present invention also provides a drug linker assembly unit, or a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated thereof, wherein the drug linker assembly unit comprises a connector and a drug, the connector end being capable of connecting to a target connector, the target connector being a substance capable of targeting and binding to a lesion site; the connector includes the structure shown in Formula A, the structure shown in Formula A being as shown in Formula E-0:

[0133]

[0134] Where X, Y, and Z are independently selected from N and N, respectively. + B0 - R0 or CH; R10 R 11 R 12 R 13 R 14 R 15 Each independently selected from hydrogen or n² is an integer from 1 to 40, R 1 It is hydrogen or methyl; N + It is a monovalent nitrogen cation, B0 - It is a monovalent negative ion, and R0 is C. 1～5 alkyl;

[0135] r7, r8, r9, r 10 r 13 r 14 Each integer r is independently selected from 1 to 5. 11 r 12 r 15 Each integer r is independently selected from 0 to 5. 16 It is 0 or 1;

[0136] r 17 r 18 r 19 r 20 Each integer is independently selected from 1 to 3;

[0137] r 22 Selected from 0 or 1;

[0138] r 24 Selected from 0 or 1, r 25 Selected from 0 or 1, and r 24 r 25 Not both are 0;

[0139] r 26 Selected from 0 or 1, r 27 Selected from 0 or 1, and r 26 r 27 Not both are 0;

[0140] K1 is selected from NR 16 N + R 16 B - R 18 CHR 20 K2 is selected from NR 17 N + R 17 B - R 18 CHR 21 ;

[0141] N + B is a monovalent nitrogen cation. -R is a monovalent negative ion. 18 Selected from C 1～3 Alkyl, C 1～3 Alkoxy, n3 is an integer from 1 to 50, R 2 Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 19 )2, R 19 Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0142] R 16 R 17 Each was independently selected from C 1～3 Alkyl, C 1～3 Alkoxy, n4 is an integer from 1 to 40, R 3 Selected from hydrogen, methyl; or, R 16 R 17 Connect into j1 is selected from integers from 0 to 5; X1 is -CO-, -CONH-, or none;

[0143] R 20 R 21 Each independently selected n' is an integer from 1 to 40, R ’ Selected from hydrogen and methyl, X0 is O or absent.

[0144] Furthermore, the structure shown in equation A is as shown in equation E-1:

[0145]

[0146] Where X, Y, and Z are independently selected from N and N, respectively. + B0 - R0 or CH; R 10 R 11 R 12 R 13 R 14 R 15 Each independently selected from hydrogen or n² is an integer from 1 to 40, R 1 It is hydrogen or methyl; N + It is a monovalent nitrogen cation, B0 -It is a monovalent negative ion, and R0 is C. 1～5 alkyl;

[0147] r7, r8, r9, r 10 r 13 r 14 Each integer r is independently selected from 1 to 5. 11 r 12 r 15 Each integer r is independently selected from 0 to 5. 16 It is 0 or 1.

[0148] Furthermore, :R 10 R 11 R 12 R 13 R 14 R 15 Both are hydrogen.

[0149] Furthermore, the structure shown in equation A is as shown in equation E-1-a or equation E-1-a':

[0150]

[0151] Where, r 13 r 14 Each integer is independently selected from 1 to 5; preferably, it is an integer from 1 to 4.

[0152] B0 - It is a monovalent negative ion, and R0 is C. 1～5 alkyl.

[0153] Furthermore, the structure shown in equation A is as shown in equation E-1-b:

[0154]

[0155] Where, r 13 r 14 Each integer is independently selected from 1 to 5.

[0156] Furthermore, the structure shown in equation A is as shown in equation E-1-c:

[0157]

[0158] Where, r 13 r 14 Each integer is independently selected from 1 to 5.

[0159] Furthermore, the structure shown in equation A is as shown in equation E-1-d:

[0160]

[0161] Where, r13 r 14 Each integer r is independently selected from 1 to 5. 15 Integers selected from 0 to 5.

[0162] Furthermore, the structure shown in equation A is as shown in equation F-1:

[0163]

[0164] Where, r 17 r 18 r 19 r 20 r 21 Each integer is independently selected from 1 to 3;

[0165] r 22 Selected from 0 or 1;

[0166] r 23 Selected from 0 or 1;

[0167] K1 is selected from NR 16 N + R 16 B - R 18 CHR 20 K2 is selected from NR 17 N + R 17 B - R 18 CHR 21 ;

[0168] N + B is a monovalent nitrogen cation. - R is a monovalent negative ion. 18 Selected from C 1～3 Alkyl, C 1～3 Alkoxy, n3 is an integer from 1 to 50, R 2 Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 19 )2, R 19 Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0169] R 16 R17 Each was independently selected from C 1～3 Alkyl, C 1～3 Alkoxy, n4 is an integer from 1 to 40, R 3 Selected from hydrogen, methyl; or, R 16 R 17 Connect into j1 is selected from integers from 0 to 5; X1 is -CO-, -CONH-, or none;

[0170] R 20 R 21 Each independently selected n' is an integer from 1 to 40, R' is selected from hydrogen and methyl, and X0 is O or none.

[0171] Furthermore, the structure shown in equation A is as shown in equation F-1-a:

[0172]

[0173] Where, r 17 r 18 r 19 r 20 Each integer is independently selected from 1 to 3;

[0174] R 16 R 17 Each was independently selected from C 1～3 Alkyl, C 1～3 Alkoxy, n4 is an integer from 1 to 40, preferably an integer from 9 to 16, R 3 Selected from hydrogen, methyl; or, R 16 R 17 Connect into j1 is selected from integers from 0 to 5; X1 is -CO-, -CONH-, or none.

[0175] Furthermore, the structure shown in equation A is as shown in equation F-1-b:

[0176]

[0177]

[0178] Where, r 17 r 18 r 19 r 20 Each integer is independently selected from 1 to 3;

[0179] n' is an integer from 1 to 40, preferably an integer from 9 to 16, and R' is selected from hydrogen and methyl.

[0180] X0 is 0 or none.

[0181] Furthermore, the structure shown in equation A is as shown in equation F-1-c:

[0182]

[0183] Where, r 17 r 18 r 19 r 20 r 21 Each integer is independently selected from 1 to 3;

[0184] r 22 Selected from 0 or 1;

[0185] G1 is selected from N, N + B - R 18 G2 is selected from N, N + B - R 18 ;

[0186] N + B is a monovalent nitrogen cation. - R is a monovalent negative ion. 18 Selected from C 1～3 Alkyl, C 1～3 Alkoxy, n3 is an integer from 1 to 50, preferably an integer from 3 to 16, R 2 Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 alkynyl, 3-10 aryl, 3-10 heteroaryl, N(R) 19 )2, R 19 Selected from hydrogen, C 1～10 Alkyl, C 1～10 Alkoxy, C2-C 10 alkenyl, C2-C 10 Alkynyl, 3-10 aryl, 3-10 heteroaryl;

[0187] R 16 R 17 Each was independently selected from C 1～3 Alkyl, C 1～3 Alkoxy, n4 is an integer from 1 to 40, preferably an integer from 9 to 16, R 3 Selected from hydrogen, methyl; or, R 16 R 17 Connect into j1 is selected from integers from 0 to 5; X1 is -CO-, -CONH-, or none.

[0188] Furthermore, the structure shown in equation A is as follows:

[0189]

[0190]

[0191]

[0192] n4 is an integer selected from 1 to 24, preferably an integer from 9 to 16, R3 is hydrogen or methyl, n' is an integer selected from 1 to 24, preferably an integer from 9 to 16, R' is hydrogen or methyl, n3 is an integer selected from 1 to 24, preferably an integer from 3 to 16, R 2 It is hydrogen or methyl, B0 - It is a monovalent negative ion.

[0193] Furthermore, the structure of the connector is shown in formula G-1 or formula G-2:

[0194]

[0195] Wherein, T is a tethering group, which can connect to a target connector; the target connector is a substance that can target and bind to the lesion site;

[0196] L1 represents a non-breakable, breakable, or non-breakable connector segment;

[0197] L2 represents a non-breakable, breakable, or non-breakable connector segment;

[0198] L2' represents a non-breakable, breakable, or non-breakable connector segment;

[0199] L3 represents a non-breakable, breakable, or non-breakable connector segment;

[0200] L3' represents a non-breakable, breakable, or non-breakable connector segment;

[0201] L4 represents a non-breakable, breakable, or non-breakable connector segment;

[0202] L4' represents a non-breakable, breakable, or non-breakable connector segment;

[0203] L5 is a three-pronged connector sub-segment that is non-breakable, breakable, or non-breakable;

[0204] Z1 is 0 or 1, Z2 is 0 or 1, and Z1 and Z2 are not both 0 at the same time;

[0205] The structure of Q is shown in equation A.

[0206] Furthermore, the structure of L5 is selected from: or

[0207] y5 is selected from integers from 1 to 10; preferably 1;

[0208] y6 is selected from integers from 1 to 50; preferably, it is an integer from 2 to 9;

[0209] y7 is selected from integers from 1 to 50; preferably integers from 2 to 9.

[0210] Furthermore, the structure of the connector is shown in Equation G-3:

[0211]

[0212] Wherein, T is a tethering group, which can connect to a target connector; the target connector is a substance that can target and bind to the lesion site;

[0213] L1 is a connective segment that can be broken or not broken;

[0214] L2 represents a non-breakable, breakable, or non-breakable connector segment;

[0215] L3 is a connective segment that can be broken or not broken;

[0216] L4 represents a non-breakable, breakable, or non-breakable connector segment;

[0217] The structure of Q is shown in equation A.

[0218] Furthermore, the structure of T is as follows:

[0219] Furthermore, the structure of L1 is as follows:

[0220] Where x is selected from an integer from 1 to 10, preferably an integer from 2 to 6;

[0221] m is selected from an integer from 1 to 25, preferably an integer from 2 to 10; more preferably an integer from 2 to 9;

[0222] x1 is selected from an integer from 1 to 10, preferably an integer from 2 to 6; more preferably 2;

[0223] x2 is selected from an integer from 1 to 10, preferably an integer from 1 to 5; more preferably 1.

[0224] x3 is selected from an integer from 0 to 50, preferably an integer from 8 to 30; more preferably 9;

[0225] R x Selected from hydrogen or methyl.

[0226] Furthermore, the structure of L2 is as follows: Or L2 is none;

[0227] The structure of L2' is Or L2' is none;

[0228] Where y and y' are each independently selected from integers from 1 to 10, preferably 1 or 2;

[0229] y1 and y3 are each independently selected from integers from 0 to 10, preferably 0, 1 or 2, more preferably 0 or 1;

[0230] y2 and y4 are each independently selected from integers from 1 to 50, preferably integers from 1 to 20; more preferably integers from 2 to 12.

[0231] Furthermore, the structures of L3 and L3' are each independently selected from:

[0232]

[0233] Where p is selected from integers from 1 to 24, and R6 is selected from hydrogen or nitro.

[0234] Furthermore, the structures of L4 and L4' are each independently selected from none,

[0235] Furthermore, the structure of the aforementioned connector is selected from:

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] -PEG9 indicates -PEG 16 express

[0259] The right side of the above structure is all It can be linked with other groups.

[0260] Furthermore, the structure of the drug linking assembly unit is shown in Formula G-4 or Formula G-5:

[0261]

[0262]

[0263] Among them, T, Q, L1, L2, L2', L3, L3', L4, L4', L5, Z1, and Z2 are as described above;

[0264] D1 is a drug;

[0265] D2 is a drug.

[0266] Furthermore, D1 and D2 are each independently selected from cytotoxic drugs, drugs for treating autoimmune toxicity, or anti-inflammatory drugs;

[0267] Preferably, D1 and D2 are each independently selected from drugs that target DNA or drugs that target tubulin;

[0268] More preferably, D1 and D2 are each independently selected from one of the following compounds or their derivatives:

[0269]

[0270] Furthermore, the structure of the drug linking assembly unit is selected from:

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352] -PEG9 indicates -PEG 16 express

[0353] The present invention also provides a target connector-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof, wherein the target connector-drug conjugate is obtained by connecting a target connector and the above-mentioned drug linking assembly unit, and the structure is shown in Formula J-1.

[0354]

[0355] Ab is the target connector; q is an integer from 1 to 20;

[0356] The target connector is a substance that can target and bind to the lesion site.

[0357] Furthermore, the structure of the target connector-drug conjugate is shown in Formula J-2 or Formula J-3:

[0358]

[0359] Where Ab is the target connector; q is an integer from 1 to 20;

[0360] T, L1, Q, L2, L3, L 4、 L2', L3', L4', L5, D1, D2, Z1, and Z2 are as described above;

[0361] Preferably, the structure of the target connector-drug conjugate is shown in Formula J-4:

[0362] Ab-(T-L1-Q-L2-L3-L4-D2) q

[0363] J-4

[0364] Where Ab is the target connector; q is an integer from 1 to 20;

[0365] T, L1, Q, L2, L3, L4, and D2 are as described above.

[0366] Furthermore, the DAR value of the target connector-drug conjugate is 1.00 to 20.00, preferably 2.0 to 8.0, and more preferably 2.0 to 5.0.

[0367] Furthermore, the structure of the target connector-drug conjugate is selected from:

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389] q is an integer from 1 to 20;

[0390] -PEG9 indicates -PEG 16 express

[0391] The specific target connector-drug conjugates listed above were labeled with DAR values ​​based on the test results of the examples.

[0392] The DAR value has an error range due to the influence of detection conditions and personnel. According to current detection methods, the DAR value error range is typically ±0.3, but this range may vary depending on the detection method. The key to target-drug conjugates lies in their structural formula; any target-drug conjugate with the same structural formula as the specific target-drug conjugates listed above is within the scope of protection of this invention.

[0393] The specific target linker-drug conjugates listed above use HER2 antibodies in the examples.

[0394] Furthermore, the targeting adapter is an antibody, antibody fragment, protein, small molecule polypeptide, glycopeptide, mimic peptide, small molecule compound, or nucleic acid oligonucleotide aptamer;

[0395] Preferably, the antibody is an antibody targeting cell surface receptors and tumor-associated antigens.

[0396] The present invention also provides a linker for a target connector-drug conjugate, or a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated thereof, wherein the structure of the linker is shown in formula K-1 or formula K-2:

[0397]

[0398] Wherein, T is a tethering group, which can connect to a target connector; the target connector is a substance that can target and bind to the lesion site;

[0399] L1 represents a non-breakable, breakable, or non-breakable connector segment;

[0400] L2 represents a non-breakable, breakable, or non-breakable connector segment;

[0401] L2' represents a non-breakable, breakable, or non-breakable connector segment;

[0402] L3 represents a non-breakable, breakable, or non-breakable connector segment;

[0403] L3' represents a non-breakable, breakable, or non-breakable connector segment;

[0404] L4 represents a non-breakable, breakable, or non-breakable connector segment;

[0405] L4' represents a non-breakable, breakable, or non-breakable connector segment;

[0406] L5 is a three-pronged connector sub-segment that is non-breakable, breakable, or non-breakable;

[0407] R0” is a leaving group;

[0408] R0' is a leaving group;

[0409] Z1 is 0 or 1, Z2 is 0 or 1, and Z1 and Z2 are not both 0 at the same time;

[0410] The structure of Q is shown in equation A above.

[0411] Furthermore, the leaving group is a hydroxyl group.

[0412] Furthermore, the structure of L5 is selected from: or

[0413] y5 is selected from integers from 1 to 10; preferably 1;

[0414] y6 is selected from integers from 1 to 50; preferably, it is an integer from 2 to 9;

[0415] y7 is selected from integers from 1 to 50; preferably integers from 2 to 9.

[0416] Furthermore, the structure of the connector is shown in Equation K-3:

[0417] T-L1-Q-L2-L3-L4-R0”

[0418] K-3

[0419] Wherein, T is a tethering group, which can connect to a target connector; the target connector is a substance that can target and bind to the lesion site;

[0420] L1 is a connective segment that can be broken or not broken;

[0421] L2 represents a non-breakable, breakable, or non-breakable connector segment;

[0422] L3 is a connective segment that can be broken or not broken;

[0423] L4 represents a non-breakable, breakable, or non-breakable connector segment;

[0424] R0” is a leaving group;

[0425] The structure of Q is shown in equation A above.

[0426] Furthermore, the structure of T is as follows:

[0427] Furthermore, the structure of L1 is as follows:

[0428] Where x is selected from an integer from 1 to 10, preferably an integer from 2 to 6;

[0429] m is selected from an integer from 1 to 25, preferably an integer from 2 to 10; more preferably an integer from 2 to 9;

[0430] x1 is selected from an integer from 1 to 10, preferably an integer from 2 to 6; more preferably 2;

[0431] x2 is selected from an integer from 1 to 10, preferably an integer from 1 to 5; more preferably 1.

[0432] x3 is selected from an integer from 0 to 50, preferably an integer from 8 to 30; more preferably 9;

[0433] R x Selected from hydrogen or methyl.

[0434] Furthermore, the structure of L2 is as follows: Or L2 is none;

[0435] The structure of L2' is Or L2' is none;

[0436] Where y and y' are each independently selected from integers from 1 to 10, preferably 1 or 2;

[0437] y1 and y3 are each independently selected from integers from 0 to 10, preferably 0, 1 or 2; more preferably 0 or 1;

[0438] y2 and y4 are each independently selected from integers from 1 to 50, preferably integers from 1 to 20; more preferably integers from 2 to 12.

[0439] Furthermore, the structures of L3 and L3' are each independently selected from:

[0440]

[0441] Where p is selected from integers from 1 to 24, and R6 is selected from hydrogen or nitro.

[0442] Furthermore, the structures of L4 and L4' are each independently selected from none,

[0443] Furthermore, the structure of the connector is as follows:

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464] -PEG9 indicates -PEG 16 express

[0465] The present invention also provides a pharmaceutical formulation for the prevention and / or treatment of tumors, which is a formulation made of the above-mentioned target linker-drug conjugate, or its stereoisomer, or its optical isomer, or its salt, or its deuterated product as the active ingredient, plus pharmaceutically acceptable excipients.

[0466] The present invention also provides the use of the above-mentioned targeted linker-drug conjugate, or its stereoisomer, or its optical isomer, or its salt, or its deuterated derivative, in the preparation of pharmaceutical formulations for the prevention and / or treatment of tumors.

[0467] Furthermore, the tumor is selected from lung cancer, urethral cancer, colorectal cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[0468] The present invention also provides a method for preparing the above-mentioned target linker-drug conjugate, or its stereoisomer, or its optical isomer, or its salt, or its deuterated derivative, the method comprising the following steps:

[0469] (1) Drugs are coupled with linkers to obtain drug linker assembly units;

[0470] (2) The drug linking assembly unit is coupled with the target adapter to obtain a target adapter-drug conjugate; or, the method includes the following steps:

[0471] (1') The target connector and the connector are coupled to obtain the target connector-connector unit;

[0472] (2') The target connector-connector subunit is coupled with the drug to obtain the target connector-drug conjugate.

[0473] The drug linking assembly unit of the present invention can be connected to a target connector, which is a substance capable of targeting and binding to a lesion site; the drug linking assembly unit consists of a connector and a drug.

[0474] Covalently linking a target linker with 1 to 20 drug linker assembly units can form a target linker-drug conjugate. When the target linker is an antibody, the target linker-drug conjugate is called an antibody-drug conjugate (ADC).

[0475] In this invention, the "DAR value" represents the average number of drug link assembly units coupled to a single target linker in a target linker-drug conjugate, equivalent to the average value of the q value. The DAR value does not have to be an integer.

[0476] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0477] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1～10 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 10 carbon atoms.

[0478] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group with a conjugated π-electron system, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent group must be on a carbon atom of a ring with a conjugated π-electron system. The aryl group can be substituted or unsubstituted.

[0479] "Heteroaryl" refers to a heteroaryl group containing one or more heteroatoms. Heteratoms include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0480] "Deuterated compounds" refer to compounds obtained by replacing one or more hydrogen atoms with deuterium.

[0481] "Targeted linker-drug conjugate" refers to a conjugate formed by linking a drug to a targeted linker that has the function of targeting and binding to lesion sites via a linker. For example, "antibody-drug conjugate" (ADC) is a type of targeted linker-drug conjugate of this invention, and its targeted linker is an antibody or antibody fragment.

[0482] The “drug linker assembly unit” is the product obtained by connecting the linker in the aforementioned target linker-drug conjugate with the drug. It is an intermediate in the preparation of the target linker-drug conjugate. By coupling it with the target linker, the target linker-drug conjugate can be obtained.

[0483] "Drug" or designated with the codes "D", "D1", or "D2" refers to a compound possessing desired biological activity (such as therapeutic effect). For example, it can be a cytotoxic compound used for cancer treatment, or a biologically active protein or polypeptide. The drugs of this invention include, but are not limited to, existing compounds with therapeutic effects, including camptothecin derivatives such as SN-38, Dxd, and Dx-8951, as well as compounds acting on microtubules, such as Eribulin, MMAE, MMAF, and maytansine.

[0484] "Targeted adaptors" are substances that can target and bind to lesion sites, such as antibodies, antibody fragments, proteins, small molecule peptides, glycopeptides, peptide mimics, small molecule compounds, or nucleic acid oligonucleotide aptamers. Targeted adaptors can bind to targets (such as antigens) at the lesion site, enabling targeted adaptor-drug conjugates to target and bind to the lesion site.

[0485] "Antibody" refers to a protective protein produced by the body in response to antigen stimulation. When the targeting linker is an antibody or antibody fragment, the resulting targeting linker-drug conjugate is an antibody-drug conjugate. The antibody or antibody fragment of this invention can bind to antigens at the lesion site, enabling the antibody-drug conjugate to target and bind to the lesion site. The antibody can be any protein or protein molecule; it can bind to, chelate with, or react with a portion of the cell population to be treated or bioengineered.

[0486] The embodiments provided by this invention use Her2 as an antibody to prepare targeted linker-drug conjugates.

[0487] In this invention, the antibodies constituting the antibody-drug conjugate preferably retain their original wild-state antigen-binding capacity. Therefore, the antibodies in this invention can specifically bind to antigens. The antigens involved include, but are not limited to, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth or differentiation, lymphokines, cytokines, molecules involved in cell circulation regulation, molecules involved in angiogenesis, and factors related to angiogenesis. Tumor-associated factors can also be cluster differentiation factors (such as CD proteins). The antigens bound by the antibodies in this invention can be one or a subset of the above categories, while other subsets include other molecules / antigens with specific properties.

[0488] Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies targeting cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known in the art and can be prepared using antibody preparation methods and information known in the art. These targets are specifically expressed on the surface of one or more cancer cells, while being expressed sparingly or not at all on the surface of one or more non-cancer cells. Typically, such tumor-associated peptides are more overexpressed on the surface of cancer cells compared to non-cancer cells. Identifying such tumor-associated factors can significantly improve the specific targeting properties of antibody-based cancer therapies.

[0489] Tumor-associated antigens include, but are not limited to, the tumor-associated antigens listed below. Antibody targeting of tumor-associated antigens includes all amino acid sequence variants and homologs, possessing at least 70%, 80%, 85%, 90%, or 95% homology with sequences identified in the references, or possessing biological properties and characteristics completely identical to those of tumor-associated antigen sequences in the cited literature.

[0490] Tumor-associated antigens include: BMPR1B (Genbank accession number: NM-001203), E16 (Genbank accession number: NM-003486), STEAP1 (Genbank accession number: NM-012449), 0772P (Genbank accession number: AF361486), MPF (Genbank accession number: NM-005823), Napi3b (Genbank accession number: NM-006424), Sema 5b (Genbank accession number: AB040878), PSCA hlg (Genbank login number: AY358628), ETBR (Genbank login number: AY275463), MSG783 (Genbank login number: NM-017763), STEAP2 (Genbank login number: AF455138), TrpM4 (Genbank login number: NM-017636), CRIPTO (Genbank login number: NP-003203 or NM-003212), CD21 (Genbank login number: M2600) 4), CD79B (Genbank login number: NM-000626), FcRH2 (Genbank login number: NM-030764), HER2 (Genbank login number: M11730), NCA (Genbank login number: M18728), MDP (Genbank login number: BC017023), IL20Rα (Genbank login number: AF184971), Brevican (Genbank login number: AF229053), EphB2R (G Genbank login number: NM-004442), GEDA (Genbank login number: AY260763), BAFF-R (Genbank login number: AF1164546), CD22 (Genbank login number: AK026467), CD79a (Genbank login number: NP-001774.1), CXCR5 (Genbank login number: NP-001701.1), HLA-DOB (Genbank login number: NP-002111.1), P2 X5 (Genbank login number: NP-002552.2), CD72 (Genbank login number: NP-001773.1), LY64 (Genbank login number: NP-005573.1), FcRH1 (Genbank login number: NP-443170.1), IRTA2 (Genbank login number: NP-112571.1), TENB2 (Genbank login number: AF179274), CEA, B7H3, Her3, 5T4, Claudin 18.2. FGFR2b, FRα, TROP2, B7H4, Nectin4, CD30, c-MET, EGFR, carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), ROR1, CD20, hepatocyte growth factor receptor (HGFR), ROR2, IL3RA, mesothelin receptor, CD74, PD-L1.

[0491] The antibody used in this invention can be an antibody corresponding to the aforementioned antigen.

[0492] A linker, also known as a connector, is a substance used to connect a therapeutic compound to a target connector that binds to the lesion site. A linker fragment refers to the structural part of the linker that has the connecting function.

[0493] According to the intracellular drug release mechanism, "linkers" can be divided into two categories: unbreakable linkers and breakable linkers.

[0494] For targeted linker-drug conjugates containing unbreakable linkers (such as antibody-drug conjugates), the drug release mechanism can be as follows: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically digested in the lysosome, releasing the drug, which is an active small molecule composed of the linker and antibody amino acid residues.

[0495] Targeted linkers-drug conjugates containing cleavable linkers (such as antibody-drug conjugates) can cleave within target cells and release the drug (such as the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically unstable linkers and enzyme-unstable linkers. Chemically unstable linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties, such as pH and glutathione concentration. Enzyme-unstable linkers, such as peptide linkers, can be effectively cleaved by lysosomal proteases, such as cathepsins or plasminogen activators. Peptide linkers are considered to be very stable in plasma.

[0496] Linkers are the core component of target linker-drug conjugates (such as antibody-drug conjugates) and can significantly influence their pharmacokinetics, therapeutic index, and efficacy.

[0497] The key to this invention lies in the improvement of the linker. Specifically, the linker incorporates the structure shown in Formula A, which, in addition to the functions of a traditional linker, significantly enhances the safety, stability, efficacy, and controllability of the prepared target linker-drug conjugates (such as antibody-drug conjugates). This can effectively promote the clinical use of target linker-drug conjugates (such as antibody-drug conjugates).

[0498] The targeted adapter-drug conjugate provided in this embodiment of the invention comprises a targeted adapter and 1 to 20 covalently linked drug linking assembly units, wherein the drug linking assembly units can be linked to thiols generated by interchain disulfide bonds in reducing antibodies and / or each linking assembly unit is linked to a thiol from a cysteine ​​residue.

[0499] To facilitate connection, drug linker assembly units are typically constructed before being connected to the target adapter. However, the construction order can be altered. For example, an assembly unit with a protecting group can be connected to the target adapter first, and then the protecting group can be removed and other drug units added after connection to the target adapter.

[0500] The targeted adapter-drug conjugate provided by this invention targets specific cells, such as tumor cells, and binds to cell surface-specific proteins (such as antigens). The conjugate can enter the cell via endocytosis, and the drug is released into the cell in an active form to exert its therapeutic effect, or the drug can be released extracellularly and penetrate into the cell to exert its therapeutic effect.

[0501] The beneficial effects of this invention are:

[0502] By introducing the structure shown in Formula A into the connecting unit, it is possible to reduce the polymer content and naked antibody percentage of the target connector-drug conjugate formed by connecting it with various different drug units and target connectors, maintain the DAR value of the target connector-drug conjugate within an appropriate range, improve the plasma stability and storage stability of the target connector-drug conjugate, and enhance its therapeutic efficacy.

[0503] The drug linking assembly unit provided by the present invention contains the above-mentioned linking unit structure, and can also reduce the polymer content and bare antibody percentage of the target linker-drug conjugate, and maintain the DAR value of the target linker-drug conjugate within an appropriate range.

[0504] The final prepared targeted linker-drug conjugates (e.g., antibody-drug conjugates) have low polymer content and naked antibody percentage, suitable DAR value, and exhibit excellent plasma stability, storage stability, and antitumor activity, showing great promise for clinical application.

[0505] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0506] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0507] Figure 1 DAR value detection results of SMP-93566a (RP-HPLC).

[0508] Figure 2 SMP-93566a polymer detection results (SE-HPLC).

[0509] Figure 3 SMP-93566a naked antibody detection results (HIC).

[0510] Figure 4 DAR value detection results of SMP-88480a (RP-HPLC).

[0511] Figure 5 SMP-88480a polymer detection results (SE-HPLC).

[0512] Figure 6 SMP-88480a naked antibody detection results (HIC).

[0513] Figure 7 Results of plasma stability of monotoxin ADC in human, monkey, and mouse plasma.

[0514] Figure 8 Plasma stability results of Dxd in human, monkey and mouse plasma in dual-toxin ADC.

[0515] Figure 9 Results of plasma stability of eribulin in human, monkey, and mouse plasma in dual-toxin ADC.

[0516] Figure 10 The in vivo tumor-inhibiting effects of monotoxin ADCs on ovarian cancer, gastric cancer, and breast cancer.

[0517] Figure 11 The in vivo tumor-inhibiting effect of dual-toxin ADC on gastric cancer.

[0518] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0519] The structure of the target linker-drug conjugate provided by this invention can be synthesized by those skilled in the art using different methods and strategies based on conventional technical knowledge and means in the field, for example:

[0520] (1) First, synthesize the complete linker structure, and then further conjugate it with the target linker and drug to obtain the target linker-drug conjugate;

[0521] Alternatively, (2) starting with a drug, gradually couple and connect sub-fragments to obtain a drug linking assembly unit, and then further couple it with a target adapter to obtain a target adapter-drug conjugate;

[0522] Alternatively, (3) starting with the target connector, gradually couple the connecting sub-fragments to obtain the target connector-connecting sub-unit, and then further couple it with the drug to obtain the target connector-drug conjugate.

[0523] The present invention provides a specific method for preparing a targeted linker-drug conjugate in the following embodiments:

[0524] Example Section

[0525] I. Preparation of Targeted Connector-Drug Conjugates

[0526] Example 1: Preparation of Intermediate A

[0527]

[0528] Step 1: Preparation of compound A-2

[0529]

[0530] Add A-1 (20.0 g, 56.49 mmol) and acetonitrile (200 mL) to a 500 mL reaction flask and stir at room temperature. After complete clarification, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.0 g, 57.38 mmol) and N-hydroxysuccinimide (7.0 g, 60.82 mmol), and continue stirring at room temperature for 12 hours. TLC showed that the reaction was complete. Filter the reaction mixture, and dry the resulting solid under vacuum to give compound A-2, weighing 22.0 g, in 86% yield.

[0531] Step 2: Preparation of compound A-3

[0532]

[0533] To a 500 mL reaction flask, add L-phenylalanine (8.0 g, 48.48 mmol), sodium bicarbonate (8.0 g, 95.24 mmol), and water (200 mL), and stir at room temperature. After complete clarification, dissolve compound A-2 (22.0 g, 48.78 mmol) in ethylene glycol dimethyl ether (50 mL) and slowly add it dropwise to the above reaction solution. After the addition is complete, continue stirring at room temperature for 12 hours. TLC shows that the reaction is complete. Concentrate under reduced pressure to remove ethylene glycol dimethyl ether. Add the remaining reaction solution dropwise to 0.5 M hydrochloric acid aqueous solution (500 mL), and a large amount of solid precipitates. Filter, and dry the obtained solid under vacuum to give compound A-3, weighing 15.0 g, with a yield of 61%.

[0534] 1 HNMR(400MHz, CDCl3)12.51(s,1H),9.04(s,1H),8.31(s,1H),7.95(s,1H),7.90(d,J=8.0Hz,2H),7.56(d,J=7.8Hz,2H),7.38-7.28(m,4H),7.19- 7.14(m,5H),4.85(t,J=8.2Hz,1H),4.71(d,J=8.2Hz,2H),4.39(t,J=8.4 Hz,1H),4.10-3.83(m,4H),3.12(d,J=9.6Hz,1H),2.85(d,J=9.6Hz,1H).

[0535] Step 3: Preparation of compound A-4

[0536]

[0537] In a 500 mL reaction flask, A-3 (15.0 g, 29.94 mmol), acetonitrile (200 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6 g, 31.30 mmol), and N-hydroxysuccinimide (4 g, 34.78 mmol) were added sequentially. The mixture was stirred at room temperature for 12 hours, and TLC showed that the reaction was complete. The reaction mixture was filtered, and the resulting solid was dried under vacuum to give compound A-4, weighing 13.0 g, with a yield of 73%.

[0538] Step 4: Preparation of compound A-5

[0539]

[0540] In a 500 mL reaction flask, glycine (3.0 g, 40.00 mmol), sodium bicarbonate (6.7 g, 79.76 mmol), and water (150 mL) were added and stirred at room temperature. After complete clarification, compound A-4 (13.0 g, 21.74 mmol) was dissolved in ethylene glycol dimethyl ether (40 mL) and slowly added dropwise to the above reaction solution. After the addition was complete, the reaction was stirred at room temperature for 12 hours. TLC showed that the reaction was complete. The organic solvent was removed by concentration under reduced pressure. The remaining reaction solution was added dropwise to 0.5 M hydrochloric acid aqueous solution (300 mL), and a large amount of solid precipitated. The solid was filtered and dried under vacuum to give compound A-5, weighing 10.0 g, with a yield of 83%.

[0541] 1HNMR(400MHz, CDCl3)13.01(s,1H),9.01(s,1H),8.27(s,1H),7.98(s,1H),7.89(d,J=8.0Hz,2H),7.54(d,J=7.8Hz,2H),7.34-7.23(m,4H),7.19- 7.14(m,5H),4.75(t,J=8.2Hz,1H),4.61(d,J=8.2Hz,2H),4.30(t,J=8.4 Hz,1H),4.04-3.83(m,6H),3.10(d,J=9.6Hz,1H),2.75(d,J=9.6Hz,1H).

[0542] Step 5: Preparation of compound A-6

[0543]

[0544] To a 25 mL reaction flask, A-5 (200.0 mg, 0.36 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (163.0 mg, 0.43 mmol), N,N-diisopropylethylamine (66.9 mg, 0.52 mmol), and N,N-dimethylformamide (5 mL) were added sequentially. The mixture was stirred at room temperature for 5 min, and then iribulin (263.1 mg, 0.36 mmol) was added. Stirring continued at room temperature for 10 min, and TLC showed complete reaction. N,N-dimethylformamide was removed by concentration under reduced pressure, and the residue was purified by thin-layer chromatography (dichloromethane:methanol = 10:1) to give compound A-6, weighing 300.0 mg, in 66% yield.

[0545] Step 6: Preparation of Compound A

[0546]

[0547] To a 25 mL reaction flask, N,N-dimethylformamide (2 mL), A-6 (20.0 mg, 0.016 mmol), and 1,8-diazobispyrocyclo[5.4.0]undec-7-ene (5.0 mg, 0.032 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to give compound A, weighing 10.0 mg, with a yield of 60%.

[0548] 1HNMR(400MHz,DMSO-d6)δ8.48(t,J=5.5Hz,1H),8.31(dd,J=10.1,4.6Hz,2H),7.98(s,3H ),7.74(t,J=5.6Hz,1H),7.30–7.21(m,4H),7.22–7.15(m,1H),5.02(d,J=22.6Hz,2H),4 .79(d,J=29.3Hz,2H),4.63(dd,J=5.7,3.7Hz,1H),4.59–4.50(m,2H),4.24(t,J=13.5Hz ,1H),4.21–4.12(m,1H),4.12–4.06(m,3H),4.02(s,1H),3.86(dd,J=16.8,5.7Hz,1H),3. 68(dd,J=16.7,5.5Hz,4H),3.54(dd,J=19.0,7.3Hz,12H),3.11(dd,J=16.3,11.0Hz,1H) ,3.07–2.99(m,2H),2.84(d,J=9.6Hz,1H),2.80–2.65(m,3H),2.62–2.54(m,1H),2.24(dd d,J=48.1,23.9,9.6Hz,6H),1.98(dd,J=29.5,15.1Hz,6H),1.73–1.57(m,5H),1.55–1.4 0(m,2H),1.37–1.25(m,3H),1.22–1.13(m,1H),1.03(d,J=6.4Hz,3H),1.00–0.89(m,1H).

[0549] Example 2: Preparation of Intermediate B

[0550]

[0551] Step 1: Preparation of compound B-2

[0552]

[0553] To a 25 mL reaction flask, N,N-dimethylformamide (5 mL), B-1 (200.0 mg, 0.31 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (118.0 mg, 0.31 mmol), and N,N-diisopropylethylamine (50.0 mg, 0.39 mmol) were added sequentially. After stirring at room temperature for 5 min, ixotecan (B-2, 112.0 mg, 0.26 mmol) was added. The mixture was stirred for 10 min, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure, and the residue was purified by thin-layer chromatography (dichloromethane:methanol = 10:1) to give compound B-3, weighing 140.0 mg, with a yield of 50%.

[0554] MS(ESI) m / z: 1063 [M+H] + .

[0555] Step 2: Preparation of Compound B

[0556]

[0557] To a 25 mL reaction flask, add N,N-dimethylformamide (3 mL), B-3 (140.0 mg, 0.13 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (30.0 mg, 0.20 mmol). Stir at room temperature for 30 minutes. TLC showed that the reaction was complete. Most of the N,N-dimethylformamide was concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound B, weighing 70.0 mg, in 64% yield.

[0558] MS(ESI) m / z: 841 [M+H] + .

[0559] 1H NMR (400MHz, DMSO-d6) δ8.70(t,J=6.6Hz,1H),8.58–8.45(m,2H),8.39(t,J=5.8Hz,1H),8.33(d,J=8.2Hz,1H),7.97(d,J=12.0Hz,3H),7.77(d,J=1 0.9Hz,1H),7.31(d,J=3.5Hz,1H),7.28–7.20(m,4H),7.20–7.12(m,1H), 6.54(s,1H),5.59(dd,J=14.3,5.8Hz,1H),5.42(s,2H),5.18(s,2H),4.64 (d,J=6.6Hz,2H),4.53(td,J=9.4,4.5Hz,1H),4.02(s,2H),3.89–3.79(m ,1H),3.79–3.65(m,3H),3.48(d,J=5.9Hz,1H),3.27–3.07(m,3H),3.03(d d,J=13.7,4.3Hz,1H),2.74(dd,J=14.3,9.2Hz,1H),2.38(s,2H),2.18(dd ,J=14.7,9.5Hz,2H),1.86(td,J=14.0,7.1Hz,2H),0.87(t,J=7.3Hz,3H).

[0560] Example 3: Preparation of Intermediate C

[0561]

[0562] Step 1: Preparation of compound C-2

[0563]

[0564] Compound C-1 (1.0 g, 4.90 mmol) and N,N-dimethylformamide (15 mL) were added sequentially to a 25 mL reaction flask and stirred at room temperature. After the reaction solution became clear, potassium carbonate (2.4 g, 17.4 mmol), potassium iodide (1.6 g, 9.64 mmol), and 2-chloroethoxyethanol (1.6 g, 12.90 mmol) were added sequentially, and the mixture was stirred at 110 °C for 12 hours. TLC showed that the reaction was complete. The reaction system was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by HPLC to give C-2, weighing 1.5 g, with a yield of 80%.

[0565] 1HNMR (400MHz, CDCl3) δ7.32-7.29(m,5H),4.51(s,2H),4.43(s,1H),3.72–3.69(m,4H),3.55–3.50(m,8H),2.65–2.50(m,6H),1.59–1.34(m,8H).

[0566] Step 2: Preparation of compound C-3

[0567]

[0568] Under ice bath conditions, dichloromethane (10 mL), C-2 (1.5 g, 3.94 mmol), and tert-butyl acrylate (2.1 g, 16.40 mmol) were added sequentially to a 25 mL reaction flask, and the mixture was stirred for 30 minutes. Then, 50% sodium hydroxide aqueous solution (2.6 g) was slowly added dropwise, and the reaction was continued for 10 minutes after the addition was complete. The ice bath was removed, and the reaction was allowed to proceed at room temperature for 3 hours. LC-MS was used to confirm the completeness of the reaction. The pH was adjusted to 5–6 with 0.5 M dilute hydrochloric acid, and the mixture was extracted with dichloromethane (10 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the target product C-, weighing 1.7 g (67% yield), which was used directly in the next step.

[0569] Step 3: Preparation of compound C-4

[0570]

[0571] To a 25 mL reaction flask, C-3 (1.0 g, 1.57 mmol), methanol (10 mL), and palladium on carbon (0.1 g) were added sequentially, followed by three purgings with hydrogen. The reaction system was stirred at room temperature under hydrogen atmosphere for 2 hours. LC-MS showed that the reaction was complete. The palladium on carbon was removed by filtration, and the filtrate was concentrated under reduced pressure to give compound C-4, weighing 800.0 mg, with a yield of 93%, which was used directly in the next step.

[0572] 1 HNMR (400MHz, CDCl3) δ3.72–3.69(m,4H),3.55–3.50(m,12H),2.65–2.42(m,10H),1.59–1.34(m,8H),1.49(s,18H).

[0573] Step 4: Preparation of compound C-5

[0574]

[0575] To a 25 mL reaction flask, C-4 (800.0 mg, 1.46 mmol), THF (10 mL), and water (2.5 mL) were added sequentially, and the mixture was stirred at room temperature. After the reaction solution became clear, sodium bicarbonate (184.0 mg, 2.19 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (529.0 mg, 1.57 mmol) were added sequentially, and the mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was subjected to column chromatography (dichloromethane:methanol = 20:1) to give compound C-5, weighing 1.0 g, with a yield of 89%.

[0576] 1 HNMR(400MHz, CDCl3)δ7.90(d,J=8.9Hz,2H),7.55(d,J=9.2Hz,2H),7.38-7.28(m,4H),4.70(d,J=11.2Hz,2H),4. 46(t,J=11.2Hz,1H),3.71–3.67(m,4H),3.53–3.48(m,12H),2.55–2.41(m,10H),1.51–1.24(m,8H),1.41(s,18H).

[0577] Step 5: Preparation of compound C-6

[0578]

[0579] Intermediate C-5 (1.0 g, 1.30 mmol), dichloromethane (5 mL), and trifluoroacetic acid (5 mL) were added sequentially to a 25 mL reaction flask. The reaction was carried out at room temperature for 2 hours, and LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give compound C-6, weighing 600.0 mg, with a yield of 70%.

[0580] MS(ESI) m / z: 657 [M+H] + .

[0581] Step 6: Preparation of compound C-7

[0582]

[0583] To a 25 mL reaction flask, N,N-dimethylformamide (3 mL), A (50.0 mg, 0.048 mmol), C-6 (65.0 mg, 0.10 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (19.0 mg, 0.050 mmol), and N,N-diisopropylethylamine (13.0 mg, 0.10 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to give compound C-7, weighing 50.0 mg, in 62% yield. MS (ESI) m / z: 844 [M / 2+H] + .

[0584] Step 7: Preparation of compound C-8

[0585]

[0586] To a 25 mL reaction flask, N,N-dimethylformamide (3 mL), C-7 (30.0 mg, 0.018 mmol), B (16.0 mg, 0.020 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (7.0 mg, 0.018 mmol), and N,N-diisopropylethylamine (3.0 mg, 0.023 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound C-8, weighing 20.0 mg, in a yield of 44%. MS (ESI) m / z: 1255 [M / 2+H] + 837 [M / 3+H] + .

[0587] Step 8: Preparation of Compound C

[0588]

[0589] To a 25 mL reaction flask, N,N-dimethylformamide (2 mL), C-8 (20.0 mg, 0.0080 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (2.45 mg, 0.016 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound C, weighing 15.0 mg, in 82% yield.

[0590] MS(ESI) m / z: 1144 [M / 2+H] + 763 [M / 3+H] + .

[0591] Example 4: Preparation of Intermediate D

[0592]

[0593] The synthesis method of intermediate D is the same as that of compound C.

[0594] Example 5: Preparation of Intermediate E

[0595]

[0596] Step 1: Preparation of compound E-2

[0597]

[0598] Compound D-1 (1.2 g, 5.61 mmol) and N,N-dimethylformamide (15 mL) were added sequentially to a 25 mL reaction flask and stirred at room temperature. After the reaction solution became clear, potassium carbonate (2.6 g, 18.84 mmol), potassium iodide (1.9 g, 11.40 mmol), and bromo-heptapolyethylene glycol-tert-butyl propionate (6.3 g, 11.22 mmol) were added sequentially. The mixture was stirred at 90 °C for 12 hours, and TLC showed that the reaction was complete. The reaction system was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by HPLC to give intermediate E-2, weighing 2.0 g, with a yield of 30%. MS (ESI) m / z: 1175 [M+H] + .

[0599] Step 2: Preparation of compound E-3

[0600]

[0601] Compound E-2 (2.0 g, 1.70 mmol) and trifluoroacetic acid (10 mL) were added to a 25 mL reaction flask and stirred at room temperature for 0.5 hours. The reaction was completed by LC-MS. The trifluoroacetic acid was removed by concentration under reduced pressure to obtain intermediate E-3, weighing 1.0 g, with a yield of 61%, which was used directly in the next step.

[0602] MS(ESI) m / z: 964 [M+H] +

[0603] Step 3: Preparation of compound E-4

[0604]

[0605] To a 25 mL reaction flask, intermediate E-3 (600.0 mg, 0.62 mmol), tetrahydrofuran (20 mL), and water (2.5 mL) were added sequentially, and the mixture was stirred at room temperature. After the reaction solution became clear, sodium bicarbonate (105.0 mg, 1.25 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (252.0 mg, 0.75 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour, and LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by HPLC to give compound E-4, weighing 550.0 mg, in 75% yield.

[0606] MS(ESI) m / z: 1185 [M+H] +

[0607] Step 4: Preparation of compound E-5

[0608]

[0609] To a 25 mL reaction flask, N,N-dimethylformamide (4 mL), A (50.0 mg, 0.048 mmol), E-4 (118.0 mg, 0.10 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (19.0 mg, 0.05 mmol), and N,N-diisopropylethylamine (13.0 mg, 0.10 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound E-5, weighing 80.0 mg, with a yield of 72%. MS (ESI) m / z: 1108 [M / 2+H] + .

[0610] Step 5: Preparation of compound E-6

[0611]

[0612] To a 25 mL reaction flask, N,N-dimethylformamide (3 mL), E-6 (35.0 mg, 0.016 mmol), B (16.0 mg, 0.019 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (7.0 mg, 0.018 mmol), and N,N-diisopropylethylamine (3.0 mg, 0.023 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound E-6, weighing 20.0 mg, with a yield of 41%.

[0613] MS(ESI) m / z: 1013 [M / 3+H] + .

[0614] Step 6: Preparation of Compound E

[0615]

[0616] To a 25 mL reaction flask, N,N-dimethylformamide (2 mL), E-6 (20.0 mg, 0.0066 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (2.0 mg, 0.014 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound E, weighing 15.0 mg, with a yield of 76%.

[0617] MS(ESI) m / z: 1408 [M / 2+H] + .

[0618] Example 6: Preparation of intermediate Fa

[0619]

[0620] Step 1: Preparation of compound F-2

[0621]

[0622] Compound D-1 (1.2 g, 5.61 mmol) and N,N-dimethylformamide (15 mL) were added sequentially to a 25 mL reaction flask and stirred at room temperature. After the reaction solution became clear, potassium carbonate (2.8 g, 0.29 mmol), potassium iodide (1.8 g, 10.84 mmol), and bromo-diethylene glycol-tert-butyl propionate (2.0 g, 6.73 mmol) were added sequentially. The mixture was stirred at 90 °C for 12 hours, and TLC showed that the reaction was complete. The reaction system was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by HPLC to obtain intermediate F-2, weighing 0.6 g, with a yield of 25%.

[0623] MS(ESI)m / z:431[M+H] + .

[0624] 1 HNMR (400MHz, CDCl3)7.64(s,1H),5.50(s,1H),3.73(m,2H),3.50-3,54(m,7H),2.72(m,2H),2.42-2.55(m,3H),1.59-1.72(m,8H),1.42(s,18H).

[0625] Step 2: Preparation of compound F-3

[0626]

[0627] Compound F-2 (600.0 mg, 1.40 mmol) and N,N-dimethylformamide (15 mL) were added sequentially to a 25 mL reaction flask and stirred at room temperature. After the reaction solution became clear, potassium carbonate (386.0 mg, 2.80 mmol), potassium iodide (232.0 mg, 1.40 mmol), and bromo-nonadenylated polyethylene glycol-tert-butyl propionate (1.8 g, 2.77 mmol) were added sequentially, and the mixture was stirred at 90 °C for 12 hours. TLC showed that the reaction was complete. The reaction system was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by HPLC to give intermediate F-3, weighing 870.0 mg, with a yield of 62%. MS (ESI) m / z: 999 [M+H] + .

[0628] Step 3: Preparation of compound F-4

[0629]

[0630] Compound F-3 (870.0 mg, 0.87 mmol) and trifluoroacetic acid (3 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 0.5 hours, and LC-MS showed that the reaction was complete. The solution was concentrated under reduced pressure to give intermediate F-4, weighing 600.0 mg, in 87% yield. This was used directly in the next step.

[0631] MS(ESI)m / z:787[M+H] +

[0632] Step 4: Preparation of compound F-5

[0633]

[0634] To a 25 mL reaction flask, F-4 (600.0 mg, 0.76 mmol), tetrahydrofuran (10 mL), and water (2.5 mL) were added sequentially, and the mixture was stirred at room temperature. After the reaction solution became clear, sodium bicarbonate (128.0 mg, 1.52 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (257.0 mg, 0.76 mmol) were added sequentially. The mixture was stirred at room temperature for 2 hours, and LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by HPLC to give compound F-5, weighing 660 mg, in 86% yield.

[0635] MS(ESI) m / z: 1009 [M+H] + .

[0636] Step 5: Preparation of compounds F-6a / F6-b

[0637]

[0638] To a 25 mL reaction flask, N,N-dimethylformamide (4 mL), A (50 mg, 0.05 mmol), F-5 (101 mg, 0.10 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (19 mg, 0.05 mmol), and N,N-diisopropylethylamine (13 mg, 0.10 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was then subjected to preparative HPLC to give compound F-6a (35 mg, 34% yield) and compound F-6b (25 mg, 25% yield).

[0639] MS(ESI) m / z: 1019 [M / 2+H] + .

[0640] Step 6: Preparation of compound F-7a

[0641]

[0642] To a 25 mL reaction flask, N,N-dimethylformamide (2 mL), F-6a (35 mg, 0.017 mmol), B (17 mg, 0.021 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (8 mg, 0.021 mmol), and N,N-diisopropylethylamine (3 mg, 0.023 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to give compound F-7a, weighing 20.0 mg, with a yield of 41%. MS (ESI) m / z: 1430 [M / 2+H] + .

[0643] Step 6: Preparation of compound Fa

[0644]

[0645] To a 25 mL reaction flask, add N,N-dimethylformamide (2 mL), F-7a (20.0 mg, 0.0070 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (2.0 mg, 0.010 mmol). Stir at room temperature for 30 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound Fa, weighing 15.0 mg, in 81% yield.

[0646] MS(ESI) m / z: 1319 [M / 2+H] + .

[0647] Example 7: Preparation of intermediate Fb

[0648]

[0649] The synthesis method of intermediate Fb is the same as that of compound Fa.

[0650] Example 8: Preparation of Intermediate G

[0651]

[0652] The synthesis method of intermediate G is the same as that of compound A.

[0653] MS(ESI)m / z:991[M+H]+.

[0654] 1HNMR (400MHz, DMSO-d6) δ8.68(d,J=7.9Hz,1H),8.45(t,J=5.6Hz,1H),7.90(s,3H),7.77(d,J=5.5Hz,1H),7.28–7.22(m,4H),7.22–7.17 (m,1H),5.03(d,J=22.6Hz,2H),4.82(s,1H),4.75(s,1H),4.64(s,3H),4.55(t,J=4.1Hz,1H),4.26(d,J=10.2Hz,1H),4.18(d,J=10.4Hz, 1H),4.10(s,3H),4.02(s,1H),3.83–3.64(m,5H),3.61–3.46(m,6H),3.26(s,4H),3.16–3.02(m,3H),2.84(d,J=9.6Hz,1H),2.79–2.66(m ,3H),2.35–2.17(m,6H),2.04–1.84(m,6H),1.74–1.57(m,6H),1.54–1.42(m,2H),1.31(s,3H),1.03(d,J=6.3Hz,3H),1.02–0.90(m,1H).

[0655] Example 9: Preparation of Intermediate H

[0656]

[0657] The synthesis method of intermediate H is the same as that of compound E.

[0658] Example 10: Preparation of Intermediate I

[0659]

[0660] Step 1: Preparation of compound I-2

[0661]

[0662] To a 25 mL reaction flask, add I-1 (1.0 g, 5.15 mmol), potassium carbonate (1.4 g, 10.14 mmol), N,N-dimethylformamide (25 mL), and PEG9-OMs (2.3 g, 4.55 mmol) all at once. Stir overnight at 60 °C. LC-MS showed the reaction was complete. Filter the reaction mixture, concentrate under reduced pressure to remove N,N-dimethylformamide, and purify the residue by thin-layer chromatography (petroleum ether:ethyl acetate = 1:1) to give compound I-2, weighing 2.5 g, in 81% yield.

[0663] MS(ESI) m / z: 605 [M+H] + .

[0664] 1 HNMR(400MHz, CDCl3)7.42(m,1H),7.33(d,J=8.0Hz,2H),7.15(d,J=7.8Hz,2H),6.75(m,1H) ,5.05(s,2H),4.21(m,1H),3.43-3.54(m,34H),3.30(s,3H),3.07(m,2H),2.66-2.75(m,4H).

[0665] Step 2: Preparation of compound I-3

[0666]

[0667] To a 25 mL reaction flask, I-2 (1.0 g, 1.66 mmol), potassium carbonate (457.0 mg, 3.31 mmol), and N,N-dimethylformamide (20 mL) were added. Then, tert-butyl bromoacetate (483.0 mg, 2.48 mmol) was added in a single batch. The mixture was stirred at room temperature for 1 hour, and LC-MS showed complete reaction. The reaction system was filtered, and N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to give compound I-3, weighing 600.0 mg, in 50% yield.

[0668] MS(ESI) m / z: 719 [M+H] + .

[0669] 1HNMR(400MHz, CDCl3)7.40(m,1H),7.35(d,J=8.0Hz,2H),7.19(d,J=7.8Hz,2H),6.76(m,1H),5. 12(s,2H),3.40-3.52(m,34H),3.30(s,3H),3.07-3.21(m,4H),2.66-2.75(m,4H),1.31(s,9H).

[0670] Step 3: Preparation of compound I-4

[0671]

[0672] I-3 (500.0 mg, 0.70 mmol), palladium on carbon (50.0 mg), and methanol (10 mL) were added to a 25 mL reaction flask. After purging with hydrogen three times, the mixture was stirred at room temperature under hydrogen atmosphere for 1 hour. LC-MS showed that the reaction was complete. The reaction system was filtered, and methanol was removed by concentration under reduced pressure to give compound I-4, weighing 360.0 mg, with a yield of 88%.

[0673] MS(ESI) m / z: 585 [M+H] +

[0674] Step 4: Preparation of compound I-5

[0675]

[0676] To a 25 mL reaction flask, I-4 (360.0 mg, 0.62 mmol), sodium bicarbonate (103.0 mg, 1.23 mmol), tetrahydrofuran (8 mL), and water (1 mL) were added. N-methoxycarbonylmaleimide (142.0 mg, 0.92 mmol) was slowly added at 0 °C. After the addition was complete, the reaction was continued at 0 °C for 10 minutes. LC-MS showed that the reaction was complete. The tetrahydrofuran and water were removed by concentration under reduced pressure. The residue was purified by HPLC to give compound I-5, weighing 180.0 mg, with a yield of 44%.

[0677] MS(ESI) m / z: 665 [M+H] + .

[0678] 1 HNMR(400MHz, CDCl3)7.86(s,1H),7.75(s,1H),3.32-3.52(m,38H),3.20(s,3H),2.66-2.75(m,4H),1.23(s,9H).

[0679] Step 5: Preparation of Compound I

[0680]

[0681] I-5 (180.0 mg, 0.27 mmol) and trifluoroacetic acid (3 mL) were added to a 25 mL reaction flask. The mixture was stirred overnight at room temperature, and LC-MS showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the residue was purified by HPLC to give compound I, weighing 120.0 mg, in 73% yield.

[0682] MS(ESI) m / z: 609 [M+H] + .

[0683] Example 11: Preparation of Intermediate J

[0684]

[0685] Step 1: Preparation of compound J-2

[0686]

[0687] To a 25 mL reaction flask, N,N-dimethylformamide (5 mL), A-1 (130.0 mg, 0.37 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (164.0 mg, 0.43 mmol), and N,N-diisopropylethylamine (70.0 mg, 0.54 mmol) were added sequentially. After stirring at room temperature for 5 minutes, glycine tert-butyl ester (56.0 mg, 0.43 mmol) was added. The reaction was continued for 25 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure, and the residue was purified by thin-layer chromatography (dichloromethane:methanol = 20:1) to give compound J-2, weighing 110.0 mg, in 65% yield.

[0688] MS(ESI) m / z: 468 [M+H] + .

[0689] 1 HNMR(400MHz, CDCl3)9.01(s,1H),8.87(s,1H),8.15(s,1H),7.90(d,J=8.0Hz,2H),7.55(d ,J=8.5Hz,2H),7.29-7.35(m,4H),4.70(d,2H),446(t,1H),4.09-4.17(m,6H),1.35(s,9H).

[0690] Step 2: Preparation of compound J-3

[0691]

[0692] Compound J-2 (100.0 mg, 0.21 mmol) and trifluoroacetic acid (2 mL) were added to a 25 mL reaction flask and stirred at room temperature for 30 minutes. LC-MS showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by HPLC to give intermediate J-3, weighing 75.0 mg, in 86% yield.

[0693] MS(ESI)m / z:412[M+H] + .

[0694] Step 3: Preparation of compound J-4

[0695]

[0696] To a 25 mL reaction flask, N,N-dimethylformamide (3 mL), J-3 (75.0 mg, 0.18 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (83.0 mg, 0.22 mmol), and N,N-diisopropylethylamine (35.0 mg, 0.27 mmol) were added sequentially. After stirring at room temperature for 5 min, iribulin (138.0 mg, 0.19 mmol) was added. Stirring was continued for 10 min, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative thin-layer chromatography (dichloromethane:methanol = 10:1) to give compound J-4, weighing 175.0 mg, with a yield of 87%.

[0697] MS(ESI) m / z: 1123 [M+H] + .

[0698] Step 4: Preparation of compound J-5

[0699]

[0700] To a 25 mL reaction flask, add N,N-dimethylformamide (2 mL), J-4 (175.0 mg, 0.16 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (49.0 mg, 0.32 mmol). Stir at room temperature for 15 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to give compound J-5, weighing 108.0 mg, with a yield of 75%.

[0701] MS(ESI)m / z:901[M+H] + .

[0702] Step 5: Preparation of compound J-6

[0703]

[0704] To a 25 mL reaction flask, N,N-dimethylformamide (3 mL), J-5 (45.0 mg, 0.05 mmol), C-6 (66.0 mg, 0.10 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (19.0 mg, 0.05 mmol), and N,N-diisopropylethylamine (13.0 mg, 0.10 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to give compound J-6, weighing 49.0 mg, with a yield of 64%. MS (ESI) m / z: 1539 [M+H] + .

[0705] Step 6: Preparation of compound J-7

[0706]

[0707] To a 25 mL reaction flask, N,N-dimethylformamide (3 mL), J-6 (40.0 mg, 0.026 mmol), B (26.0 mg, 0.031 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (12.0 mg, 0.031 mmol), and N,N-diisopropylethylamine (5.0 mg, 0.039 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure, and the residue was purified by thin-layer chromatography (dichloromethane:methanol = 12:1) to give compound J-6, weighing 32.0 mg, in 52% yield.

[0708] MS(ESI) m / z: 1181 [M / 2+H] + .

[0709] Step 7: Preparation of compound J

[0710]

[0711] To a 25 mL reaction flask, N,N-dimethylformamide (2 mL), J-7 (32.0 mg, 0.014 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (3.0 mg, 0.020 mmol) were added sequentially. The mixture was stirred at room temperature for 15 minutes, and TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound J, weighing 25.0 mg, in 86% yield.

[0712] MS(ESI) m / z: 1070 [M / 2+H] + .

[0713] Example 12: Preparation of Intermediate K

[0714]

[0715] Step 1: Preparation of compound K-2

[0716]

[0717] Compound K-1 (601.0 mg, 1.00 mmol) and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask, followed by N,N-diisopropylethylamine (258.0 mg, 2.00 mmol). Then, di(p-nitrobenzene) carbonate (456.0 mg, 1.50 mmol) was added in a single addition. The mixture was stirred overnight at room temperature. TLC showed complete reaction. The reaction solution was slowly added dropwise to methyl tert-butyl ether (25 mL), precipitating a pale yellow solid. The solid was filtered and dried under vacuum to give compound K-2, weighing 580.0 mg, with a yield of 76%. MS (ESI) m / z: 767 [M+H] + .

[0718] Step 2: Preparation of compound K-3

[0719]

[0720] To a 25 mL reaction flask, compound K-2 (300.0 mg, 0.39 mmol) and N,N-dimethylformamide (5 mL) were added and stirred at room temperature. After complete clarification, N,N-diisopropylethylamine (101.0 mg, 0.78 mmol) and 1-hydroxybenzotriazole (27 mg, 0.20 mmol) were added, followed by a single-dose addition of iribulin (292.0 mg, 0.40 mmol). The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by thin-layer chromatography (dichloromethane:methanol = 15:1) to give compound K-3, weighing 478.0 mg, in 90% yield.

[0721] MS(ESI) m / z: 1357 [M+H] + .

[0722] Step 3: Preparation of compound K

[0723]

[0724] To a 25 mL reaction flask, add N,N-dimethylformamide (4 mL), K-3 (245 mg, 0.18 mmol), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (54.0 mg, 0.36 mmol). Stir at room temperature for 20 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound K, weighing 180.0 mg, with a yield of 88%.

[0725] MS(ESI) m / z: 1135 [M+H] + .

[0726] Example 13: Preparation of compound SMP-22682

[0727]

[0728] Step 1: Preparation of compound SMP-22682-2

[0729]

[0730] Add SMP-22682-1 (3.0 g, 11.28 mmol) and dichloromethane (30 mL) to a 250 mL reaction flask. Under ice-water bath and nitrogen protection, add oxaloyl chloride (5.7 g, 44.88 mol), followed by slow dropwise addition of N,N-dimethylformamide (0.1 mL). After the addition is complete, react at room temperature for 16 hours. LC-MS showed that the starting material was essentially completely reacted. Remove the solvent and excess oxaloyl chloride by direct vacuum distillation. After concentration, add a small amount of toluene, and distill again under vacuum to obtain 3.0 g of crude product, which was directly used in the next step.

[0731] Step 2: Preparation of compound SMP-22682-4

[0732]

[0733] Add SMP-22682-3 (2.4 g, 0.01 mol), triethylamine (4.0 g, 0.040 mol), and dichloromethane (60 mL) to a 250 mL reaction flask. Under an ice-water bath, slowly add a dichloromethane suspension of SMP-22682-2 (3.0 g, crude product mixed in 20 mL dichloromethane). After the addition is complete, continue stirring for 10 minutes, slowly raise the temperature to room temperature, and maintain the reaction at room temperature for 1 hour. LC-MS showed the target compound molecular weight. Concentrate the reaction system, and the crude product was subjected to column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-22682-4, weighing 0.90 g, with a two-step yield of 17%.

[0734] MS(ESI) m / z: 467 [M+H]+ .

[0735] 1 HNMR(400MHz, CDCl3)7.41(s,1H),7.12(s,1H),4.26-4.32(m,4H),3.31-3.56(m,32H).

[0736] Step 3: Preparation of compound SMP-22682-5

[0737]

[0738] Add SMP-22682-4 (0.90 g, 1.93 mmol) to a 100 mL reaction flask, followed by 20 mL of a 1 M lithium aluminum hydride tetrahydrofuran solution. The reaction mixture was heated under reflux for 16 hours. LC-MS showed that most of the molecules were of the target compound. Add sodium sulfate decahydrate (5.0 g) to the reaction solution in portions, and filter. Concentrate the filtrate, and the crude product was subjected to column chromatography (ethyl acetate:methanol = 5:1) to give compound SMP-22682-5, weighing 0.50 g, with a yield of 59%.

[0739] MS(ESI) m / z: 439 [M+H] + .

[0740] 1 HNMR(400MHz, CDCl3)5.91(s,1H),5.32(s,1H),4.26-4.32(m,4H),3.49-3.56(m,32H),2.66-2.72(m,8H).

[0741] Step 4: Preparation of compound SMP-22682-6

[0742]

[0743] To a 100 mL reaction flask, add acetonitrile (10 mL), SMP-22682-5 (500.0 mg, 1.14 mmol), tert-butyl bromoacetate (178.0 mg, 0.91 mmol), and potassium carbonate (126.0 mg, 0.91 mmol). Stir at room temperature for 1 hour. LC-MS showed that most of the product was of the target compound molecular weight. The reaction solution was concentrated, and the crude product was subjected to column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-22682-6, weighing 240.0 mg, with a yield of 48%.

[0744] MS(ESI) m / z: 553 [M+H] + .

[0745] 1HNMR(400MHz, CDCl3)5.91(s,1H),3.33-3.54(m,34H),2.52-2.70(m,8H),1.33(s,9H).

[0746] Step 5: Preparation of compound SMP-22682-7

[0747]

[0748] To a 10 mL reaction flask, N,N-dimethylformamide (2 mL), SMP-22682-6 (75.0 mg, 0.14 mmol), Mal-PEG4-acid (56.0 mg, 0.16 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (62.0 mg, 0.16 mmol), and N,N-diisopropylethylamine (26.0 mg, 0.20 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-22682-7, weighing 65 mg, with a yield of 56%.

[0749] MS(ESI) m / z: 880 [M+H] + .

[0750] Step 6: Preparation of compound SMP-22682-8

[0751]

[0752] SMP-22682-7 (65.0 mg, 0.074 mmol), dichloromethane (2 mL), and trifluoroacetic acid (2 mL) were added sequentially to a 10 mL reaction flask. The mixture was stirred at room temperature for 16 hours. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by preparative HPLC to obtain compound SMP-22682-8, weighing 40.0 mg, with a yield of 66%.

[0753] MS(ESI) m / z: 824 [M+H] + .

[0754] Step 7: Preparation of compound SMP-22682

[0755]

[0756] To a 10 mL reaction flask, N,N-dimethylformamide (1 mL), SMP-22682-8 (10.0 mg, 0.012 mmol), D (8.0 mg, 0.0037 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.0 mg, 0.0053 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.016 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-22682, weighing 2.2 mg, with a yield of 20%.

[0757] MS(ESI) m / z: 999 [M / 3+H] + .

[0758] Example 14: Preparation of compound SMP-07861

[0759]

[0760] Step 1: Preparation of compound SMP-07861-1

[0761]

[0762] To a 10 mL reaction flask, N,N-dimethylformamide (2 mL), SMP-22682-6 (50.0 mg, 0.09 mmol), maleimide hexanoic acid (23.0 mg, 0.11 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (41.0 mg, 0.11 mmol), and N,N-diisopropylethylamine (18.0 mg, 0.14 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-07861-1, weighing 40.0 mg, with a yield of 59%.

[0763] MS(ESI) m / z: 746 [M+H] + .

[0764] Step 2: Preparation of compound SMP-07861-2

[0765]

[0766] SMP-07861-1 (40.0 mg, 0.054 mmol), dichloromethane (2 mL), and trifluoroacetic acid (2 mL) were added sequentially to a 10 mL reaction flask. The mixture was stirred at room temperature for 16 hours. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by preparative HPLC to obtain compound SMP-07861-2, weighing 30.0 mg, with a yield of 82%.

[0767] MS(ESI) m / z: 690 [M+H] + .

[0768] Step 3: Preparation of compound SMP-07861

[0769]

[0770] To a 10 mL reaction flask, N,N-dimethylformamide (1 mL), SMP-07861-2 (8.0 mg, 0.012 mmol), D (8.0 mg, 0.0037 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.0 mg, 0.0053 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.016 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-07861, weighing 2.2 mg, with a yield of 21%.

[0771] MS(ESI) m / z: 954 [M / 3+H] + .

[0772] Example 15: Preparation of compound SMP-61694

[0773]

[0774] Step 1: Preparation of compound SMP-61694-1

[0775]

[0776] To a 10 mL reaction flask, N,N-dimethylformamide (2 mL), SMP-22682-6 (50.0 mg, 0.09 mmol), Mal-PEG8-acid (57.0 mg, 0.11 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (41.0 mg, 0.11 mmol), and N,N-diisopropylethylamine (18.0 mg, 0.14 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-61694-1, weighing 20.0 mg, with a yield of 21%.

[0777] MS(ESI) m / z: 1056 [M+H] + .

[0778] Step 2: Preparation of compound SMP-61694-2

[0779]

[0780] Add SMP-61694-1 (20.0 mg, 0.019 mmol), dichloromethane (1 mL), and trifluoroacetic acid (1 mL) sequentially to a 10 mL reaction flask. Stir at room temperature for 20 minutes. LC-MS showed that most of the product was of the target compound molecular weight. Concentrate to obtain crude product SMP-61694-2, weighing 15.0 mg, and proceed directly to the next step.

[0781] MS(ESI) m / z: 1000 [M+H] + .

[0782] Step 3: Preparation of compound SMP-61694

[0783]

[0784] To a 10 mL reaction flask, N,N-dimethylformamide (1 mL), SMP-61694-2 (6.0 mg, 0.0060 mmol), D (10.0 mg, 0.0046 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.0 mg, 0.0053 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.016 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-61694, weighing 6.8 mg, with a yield of 47%.

[0785] MS(ESI) m / z: 1057 [M / 3+H] + .

[0786] Example 16: Preparation of compound SMP-45539

[0787]

[0788] Step 1: Preparation of compound SMP-45539

[0789]

[0790] To a 10 mL reaction flask, N,N-dimethylformamide (1 mL), SMP-07861-2 (5.0 mg, 0.0073 mmol), C (10.0 mg, 0.0044 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (2.0 mg, 0.0053 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.016 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-45539, weighing 2.6 mg, with a yield of 20%.

[0791] MS(ESI) m / z: 986 [M / 3+H] + .

[0792] Example 17: Preparation of compound SMP-59034

[0793]

[0794] Step 1: Preparation of compound SMP-59034

[0795]

[0796] To a 10 mL reaction flask, N,N-dimethylformamide (1 mL), SMP-61694-2 (6.0 mg, 0.0060 mmol), C (10.0 mg, 0.0044 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (2.0 mg, 0.0053 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.016 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-45539, weighing 2.8 mg, with a yield of 20%.

[0797] MS(ESI) m / z: 1090 [M / 3+H] + .

[0798] Example 18: Preparation of compound SMP-29438

[0799]

[0800] Step 1: Preparation of compound SMP-29438

[0801]

[0802] To a 10 mL reaction flask, N,N-dimethylformamide (1 mL), SMP-22682-8 (5.0 mg, 0.0061 mmol), C (10.0 mg, 0.0044 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (2.0 mg, 0.0053 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.016 mmol) were added sequentially. The reaction was carried out at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated, and the crude product was purified by HPLC to obtain compound SMP-45539, weighing 3.9 mg, with a yield of 29%.

[0803] MS(ESI) m / z: 1031 [M / 3+H] + .

[0804] Example 19: Preparation of compound SMP-49182

[0805]

[0806] Step 1: Preparation of compound SMP-49182-2

[0807]

[0808] Add SMP-49182-1 (6.0 g, 27.03 mmol) and toluene (60 mL) to a 250 mL reaction flask. Under nitrogen protection in an ice bath, add oxaloyl chloride (13.7 g, 108.11 mol), followed by dropwise addition of N,N-dimethylformamide (0.1 mL). After the addition is complete, heat to 60 °C and react for 6 hours. LC-MS showed that the starting material was essentially completely reacted. Remove the solvent and excess oxaloyl chloride by direct vacuum distillation to obtain 6.0 g of crude product, which was used directly in the next step.

[0809] Step 2: Preparation of compound SMP-49182-4

[0810]

[0811] To a 250 mL reaction flask, add SMP-49182-3 (4.4 g, 23.16 mmol), N,N-diisopropylethylamine (11.9 g, 92.66 mmol), and dichloromethane (100 mL). Under an ice-water bath, slowly add a dichloromethane suspension of SMP-49182-2 (6.0 g, crude product mixed in 30 mL of dichloromethane). After stirring for 10 minutes, slowly heat to room temperature and maintain the reaction at room temperature for 1 hour. LC-MS showed the target compound molecular weight. Concentrate the reaction system. Column chromatography (dichloromethane:methanol = 10:1) yielded compound SMP-49182-4, weighing 1.6 g, with a two-step yield of 16%.

[0812] MS(ESI) m / z: 379 [M+H] + .

[0813] Step 3: Preparation of compound SMP-49182-5

[0814]

[0815] In a 100 mL reaction flask, intermediate SMP-49182-4 (1.2 g, 3.17 mmol) was added, followed by 30 mL of 1 M lithium aluminum hydride tetrahydrofuran solution. The mixture was refluxed in an oil bath for 16 hours. LC-MS showed that most of the molecules were of the target compound. After cooling the reaction solution to room temperature, sodium sulfate decahydrate (3.0 g) was added to the system, and stirring was continued for 30 minutes. The mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding a crude product. The crude product was subjected to column chromatography (ethyl acetate:methanol = 5:1, with the addition of 5% ammonia water by volume) to obtain compound SMP-49182-5, weighing 600 mg, with a yield of 53.9%.

[0816] MS(ESI) m / z: 351 [M+H] + .

[0817] Step 4: Preparation of compound SMP-49182-6

[0818]

[0819] In a 100 mL reaction flask, intermediate SMP-49182-5 (0.6 g, 1.71 mmol), tert-butyl bromoacetate (0.3 g, 1.37 mmol), potassium carbonate (0.2 g, 1.37 mmol), and acetonitrile (10 mL) were added. The mixture was reacted at room temperature for 1 hour. LC-MS showed that most of the molecules were of the target compound. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain compound SMP-49182-6, weighing 0.3 g, with a yield of 47.1%.

[0820] MS(ESI) m / z: 465 [M+H] + .

[0821] Step 5: Preparation of compound SMP-49182-7

[0822]

[0823] In a 10 mL reaction flask, intermediate SMP-49182-6 (110.0 mg, 0.23 mmol), N,N-dimethylformamide (5 mL), maleimide hexanoic acid (60.00 mg, 0.28 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (108.0 mg, 0.28 mmol), and N,N-diisopropylethylamine (46.0 mg, 0.35 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-49182-7, weighing 90.0 mg, with a yield of 59.5%.

[0824] MS(ESI) m / z: 658 [M+H] + .

[0825] Step 6: Preparation of compound SMP-49182-8

[0826]

[0827] In a 10 mL reaction flask, SMP-49182-7 (90.0 mg, 0.13 mmol), dichloromethane (2 mL), and trifluoroacetic acid (2 mL) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the target compound's molecular weight was present. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in a small amount of acetonitrile and purified by HPLC to obtain compound SMP-49182-8, weighing 40.0 mg, with a yield of 51.1%. MS (ESI) m / z: 602 [M+H] + .

[0828] Step 7: Preparation of compound SMP-49182

[0829]

[0830] In a 10 mL reaction flask, SMP-49182-8 (2.5 mg, 0.042 mmol), intermediate C (8.0 mg, 0.0035 mmol), N,N-dimethylformamide (1 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.6 mg, 0.0042 mmol), and N,N-diisopropylethylamine (0.7 mg, 0.053 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to remove most of the solvent, yielding a crude product. The crude product was purified by HPLC to obtain compound SMP-49182, weighing 6.4 mg, with a yield of 63.7%.

[0831] MS(ESI) m / z: 957 [M / 3+H] + .

[0832] Example 20: Preparation of compound SMP-40359

[0833]

[0834] Step 1: Preparation of compound SMP-40359-1

[0835]

[0836] In a 10 mL reaction flask, intermediate SMP-49182-6 (120.0 mg, 0.26 mmol), Mal-PEG4-acid (107.0 mg, 0.31 mmol), N,N-dimethylformamide (5 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (118.0 mg, 0.31 mmol), and N,N-diisopropylethylamine (50.0 mg, 0.38 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-40359-1, weighing 80.0 mg, with a yield of 38.9%.

[0837] MS(ESI) m / z: 792 [M+H] + .

[0838] Step 2: Preparation of compound SMP-40359-2

[0839]

[0840] In a 10 mL reaction flask, SMP-40359-1 (80.0 mg, 0.10 mmol), dichloromethane (2 mL), and trifluoroacetic acid (2 mL) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the target compound's molecular weight was present. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in a small amount of acetonitrile and purified by HPLC to obtain compound SMP-40359-2, weighing 40.0 mg, with a yield of 54.4%. MS (ESI) m / z: 736 [M+H] + .

[0841] Step 7: Preparation of compound SMP-40359

[0842]

[0843] In a 10 mL reaction flask, SMP-40359-2 (5.8 mg, 0.0078 mmol), intermediate C (15.0 mg, 0.0066 mmol), N,N-dimethylformamide (1 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.0 mg, 0.0078 mmol), and N,N-diisopropylethylamine (1.3 mg, 0.0098 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-49182, weighing 11.6 mg, with a yield of 58.5%.

[0844] MS(ESI) m / z: 1002 [M / 3+H] + .

[0845] Example 21: Preparation of compound SMP-93566

[0846]

[0847] Step 1: Preparation of compound SMP-93566-2

[0848]

[0849] In a 100 mL reaction flask, intermediate SMP-93566-1 (1.0 g, 5.75 mmol), tert-butyl bromoacetate (0.9 g, 4.60 mmol), potassium carbonate (0.6 g, 4.60 mmol), and N,N-dimethylformamide (20 mL) were added sequentially. The reaction was carried out at room temperature for 2 hours. LC-MS showed that most of the molecules were of the target compound. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 12:1) to obtain compound SMP-93566-2, weighing 0.2 g, with a yield of 15.1%. MS (ESI) m / z: 289 [M+H] + .

[0850] 1 HNMR (400MHz, CDCl3) δ5.91(s,1H),3.40-3.49(m,8H),323(s,2H),2.54(m,4H),2.70(m,4H),1.33(s,9H).

[0851] Step 2: Preparation of compound SMP-93566-3

[0852]

[0853] In a 10 mL reaction flask, intermediate SMP-93566-2 (200.0 mg, 0.69 mmol), maleimide propionic acid (140.8 mg, 0.83 mmol), N,N-dimethylformamide (5 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (315.4 mg, 0.83 mmol), and N,N-diisopropylethylamine (134.2 mg, 1.04 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-93566-3, weighing 220.0 mg, with a yield of 72.6%.

[0854] MS(ESI) m / z: 440 [M+H] + .

[0855] Step 3: Preparation of compound SMP-93566-4

[0856]

[0857] In a 25 mL reaction flask, SMP-93566-3 (220.0 mg, 0.50 mmol), dichloromethane (5 mL), and trifluoroacetic acid (5 mL) were added sequentially. The reaction was carried out at room temperature for 2 hours. LC-MS showed that most of the target compound's molecular weight was present. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in a small amount of acetonitrile and purified by preparative HPLC to obtain compound SMP-93566-4, weighing 90.0 mg, with a yield of 47.0%. MS (ESI) m / z: 384 [M+H] + .

[0858] Step 4: Preparation of compound SMP-93566

[0859]

[0860] In a 10 mL reaction flask, SMP-93566-4 (8.8 mg, 0.023 mmol), intermediate A (20 mg, 0.019 mmol), N,N-dimethylformamide (2 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (8.7 mg, 0.023 mmol), and N,N-diisopropylethylamine (3.7 mg, 0.029 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-93566, weighing 18.0 mg, with a yield of 67.0%.

[0861] MS(ESI) m / z: 1414 [M+H] + 707 [M / 2+H] + .

[0862] 1H NMR (400MHz, DMSO-d6) δ8.89(s,1H),8.29(t,J=5.6Hz,1H),8.19(d,J=8.2Hz,1H),8.12(t,J=5.6Hz,1H),7.95(s,1H) ,7.72(s,1H),7.31–7.09(m,6H),6.98(s,2H),5.02(d,J=21.4Hz,2H),4.82(s,1H),4.75(s,1H),4.63(s,1H),4.58–4 .48(m,2H),4.28–4.06(m,10H),3.87–3.47(m,30H),3.16–2.99(m,5H),2.85–2.64(m,6H),2.62–2.53(m,2H),2.34–2 .16(m,6H),2.01–1.87(m,6H),1.73–1.58(m,6H),1.31(s,3H),1.19(m,1H),1.03(d,J=6.3Hz,3H),1.00–0.91(m,1H).

[0863] Example 22: Preparation of compound SMP-88480

[0864]

[0865] Step 1: Preparation of compound SMP-88480

[0866]

[0867] In a 10 mL reaction flask, SMP-93566-4 (9.8 mg, 0.026 mmol), intermediate B (18.0 mg, 0.021 mmol), N,N-dimethylformamide (2 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (9.8 mg, 0.026 mmol), and N,N-diisopropylethylamine (4.1 mg, 0.032 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-88480, weighing 12.0 mg, with a yield of 47.4%.

[0868] MS(ESI) m / z: 1206 [M+H] + 603 [M / 2+H] + .

[0869] 1HNMR(400MHz, DMSO-d6)δ8.65(s,1H),8.51(d,J=8.5Hz,1H),8.33(d,J=24.8Hz,1H),8.22–7.92(m,3H),7.77(d,J=10.9Hz,1H),7.3 0(s,1H),7.28–7.12(m,5H),6.98(s,2H),6.53(s,1H),5.67–5.54(m,1H),5.49–5.32(m,2H),5.18(s,2H),4.64(d,J=6.5Hz,2H),4.4 8(s,1H),4.13(s,1H),4.02(s,2H),3.90(s,1H),3.61(dd,J=41.9,31.1Hz,15H),3.39(m,5H),3.17(dd,J=35.2,18.2Hz,4H),3.01( dd,J=13.6,4.2Hz,1H),2.71(dd,J=41.8,13.0Hz,5H),2.37(s,3H),2.18(d,J=5.4Hz,2H),1.92–1.74(m,2H),0.86(t,J=7.3Hz,3H).

[0870] Example 23: Preparation of compound SMP-34048

[0871]

[0872] Step 1: Preparation of compound SMP-34048-2

[0873]

[0874] In a 250 mL reaction flask, SMP-34048-1 (1.0 g, 2.55 mmol) and dichloromethane (75 mL) were added. Then, di-tert-butyl dicarbonate (0.7 g, 3.32 mmol) and pyridine (6.1 mL, 75 mmol) were added, and the mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The reaction solution was washed successively with 0.5 N hydrochloric acid aqueous solution (20 mL * 3), followed by washing with saturated sodium carbonate (20 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product SMP-34048-1, weighing 1.2 g, with a yield of 95.6%.

[0875] MS(ESI) m / z: 493 [M+H] + .

[0876] Step 2: Preparation of compound SMP-34048-3

[0877]

[0878] In a 250 mL reaction flask, SMP-34048-2 (1.2 g, 2.44 mmol) and dichloromethane (100 mL) were added and stirred at room temperature. Then, p-nitrophenyl chloroformate (0.5 g, 2.68 mmol) was added in portions, followed by 4-dimethylaminopyridine (0.3 g, 2.44 mmol). The reaction was continued under these conditions with stirring for 2 hours. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was then subjected to column chromatography (dichloromethane:methanol = 15:1) to give SMP-34048-3, weighing 360.0 mg, with a yield of 22.4%.

[0879] MS(ESI) m / z: 658 [M+H] + .

[0880] Step 3: Preparation of compound SMP-34048-4

[0881]

[0882] In a 50 mL reaction flask, SMP-34048-3 (360.0 mg, 0.55 mmol), methyl (2-(methylamino)ethyl)carbamate tert-butyl ester (155.0 mg, 0.82 mmol), N,N-dimethylformamide (5 mL), 1-hydroxybenzotriazole (74.2 mg, 0.55 mmol), and N,N-diisopropylethylamine (140.6 mg, 1.09 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane:methanol = 12:1) to obtain SMP-34048-4, weighing 300.0 mg, with a yield of 77.2%.

[0883] MS(ESI) m / z: 707 [M+H] + .

[0884] Step 4: Preparation of compound SMP-34048-5

[0885]

[0886] In a 250 mL reaction flask, SMP-34048-4 (300.0 mg, 0.42 mmol) and dioxane hydrochloride solution (10 mL) were added sequentially, and the mixture was stirred in an oil bath at 70 °C for 1.5 hours. TLC showed that the reaction was complete. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to obtain crude product SMP-34048-5, weighing 200.0 mg, with a yield of 94.1%.

[0887] MS(ESI) m / z: 507 [M+H]+ .

[0888] Step 5: Preparation of compound SMP-34048-6

[0889]

[0890] In a 25 mL reaction flask, intermediate B-1 (225.7 mg, 0.35 mmol), 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (182.0 mg, 0.35 mmol), N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (59.8 mg, 0.46 mmol) were added sequentially. The mixture was stirred at room temperature for 5 minutes, and then SMP-34048-5 (150.0 mg, 0.30 mmol) was added. The reaction mixture was stirred for another 25 minutes, and the reaction was confirmed to be complete by TLC. The N,N-dimethylformamide was removed by vacuum distillation, and the residue was purified by column chromatography (dichloromethane:methanol-10:1) to obtain compound SMP-34048-6, weighing 200.0 mg, with a yield of 58.8%.

[0891] MS(ESI) m / z: 1134 [M+H] + 567 [M / 2+H] + .

[0892] Step 6: Preparation of compound SMP-34048-7

[0893]

[0894] In a 25 mL reaction flask, SMP-34048-6 (200.0 mg, 0.18 mmol), N,N-dimethylformamide (2 mL), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (40.2 mg, 0.26 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. The reaction was monitored by TLC until it was complete. The reaction solution was concentrated under reduced pressure to remove N,N-dimethylformamide. The residue was purified by HPLC to obtain compound SMP-34048-7, weighing 100.0 mg, with a yield of 61.0%.

[0895] MS(ESI) m / z: 912 [M+H] + .

[0896] Step 7: Preparation of compound SMP-34048

[0897]

[0898] In a 10 mL reaction flask, SMP-93566-4 (9.2 mg, 0.024 mmol), SMP-34048-7 (18 mg, 0.020 mmol), N,N-dimethylformamide (2 mL), 1H-benzotriazol-1-yloxytripyrrolyl hexafluorophosphate (12.5 mg, 0.024 mmol), and N,N-diisopropylethylamine (3.9 mg, 0.030 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC to obtain compound SMP-34048, weighing 8.0 mg, with a yield of 31.3%.

[0899] MS(ESI) m / z: 1277 [M+H] + 639 [M / 2+H] + .

[0900] Example 24: Preparation of compound SMP-97962

[0901]

[0902] Step 1: Preparation of compound SMP-97962

[0903]

[0904] In a 10 mL reaction flask, SMP-93566-4 (4.6 mg, 0.012 mmol), intermediate G (10 mg, 0.010 mmol), N,N-dimethylformamide (2 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.6 mg, 0.012 mmol), and N,N-diisopropylethylamine (2.6 mg, 0.020 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-97962, weighing 5.0 mg, with a yield of 36.8%.

[0905] MS(ESI) m / z: 679 [M / 2+H] + .

[0906] 1HNMR(400MHz, DMSO-d6)δ8.79(s,1H),8.30(d,J=5.2Hz,2H),7.96(s,1H),7.73(s,1H),7.26–7.19(m,4H),7.17(t,J=6.3Hz,1H),6. 99(s,2H),5.02(d,J=21.6Hz,2H),4.79(d,J=29.1Hz,2H),4.63(s,1H),4.59–4.51(m,2H),4.26(d,J=11.4Hz,1H),4.19–4.05(m,6H) ,4.02(s,1H),3.92(m,1H),3.84–3.46(m,28H),3.22–2.98(m,6H),2.75(ddd,J=20.7,18.1,5.9Hz,5H),2.61–2.53(m,1H),2.34–2.1 6(m,6H),2.02–1.88(m,6H),1.73–1.56(m,6H),1.49(d,J=12.7Hz,2H),1.39–1.13(m,5H),1.03(d,J=6.3Hz,3H),1.00–0.87(m,1H).

[0907] Example 25: Preparation of compound SMP-00171

[0908]

[0909] Step 1: Preparation of compound SMP-00171

[0910]

[0911] In a 10 mL reaction flask, SMP-93566-4 (19.4 mg, 0.051 mmol), SMP-58054-2 (50.0 mg, 0.042 mmol), N,N-dimethylformamide (4 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (19.4 mg, 0.051 mmol), and N,N-diisopropylethylamine (8.2 mg, 0.064 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-00171, weighing 59.0 mg, with a yield of 90.6%.

[0912] MS(ESI) m / z: 776 [M / 2+H] + .

[0913] Example 26: Preparation of compound SMP-24600

[0914]

[0915] Step 1: Preparation of compound SMP-24600-1

[0916]

[0917] In a 10 mL reaction flask, SMP-93566-1 (100.0 mg, 0.57 mmol), monotert-butyl succinate (99.2 mg, 0.57 mmol), N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (89.0 mg, 0.69 mmol) were added sequentially. Then, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (197.6 mg, 0.52 mmol, dissolved in 1 mL of N,N-dimethylformamide) was slowly added dropwise at room temperature. After the addition was complete, the reaction was continued for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-24600-1, weighing 120.0 mg, with a yield of 70.0%.

[0918] MS(ESI)m / z:331[M+H] + .

[0919] 1 HNMR (400MHz, CDCl3) δ5.51(s,1H),3.49-3.60(m,8H),3.23(m,4H),2.49-2.65(m,8H),1.35(s,9H).

[0920] Step 2: Preparation of compound SMP-24600-2

[0921]

[0922] In a 10 mL reaction flask, SMP-24600-1 (120.0 mg, 0.36 mmol), maleimide propionic acid (73.7 mg, 0.44 mmol), N,N-dimethylformamide (3 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (165.8 mg, 0.44 mmol), and N,N-diisopropylethylamine (71.0 mg, 0.55 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-24600-2, weighing 95.0 mg, with a yield of 54.8%.

[0923] MS(ESI) m / z: 482 [M+H] + .

[0924] Step 3: Preparation of compound SMP-24600-3

[0925]

[0926] SMP-24600-2 (55.0 mg, 0.11 mmol) and trifluoroacetic acid (3 mL) were added sequentially to a 25 mL reaction flask. The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain compound SMP-24600-3, weighing 25.0 mg, with a yield of 53.5%.

[0927] MS(ESI) m / z: 426 [M+H] + .

[0928] Step 4: Preparation of compound SMP-24600

[0929]

[0930] In a 10 mL reaction flask, SMP-24600-3 (7.2 mg, 0.017 mmol), intermediate A (15.0 mg, 0.014 mmol), N,N-dimethylformamide (2 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (6.5 mg, 0.017 mmol), and N,N-diisopropylethylamine (2.7 mg, 0.021 mmol) were added sequentially. The mixture was reacted at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-24600, weighing 12.0 mg, with a yield of 59.0%.

[0931] MS(ESI) m / z: 728 [M / 2+H] + .

[0932] Example 27: Preparation of compound SMP-36297

[0933]

[0934] Step 1: Preparation of compound SMP-36297-1

[0935]

[0936] In a 10 mL reaction flask, SMP-22682-6 (55.0 mg, 0.10 mmol), intermediate I (73.0 mg, 0.12 mmol), N,N-dimethylformamide (2 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (45.6 mg, 0.12 mmol), and N,N-diisopropylethylamine (19.4 mg, 0.15 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-36297-1, weighing 50.0 mg, with a yield of 43.7%.

[0937] MS(ESI) m / z: 572 [M / 2+H] + .

[0938] Step 2: Preparation of compound SMP-36297-2

[0939]

[0940] In a 25 mL reaction flask, intermediate SMP-36297-1 (50 mg, 0.044 mmol), dichloromethane (2 mL), and trifluoroacetic acid (2 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse-phase HPLC to obtain compound SMP-36297-2, weighing 30.0 mg, with a yield of 62.7%.

[0941] MS(ESI) m / z: 543.54 [M / 2+H] + .

[0942] Step 3: Preparation of compound SMP-36297

[0943]

[0944] In a 10 mL reaction flask, SMP-36297-2 (6.0 mg, 0.055 mmol), intermediate D (10 mg, 0.0045 mmol), N,N-dimethylformamide (1 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.1 mg, 0.0055 mmol), and N,N-diisopropylethylamine (0.9 mg, 0.0068 mmol) were added sequentially. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-36297, weighing 6.6 mg, with a yield of 45.0%.

[0945] MS(ESI) m / z: 1087 [M / 3+H] + 815 [M / 4+H] + .

[0946] Example 28: Preparation of compound SMP-12395

[0947]

[0948] Step 1: Preparation of compound SMP-12395-2

[0949]

[0950] In a 100 mL reaction flask, add intermediate diglycinate (644.0 mg, 4.80 mmol), sodium bicarbonate (806.4 mg, 9.60 mmol), water (2 mL), and tetrahydrofuran (20 mL). Stir at room temperature until the solution is completely clear. Dissolve compound A-2 (2.2 g, 4.80 mmol) in ethylene glycol dimethyl ether (5 mL) and slowly add it to the above reaction solution. Continue stirring overnight. TLC shows that the reaction is complete. Concentrate the reaction solution under reduced pressure. Add the residue dropwise to 0.5 M hydrochloric acid aqueous solution (200 mL), and a large amount of solid precipitates. Filter and dry the solid under vacuum to give compound SMP-12395-2, weighing 1.5 g, with a yield of 66.7%. MS (ESI) m / z: 469 [M+H] + .

[0951] 1 HNMR(400MHz, CDCl3)δ13.01(s,1H),9.01(s,1H),8.76(s,1H),8.55(s,1H),8.43(s,1H), 7.90(d,2H),7.55(d,2H),7.28-7.38(m,4H),4.71(d,2H),4.45(t,1H),3.85-4.01(m,8H).

[0952] Step 2: Preparation of compound SMP-12395-3

[0953]

[0954] Compound SMP-12395-2 (420.0 mg, 0.90 mmol) and tetrahydrofuran (5 mL) solvent were added to a 25 mL reaction flask. The mixture was stirred at room temperature until completely clear. Lead tetraacetate (480.0 mg, 1.07 mmol) and pyridine (107.2 mg, 1.34 mmol) were added sequentially. The mixture was stirred overnight at 70 °C. TLC showed complete reaction. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:ethyl acetate = 2:1) to give intermediate SMP-12395-3, weighing 290.0 mg, with a yield of 67.0%. MS (ESI) m / z: 482 [M+H] + .

[0955] 1 HNMR(400MHz, CDCl3)δ9.04(s,1H),8.86(s,1H),8.75(s,1H),8.23(s,1H),7.87(d,2H),7.52(d ,2H),7.26-7.39(m,4H),5.99(s,2H),4.73(d,2H),4.41(t,1H),3.83-4.02(m,8H),2.11(s,3H).

[0956] Step 3: Preparation of compound SMP-12395-4

[0957]

[0958] Compound SMP-12395-3 (290.0 mg, 0.60 mmol), tetrahydrofuran (25 mL), and glycolic acid (50.0 mg, 0.66 mmol) were added to a 100 mL reaction flask. After the reaction solution became clear, p-toluenesulfonic acid (51.0 mg, 0.30 mmol) was added in one go at 0 °C, and the mixture was stirred at room temperature for 1.5 hours. TLC showed that the reaction was complete. The mixture was filtered and concentrated under reduced pressure to remove excess tetrahydrofuran. The residue was purified by HPLC to obtain intermediate SMP-12395-4, weighing 190.0 mg, with a yield of 63%.

[0959] MS(ESI) m / z: 499 [M+H] + .

[0960] Step 4: Preparation of compound SMP-12395-5

[0961]

[0962] In a 25 mL reaction flask, intermediate SMP-12395-4 (168.0 mg, 0.34 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (129.0 mg, 0.34 mmol), N,N-diisopropylethylamine (55.0 mg, 0.43 mmol), and N,N-dimethylformamide (8 mL) were added. After stirring at room temperature for 5 minutes, eczemab mesylate (150.0 mg, 0.28 mmol) was added, and stirring was continued for 25 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by thin-layer chromatography (dichloromethane:methanol = 8:1) to give compound SMP-12395-5, weighing 200.0 mg, with a yield of 78%.

[0963] MS(ESI) m / z: 916 [M+H] + .

[0964] Step 5: Preparation of compound SMP-12395-6

[0965]

[0966] In a 25 mL reaction flask, intermediate SMP-12395-5 (120.0 mg, 0.13 mmol), 1,8-diazobispirocyclo[5.4.0]undec-7-ene (40.0 mg, 0.26 mmol), and N,N-dimethylformamide (3 mL) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain compound SMP-12395-6, weighing 65.0 mg, with a yield of 72%. MS (ESI) m / z: 694 [M+H] + .

[0967] Step 6: Preparation of compound SMP-12395

[0968]

[0969] Compound SMP-93566-4 (9.0 mg, 0.020 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.0 mg, 0.024 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.03 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound SMP-12395-6 (13.0 mg, 0.019 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC and lyophilized to give a white solid SMP-12395, weighing 5.8 mg, with a yield of 29%.

[0970] MS(ESI) m / z: 1059 [M+H] + .

[0971] 1 HNMR(400MHz,DMSO-d6)δ8.93(s,1H),8.72(t,J=7.3Hz,1H),8.52(d,J=8.8Hz,1H),8.28(s,1H),8.19(t,J=5.7Hz,1H),7 .95(s,1H),7.79(d,J=11.0Hz,1H),7.32(d,J=8.3Hz,1H),6.99(s,2H),6.54(s,1H),5.60(dd,J=14.0,5.8Hz,1H),5.40(d ,J=16.8Hz,2H),5.20(s,2H),4.62(d,J=6.5Hz,2H),4.15(s,2H),4.00(s,2H),3.94(s,2H),3.85–3.69(m,8H),3.69–3.4 8(m,14H),2.67(s,1H),2.39(s,4H),2.17(dt,J=15.8,6.2Hz,2H),1.91–1.79(m,2H),1.23(s,1H),0.87(t,J=7.2Hz,3H).

[0972] Example 29: Preparation of compound SMP-56822

[0973]

[0974] Step 1: Preparation of compound SMP-56822-2

[0975]

[0976] Compound SMP-56822-1 (2.0 g, 4.4 mmol) and N,N-dimethylformamide (30 mL) were added sequentially to a 100 mL reaction flask. Sodium hydride (60 wt%) (0.53 g, 13.20 mmol) was added at 0 °C. After reacting for 10 minutes at room temperature, PEG9-OMs (4.9 g, 9.70 mmol) were added in a single batch. The mixture was stirred at 50 °C for 12 hours, and TLC showed complete reaction. The reaction was quenched by slowly adding water (15 mL) at 0 °C. Extraction was performed using dichloromethane (100 mL)*3. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:ethyl acetate = 2:1) to give intermediate SMP-56822-2, weighing 4.0 g, with a yield of 71%.

[0977] MS(ESI) m / z: 1275 [M+H] + .

[0978] Step 2: Preparation of compound SMP-56822-3

[0979]

[0980] Compound SMP-56822-2 (4.0 g, 3.1 mmol) and tetrahydrofuran (32 mL) were added sequentially to a 100 mL reaction flask. A 1 M lithium aluminum hydride solution in tetrahydrofuran (18.6 mL, 18.6 mmol) was slowly added dropwise at 0 °C. After the temperature returned to room temperature, the mixture was heated to 50 °C and stirred for 2 hours. LC-MS analysis showed that the reaction was complete. Sodium sulfate decahydrate (5.0 g) was added in portions at 0 °C. The mixture was filtered, and the solution was concentrated under reduced pressure to remove excess tetrahydrofuran, yielding intermediate SMP-56822-3, weighing 2.4 g (79% yield).

[0981] MS(ESI) m / z: 967 [M+H] + .

[0982] Step 3: Preparation of compound SMP-56822-4

[0983]

[0984] Compound SMP-56822-3 (630.0 mg, 0.65 mmol) and solvent N,N-dimethylformamide (5 mL) were added sequentially to a 25 mL reaction flask. After the mixture was completely clear, potassium carbonate (180.0 mg, 1.30 mmol) and tert-butyl bromoacetate (127.0 mg, 0.65 mmol) were added sequentially. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. After filtration, the mixture was concentrated under reduced pressure to remove excess N,N-dimethylformamide. The residue was purified by column chromatography (dichloromethane:methanol = 15:1) to obtain intermediate SMP-56822-4, weighing 170.0 mg, with a yield of 24%.

[0985] MS(ESI) m / z: 1081 [M+H] + .

[0986] Step 4: Preparation of compound SMP-56822-5

[0987]

[0988] Maleimide hexanoic acid (24.0 mg, 0.11 mmol) and N,N-dimethylformamide (4 mL) were added to a 25 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (42.0 mg, 0.11 mmol) and N,N-diisopropylethylamine (17.0 mg, 0.13 mmol) were added sequentially. After reacting at room temperature for 5 minutes, SMP-56822-4 (102.0 mg, 0.094 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 20:1) to obtain intermediate SMP-56822-5, weighing 100.0 mg, with a yield of 83%.

[0989] MS(ESI) m / z: 1274 [M+H] + .

[0990] Step 5: Preparation of compound SMP-56822-6

[0991]

[0992] Compound SMP-56822-5 (100.0 mg, 0.08 mmol) and trifluoroacetic acid (5 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-56822-6, weighing 80.0 mg, with a yield of 83%.

[0993] MS(ESI) m / z: 1218 [M+H] + .

[0994] Step 6: Preparation of compound SMP-56822

[0995]

[0996] Compound SMP-56822-6 (30.0 mg, 0.025 mmol) and N,N-dimethylformamide (4 mL) were added to a 25 mL reaction flask. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.0 mg, 0.024 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.032 mmol) were added sequentially. The mixture was reacted at room temperature for 5 minutes, and then compound D (45.0 mg, 0.020 mmol) was added and the mixture was stirred for 25 minutes. The reaction was complete by TLC. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC and lyophilized to give a white solid SMP-56822, weighing 40.0 mg, with a yield of 59%.

[0997] MS(ESI) m / z: 1131 [M / 3+H] + .

[0998] Example 30: Preparation of compound SMP-82891

[0999]

[1000] Step 1: Preparation of compound SMP-82891-2

[1001]

[1002] Compound SMP-56822-1 (2.0 g, 4.40 mmol) and solvent N,N-dimethylformamide (30 mL) were added sequentially to a 100 mL reaction flask. Sodium hydride (60 wt%) (0.5 g, 13.2 mmol) was added at 0 °C. After reacting at room temperature for 10 minutes, PEG was added all at once. 16 -OMs (7.5 g, 9.7 mmol) were stirred at 80 °C for 12 hours, and the reaction was complete according to TLC. The reaction was quenched by slowly adding water (15 mL) at 0 °C, and extracted with dichloromethane (100 mL * 3). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 20:1) to give intermediate SMP-82891-2, weighing 5.0 g, with a yield of 63%.

[1003] MS(ESI) m / z: 1804 [M+H] + .

[1004] Step 2: Preparation of compound SMP-82891-3

[1005]

[1006] Compound SMP-82891-2 (4.0 g, 2.2 mmol) and tetrahydrofuran (40 mL) were added sequentially to a 100 mL reaction flask. A 1 M solution of lithium aluminum hydride in tetrahydrofuran (13.2 mL, 13.2 mmol) was slowly added dropwise at 0 °C. After returning to room temperature, the mixture was heated to 50 °C and stirred for 2 hours. LC-MS analysis showed the reaction was complete. Sodium sulfate decahydrate (5.0 g) was added in portions at 0 °C. The mixture was filtered, concentrated under reduced pressure to remove excess tetrahydrofuran, and the residue was purified by column chromatography (dichloromethane:methanol = 8:1) to give intermediate SMP-82891-3, weighing 2.4 g, with a yield of 73%.

[1007] MS(ESI) m / z: 1495 [M+H] + .

[1008] Step 3: Preparation of compound SMP-82891-4

[1009]

[1010] Compound SMP-82891-3 (2.4 g, 1.61 mmol) was added sequentially to a 100 mL reaction flask along with N,N-dimethylformamide (35 mL), followed by potassium carbonate (443.0 mg, 3.21 mmol) and tert-butyl bromoacetate (314 mg, 1.61 mmol). The mixture was stirred at room temperature for 2 hours. TLC showed complete reaction. After filtration, excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 12:1) to give intermediate SMP-82891-4, weighing 360.0 mg, with a yield of 14%. MS (ESI) m / z: 1609 [M+H] + .

[1011] Step 4: Preparation of compound SMP-82891-5

[1012]

[1013] In a 25 mL reaction flask, compound 3-(2-(2,5-dioxy-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionic acid (32.0 mg, 0.13 mmol) and solvent N,N-dimethylformamide (8 mL) were added. After the reaction solution became completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (49.0 mg, 0.13 mmol) and N,N-diisopropyl... 21.0 mg, 0.16 mmol of N,N-dimethylformamide was reacted at room temperature for 5 minutes, followed by the addition of compound SMP-82891-4 (170.0 mg, 0.11 mmol). The mixture was stirred for 25 minutes, and TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 15:1) to give intermediate SMP-82891-5, weighing 150.0 mg, with a yield of 74%.

[1014] MS(ESI) m / z: 1849 [M+H] + .

[1015] Step 5: Preparation of compound SMP-82891-6

[1016]

[1017] Compound SMP-82891-5 (150.0 mg, 0.08 mmol) and trifluoroacetic acid (4 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-82891-6, weighing 130.0 mg, with a yield of 90%.

[1018] MS(ESI) m / z: 1793 [M+H] + .

[1019] Step 6: Preparation of compound SMP-82891

[1020]

[1021] Compound SMP-82891-6 (20.0 mg, 0.01 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 25 mL reaction flask. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.03 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.03 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound D (22.0 mg, 0.01 mmol) was added and the mixture was stirred for 25 minutes. The reaction was complete by TLC. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-82891, weighing 30.0 mg, with a yield of 76%.

[1022] MS(ESI) m / z: 1322 [M / 3+H] + .

[1023] Example 31: Preparation of compound SMP-51013-A

[1024]

[1025] Step 1: Preparation of compound SMP-51013-2

[1026]

[1027] In a 25 mL reaction flask, maleimide-diethylene glycol-carboxylic acid (29.0 mg, 0.11 mmol) and N,N-dimethylformamide (3 mL) were added. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (41.0 mg, 0.11 mmol) and N,N-diisopropylethylamine (17.0 mg, 0.13 mmol) were added sequentially. After reacting at room temperature for 5 minutes, SMP-56822-4 (102.0 mg, 0.092 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 12:1) to obtain intermediate SMP-51013-2, weighing 100.0 mg, with a yield of 84%.

[1028] MS(ESI) m / z: 1320 [M+H] + .

[1029] Step 2: Preparation of compound SMP-51013-3

[1030]

[1031] Compound SMP-51013-2 (100.0 mg, 0.076 mmol) and trifluoroacetic acid (4 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 30 minutes. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-51013-3, weighing 80.0 mg, with a yield of 83%.

[1032] MS(ESI) m / z: 1264 [M+H] + .

[1033] Step 3: Preparation of compound SMP-51013-A

[1034]

[1035] Compound SMP-51013-3 (20.0 mg, 0.016 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.029 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound Fa (35.0 mg, 0.013 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-51013-A, weighing 30.0 mg, with a yield of 62%. MS (ESI) m / z: 1295 [M / 3+H] + .

[1036] Example 32: Preparation of compound SMP-51013-B

[1037]

[1038] Step 1: Preparation of compound SMP-51013-B

[1039]

[1040] Compound SMP-51013-3 (20.0 mg, 0.016 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.029 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound Fb (35.0 mg, 0.013 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-51013-B, weighing 28.0 mg, with a yield of 72%. MS (ESI) m / z: 1295 [M / 3+H] + .

[1041] Example 33: Preparation of compound SMP-36256

[1042]

[1043] Step 1: Preparation of compound SMP-36256

[1044]

[1045] Compound SMP-56822-6 (20.0 mg, 0.016 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 25 mL reaction flask. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.029 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound C (45.0 mg, 0.020 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-36256, weighing 40.0 mg, with a yield of 57%.

[1046] MS(ESI) m / z: 1162 [M / 3+H] + .

[1047] Example 34: Preparation of compound SMP-45582

[1048]

[1049] Step 1: Preparation of compound SMP-45582

[1050]

[1051] Compound SMP-51013-3 (25.0 mg, 0.020 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.029 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound E (60.0 mg, 0.021 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-45582, weighing 48.0 mg, with a yield of 59%. MS (ESI) m / z: 1354 [M / 3+H] + .

[1052] Example 35: Preparation of compound SMP-01984

[1053]

[1054] Step 1: Preparation of compound SMP-01984-1

[1055]

[1056] In a 25 mL reaction flask, maleimide-8 polyethylene glycol-carboxylic acid (57.0 mg, 0.11 mmol) and N,N-dimethylformamide (5 mL) were added sequentially. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (41.0 mg, 0.11 mmol) and N,N-diisopropylethylamine (17.0 mg, 0.14 mmol) were added. After reacting at room temperature for 5 minutes, SMP-56822-4 (102.0 mg, 0.094 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 15:1) to obtain intermediate SMP-01984-1, weighing 100.0 mg, with a yield of 67%.

[1057] MS(ESI) m / z: 1584 [M+H] + .

[1058] Step 2: Preparation of compound SMP-01984-2

[1059]

[1060] Compound SMP-01984-1 (100.0 mg, 0.063 mmol) and trifluoroacetic acid (3 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-01984-2, weighing 82.0 mg, with a yield of 85%.

[1061] MS(ESI) m / z: 1528 [M+H] + .

[1062] Step 3: Preparation of compound SMP-01984

[1063]

[1064] Compound SMP-01984-2 (35.0 mg, 0.023 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.029 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound C (60.0 mg, 0.026 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-01984, weighing 48.0 mg, with a yield of 55%. MS (ESI) m / z: 1266 [M / 3+H] + .

[1065] Example 36: Preparation of compound SMP-30920

[1066]

[1067] Step 1: Preparation of compound SMP-30920-1

[1068]

[1069] In a 25 mL reaction flask, maleimide-6 polyethylene glycol-carboxylic acid (48.0 mg, 0.11 mmol) and N,N-dimethylformamide (5 mL) were added sequentially. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (41.0 mg, 0.11 mmol) and N,N-diisopropylethylamine (17.0 mg, 0.13 mmol) were added. After reacting at room temperature for 5 minutes, SMP-56822-4 (102 mg, 0.094 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 15:1) to give intermediate SMP-30920-1, weighing 98.0 mg, with a yield of 70%.

[1070] MS(ESI) m / z: 1496 [M+H] + .

[1071] Step 2: Preparation of compound SMP-30920-2

[1072]

[1073] Compound SMP-30920-1 (95.0 mg, 0.063 mmol) and trifluoroacetic acid (3 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-30920-2, weighing 80.0 mg, with a yield of 88%.

[1074] MS(ESI) m / z: 1440 [M+H] + .

[1075] Step 3: Preparation of compound SMP-30920

[1076]

[1077] Compound SMP-30920-2 (29.0 mg, 0.020 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.03 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound C (60.0 mg, 0.026 mmol) was added and the mixture was stirred for 25 minutes. The reaction was complete by TLC. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-30920, weighing 43.0 mg, with a yield of 58%.

[1078] MS(ESI) m / z: 1236 [M / 3+H] + .

[1079] Example 37: Preparation of compound SMP-14324

[1080]

[1081] Step 1: Preparation of compound SMP-14324

[1082]

[1083] Compound SMP-51013-3 (13.0 mg, 0.010 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.0 mg, 0.029 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound C (22.0 mg, 0.0096 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-14324, weighing 26.0 mg, with a yield of 73%. MS (ESI) m / z: 1178 [M / 3+H] + .

[1084] Example 38: Preparation of compound SMP-61393

[1085]

[1086] Step 1: Preparation of compound SMP-61393

[1087]

[1088] Compound SMP-51013-3 (13.0 mg, 0.011 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.0 mg, 0.011 mmol) and N,N-diisopropylethylamine (2.0 mg, 0.014 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound H (22.0 mg, 0.009 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-61393, weighing 24.0 mg, with a yield of 74%. MS (ESI) m / z: 1207 [M / 3+H] + .

[1089] Example 39: Preparation of compound SMP-33693

[1090]

[1091] Step 1: Preparation of compound SMP-33693-2

[1092]

[1093] Compound SMP-33693-1 (1.0 g, 1.75 mmol) and 33% acetic acid bromide solution (15 mL) were added sequentially to a 50 mL reaction flask. After the reaction solution became clear, phenol (494.0 mg, 5.25 mmol) was added. The reaction was carried out at room temperature for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was slowly added dropwise to methyl tert-butyl ether (20 mL), and a white solid precipitated. The mixture was filtered, the filter cake was collected, and dried to obtain the target product SMP-33693-2, weighing 300.0 mg, with a yield of 66%.

[1094] MS(ESI) m / z: 263 [M+H] + .

[1095] Step 2: Preparation of compound SMP-33693-3

[1096]

[1097] Compound SMP-33693-2 (300.0 mg, 1.15 mmol), N,N-dimethylformamide (10 mL), potassium carbonate (316.0 mg, 2.30 mmol), and tert-butyl bromoacetate (224.0 mg, 1.15 mmol) were added sequentially to a 25 mL reaction flask. The mixture was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. After filtration, the mixture was concentrated under reduced pressure to remove excess N,N-dimethylformamide. The residue was purified by column chromatography (dichloromethane:methanol = 12:1) to give intermediate SMP-33693-3, weighing 130.0 mg, with a yield of 30%. MS (ESI) m / z: 377 [M+H] + .

[1098] 1 HNMR (400MHz, CDCl3) δ5.51(s,1H),3.33-3.56(m,18H),2.52-2.70(m,8H),1.33(s,9H).

[1099] Step 3: Preparation of compound SMP-33693-4

[1100]

[1101] Maleimide hexanoic acid (87.0 mg, 0.41 mmol) and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask. After the reaction solution became clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (156.0 mg, 0.41 mmol) and N,N-diisopropylethylamine (66.0 mg, 0.51 mmol) were added sequentially. The reaction was carried out at room temperature for 5 minutes, and then SMP-33693-3 (130.0 mg, 0.34 mmol) was added. The mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 20:1) to obtain intermediate SMP-33693-4, weighing 150.0 mg, with a yield of 77%.

[1102] MS(ESI) m / z: 570 [M+H] + .

[1103] 1 HNMR (400MHz, CDCl3) δ7.84(s,1H),7.64(s,1H),4.05(t,2H),3.32-3.63(m,22H),2.52(m,4H),2.25(m,2H),1.32-1.63(m,6H),1.33(s,9H).

[1104] Step 4: Preparation of compound SMP-33693-5

[1105]

[1106] Compound SMP-33693-4 (100.0 mg, 0.18 mmol) and trifluoroacetic acid (3 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-33693-5, weighing 80.0 mg, with a yield of 89%.

[1107] MS(ESI) m / z: 514 [M+H] + .

[1108] Step 5: Preparation of compound SMP-33693

[1109]

[1110] Compound SMP-33693-5 (20.0 mg, 0.039 mmol) and N,N-dimethylformamide (4 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.0 mg, 0.039 mmol) and N,N-diisopropylethylamine (6.0 mg, 0.047 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound A (40.0 mg, 0.038 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-33693, weighing 48.0 mg, with a yield of 78%.

[1111] MS(ESI) m / z: 1544 [M+H] + .

[1112] Example 40: Preparation of compound SMP-52573

[1113]

[1114] Step 1: Preparation of compound SMP-52573

[1115]

[1116] Compound SMP-33693-5 (20.0 mg, 0.039 mmol) and solvent N,N-dimethylformamide (2 mL) were added to a 10 mL reaction flask. After the reaction solution was completely clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.0 mg, 0.039 mmol) and N,N-diisopropylethylamine (6.0 mg, 0.047 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound G (40.0 mg, 0.040 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain the target product SMP-52573, weighing 45.0 mg, with a yield of 75%.

[1117] MS(ESI) m / z: 1487 [M+H] + .

[1118] Example 41: Preparation of compound SMP-42930

[1119]

[1120] Step 1: Preparation of compound SMP-42930-1

[1121]

[1122] Maleimide butyric acid (103.0 mg, 0.56 mmol) and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (212.0 mg, 0.55 mmol) and N,N-diisopropylethylamine (91.0 mg, 0.71 mmol) were added sequentially. After reacting at room temperature for 5 minutes, SMP-33693-3 (176.0 mg, 0.47 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 20:1) to give intermediate SMP-42930-1, weighing 185.0 mg, with a yield of 72%.

[1123] MS(ESI) m / z: 542 [M+H] + .

[1124] Step 2: Preparation of compound SMP-42930-2

[1125]

[1126] Compound SMP-42930-1 (100.0 mg, 0.18 mmol) and trifluoroacetic acid (4 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. Excess trifluoroacetic acid was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-42930-2, weighing 80.0 mg, with a yield of 89%.

[1127] MS(ESI) m / z: 486 [M+H] + .

[1128] Step 3: Preparation of compound SMP-42930.

[1129]

[1130] Compound SMP-42930-2 (19.0 mg, 0.039 mmol) and N,N-dimethylformamide (2 mL) were added to a 10 mL reaction flask. After the reaction solution became clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.0 mg, 0.039 mmol) and N,N-diisopropylethylamine (6.0 mg, 0.047 mmol) were added sequentially. The mixture was reacted at room temperature for 5 minutes, and then compound A (40.0 mg, 0.038 mmol) was added. The mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-42930, weighing 42.0 mg, with a yield of 73%.

[1131] MS(ESI) m / z: 1516 [M+H] + .

[1132] Example 42: Preparation of compound SMP-80439

[1133]

[1134] Step 1: Preparation of compound SMP-80439

[1135]

[1136] Compound SMP-42930-2 (20.0 mg, 0.041 mmol) and N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask. After the reaction solution became clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (15.0 mg, 0.039 mmol) and N,N-diisopropylethylamine (6.0 mg, 0.047 mmol) were added sequentially. The mixture was reacted at room temperature for 5 minutes, and then compound G (40.0 mg, 0.038 mmol) was added and the mixture was stirred for 25 minutes. The reaction was complete by TLC. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-80439, weighing 42.0 mg, with a yield of 76%.

[1137] MS(ESI) m / z: 1458 [M+H] + .

[1138] Example 43: Preparation of compound SMP-81404

[1139]

[1140] Step 1: Preparation of compound SMP-81404

[1141]

[1142] Compound SMP-01984-2 (42.0 mg, 0.024 mmol) and N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.0 mg, 0.024 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound J (43.0 mg, 0.020 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-81404, weighing 57.0 mg, with a yield of 78%. MS (ESI) m / z: 1217 [M / 3+H] + .

[1143] Example 44: Preparation of compound SMP-64906

[1144]

[1145] Step 1: Preparation of compound SMP-64906

[1146]

[1147] Intermediate SMP-66348-2 (39.0 mg, 0.024 mmol) and solvent N,N-dimethylformamide (4 mL) were added to a 10 mL reaction flask. After the reaction solution became clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.0 mg, 0.024 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. The mixture was reacted at room temperature for 5 minutes, and then intermediate Fa (52.0 mg, 0.020 mmol) was added. The mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-64906, weighing 55.0 mg, with a yield of 65%.

[1148] MS(ESI) m / z: 1413 [M / 3+H] + .

[1149] Example 45: Preparation of compound SMP-66348

[1150]

[1151] Step 1: Preparation of compound SMP-66348-1

[1152]

[1153] Intermediate I (61.0 mg, 0.10 mmol) and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (38.0 mg, 0.10 mmol) and N,N-diisopropylethylamine (15.0 mg, 0.12 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound SMP-56822-4 (95.0 mg, 0.08 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 12:1) to obtain the target product SMP-66348-1, weighing 80.0 mg, with a yield of 60%.

[1154] MS(ESI) m / z: 1671 [M+H] + .

[1155] Step 2: Preparation of compound SMP-66348-2

[1156]

[1157] In a 25 mL reaction flask, intermediate SMP-66348-1 (50.0 mg, 0.03 mmol) and trifluoroacetic acid (3 mL) were added. The mixture was stirred at room temperature for 30 minutes. LC-MS showed that the reaction was complete. After filtration, the mixture was concentrated under reduced pressure to remove trifluoroacetic acid. The residue was then subjected to HPLC to prepare compound SMP-66348-2, weighing 35.0 mg, with a yield of 72%.

[1158] MS(ESI) m / z: 1615 [M+H] + .

[1159] Step 3: Preparation of compound SMP-66348

[1160]

[1161] Intermediate SMP-66348-2 (39.0 mg, 0.024 mmol) and solvent N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask. After the reaction solution became clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.0 mg, 0.024 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. The reaction was carried out at room temperature for 5 minutes, and then intermediate Fb (52.0 mg, 0.020 mmol) was added. The mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-66348, weighing 60.0 mg, with a yield of 71%.

[1162] MS(ESI) m / z: 1413 [M / 3+H] + .

[1163] Example 46: Preparation of compound SMP-34421

[1164]

[1165] Step 1: Preparation of compound SMP-34421-2

[1166]

[1167] In a 50 mL reaction flask, intermediate SMP-34421-1 (200.0 mg, 0.50 mmol) and N,N-dimethylformamide (10 mL) were added. After complete clarification, PEG9-OMs (253.0 mg, 0.50 mmol) and potassium carbonate (207.0 mg, 1.50 mmol) were added. The mixture was stirred at 80 °C for 20 hours. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-34421-2, weighing 60.0 mg, with a yield of 15%.

[1168] MS(ESI)m / z:811[M+H] + .

[1169] 1 HNMR (400MHz, CDCl3) δ4.31(s,1H),3.33-3.54(m,38H),3.11(s,3H),2.46-2.65(m,16H),1.35(s,18H).

[1170] Step 2: Preparation of compound SMP-34421-3

[1171]

[1172] In a 25 mL reaction flask, the intermediate maleimide-diethylene glycol-carboxylic acid (22.0 mg, 0.086 mmol) and N,N-dimethylformamide (4 mL) were added. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (34.0 mg, 0.089 mmol), N,N-diisopropylethylamine (30.0 mg, 0.22 mmol), and SMP-34421-2 (60.0 mg, 0.074 mmol) were added. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34421-3, weighing 50.0 mg, with a yield of 64%.

[1173] MS(ESI) m / z: 526 [M / 2+H] + .

[1174] Step 3: Preparation of compound SMP-34421-4

[1175]

[1176] SMP-34421-3 (50.0 mg, 0.048 mmol) was placed in a 25 mL reaction flask, and trifluoroacetic acid (3 mL) was added. The mixture was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the resulting compound, SMP-34421-4, was obtained by HPLC purification, weighing 25.0 mg, with a yield of 55.97%. MS (ESI) m / z: 470 [M / 2+H] + .

[1177] Step 4: Preparation of compound SMP-34421-5

[1178]

[1179] SMP-34421-4 (25.0 mg, 0.027 mmol) was dissolved in N,N-dimethylformamide (2 mL). After complete clarification, intermediate A (28.0 mg, 0.027 mmol) and N,N-diisopropylethylamine (7.0 mg, 0.054 mmol) were added sequentially. After completion, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (10.0 mg, 0.027 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34421-5, weighing 20.0 mg, with a yield of 38.2%.

[1180] MS(ESI) m / z: 984 [M / 2+H] + .

[1181] Step 5: Preparation of compound SMP-34421

[1182]

[1183] SMP-34421-5 (20.0 mg, 0.010 mmol) was dissolved in N,N-dimethylformamide (2 mL). After complete clarification, intermediate B (9.0 mg, 0.010 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.0 mg, 0.010 mmol), and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34421, weighing 13.0 mg, with a yield of 45.8%.

[1184] MS(ESI) m / z: 1395 [M / 2+H] + .

[1185] Example 47: Preparation of compound SMP-53612

[1186]

[1187] Step 1: Preparation of compound SMP-53612-1

[1188]

[1189] SMP-34421-2 (70.0 mg, 0.086 mmol) was dissolved in N,N-dimethylformamide (2 mL). After complete clarification, 6-maleimide hexanoic acid (22.0 mg, 0.10 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (39.0 mg, 0.10 mmol), and N,N-diisopropylethylamine (17.0 mg, 0.13 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53612-1, weighing 50 mg, with a yield of 57.7%. MS (ESI) m / z: 503 [M / 2+H] + .

[1190] Step 2: Preparation of compound SMP-53612-2

[1191]

[1192] SMP-53612-1 (50.0 mg, 0.05 mmol) was dissolved in trifluoroacetic acid (3 mL), and the reaction was stirred at room temperature for 3 hours. The reaction was complete by TLC. The trifluoroacetic acid was removed by concentration under reduced pressure, and SMP-53612-2 was obtained by HPLC purification, weighing 34.0 mg, with a yield of 79.6%.

[1193] MS(ESI) m / z: 447 [M / 2+H] + .

[1194] Step 3: Preparation of compound SMP-53612-3

[1195]

[1196] SMP-53612-2 (34.0 mg, 0.038 mmol) was dissolved in N,N-dimethylformamide (2 mL). After complete clarification, intermediate A (40.0 mg, 0.038 mmol) and N,N-diisopropylethylamine (15.0 mg, 0.11 mmol) were added sequentially. After completion, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (15.0 mg, 0.039 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53612-3, weighing 24.0 mg, with a yield of 32.8%.

[1197] MS(ESI) m / z: 962 [M / 2+H] + .

[1198] Step 4: Preparation of compound SMP-53612

[1199]

[1200] SMP-53612-3 (24.0 mg, 0.012 mmol) was dissolved in N,N-dimethylformamide (2 mL). After complete clarification, intermediate B (10.0 mg, 0.012 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (5.0 mg, 0.012 mmol), and N,N-diisopropylethylamine (5.0 mg, 0.039 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53612, weighing 10.0 mg, with a yield of 29.2%.

[1201] MS(ESI) m / z: 1373 [M / 2+H] + .

[1202] 1HNMR(400MHz, DMSO-d6)δ8.68(t,J=6.6Hz,1H),8.50(t,J=14.4Hz,3H),8.36(s,1H),8. 28(s,1H),8.18(s,2H),8.12(d,J=11.6Hz,2H),7.79(d,J=10.9Hz,1H),7.71(s,1H),7.3 1(s,1H),7.26–7.12(m,10H),7.00(s,2H),5.63–5.56(m,1H),5.41(s,2H),5.20(s,2H), 5.02(d,J=21.8Hz,2H),4.82(s,1H),4.74(s,1H),4.64(d,J=6.1Hz,3H),4.54(dd,J=9.8 ,5.5Hz,4H),4.45–3.84(m,36H),3.69(m,24H),3.38(m,12H),3.24(d,J=12.4Hz,10H),3 .17–2.96(m,8H),2.92–2.62(m,13H),2.38(s,3H),2.33–2.05(m,12H),2.04–1.81(m,8H ),1.67(dd,J=25.2,13.1Hz,6H),1.46(dt,J=15.1,7.7Hz,6H),1.31(dd,J=22.5,8.0Hz, 3H), 1.19 (dd, J=21.3, 13.8Hz, 4H), 0.99 (dd, J=24.2, 8.9Hz, 4H), 0.86 (t, J=7.4Hz, 3H).

[1203] Example 48: Preparation of compound SMP-10215

[1204]

[1205] Step 1: Preparation of compound SMP-10215-1

[1206]

[1207] SMP-34421-2 (120.0 mg, 0.15 mmol), maleimide-tetraethylene glycol-carboxylic acid (51.0 mg, 0.15 mmol), and N,N-dimethylformamide (4 mL) were mixed and clarified. Then, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (56.0 mg, 0.15 mmol) and N,N-diisopropylethylamine (57.0 mg, 0.45 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-10215-1, weighing 90.0 mg, with a yield of 53.4%.

[1208] MS(ESI) m / z: 570 [M / 2+H] + .

[1209] Step 2: Preparation of compound SMP-10215-2

[1210]

[1211] SMP-10215-1 (90.0 mg, 0.079 mmol) was dissolved in trifluoroacetic acid (3 mL), and the reaction was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. Trifluoroacetic acid was removed under reduced pressure, and the reaction solution was purified by preparative HPLC to obtain compound SMP-10215-2, weighing 20.0 mg, with a yield of 24.6%.

[1212] MS(ESI) m / z: 513 [M / 2+H] + .

[1213] Step 3: Preparation of compound SMP-10215-3

[1214]

[1215] SMP-10215-2 (20.0 mg, 0.02 mmol) was dissolved in N,N-dimethylformamide (1 mL). After complete dissolution, intermediate A (20.0 mg, 0.02 mmol) and N,N-diisopropylethylamine (7.0 mg, 0.06 mmol) were added sequentially. After completion, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (7.0 mg, 0.02 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-10215-3, weighing 19.0 mg, with a yield of 48.4%.

[1216] MS(ESI) m / z: 1027 [M / 2+H]+ .

[1217] Step 4: Preparation of compound SMP-10215

[1218]

[1219] SMP-10215-3 (19.0 mg, 0.0093 mmol) and intermediate B (8.0 mg, 0.0095 mmol) were miscible in N,N-dimethylformamide (1 mL). After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.0 mg, 0.011 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was subjected to preparative HPLC to obtain compound SMP-10215, weighing 12.0 mg, with a yield of 43.8%.

[1220] MS(ESI) m / z: 1439 [M / 2+H] + .

[1221] Example 49: Preparation of compound SMP-34710

[1222]

[1223] Step 1: Preparation of compound SMP-34710-1

[1224]

[1225] SMP-10215-2 (60.0 mg, 0.06 mmol), intermediate tert-butyl 9-amino-4,7-dioxanonate (27.0 mg, 0.12 mmol), was miscible in N,N-dimethylformamide (2 mL). After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (44.0 mg, 0.12 mmol) and N,N-diisopropylethylamine (30.0 mg, 0.24 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34710-1, weighing 45.0 mg, with a yield of 52.9%.

[1226] MS(ESI) m / z: 728 [M / 2+H] + .

[1227] Step 2: Preparation of compound SMP-34710-2

[1228]

[1229] SMP-34710-1 (45.0 mg, 0.03 mmol) was dissolved in trifluoroacetic acid (3 mL). The reaction was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure. The reaction solution was purified by preparative HPLC to obtain compound SMP-34710-2, weighing 20.0 mg, with a yield of 48.2%.

[1230] MS(ESI) m / z: 672 [M / 2+H] + .

[1231] Step 3: Preparation of compound SMP-34710-3

[1232]

[1233] SMP-34710-2 (20.0 mg, 0.015 mmol), intermediate A (16.0 mg, 0.015 mmol) was miscible in N,N-dimethylformamide (2 mL). After N was completely clear, N,N-diisopropylethylamine (4.0 mg, 0.031 mmol) was added. After that, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (5.7 mg, 0.015 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34710-3, weighing 12.0 mg, with a yield of 34.0%.

[1234] MS(ESI) m / z: 1187 [M / 2+H] + .

[1235] Step 4: Preparation of compound SMP-34710

[1236]

[1237] SMP-34170-3 (12.0 mg, 0.005 mmol) and intermediate B (4.2 mg, 0.005 mmol) were miscible in N,N-dimethylformamide (2 mL). After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.9 mg, 0.005 mmol) and N,N-diisopropylethylamine (2.0 mg, 0.015 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. Compound SMP-34710 was prepared by reverse phase reaction, weighing 9.0 mg, with a yield of 55.7%.

[1238] MS(ESI) m / z: 1598 [M / 2+H]+ .

[1239] Example 50: Preparation of compound SMP-44419

[1240]

[1241] Step 1: Preparation of compound SMP-44419-1

[1242]

[1243] SMP-10215-2 (60.0 mg, 0.06 mmol), intermediate tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate (38.0 mg, 0.12 mmol), was miscible in N,N-dimethylformamide (2 mL). After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (45.6 mg, 0.12 mmol) and N,N-diisopropylethylamine (30.9 mg, 0.24 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-4419-1, weighing 48.0 mg, with a yield of 50.3%.

[1244] MS(ESI) m / z: 816 [M / 2+H] + .

[1245] Step 2: Preparation of compound SMP-44419-2

[1246]

[1247] Intermediate SMP-44419-1 (48.0 mg, 0.029 mmol) was dissolved in trifluoroacetic acid (3 mL), and the reaction was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and compound SMP-44419-2 was obtained by HPLC purification, weighing 20.0 mg, with a yield of 44.7%.

[1248] MS(ESI) m / z: 760 [M / 2+H] + .

[1249] Step 3: Preparation of compound SMP-44419-3

[1250]

[1251] In a 10 mL reaction flask, intermediate SMP-44419-2 (20.0 mg, 0.013 mmol), intermediate A (14.0 mg, 0.013 mmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (5.0 mg, 0.039 mmol) were added. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (5.0 mg, 0.013 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-44419-3, weighing 10.0 mg, with a yield of 29.8%.

[1252] MS(ESI) m / z: 1274 [M / 2+H] + .

[1253] Step 4: Preparation of compound SMP-44419

[1254]

[1255] In a 10 mL reaction flask, intermediate SMP-44419-3 (9.0 mg, 0.004 mmol), intermediate B (3.4 mg, 0.004 mmol), and N,N-dimethylformamide (1 mL) were added. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.0 mg, 0.004 mmol) and N,N-diisopropylethylamine (1.6 mg, 0.012 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-44419, weighing 2.0 mg, with a yield of 16.8%.

[1256] MS(ESI) m / z: 1124 [M / 3+H] + .

[1257] Example 51: Preparation of compound SMP-35055

[1258]

[1259] Step 1: Preparation of compound SMP-35055-1

[1260]

[1261] In a 10 mL reaction flask, intermediate SMP-10215-2 (60.0 mg, 0.06 mmol), amino-octaethylene glycol-tert-butyl propionate (58.0 mg, 0.12 mmol), and N,N-dimethylformamide (3 mL) were added. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (45.6 mg, 0.12 mmol) and N,N-diisopropylethylamine (30.0 mg, 0.24 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-35055-1, weighing 50.0 mg, with a yield of 43.1%.

[1262] MS(ESI) m / z: 993 [M / 2+H] + .

[1263] Step 2: Preparation of compound SMP-35055-2

[1264]

[1265] SMP-35055-1 (50.0 mg, 0.03 mmol) was dissolved in trifluoroacetic acid (3 mL), and the reaction was stirred at room temperature for 2 hours. The reaction was complete by TLC. The trifluoroacetic acid was removed by concentration under reduced pressure. The reaction solution was purified by preparative HPLC to obtain compound SMP-35055-2, weighing 20.0 mg, with a yield of 42.4%.

[1266] MS(ESI) m / z: 937 [M / 2+H] + .

[1267] Step 3: Preparation of compound SMP-35055-3

[1268]

[1269] In a 10 mL reaction flask, intermediate SMP-35055-2 (20.0 mg, 0.011 mmol), intermediate A (11.5 mg, 0.011 mmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (4.0 mg, 0.032 mmol) were added and completely dissolved. Then, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.2 mg, 0.011 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-35055-3, weighing 8.0 mg, with a yield of 25.8%.

[1270] MS(ESI) m / z: 968 [M / 3+H] + .

[1271] Step 4: Preparation of compound SMP-35055

[1272]

[1273] In a 10 mL reaction flask, intermediate SMP-35055-3 (8.0 mg, 0.003 mmol), intermediate B (2.5 mg, 0.003 mmol), and N,N-dimethylformamide (1 mL) were added. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.1 mg, 0.003 mmol) and N,N-diisopropylethylamine (1.0 mg, 0.008 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-35055, weighing 1.2 mg, with a yield of 11.7%.

[1274] MS(ESI) m / z: 1242 [M / 2+H] + .

[1275] Example 52: Preparation of compound SMP-94170-A

[1276]

[1277] Step 1: Preparation of compound SMP-94170-1

[1278]

[1279] Intermediate SMP-10215-2 (250.0 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL). Then, intermediate 9-amino-4,7-dioxanonate tert-butyl ester (57.0 mg, 0.24 mmol) and N,N-diisopropylethylamine (94.0 mg, 0.73 mmol) were added sequentially. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (93.0 mg, 0.24 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed complete reaction. The reaction solution was purified by preparative HPLC to give compound SMP-94170-1, weighing 110 mg, with a yield of 36.4%.

[1280] MS(ESI) m / z: 621 [M / 2+H] + .

[1281] Step 2: Preparation of compound SMP-94170-2

[1282]

[1283] In a 10 mL reaction flask, intermediate SMP-94170-1 (110.0 mg, 0.09 mmol), amino-dodecyl polyethylene glycol-tert-butyl propionate (59.0 mg, 0.09 mmol), and N,N-dimethylformamide (3 mL) were added. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (34.2 mg, 0.09 mmol) and N,N-diisopropylethylamine (34.8 mg, 0.27 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-94170-2, weighing 110 mg, with a yield of 65.5%.

[1284] MS(ESI) m / z: 948 [M / 2+H] + .

[1285] Step 3: Preparation of compound SMP-94170-3

[1286]

[1287] SMP-94170-2 (110.0 mg, 0.06 mmol) was dissolved in trifluoroacetic acid (3 mL). The reaction was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the compound SMP-94170-3 was obtained by HPLC purification, weighing 70 mg, with a yield of 67.6%.

[1288] MS(ESI) m / z: 893 [M / 2+H] + .

[1289] Step 4: Preparation of compounds SMP-94170-A-1 and SMP-94170-B-1

[1290]

[1291] SMP-94170-3 (70.0 mg, 0.04 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of A (41.0 mg, 0.04 mmol) and N,N-diisopropylethylamine (15.0 mg, 0.12 mmol). After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (15.0 mg, 0.04 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes, and TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-94170-A-1, weighing 18.0 mg (yield 16.3%), and compound SMP-94170-B-1, weighing 30.0 mg (yield 27.2%).

[1292] MS(ESI) m / z: 1407 [M / 2+H] + .

[1293] Step 5: Preparation of compound SMP-94170-A

[1294]

[1295] In a 10 mL reaction flask, intermediate SMP-94170-A-1 (18.0 mg, 0.006 mmol), intermediate B (5.0 mg, 0.006 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.3 mg, 0.006 mmol) and N,N-diisopropylethylamine (2.5 mg, 0.019 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-94170-A, weighing 10.0 mg, with a yield of 43.0%.

[1296] MS(ESI) m / z: 1212 [M / 3+H] + .

[1297] Example 53: Preparation of compound SMP-94170-B

[1298]

[1299] Step 1: Preparation of compound SMP-94170-B

[1300]

[1301] SMP-94170-B-1 (30.0 mg, 0.011 mmol) was dissolved in N,N-dimethylformamide (2 mL). After complete dissolution, intermediate B (9.0 mg, 0.011 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.0 mg, 0.011 mmol), and N,N-diisopropylethylamine (4.3 mg, 0.033 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-94170-B, weighing 15 mg, with a yield of 38.7%.

[1302] MS(ESI) m / z: 1212 [M / 3+H] + .

[1303] Example 54: Preparation of compound SMP-46940

[1304]

[1305] Step 1: Preparation of compound SMP-46940-1

[1306]

[1307] SMP-34421-2 (300.0 mg, 0.37 mmol) was dissolved in N,N-dimethylformamide (5 mL). After complete dissolution, maleimide-octaethylene glycol-carboxylic acid (193.0 mg, 0.37 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (141.0 mg, 0.37 mmol), and N,N-diisopropylethylamine (143.0 mg, 1.11 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-46940-1, weighing 132.0 mg, with a yield of 27.2%.

[1308] MS(ESI) m / z: 657 [M / 2+H] + .

[1309] Step 2: Preparation of compound SMP-46940-2

[1310]

[1311] In a 10 mL reaction flask, intermediate SMP-46940-1 (132.0 mg, 0.10 mmol) and trifluoroacetic acid (3 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure. The reaction solution was purified by preparative HPLC to obtain compound SMP-46940-2, weighing 60.0 mg, with a yield of 49.7%.

[1312] MS(ESI) m / z: 601 [M / 2+H] + .

[1313] Step 3: Preparation of compound SMP-46940-3

[1314]

[1315] In a 10 mL reaction flask, intermediate SMP-46940-3 (60.0 mg, 0.05 mmol), tert-butyl 9-amino-4,7-dioxanonate (23.0 mg, 0.10 mmol), and N,N-dimethylformamide (2 mL) were added. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (38.0 mg, 0.10 mmol) and N,N-diisopropylethylamine (39.0 mg, 0.30 mmol) were added sequentially. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-46940-3, weighing 40.0 mg, with a yield of 49.1%.

[1316] MS(ESI) m / z: 816 [M / 2+H] + .

[1317] Step 4: Preparation of compound SMP-46940-4

[1318]

[1319] In a 10 mL reaction flask, intermediate SMP-46940-3 (40.0 mg, 0.025 mmol) and trifluoroacetic acid (3 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the compound SMP-46940-4 was obtained by HPLC purification, weighing 25.0 mg, with a yield of 67.1%.

[1320] MS(ESI) m / z: 760 [M / 2+H] + .

[1321] Step 5: Preparation of compound SMP-46940-5

[1322]

[1323] SMP-46940-4 (25.0 mg, 0.016 mmol) was dissolved in N,N-dimethylformamide (2 mL), followed by the sequential addition of intermediate A (17.0 mg, 0.016 mmol) and N,N-diisopropylethylamine (6.2 mg, 0.048 mmol). After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (6.0 mg, 0.016 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes, and TLC showed complete reaction. The reaction solution was purified by preparative HPLC to obtain compound SMP-46940-5, weighing 14.0 mg, with a yield of 33.4%.

[1324] MS(ESI) m / z: 1275 [M / 2+H] + .

[1325] Step 5: Preparation of compound SMP-46940

[1326]

[1327] In a 10 mL reaction flask, intermediate SMP-46940-5 (14.0 mg, 0.005 mmol), intermediate B (5.0 mg, 0.005 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.0 mg, 0.005 mmol) and N,N-diisopropylethylamine (2.0 mg, 0.015 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-46940, weighing 9.0 mg, with a yield of 48.6%.

[1328] MS(ESI) m / z: 1124 [M / 3+H] + .

[1329] Example 55: Preparation of compound SMP-13069

[1330]

[1331] Step 1: Preparation of compound SMP-13069-1

[1332]

[1333] In a 25 mL reaction flask, intermediate SMP-34421-2 (300.0 mg, 0.37 mmol), maleimide-hexaethylene glycol-carboxylic acid (160.0 mg, 0.37 mmol), and N,N-dimethylformamide (5 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (140.7 mg, 0.37 mmol) and N,N-diisopropylethylamine (143.2 mg, 1.11 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-13069-1, weighing 153.0 mg, with a yield of 33.7%.

[1334] MS(ESI) m / z: 613 [M / 2+H] + .

[1335] Step 2: Preparation of compound SMP-13069-2

[1336]

[1337] In a 10 mL reaction flask, intermediate SMP-13069-1 (153.0 mg, 0.12 mmol) and trifluoroacetic acid (3 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure. The reaction solution was purified by preparative HPLC to obtain compound SMP-13069-2, weighing 80.0 mg, with a yield of 57.6%.

[1338] MS(ESI) m / z: 557 [M / 2+H] + .

[1339] Step 3: Preparation of compound SMP-13069-3

[1340]

[1341] In a 10 mL reaction flask, intermediate SMP-13069-3 (80.0 mg, 0.072 mmol), tert-butyl 9-amino-4,7-dioxanonate (33.6 mg, 0.144 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (54.0 mg, 0.144 mmol) and N,N-diisopropylethylamine (55.0 mg, 0.43 mmol) were added sequentially. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-13069-3, weighing 60.0 mg, with a yield of 53.8%.

[1342] MS(ESI) m / z: 772 [M / 2+H] + .

[1343] Step 4: Preparation of compound SMP-13069-4

[1344]

[1345] In a 10 mL reaction flask, intermediate SMP-13069-3 (60.0 mg, 0.039 mmol) and trifluoroacetic acid (3 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the compound SMP-13069-4 was obtained by HPLC purification, weighing 35.0 mg, with a yield of 62.9%. MS (ESI) m / z: 716 [M / 2+H] + .

[1346] Step 5: Preparation of compound SMP-13069-5

[1347]

[1348] In a 10 mL reaction flask, intermediate SMP-13069-4 (35.0 mg, 0.024 mmol), intermediate A (25.0 mg, 0.024 mmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (9.3 mg, 0.072 mmol) were added sequentially. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (9.0 mg, 0.024 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-13069-5, weighing 18.0 mg, with a yield of 29.9%.

[1349] MS(ESI) m / z: 1231 [M / 2+H] + .

[1350] Step 6: Preparation of compound SMP-13069

[1351]

[1352] In a 10 mL reaction flask, intermediate SMP-13069-5 (18.0 mg, 0.007 mmol), intermediate B (6.0 mg, 0.007 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.0 mg, 0.007 mmol) and N,N-diisopropylethylamine (3.0 mg, 0.021 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-13069, weighing 9.5 mg, with a yield of 39.6%.

[1353] MS(ESI) m / z: 1095 [M / 3+H] + .

[1354] Example 56: Preparation of compound SMP-28945

[1355]

[1356] Step 1: Preparation of compound SMP-28945-1

[1357]

[1358] In a 50 mL reaction flask, add intermediate SMP-34421-1 (250.0 mg, 0.62 mmol) and N,N-dimethylformamide (10 mL) sequentially. After complete clarification, add PEG. 16 -OMs (525.0 mg, 0.62 mmol), potassium carbonate (258.0 mg, 1.87 mmol). The reaction was stirred at 80 °C for 20 hours. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-34421-2, weight 180.0 mg, yield 25.8%.

[1359] MS(ESI) m / z: 560 [M / 2+H] + .

[1360] Step 2: Preparation of compound SMP-28945-2

[1361]

[1362] In a 25 mL reaction flask, intermediate SMP-28945-1 (180.0 mg, 0.16 mmol) and N,N-dimethylformamide (5 mL) were added sequentially. After complete dissolution, 6-maleimide hexanoic acid (34.0 mg, 0.16 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (61.0 mg, 0.16 mmol), and N,N-diisopropylethylamine (62.0 mg, 0.48 mmol) were added sequentially. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-28945-2, weighing 120.0 mg, with a yield of 56.9%.

[1363] MS(ESI) m / z: 656 [M / 2+1] + .

[1364] Step 3: Preparation of compound SMP-28945-3

[1365]

[1366] In a 10 mL reaction flask, intermediate SMP-28945-2 (120.0 mg, 0.092 mmol) and trifluoroacetic acid (3 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the compound SMP-28945-3 was obtained by HPLC purification, weighing 80.0 mg, with a yield of 72.9%.

[1367] MS(ESI) m / z: 600 [M / 2+H] + .

[1368] Step 4: Preparation of compound SMP-28945-4

[1369]

[1370] In a 10 mL reaction flask, intermediate SMP-28945-3 (30.0 mg, 0.025 mmol), intermediate A (26.0 mg, 0.025 mmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (10.0 mg, 0.075 mmol) were added sequentially. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (10 mg, 0.025 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-28945-4, weighing 20.0 mg, with a yield of 35.9%.

[1371] MS(ESI) m / z: 1115 [M / 2+H] + .

[1372] Step 5: Preparation of compound SMP-28945

[1373]

[1374] In a 10 mL reaction flask, intermediate SMP-28945-4 (20.0 mg, 0.009 mmol), intermediate B (7.6 mg, 0.009 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.4 mg, 0.009 mmol) and N,N-diisopropylethylamine (3.5 mg, 0.027 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-28945, weighing 2.6 mg, with a yield of 9.5%.

[1375] MS(ESI) m / z: 1017 [M / 3+H] + .

[1376] Example 57: Preparation of compound SMP-28823

[1377]

[1378] Step 1: Preparation of compound SMP-28823-1

[1379]

[1380] In a 50 mL reaction flask, aminononethylene glycol monomethyl ether (300.0 mg, 0.70 mmol), triphosgene (206.0 mg, 0.70 mmol), and dichloromethane (10 mL) were added sequentially. After complete clarification, triethylamine (1 mL) was added. The reaction was stirred at 0 °C for 20 minutes. TLC showed that the reaction was complete. The dichloromethane was removed by concentration under reduced pressure to give the crude compound SMP-28823-1, weighing 300.0 mg. MS (ESI) m / z: 454 [M+H] + .

[1381] Step 2: Preparation of compound SMP-28823-2

[1382]

[1383] In a 50 mL reaction flask, crude intermediate SMP-28823-1 (300.0 mg, 0.66 mmol), intermediate SMP-34421-1 (300.0 mg, 0.75 mmol), and dichloromethane (10 mL) were added sequentially. After complete clarification, triethylamine (1 mL) was added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The dichloromethane was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain compound SMP-28823-1, weighing 300.0 mg, with a yield of 53.1%.

[1384] MS(ESI) m / z: 854 [M+H] + .

[1385] Step 3: Preparation of compound SMP-28823-3

[1386]

[1387] In a 25 mL reaction flask, intermediate SMP-28823-2 (230.0 mg, 0.27 mmol) and N,N-dimethylformamide (5 mL) were added and completely dissolved. Then, maleimide-diethylene glycol-carboxylic acid (69.4 mg, 0.27 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (102.7 mg, 0.27 mmol), and N,N-diisopropylethylamine (104.5 mg, 0.81 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-28823-3, weighing 200.0 mg, with a yield of 67.9%.

[1388] MS(ESI) m / z: 547 [M+H] + .

[1389] Step 4: Preparation of compound SMP-28823-4

[1390]

[1391] In a 10 mL reaction flask, intermediate SMP-28823-3 (200 mg, 0.18 mmol) and trifluoroacetic acid (3 mL) were added sequentially. The reaction was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and compound SMP-28823-4 was obtained by HPLC, weighing 40.0 mg, with a yield of 22.3%. MS (ESI) m / z: 491 [M / 2+H] + .

[1392] Step 5: Preparation of compound SMP-28823-5

[1393]

[1394] In a 10 mL reaction flask, intermediate SMP-28823-4 (40.0 mg, 0.041 mmol), intermediate A (42.9 mg, 0.041 mmol), N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (16.8 mg, 0.13 mmol) were added sequentially. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (15.7 mg, 0.041 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-28823-5, weighing 17.0 mg, with a yield of 20.7%. MS (ESI) m / z: 1005 [M / 2+H] + .

[1395] Step 6: Preparation of compound SMP-28823

[1396]

[1397] In a 10 mL reaction flask, intermediate SMP-28823-5 (17.0 mg, 0.0085 mmol), intermediate B (7.1 mg, 0.0085 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.3 mg, 0.0085 mmol) and N,N-diisopropylethylamine (3.3 mg, 0.026 mmol) were added. The reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-28823, weighing 12 mg, with a yield of 50.1%.

[1398] MS(ESI) m / z: 944 [M / 3+H] + .

[1399] Example 58: Preparation of compound SMP-53816

[1400]

[1401] Step 1: Preparation of compound SMP-53816-1

[1402]

[1403] In a 100 mL reaction flask, cyclohexane (1.0 g, 5.81 mmol), benzooxycarbonyl succinimide (2.9 g, 11.62 mmol), and chloroform (20 mL) were added sequentially. The reaction was stirred at room temperature for 24 hours. TLC showed that the reaction was complete. Excess chloroform was removed under reduced pressure. The reaction solution was purified by preparative HPLC to obtain compound SMP-53816-1, weighing 2.0 g, with a yield of 78.1%.

[1404] MS(ESI)m / z:441[M+H] + .

[1405] Step 2: Preparation of compound SMP-53816-2

[1406]

[1407] In a 50 mL reaction flask, intermediate SMP-53816-1 (1.0 g, 2.27 mmol), N-tert-butoxycarbonyl-7-aminoheptanoic acid (556.2 mg, 2.27 mmol), and N,N-dimethylformamide (20 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (863.2 mg, 2.27 mol) and N,N-diisopropylethylamine (879.0 mg, 6.81 mol) were added. The reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. Compound SMP-53816-2 was prepared by reverse phase reaction, weighing 300.0 mg, with a yield of 19.7%.

[1408] MS(ESI) m / z: 668 [M+H] + .

[1409] Step 3: Preparation of compound SMP-53816-3

[1410]

[1411] In a 25 mL reaction flask, intermediate SMP-53816-2 (300.0 mg, 0.45 mmol), maleimide-nonadenylated glycol-carboxylic acid (286.0 mg, 0.45 mmol), and N,N-dimethylformamide (5 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (171.1 mg, 0.45 mol) and N,N-diisopropylethylamine (174.2 mg, 1.35 mmol) were added. The reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53816-3, weighing 300.0 mg, with a yield of 60.4%.

[1412] MS(ESI) m / z: 553 [M / 2+H] + .

[1413] Step 4: Preparation of compound SMP-53816-4

[1414]

[1415] In a 25 mL reaction flask, intermediate SMP-53816-3 (300.0 mg, 0.27 mmol) and methanol (5 mL) were added sequentially. After complete clarification, palladium on carbon (60 mg) was added, and the air was replaced with hydrogen gas. The mixture was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated to give compound SMP-53816-4, weighing 220.0 mg, with a yield of 96.8%.

[1416] MS(ESI) m / z: 838 [M+H] + .

[1417] Step 5: Preparation of compound SMP-53816-5

[1418]

[1419] In a 25 mL reaction flask, intermediate SMP-53816-4 (220 mg, 0.26 mmol), carboxylic acid-tetraethylene glycol-tert-butyl ester (182.0 mg, 0.52 mmol), and N,N-dimethylformamide (3 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (197.8 mg, 0.52 mol) and N,N-diisopropylethylamine (100.6 mg, 0.78 mol) were added. The reaction was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53816-5, weighing 360 mg, with a yield of 91.2%.

[1420] MS(ESI) m / z: 751 [M / 2+H] + .

[1421] Step 6: Preparation of compound SMP-53816-6

[1422]

[1423] In a 10 mL reaction flask, intermediate SMP-53816-5 (360.0 mg, 0.24 mmol) and trifluoroacetic acid (5 mL) were added sequentially. The mixture was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the compound SMP-53816-6 was obtained by HPLC purification, weighing 300.0 mg, with a yield of 90.0%.

[1424] MS(ESI) m / z: 695 [M / 2+H] + .

[1425] Step 7: Preparation of compound SMP-53816-7

[1426]

[1427] In a 25 mL reaction flask, intermediate SMP-53816-4 (300.0 mg, 0.22 mmol), N-methoxycarbonylcis-butenediamide (68.2 mg, 0.44 mmol), THF (3 mL), and saturated sodium bicarbonate aqueous solution (3 mL) were added sequentially. The reaction was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53816-7, weighing 260.0 mg, with a yield of 87.9%. MS (ESI) m / z: 685 [M / 2+H] + .

[1428] Step 8: Preparation of compound SMP-53816-8

[1429]

[1430] In a 25 mL reaction flask, intermediate SMP-53816-7 (100.0 mg, 0.073 mmol), intermediate A (74.3 mg, 0.073 mmol), N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (28.2 mg, 0.22 mmol) were added sequentially. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (27.8 mg, 0.073 mmol) in N,N-dimethylformamide (3 mL) was added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53816-8, weighing 40.0 mg, with a yield of 22.8%.

[1431] MS(ESI) m / z: 1200 [M / 2+H] + .

[1432] Step 9: Preparation of compound SMP-53816

[1433]

[1434] In a 10 mL reaction flask, intermediate SMP-53816-8 (40.0 mg, 0.017 mmol), intermediate B (14.3 mg, 0.017 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (6.5 mg, 0.017 mmol) and N,N-diisopropylethylamine (6.5 mg, 0.051 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-53816, weighing 14.0 mg, with a yield of 26.0%.

[1435] MS(ESI) m / z: 1074 [M / 3+H] + .

[1436] Example 59: Preparation of compound SMP-23135

[1437]

[1438] Step 1: Preparation of compound SMP-23135-2

[1439]

[1440] In a 100 mL reaction flask, intermediate SMP-23135-1 (2.0 g, 3.64 mmol), 6-maleimide hexanoic acid (768.0 mg, 3.64 mmol), and N,N-dimethylformamide (30 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (1.4 g, 3.64 mmol) and N,N-diisopropylethylamine (1.4 g, 10.92 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-23135-2, weighing 1.1 g, with a yield of 40.7%.

[1441] MS(ESI) m / z: 743 [M+H] + .

[1442] Step 2: Preparation of compound SMP-23135-3

[1443]

[1444] In a 100 mL reaction flask, intermediate SMP-23135-2 (1.1 g, 1.48 mmol), phenol (139.0 mg, 1.48 mmol), and 33% hydrobromic acid-acetic acid solution (30 mL) were added sequentially. The mixture was stirred overnight at 50 °C. LC-MS showed that the reaction was complete. After cooling the reaction solution, it was added dropwise to a single-necked flask containing methyl tert-butyl ether (300 mL). A solid precipitated out, which was filtered and dried under vacuum to give compound SMP-23135-3, weighing 350.0 mg, with a yield of 54.4%.

[1445] MS(ESI) m / z: 435 [M+H] + .

[1446] Step 3: Preparation of compound SMP-23135-4

[1447]

[1448] In a 50 mL reaction flask, intermediate SMP-23135-3 (350.0 mg, 0.81 mmol), tert-butyl bromoacetate (473.9 mg, 2.43 mmol), N,N-dimethylformamide (10 mL), and potassium carbonate (335.3 mg, 2.43 mmol) were added sequentially. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the reaction was complete. The mixture was filtered, and the filtrate was purified by HPLC to give compound SMP-23135-4, weighing 85.0 mg, with a yield of 15.9%. MS (ESI) m / z: 663 [M+H] + .

[1449] Step 4: Preparation of compound SMP-23135-5

[1450]

[1451] In a 25 mL reaction flask, intermediate SMP-23135-4 (85.0 mg, 0.13 mmol) and trifluoroacetic acid (5 mL) were added sequentially. The mixture was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure, and the compound SMP-23135-5 was obtained by HPLC purification, weighing 50.0 mg, with a yield of 70.8%. MS (ESI) m / z: 551 [M+H] + .

[1452] Step 5: Preparation of compound SMP-23135-6

[1453]

[1454] In a 10 mL reaction flask, intermediate SMP-23135-5 (50.0 mg, 0.091 mmol), intermediate A (95.4 mg, 0.091 mmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (35.2 mg, 0.27 mmol) were added sequentially. After complete clarification, a solution of N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (34.6 mg, 0.091 mmol) in N,N-dimethylformamide (1 mL) was slowly added dropwise. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-23135-6, weighing 23.0 mg, with a yield of 16.0%.

[1455] MS(ESI) m / z: 790 [M / 2+H] + .

[1456] Step 6: Preparation of compound SMP-23135

[1457]

[1458] In a 10 mL reaction flask, intermediate SMP-23135-6 (23.0 mg, 0.014 mmol), intermediate B (12.0 mg, 0.014 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (5.3 mg, 0.014 mmol) and N,N-diisopropylethylamine (5.4 mg, 0.042 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-23135, weighing 12.0 mg, with a yield of 34.3%.

[1459] MS(ESI)m / z:1201[M / 2+H] + .

[1460] Example 60: Preparation of compound SMP-21139

[1461]

[1462] Step 1: Preparation of compounds SMP-21139-1 and SMP-68691-1

[1463]

[1464] In a 25 mL reaction flask, intermediate K (56.0 mg, 0.05 mmol), intermediate F-5 (85.0 mg, 0.10 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (19.0 mg, 0.05 mmol), N,N-diisopropylethylamine (13.0 mg, 0.10 mmol), and N,N-dimethylformamide (4 mL) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-21139-1 (20.0 mg) and compound SMP-68691-1 (18.0 mg), with an overall yield of 36%.

[1465] MS(ESI) m / z: 1059 [M / 2+H] + .

[1466] Step 2: Preparation of compound SMP-21139-2

[1467]

[1468] In a 10 mL reaction flask, intermediate SMP-21139-1 (20.0 mg, 0.010 mmol), intermediate B (10.0 mg, 0.010 mmol), N,N-dimethylformamide (2 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.0 mg, 0.010 mmol), and N,N-diisopropylethylamine (2.0 mg, 0.014 mmol) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-21139-2, weighing 15.0 mg, with a yield of 61%.

[1469] MS(ESI) m / z: 1363 [M / 2+H] + .

[1470] Step 3: Preparation of compound SMP-21139-3

[1471]

[1472] In a 10 mL reaction flask, intermediate SMP-21139-2 (15.0 mg, 0.0055 mmol), 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (2.0 mg, 0.013 mmol), and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-21139-3, weighing 8.0 mg, with a yield of 58%.

[1473] MS(ESI) m / z: 1252 [M / 2+H] + .

[1474] Step 4: Preparation of compound SMP-21139

[1475]

[1476] Compound SMP-51013-3 (6.0 mg, 0.0047 mmol) and N,N-dimethylformamide (2 mL) were added to a 10 mL reaction flask. After the reaction solution became clear, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.0 mg, 0.0052 mmol) and N,N-diisopropylethylamine (1.0 mg, 0.0077 mmol) were added sequentially. The mixture was reacted at room temperature for 5 minutes, and then compound SMP-21139-3 (8.0 mg, 0.0032 mmol) was added. The mixture was stirred for 25 minutes, and TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-21139, weighing 4.5 mg, with a yield of 34%.

[1477] MS(ESI) m / z: 1324 [M / 3+H] + .

[1478] Example 61: Preparation of compound SMP-68691

[1479]

[1480] Step 1: Preparation of compound SMP-68691-2

[1481]

[1482] In a 10 mL reaction flask, intermediate SMP-68691-1 (18.0 mg, 0.008 mmol), intermediate B (8.0 mg, 0.010 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (4.0 mg, 0.010 mmol), N,N-diisopropylethylamine (2.0 mg, 0.013 mmol), and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-68691-2, weighing 12.0 mg (55% yield).

[1483] MS(ESI) m / z: 1363 [M / 2+H] + .

[1484] Step 2: Preparation of compound SMP-68691-3

[1485]

[1486] In a 10 mL reaction flask, intermediate SMP-68691-2 (12.0 mg, 0.0044 mmol), 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (1.0 mg, 0.007 mmol), and N,N-dimethylformamide (2 mL) were added. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-68691-3, weighing 8.0 mg, with a yield of 73%.

[1487] MS(ESI) m / z: 1252 [M / 2+H] + .

[1488] Step 3: Preparation of compound SMP-68691

[1489]

[1490] Compound SMP-51013-3 (6.0 mg, 0.0047 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.0 mg, 0.0052 mmol) and N,N-diisopropylethylamine (1.0 mg, 0.0077 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound SMP-68691-3 (8.0 mg, 0.0029 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure, and the residue was purified by HPLC to obtain the target product SMP-68691, weighing 4.0 mg, with a yield of 33%.

[1491] MS(ESI) m / z: 1324 [M / 3+H] + .

[1492] Example 62: Preparation of compound SMP-60226

[1493]

[1494] Step 1: Preparation of compound SMP-60226-1

[1495]

[1496] Intermediate SMP-93566-2 (100.0 mg, 0.35 mmol), intermediate I (212.0 mg, 0.35 mmol), and N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (133.0 mg, 0.35 mmol) and N,N-diisopropylethylamine (135.4 mg, 1.05 mmol) were added. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. Compound SMP-60226-1 was prepared and purified by HPLC, weighing 200.0 mg, with a yield of 65%.

[1497] MS(ESI) m / z: 878 [M+H] + .

[1498] Step 2: Preparation of compound SMP-60226-2

[1499]

[1500] Intermediate SMP-60226-1 (201.9 mg, 0.23 mmol) and trifluoroacetic acid (3 mL) were added to a 10 mL reaction flask. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The trifluoroacetic acid was removed by concentration under reduced pressure. The residue was dissolved in acetonitrile and purified by HPLC to obtain compound SMP-60226-2, weighing 80.0 mg, with a yield of 43%.

[1501] MS(ESI) m / z: 823 [M+H] + .

[1502] Step 3: Preparation of compound SMP-60226

[1503]

[1504] Intermediate SMP-60226-4 (32.9 mg, 0.04 mmol), intermediate B (33.6 mg, 0.04 mmol), and N,N-dimethylformamide (2 mL) were added to a 10 mL reaction flask. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (15.2 mg, 0.04 mmol) and N,N-diisopropylethylamine (10.3 mg, 0.08 mmol) were added. The mixture was stirred at room temperature for 20 minutes. LC-MS analysis showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-60226, weighing 35.0 mg, with a yield of 53%.

[1505] MS(ESI) m / z: 823 [M / 2+H] + .

[1506] Example 63: Preparation of compound SMP-37860

[1507]

[1508] Step 1: Preparation of compound SMP-37860-2

[1509]

[1510] In a 250 mL reaction flask, polyethylene glycol monomethyl ether (7.0 g, 16.35 mmol) was dissolved in dichloromethane (80 mL), and triethylamine (3.3 g, 32.67 mmol) was added. Methylsulfonyl chloride (2.2 g, 19.60 mmol) was then slowly added to the reaction solution under ice bath conditions. The mixture was stirred at room temperature for 20 hours, and TLC showed complete reaction. The solution was washed with water (50 mL), and the organic phase was then washed with saturated ammonium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography (dichloromethane:methanol = 20:1) to give compound SMP-37860-2, weighing 8.1 g, with a yield of 98%.

[1511] MS(ESI) m / z: 507 [M+H] + .

[1512] 1 HNMR (400MHz, CDCl3) δ3.52-3.70(m,32H),3.40(s,3H),3.16(s,3H).

[1513] Step 2: Preparation of compound SMP-37860-3

[1514]

[1515] In a 100 mL reaction flask, 1,7-di-(N-tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (2.1 g, 5.33 mmol) and compound SMP-37860-2 (8.1 g, 16.01 mmol) were dissolved in acetonitrile (30 mL), and potassium carbonate (2.4 g, 17.07 mmol) was added. The mixture was heated to 55 °C and reacted for 20 hours. The reaction was confirmed to be complete by LC-MS. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was subjected to column chromatography (dichloromethane:methanol = 5:1) to give compound SMP-37860-3, weighing 3.2 g, with a yield of 64%. MS (ESI) m / z: 612 [M / 2+H] + .

[1516] 1 HNMR (400MHz, CDCl3) δ3.52-3.55(m,60H),3.40(s,6H),3.32(s,4H),2.51(t,4H),2.46(s,16H),1.40(s,18H).

[1517] Step 3: Preparation of compound SMP-37860-4

[1518]

[1519] Compound SMP-37860-3 (90.0 mg, 0.079 mmol) was added to a 10 mL reaction flask, followed by 2 mL of trifluoroacetic acid. The mixture was stirred at room temperature for 3 hours. LC-MS confirmed the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was dissolved in a small amount of acetonitrile before HPLC purification to obtain compound SMP-37860-4, weighing 20 mg, with a yield of 25%.

[1520] MS(ESI) m / z: 555 [M / 2+H] + .

[1521] Step 4: Preparation of compound SMP-37860-5

[1522]

[1523] In a 50 mL reaction flask, 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (95.0 mg, 0.54 mmol) and compound SMP-37860-4 (500.0 mg, 0.45 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (203.8 mg, 1.58 mmol) was added. N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (171.0 mg, 0.45 mmol) was dissolved in N,N-dimethylformamide (10 mL) and added dropwise to the above solution. The mixture was stirred at room temperature for 3 hours. After the reaction was completed as monitored by LC-MS, the reaction solution was purified by preparative HPLC to obtain compound SMP-37860-5, weighing 275.0 mg, with a yield of 50%.

[1524] MS(ESI) m / z: 616 [M / 2+H] + .

[1525] Step 5: Preparation of compound SMP-37860-6

[1526]

[1527] In a 25 mL reaction flask, intermediate A-5 (200.0 mg, 0.36 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (163.4 mg, 0.43 mmol), N,N-diisopropylethylamine (69.7 mg, 0.54 mmol), and N,N-dimethylformamide (5 mL) were added. After stirring at room temperature for 5 minutes, methylaurestatin E (258.1 mg, 0.36 mmol) was added, and the reaction was continued at room temperature for 30 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-37860-6, weighing 300.0 mg, with a yield of 65%. MS (ESI) m / z: 630 [M / 2+H] + .

[1528] Step 6: Preparation of compound SMP-37860-7

[1529]

[1530] In a 50 mL reaction flask, compound SMP-37860-6 (302.4 mg, 0.24 mmol), 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (41.3 mg, 0.27 mmol), and N,N-dimethylformamide (10 mL) were added. The mixture was stirred at room temperature for 30 minutes, and TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by HPLC to give compound SMP-37860-7, weighing 200.0 mg, with a yield of 81%.

[1531] MS(ESI) m / z: 1037 [M+H] + .

[1532] Step 7: Preparation of compound SMP-37860

[1533]

[1534] In a 25 mL reaction flask, intermediates SMP-37860-5 (20.7 mg, 0.02 mmol), SMP-37860-7 (20.4 mg, 0.02 mmol), N,N-dimethylformamide (3 mL), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (15.2 mg, 0.04 mmol), and N,N-diisopropylethylamine (6.5 mg, 0.05 mmol) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-37860, weighing 20.0 mg, with a yield of 47%.

[1535] MS(ESI) m / z: 1126 [M / 2+H] + .

[1536] Example 64: Preparation of compound SMP-18777

[1537]

[1538] Step 1: Preparation of compound SMP-18777-1

[1539]

[1540] Intermediate A-5 (2.5 g, 4.38 mmol) and 4-aminobenzyl alcohol (1.1 g, 8.77 mmol) were dissolved in a 100 mL reaction flask with a mixture of dichloromethane (30 mL) and methanol (10 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (2.2 g, 8.77 mmol) was added, and the mixture was stirred at room temperature for 20 hours. TLC showed that the reaction was complete. The solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 15:1) to give compound SMP-18777-1, weighing 900.0 mg, with a yield of 31%.

[1541] MS(ESI) m / z: 664 [M+H] + .

[1542] Step 2: Preparation of compound SMP-18777-2

[1543]

[1544] In a 25 mL reaction flask, intermediate SMP-18777-1 (1.0 g, 1.51 mmol) was dissolved in N,N-dimethylformamide (10 mL). Di-p-nitrophenyl carbonate (163.0 mg, 0.43 mmol) and triethylamine (1979 mg, 1.96 mmol) were added, and the mixture was stirred at room temperature for 5 hours. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 12:1) to give compound SMP-18777-2, weighing 300.0 mg, with a yield of 24%.

[1545] MS(ESI) m / z: 829 [M+H] + .

[1546] Step 3: Preparation of compound SMP-18777-3

[1547]

[1548] In a 25 mL reaction flask, doxorubicin hydrochloride (52.2 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (10 mL). Compound SMP-18777-2 (82.9 mg, 0.10 mmol) and triethylamine (17.2 mg, 0.17 mmol) were added, and the mixture was stirred at room temperature for 18 hours. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to give compound SMP-18777-3, weighing 80.0 mg, with a yield of 77%.

[1549] MS(ESI) m / z: 614, 425 fragment peaks.

[1550] Step 4: Preparation of compound SMP-18777

[1551]

[1552] In a 25 mL reaction flask, compounds SMP-18777-3 (20.0 mg, 0.02 mmol) and SMP-37860-5 (9.0 mg, 0.024 mmol) were dissolved in N,N-dimethylformamide (2 mL). Then, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (10.4 mg, 0.02 mmol) and triethylamine (4.0 mg, 0.04 mmol) were added. The mixture was stirred at room temperature for 5 hours, and LC-MS showed complete reaction. The reaction solution was purified by preparative HPLC to obtain compound SMP-18777, weighing 1.7 mg, with a yield of 6%.

[1553] MS(ESI) m / z: 819, 914 fragment peaks.

[1554] Example 66: Preparation of compound SMP-58054

[1555]

[1556] Step 1: Preparation of compound SMP-58054-1

[1557]

[1558] In a 25 mL reaction flask, methylaurestatin E (200.7 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (5 mL). Compound SMP-18777-2 (200.0 mg, 0.24 mmol), triethylamine (48.5 mg, 0.48 mmol), and 1-hydroxybenzotriazole (37.8 mg, 0.28 mmol) were added. The mixture was stirred at room temperature for 18 hours, and TLC showed complete reaction. The reaction was purified by HPLC to give compound SMP-58054-1, weighing 80.0 mg, in a yield of 24%.

[1559] MS(ESI) m / z: 425 fragment peak, 1186 [M+H] + .

[1560] Step 2: Preparation of compound SMP-58054

[1561]

[1562] In a 25 mL reaction flask, intermediates SMP-58054-1 (15.0 mg, 0.013 mmol), SMP-37860-6 (16.0 mg, 0.013 mmol), N,N-dimethylformamide (1 mL), 1-hydroxybenzotriazole (1.3 mg, 0.014 mmol), and triethylamine (2.0 mg, 0.019 mmol) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-58054, weighing 20.0 mg, with a yield of 67%.

[1563] MS(ESI) m / z: 1200 [M / 2+H] + .

[1564] Example 67: Preparation of compound SMP-25274

[1565]

[1566] Step 1: Preparation of compound SMP-25274-1

[1567]

[1568] In a 25 mL reaction flask, compound A-5 (50.0 mg, 0.089 mmol) and doxorubicin hydrochloride (46.4 mg, 0.075 mmol) were dissolved in N,N-dimethylformamide (4 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (30.4 mg, 0.08 mmol) and triethylamine (8.0 mg, 0.08 mmol) were added. The mixture was stirred at room temperature for 5 hours, and TLC showed complete reaction. The reaction solution was purified by preparative HPLC to give compound SMP-25274-1, weighing 70.0 mg, with a yield of 74%.

[1569] MS(ESI) m / z: 1084 [M+H] + .

[1570] Step 2: Preparation of compound SMP-25274-2

[1571]

[1572] In a 10 mL reaction flask, compound SMP-25274-1 (80.0 mg, 0.07 mmol), 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (10.7 mg, 0.07 mmol), and N,N-dimethylformamide (3 mL) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by HPLC to obtain compound SMP-25274-2, weighing 50.0 mg, with a yield of 79%.

[1573] MS(ESI) m / z: 862 [M+H] + .

[1574] Step 3: Preparation of compound SMP-25274

[1575]

[1576] In a 25 mL reaction flask, compounds SMP-25274-2 (20.0 mg, 0.02 mmol) and SMP-37860-5 (30.0 mg, 0.024 mmol) were dissolved in N,N-dimethylformamide (1.5 mL). Then, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (10.4 mg, 0.021 mmol) and triethylamine (4.0 mg, 0.04 mmol) were added. The mixture was stirred at room temperature for 5 hours, and TLC showed complete reaction. The reaction solution was purified by preparative HPLC to give compound SMP-25274, weighing 14.0 mg, with a yield of 34%.

[1577] MS(ESI) m / z: 1038 [M / 2+H] + .

[1578] Example 68: Preparation of compound SMP-56875

[1579]

[1580] Step 1: Preparation of compound SMP-56875-1

[1581]

[1582] In a 25 mL reaction flask, 1-(29-amino-3,6,9,12,15,18,21,24,27-oxanonadecanyl)-1H-pyrrole-2,5-dione (90.0 mg, 0.17 mmol) and compound SMP-37860-4 (214.0 mg, 0.19 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (54.2 mg, 0.42 mmol) was added. N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (64.6 mg, 0.17 mmol) was dissolved in N,N-dimethylformamide (4 mL) and added dropwise to the above solution. The reaction was stirred at room temperature for 3 hours, and LC-MS showed that the reaction was complete. The reaction solution was purified by HPLC to obtain compound SMP-56875-1, weighing 70.0 mg, with a yield of 26%.

[1583] MS(ESI) m / z: 638 [M / 2+H] + .

[1584] Step 2: Preparation of compound SMP-56875

[1585]

[1586] In a 4 mL reaction flask, compound SMP-56875-1 (11.0 mg, 0.005 mmol) and intermediate D (16.0 mg, 0.01 mmol) were dissolved in N,N-dimethylformamide (0.5 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (0.8 mg, 0.01 mmol) were added. The mixture was stirred at room temperature for 2 hours, and TLC showed complete reaction. Compound SMP-56875 was prepared and purified by HPLC, yielding 7.0 mg (35%).

[1587] MS(ESI) m / z: 1267 [M / 3+H] + .

[1588] Example 69: Preparation of compound SMP-82444

[1589]

[1590] Step 1: Preparation of compound SMP-82444-1

[1591]

[1592] The synthesis method of compound SMP-82444-1 is based on the synthesis of intermediate C.

[1593] MS(ESI) m / z: 1184 [M / 2+H] + .

[1594] Step 2: Preparation of compound SMP-82444

[1595]

[1596] In a 4 mL reaction flask, compounds SMP-82444-1 (9.0 mg, 0.004 mmol) and SMP-37860-5 (8.0 mg, 0.007 mmol) were dissolved in N,N-dimethylformamide (0.5 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (1.0 mg, 0.011 mmol) were added. The mixture was stirred at room temperature for 2 hours, and TLC showed complete reaction. The reaction solution was purified by preparative HPLC to give compound SMP-82444, weighing 3.2 mg, with a yield of 24%. MS (ESI) m / z: 1194 [M / 3+H] + .

[1597] Example 70: Preparation of compound SMP-50309

[1598]

[1599] Step 1: Preparation of compound SMP-50309-1

[1600]

[1601] The synthesis method of compound SMP-50309-1 is based on the synthesis of intermediate C.

[1602] MS(ESI) m / z: 907 [M / 3+H] + .

[1603] Step 2: Preparation of compound SMP-50309

[1604]

[1605] In a 4 mL reaction flask, compounds SMP-82444-1 (9.0 mg, 0.003 mmol) and SMP-37860-5 (8.0 mg, 0.007 mmol) were dissolved in N,N-dimethylformamide (0.5 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (1.0 mg, 0.011 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was purified by preparative HPLC to give compound SMP-37860, weighing 3.0 mg, with a yield of 29%.

[1606] MS(ESI) m / z: 1281 [M / 3+H] + .

[1607] Example 71: Preparation of compound SMP-20094

[1608]

[1609] Step 1: Preparation of compound SMP-20094-1

[1610]

[1611] In a 4 mL reaction flask, intermediate D (10.0 mg, 0.005 mmol) and compound SMP-37860-4 (14.0 mg, 0.013 mmol) were dissolved in N,N-dimethylformamide (1.5 mL), and triethylamine (1.0 mg, 0.01 mmol) was added. N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) was dissolved in N,N-dimethylformamide (1.5 mL) and slowly added dropwise to the above solution. The mixture was stirred at room temperature for 1.5 hours. LC-MS showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-20094-1, weighing 15.0 mg, with a yield of 91%.

[1612] MS(ESI) m / z: 1094 [M / 3+H] + .

[1613] Step 2: Preparation of compound SMP-20094

[1614]

[1615] In a 4 mL reaction flask, 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione (2.0 mg, 0.009 mmol) and compound SMP-20094-1 (15.0 mg, 0.005 mmol) were dissolved in N,N-dimethylformamide (3 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and pyridine (0.9 mg, 0.01 mmol) were added, and the mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-20094, weighing 1.5 mg, with a yield of 10%.

[1616] MS(ESI) m / z: 1164 [M / 3+H] + .

[1617] Example 72: Preparation of compound SMP-99559

[1618]

[1619] Step 1: Preparation of compound SMP-99559-1

[1620]

[1621] The synthesis method of compound SMP-99559-1 is the same as that of SMP-37860-5.

[1622] MS(ESI) m / z: 924 [M / 2+H] + .

[1623] Step 2: Preparation of compound SMP-99559

[1624]

[1625] In a 4 mL reaction flask, compound SMP-99559-1 (9.0 mg, 0.005 mmol) and intermediate D (10.0 mg, 0.005 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (1.0 mg, 0.012 mmol) were added, and the mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-99559, weighing 6.0 mg, with a yield of 30%.

[1626] MS(ESI) m / z: 1005 [M / 4+H] + .

[1627] Example 73: Preparation of compound SMP-59623

[1628]

[1629] Step 1: Preparation of compound SMP-59623-1

[1630]

[1631] The synthesis method of compound SMP-59623-1 is the same as that of SMP-37860-5.

[1632] MS(ESI) m / z: 572 [M / 2+H] + .

[1633] Step 2: Preparation of compound SMP-59623

[1634]

[1635] In a 4 mL reaction flask, compound SMP-59623-1 (7.0 mg, 0.006 mmol) and intermediate D (10.0 mg, 0.005 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (2.0 mg, 0.02 mmol) were added, and the mixture was stirred at room temperature for 2 hours. TLC showed complete reaction. Preparative HPLC of the reaction solution yielded compound SMP-59623, weighing 7.2 mg, with a yield of 36%.

[1636] MS(ESI) m / z: 1105 [M / 3+H] + .

[1637] Example 74: Preparation of compound SMP-47610

[1638]

[1639] Step 1: Preparation of compound SMP-47610

[1640]

[1641] In a 4 mL reaction flask, compound SMP-59623-1 (6.0 mg, 0.005 mmol) and intermediate C (10.0 mg, 0.004 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (2 mg, 0.02 mmol) were added, and the mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was purified by preparative HPLC to give compound SMP-47610, weighing 6.5 mg, with a yield of 38%.

[1642] MS(ESI) m / z: 1138 [M / 3+H] + .

[1643] Example 75: Preparation of compound SMP-86467

[1644]

[1645] Step 1: Preparation of compound SMP-86467-1

[1646]

[1647] In a 500 mL reaction flask, 6-((tert-butoxycarbonyl)amino)hexanoic acid (584.0 mg, 2.38 mmol) and compound SMP-53816-1 (1500 mg, 2.15 mmol) were dissolved in N,N-dimethylformamide (20 mL), and triethylamine (325.2 mg, 3.22 mmol) was added. N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (817.0 mg, 2.15 mmol) was dissolved in N,N-dimethylformamide (20 mL) and added dropwise to the above solution. The mixture was stirred at room temperature for 2 hours. TLC showed that the reaction was complete. After concentration under reduced pressure, the reaction solution was purified by preparative HPLC to give compound SMP-86467-1, weighing 788.0 mg, with a yield of 55%.

[1648] MS(ESI) m / z: 654 [M+H] + .

[1649] Step 2: Preparation of compound SMP-86467-2

[1650]

[1651] In a 100 mL reaction flask, tert-butyl bromoacetate (312.0 mg, 1.60 mmol) and compound SMP-86467-1 (788.0 mg, 1.20 mmol) were dissolved in acetonitrile (20 mL), and potassium carbonate (828.0 mg, 6.00 mmol) was added. The mixture was heated to 55 °C and reacted for 20 hours. TLC showed that the reaction was complete. After cooling the reaction solution to room temperature, it was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound SMP-86467-2, weighing 900.0 mg, with a yield of 97%.

[1652] MS(ESI) m / z: 768 [M+H] + .

[1653] Step 3: Preparation of compound SMP-86467-3

[1654]

[1655] In a 100 mL reaction flask, compound SMP-86467-2 (900.0 mg, 1.17 mmol) was dissolved in methanol (20 mL), and palladium-on-carbon catalyst (90.0 mg) was added. The air in the flask was purged with a hydrogen balloon, and the reaction was stirred at room temperature for 20 hours. LC-MS showed that the reaction was complete. The mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain compound SMP-86467-3, weighing 467.0 mg, with a yield of 80%.

[1656] MS(ESI) m / z: 500 [M+H] + 400 [M+H-100] + .

[1657] Step 4: Preparation of compound SMP-86467-4

[1658]

[1659] In a 50 mL reaction flask, compounds SMP-86467-3 (467.0 mg, 0.93 mmol) and SMP-37860-2 (1.01 g, 2.00 mmol) were dissolved in acetonitrile (12 mL), and potassium carbonate (552.0 mg, 4.00 mmol) was added. The reaction was carried out at room temperature for 20 hours. LC-MS was used to confirm the completeness of the reaction. The mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. Silica gel column chromatography (dichloromethane:methanol = 12:1) was performed to give compound SMP-86467-4, weighing 737.0 mg, with a yield of 60%.

[1660] MS(ESI) m / z: 661 [M / 2+H] + .

[1661] Step 5: Preparation of compound SMP-86467-5

[1662]

[1663] Compound SMP-86467-4 (737.0 mg, 0.56 mmol) was added to a 10 mL reaction flask, followed by 4 mL of trifluoroacetic acid. The mixture was stirred at room temperature for 3 hours. The reaction was confirmed by LC-MS. The trifluoroacetic acid was removed by concentration under reduced pressure to obtain compound SMP-86467-5, weighing 609.0 mg, with a yield of 90%. MS (ESI) m / z: 1165 [M+H] + 583 [M / 2+H] + .

[1664] Step 6: Preparation of compound SMP-86467-6

[1665]

[1666] In a 25 mL reaction flask, compound SMP-86467-5 (609.0 mg, 0.52 mmol) was dissolved in 6 mL of pure water, and sodium bicarbonate (189.0 mg, 2.25 mmol) was added. N-methoxycarbonylcis-butenediamide (124 mg, 0.80 mmol) was dissolved in 3 mL of tetrahydrofuran and added dropwise to the above solution. The reaction was allowed to proceed for 2 hours. LC-MS was used to detect the completion of the reaction. The tetrahydrofuran was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-86467-6, weighing 620.0 mg, with a yield of 95%.

[1667] MS(ESI) m / z: 623 [M / 2+H] + .

[1668] Step 7: Preparation of compound SMP-86467

[1669]

[1670] In a 4 mL reaction flask, compound SMP-86467-6 (15.0 mg, 0.012 mmol) and intermediate D (20.0 mg, 0.009 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.012 mmol) and triethylamine (2.0 mg, 0.015 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by LC-MS. The reaction solution was purified by preparative HPLC to give compound SMP-59623, weighing 11.0 mg, with a yield of 36%. MS (ESI) m / z: 1139 [M / 3+H]+ .

[1671] Example 76: Preparation of compound SMP-76899

[1672]

[1673] Step 1: Preparation of compound SMP-76899-1

[1674]

[1675] In a 50 mL reaction flask, 3-[(23-methoxy-3,6,9,12,15,18,21-heptaoxatrichodo-1-yl)oxy]propionic acid (500.0 mg, 1.09 mmol) and 1,7-di-(N-tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane (181.0 mg, 0.45 mmol) were dissolved in N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (437.0 mg, 1.15 mmol) and triethylamine (141.4 mg, 1.40 mmol) were added, and the mixture was stirred at room temperature for 7 hours. The reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC to obtain compound SMP-76899, weighing 448.0 mg, with a yield of 78%.

[1676] MS(ESI) m / z: 639 [M / 2+H] + .

[1677] Step 2: Preparation of compound SMP-76899-2

[1678]

[1679] Compound SMP-76899-1 (448.0 mg, 0.34 mmol) was added to a 10 mL reaction flask, followed by 4 mL of trifluoroacetic acid. The mixture was stirred at room temperature for 3 hours. LC-MS confirmed the reaction was complete. Trifluoroacetic acid was removed by vacuum distillation. The remaining reaction solution was purified by preparative HPLC to obtain compound SMP-76899-2, weighing 350.0 mg, with a yield of 86%.

[1680] MS(ESI) m / z: 583 [M / 2+H] + .

[1681] Step 3: Preparation of compound SMP-76899-3

[1682]

[1683] In a 25 mL reaction flask, 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (39.0 mg, 0.22 mmol) and compound SMP-76899-2 (350.0 mg, 0.29 mmol) were dissolved in N,N-dimethylformamide (5 mL), and triethylamine (50.5 mg, 0.50 mmol) was added. N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (91.2 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (3 mL) and added dropwise to the above solution. The mixture was stirred at room temperature for 3 hours. LC-MS confirmed the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-76899-3, weighing 250.0 mg, with a yield of 65%.

[1684] MS(ESI) m / z: 644 [M / 2+H] + .

[1685] Step 4: Preparation of compound SMP-76899

[1686]

[1687] In a 4 mL reaction flask, compound SMP-76899-3 (9.0 mg, 0.007 mmol) and intermediate C (15.0 mg, 0.006 mmol) were dissolved in N,N-dimethylformamide (1.0 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and triethylamine (2 mg, 0.02 mmol) were added, and the mixture was stirred at room temperature for 2 hours. LC-MS analysis confirmed the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-76899, weighing 6.4 mg, with a yield of 28%.

[1688] MS(ESI) m / z: 1186 [M / 3+H] + .

[1689] Example 77: Preparation of compound SMP-27341

[1690]

[1691] Step 1: Preparation of compound SMP-27341-1

[1692]

[1693] In a 25 mL reaction flask, compound SMP-27341-0 (300.0 mg, 0.40 mmol) and N,N-dimethylformamide (8 mL) were added. After complete clarification, N,N-diisopropylethylamine (7.8 mg, 0.78 mmol) and 1-hydroxybenzotriazole (27.0 mg, 0.20 mmol) were added, followed by the addition of methylaurestatin E (286.0 mg, 0.4 mmol). The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-27341-1, weighing 475.0 mg, with a yield of 89%.

[1694] MS(ESI) m / z: 1344 [M+H] +

[1695] Step 2: Preparation of compound SMP-27341-2

[1696]

[1697] In a 25 mL reaction flask, intermediate SMP-27341-2 (241.0 mg, 0.18 mmol), 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (54.0 mg, 0.36 mmol), and N,N-dimethylformamide (6 mL) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain compound SMP-27341-2, weighing 172.0 mg, with a yield of 86%.

[1698] MS(ESI) m / z: 1122 [M+H] + .

[1699] Step 3: Preparation of compound SMP-27341

[1700]

[1701] Compound SMP-37860-5 (25.0 mg, 0.02 mmol) and solvent N,N-dimethylformamide (2 mL) were added to a 4 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.4 mg, 0.03 mmol) and N,N-diisopropylethylamine (3.9 mg, 0.03 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound SMP-27341-2 (22.0 mg, 0.02 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain the target compound SMP-27341, weighing 23.0 mg, with a yield of 51%.

[1702] MS(ESI) m / z: 1168 [M / 2+H] + .

[1703] Example 78: Preparation of compound SMP-39205

[1704]

[1705] Step 1: Preparation of compound SMP-39205

[1706]

[1707] Compound SMP-30920-2 (35.0 mg, 0.024 mmol) and N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.0 mg, 0.024 mmol) and N,N-diisopropylethylamine (4.0 mg, 0.03 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound J (43.0 mg, 0.020 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-39205, weighing 51.0 mg, with a yield of 71%. MS (ESI) m / z: 1187 [M / 3+H] + .

[1708] Example 80: Preparation of compound SMP-68962

[1709]

[1710] Step 1: Preparation of compound SMP-68962-1

[1711]

[1712] In a 10 mL reaction flask, SMP-68962-0 (33.5 mg, 0.060 mmol), intermediate G (30.0 mg, 0.030 mmol), N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (19.4 mg, 0.150 mmol) were added sequentially. Under ice-water conditions, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (11.4 mg, 0.030 mmol, dissolved in 0.5 mL of N,N-dimethylformamide) was slowly added dropwise. After the addition was complete, the reaction was continued for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-68962-1, weighing 25.0 mg, with a yield of 54.4%.

[1713] MS(ESI) m / z: 767 [M / 2+H] + .

[1714] Step 2: Preparation of compound SMP-68962-2

[1715]

[1716] In a 10 mL reaction flask, SMP-68962-1 (25.0 mg, 0.016 mmol), intermediate B (16.5 mg, 0.019 mmol), N,N-diisopropylethylamine (3.1 mg, 0.024 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. Then, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (7.4 mg, 0.019 mmol) was added. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-68962-2, weighing 20.0 mg, with a yield of 53.1%.

[1717] MS(ESI) m / z: 785 [M / 3+H] + .

[1718] Step 3: Preparation of compound SMP-68962-3

[1719]

[1720] In a 10 mL reaction flask, SMP-68962-2 (20.0 mg, 0.008 mmol), N,N-dimethylformamide (1.5 mL), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (2.0 mg, 0.012 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-68962-3, weighing 12.0 mg, with a yield of 70.4%.

[1721] MS(ESI) m / z: 711 [M / 3+H] + 1067 [M / 2+H] + .

[1722] Step 4: Preparation of compound SMP-68962

[1723]

[1724] In a 10 mL reaction flask, SMP-68962-3 (12.0 mg, 0.0056 mmol), SMP-07861-2 (5.0 mg, 0.0067 mmol), N,N-diisopropylethylamine (1.1 mg, 0.0084 mmol), and N,N-dimethylformamide (1.5 mL) were added sequentially. Then, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.5 mg, 0.0067 mmol) was added. The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-68962, weighing 9.6 mg, with a yield of 60.0%. MS (ESI) m / z: 953 [M / 3+H] + 1430 [M / 2+H] + .

[1725] Example 81: Preparation of compound SMP-31135

[1726]

[1727] Step 1: Preparation of compound SMP-31135-1

[1728]

[1729] In a 10 mL reaction flask, SMP-31135-0 (39.4 mg, 0.060 mmol), intermediate G (30.0 mg, 0.030 mmol), N,N-diisopropylethylamine (19.4 mg, 0.150 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. Under ice-water conditions, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (11.4 mg, 0.030 mmol, dissolved in 0.5 mL of N,N-dimethylformamide) was slowly added dropwise. After the addition was complete, the reaction was continued at this temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound molecular weight. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-31135-1, weighing 27 mg, with a yield of 55.3%.

[1730] MS(ESI) m / z: 815 [M / 2+H] + .

[1731] Step 2: Preparation of compound SMP-31135-2

[1732]

[1733] In a 10 mL reaction flask, SMP-31135-1 (27.0 mg, 0.016 mmol), B (16.0 mg, 0.019 mmol), N,N-diisopropylethylamine (3.1 mg, 0.024 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. The mixture was stirred at room temperature, and then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (7.2 mg, 0.019 mmol) was added. The mixture was reacted for 20 minutes under these conditions. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-31135-2, weighing 22.0 mg, with a yield of 56.1%.

[1734] MS(ESI) m / z: 818 [M / 3+H] + .

[1735] Step 3: Preparation of compound SMP-31135-3

[1736]

[1737] In a 10 mL reaction flask, SMP-31135-2 (22.0 mg, 0.009 mmol), N,N-dimethylformamide (1.5 mL), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (2.0 mg, 0.013 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-31135-3, weighing 15.0 mg, with a yield of 74.8%.

[1738] MS(ESI) m / z: 744 [M / 3+H] + 1115[M / 2+H] + .

[1739] Step 4: Preparation of compound SMP-31135

[1740]

[1741] In a 10 mL reaction flask, SMP-31135-3 (15 mg, 0.0067 mmol), SMP-61694-2 (8.1 mg, 0.0081 mmol), N,N-diisopropylethylamine (1.3 mg, 0.010 mmol), and N,N-dimethylformamide (1.5 mL) were added sequentially, followed by N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.1 mg, 0.0081 mmol). The mixture was reacted at room temperature for 20 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-31135, weighing 11.2 mg, with a yield of 52.1%. MS (ESI) m / z: 1071 [M / 3+H] + 803 [M / 4+H] + .

[1742] Example 82: Preparation of compound SMP-64791

[1743]

[1744] Step 1: Preparation of compound SMP-64791-1

[1745]

[1746] In a 10 mL reaction flask, SMP-49182-6 (50.0 mg, 0.11 mmol), intermediate I (79 mg, 0.13 mmol), N,N-diisopropylethylamine (20.6 mg, 0.16 mmol), and N,N-dimethylformamide (4 mL) were added sequentially. The mixture was stirred at room temperature, and then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (49.4 mg, 0.13 mmol) was added. The mixture was reacted for 20 minutes under these conditions. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-64791-1, weighing 45.0 mg, with a yield of 38.8%.

[1747] MS(ESI) m / z: 1055 [M+H] + .

[1748] Step 2: Preparation of compound SMP-64791-2

[1749]

[1750] In a 10 mL reaction flask, SMP-64791-1 (45.0 mg, 0.043 mmol), dichloromethane (2 mL), and trifluoroacetic acid (2 mL) were added sequentially. The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain compound SMP-64791-2, weighing 30.0 mg, with a yield of 70.0%.

[1751] MS(ESI)m / z:999[M+H] + .

[1752] Step 3: Preparation of compound SMP-64791

[1753]

[1754] In a 10 mL reaction flask, SMP-64791-2 (6.3 mg, 0.0063 mmol), intermediate C (12.0 mg, 0.0052 mmol), N,N-diisopropylethylamine (1.0 mg, 0.0078 mmol), and N,N-dimethylformamide (1.5 mL) were added sequentially. The mixture was stirred at room temperature, and then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (2.4 mg, 0.0063 mmol) was added. The mixture was reacted for 20 minutes under these conditions. LC-MS showed that most of the molecules were of the target compound. The mixture was concentrated to obtain the crude product. The crude product was purified by HPLC to obtain compound SMP-64791, weighing 8.6 mg, with a yield of 52%.

[1755] MS(ESI) m / z: 1089 [M / 3+H] + .

[1756] Example 83: Preparation of compound SMP-26598

[1757]

[1758] Step 1: Preparation of compound SMP-26598-2

[1759]

[1760] In a 500 mL reaction flask, intermediate SMP-26598-1 (21.5 g, 56.49 mmol) and acetonitrile (200 mL) were added and stirred at room temperature. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.8 g, 56.49 mmol) and N-hydroxysuccinimide (6.5 g, 56.49 mmol) were added sequentially. The mixture was stirred and reacted overnight under these conditions. TLC showed that the reaction was complete. The reaction solution was filtered, and the resulting solid was dried under vacuum to give compound SMP-26598-2, weighing 22.0 g, with a yield of 81%.

[1761] MS(ESI) m / z: 478 [M+H] + .

[1762] Step 3: Preparation of compound SMP-26598-3

[1763]

[1764] In a 500 mL reaction flask, intermediate L-aspartic acid (6.0 g, 46.12 mmol), sodium bicarbonate (7.7 g, 92.24 mmol), water (20 mL), and tetrahydrofuran (200 mL) were added sequentially. The mixture was stirred at room temperature until completely clear. Then, compound SMP-26598-2 (22 g, 46.12 mmol) was dissolved in ethylene glycol dimethyl ether (50 mL) and slowly added to the above reaction solution. The reaction was continued with stirring overnight. TLC showed complete reaction. The reaction solution was concentrated under reduced pressure, and the residue was slowly added dropwise to 0.5 M hydrochloric acid aqueous solution (500 mL), precipitating a large amount of solid. This solid was filtered, dried under vacuum, and yielded compound SMP-26598-3, weighing 15.0 g, with a yield of 65%.

[1765] MS(ESI) m / z: 497 [M+H] + .

[1766] Step 4: Preparation of compound SMP-26598-4

[1767]

[1768] Compound SMP-26598-3 (450.0 mg, 0.90 mmol) and solvent N,N-dimethylformamide (8 mL) were added to a 25 mL reaction flask. The mixture was stirred at room temperature until the solution was completely clear. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (410.4 mg, 1.08 mmol) and N,N-diisopropylethylamine (175.4 mg, 1.36 mmol) were added sequentially. After reacting for 5 minutes, compound glycine tert-butyl ester (141.5 mg, 1.08 mmol) was added, and the reaction was stirred for another 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 15:1) to give intermediate SMP-26598-4, weighing 330.0 mg, with a yield of 60.2%.

[1769] MS(ESI) m / z: 610 [M+H] + .

[1770] Step 5: Preparation of compound SMP-26598-5

[1771]

[1772] Compound SMP-26598-4 (330.0 mg, 0.54 mmol) and trifluoroacetic acid (4 mL) were added to a 25 mL reaction flask and stirred at room temperature for 30 minutes. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give intermediate SMP-26598-5, weighing 200.0 mg, with a yield of 67.0%.

[1773] MS(ESI) m / z: 554 [M+H] + .

[1774] Step 6: Preparation of compound SMP-26598-6

[1775]

[1776] Compound SMP-26598-5 (200.0 mg, 0.36 mmol) was added to a 50 mL reaction flask along with 20 mL of tetrahydrofuran solvent. The mixture was stirred at room temperature until completely clear. Lead tetraacetate (191.8 mg, 0.44 mmol) and pyridine (44.8 mg, 0.56 mmol) were then added sequentially. The mixture was stirred overnight at 70 °C. TLC showed complete reaction. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 12:1) to give intermediate SMP-26598-6, weighing 132.0 mg, with a yield of 64.7%. MS (ESI) m / z: 568 [M+H] + .

[1777] Step 7: Preparation of compound SMP-26598-7

[1778]

[1779] Compound SMP-26598-6 (132.0 mg, 0.23 mmol), tetrahydrofuran (10 mL), and glycolic acid (21.0 mg, 0.28 mmol) were added to a 25 mL reaction flask. The mixture was cooled to 0 °C in an ice-water bath. Once the temperature reached 0 °C, p-toluenesulfonic acid (21.0 mg, 0.12 mmol) was added in one go. The mixture was then slowly heated to room temperature and stirred for 1.5 hours. TLC showed that the reaction was complete. The reaction solution was filtered and concentrated under reduced pressure. The residue was purified by HPLC to obtain intermediate SMP-26598-7, weighing 86.0 mg, with a yield of 64.1%.

[1780] MS(ESI) m / z: 584 [M+H] + .

[1781] Step 8: Preparation of compound SMP-26598-8

[1782]

[1783] Compound SMP-26598-7 (62.2 mg, 0.107 mmol) and N,N-dimethylformamide (3 mL) were added to a 25 mL reaction flask and stirred at room temperature. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (40.5 mg, 0.107 mmol) and N,N-diisopropylethylamine (17.2 mg, 0.133 mmol) were added sequentially. After reacting for 5 minutes, compound SMP-34048-5 (45 mg, 0.089 mmol) was added, and the reaction was continued for another 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain intermediate SMP-26598-8, weighing 61.0 mg, with a yield of 64.0%. MS (ESI) m / z: 1072 [M+H] + .

[1784] Step 9: Preparation of compound SMP-26598-9

[1785]

[1786] In a 25 mL reaction flask, intermediate SMP-26598-8 (61.0 mg, 0.057 mmol), N,N-dimethylformamide (3 mL), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (17.3 mg, 0.114 mmol) were added. The mixture was stirred at room temperature for 30 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain compound SMP-26598-9, weighing 33.0 mg, with a yield of 68.2%. MS (ESI) m / z: 850 [M+H] + .

[1787] Step 10: Preparation of compound SMP-26598

[1788]

[1789] Compound SMP-93566-4 (17.8 mg, 0.046 mmol) and N,N-dimethylformamide (3 mL) were added to a 25 mL reaction flask and stirred at room temperature. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (17.8 mg, 0.046 mmol) and N,N-diisopropylethylamine (7.5 mg, 0.058 mmol) were added sequentially, and the mixture was reacted at this temperature for 5 minutes. Then, compound SMP-26598-9 (33.0 mg, 0.039 mmol) was added, and the reaction was continued for another 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC to give intermediate SMP-26598, weighing 27.0 mg, with a yield of 57.0%.

[1790] MS(ESI) m / z: 1215 [M+H] + .

[1791] Example 85: Preparation of compound SMP-09168

[1792]

[1793] Step 1: Preparation of compound SMP-09168-1

[1794]

[1795] In a 25 mL reaction flask, maleimide-tetraethylene glycol-carboxylic acid (30.0 mg, 0.087 mmol) and N,N-dimethylformamide (3 mL) were added. The mixture was stirred at room temperature, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (39.6 mg, 0.104 mmol) and N,N-diisopropylethylamine (16.8 mg, 0.13 mmol) were added sequentially. After reacting for 5 minutes, SMP-93566-2 (25.0 mg, 0.087 mmol) was added, and the reaction was continued for another 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give intermediate SMP-09168-1, weighing 38.0 mg, with a yield of 71.0%.

[1796] MS(ESI) m / z: 616 [M+H] + .

[1797] Step 2: Preparation of compound SMP-09168-2

[1798]

[1799] Compound SMP-09168-1 (38.0 mg, 0.062 mmol) and trifluoroacetic acid (1 mL) were added to a 25 mL reaction flask and stirred at room temperature for 20 minutes. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to remove excess trifluoroacetic acid. The residue was purified by preparative HPLC to obtain intermediate SMP-09168-2, weighing 29.0 mg, with a yield of 83.5%.

[1800] MS(ESI) m / z: 560 [M+H] + .

[1801] Step 3: Preparation of compound SMP-09168

[1802]

[1803] Compound SMP-09168-2 (11.7 mg, 0.021 mmol) and N,N-dimethylformamide (2 mL) were added to a 25 mL reaction flask and stirred at room temperature. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.0 mg, 0.021 mmol) and N,N-diisopropylethylamine (3.3 mg, 0.026 mmol) were added sequentially. After reacting for 5 minutes, compound SMP-58054-2 (20.0 mg, 0.017 mmol) was added, and the reaction was continued for another 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain intermediate SMP-09168, weighing 23.0 mg, with a yield of 78.4%. MS (ESI) m / z: 1726 [M+H] + .

[1804] Example 86: Preparation of compound SMP-87055

[1805]

[1806] Step 1: Preparation of compound SMP-87055-2

[1807]

[1808] In a 500 mL reaction flask, intermediate SMP-87055-1 (13.5 g, 45.45 mmol) and acetonitrile (200 mL) were added and stirred at room temperature. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.5 g, 54.54 mmol) and N-hydroxysuccinimide (6.3 g, 54.54 mmol) were added sequentially. The reaction was continued at this temperature with stirring overnight. TLC showed that the reaction was complete. The reaction solution was filtered, and the resulting solid was dried under vacuum to give compound SMP-26598-2, weighing 14.0 g, with a yield of 78.4%.

[1809] MS(ESI) m / z: 393 [M+H] + .

[1810] Step 2: Preparation of compound SMP-87055-3

[1811]

[1812] In a 500 mL reaction flask, intermediate L-phenylalanine (6.4 g, 35.71 mmol), sodium bicarbonate (6.0 g, 71.42 mmol), water (20 mL), and tetrahydrofuran (200 mL) were added. The mixture was stirred at room temperature until the solution was completely clear. Compound SMP-87055-2 (14.0 g, 35.71 mmol) was dissolved in ethylene glycol dimethyl ether (50 mL) and slowly added to the above reaction solution. The reaction was continued overnight with stirring. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was slowly added dropwise to 0.5 M hydrochloric acid aqueous solution (500 mL), precipitating a large amount of solid. The solid was filtered and dried under vacuum to give compound SMP-87055-3, weighing 13.0 g, with a yield of 82.0%.

[1813] MS(ESI) m / z: 445 [M+H] + .

[1814] Step 3: Preparation of compound SMP-87055-4

[1815]

[1816] Compound SMP-87055-3 (13.0 g, 29.28 mmol) and N,N-dimethylformamide (30 mL) were added to a 25 mL reaction flask and stirred at room temperature. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.1 g, 29.28 mmol) and N,N-diisopropylethylamine (5.6 g, 43.81 mmol) were added sequentially. After reacting for 5 minutes, glycine tert-butyl ester (4.6 g, 35.05 mmol) was added, and the reaction was continued for another 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 15:1) to obtain intermediate SMP-87055-4, weighing 9.1 g, with a yield of 55.8%.

[1817] MS(ESI) m / z: 558 [M+H] + .

[1818] Step 4: Preparation of compound SMP-87055-5

[1819]

[1820] Compound SMP-87055-4 (9.1 g, 16.34 mmol) and trifluoroacetic acid (30 mL) were added to a 25 mL reaction flask and stirred at room temperature for 30 minutes. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain intermediate SMP-87055-5, weighing 6.7 g, with a yield of 81.8%.

[1821] MS(ESI)m / z:502[M+H] + .

[1822] Step 5: Preparation of compound SMP-87055-6

[1823]

[1824] Intermediate SMP-87055-5 (2.1 g, 4.19 mmol), 4-aminobenzyl alcohol (1.0 g, 8.38 mmol), and N,N-dimethylformamide (3 mL) were added to a 100 mL reaction flask and stirred at room temperature. Then, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (2.1 g, 8.38 mmol) was added, and the reaction was stirred for another 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was subjected to silica gel column chromatography (dichloromethane:methanol = 15:1) to give compound SMP-87055-6, weighing 1.7 g, with a yield of 67.0%.

[1825] MS(ESI) m / z: 607 [M+H] + .

[1826] Step 6: Preparation of compound SMP-87055-7

[1827]

[1828] In a 25 mL reaction flask, intermediate SMP-87055-6 (900.0 mg, 1.48 mmol) was dissolved in 10 mL of N,N-dimethylformamide. The mixture was stirred at room temperature. After dissolution, di-p-nitrophenyl carbonate (676.1 mg, 2.22 mmol) and triethylamine (298.7 mg, 2.96 mmol) were added, and the reaction was continued overnight with stirring. TLC was used to monitor the reaction until complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound SMP-87055-7, weighing 740.0 mg, with a yield of 64.7%. MS (ESI) m / z: 772 [M+H] + .

[1829] Step 7: Preparation of compound SMP-87055-8

[1830]

[1831] In a 25 mL reaction flask, compound SMP-87055-7 (461 mg, 0.60 mmol) and N,N-dimethylformamide (5 mL) were added. The mixture was stirred at room temperature until the solution was completely clear. Then, N,N-diisopropylethylamine (103.2 mg, 0.80 mmol), 1-hydroxybenzotriazole (27.0 mg, 0.20 mmol), and methylaurestatin E (286.8 mg, 0.40 mmol) were added sequentially, and the mixture was stirred at room temperature for another 2 hours. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (dichloromethane:methanol = 10:1) to give compound SMP-87055, weighing 480.0 mg, with a yield of 88.9%.

[1832] MS(ESI) m / z: 1351 [M+H] + .

[1833] Step 8: Preparation of compound SMP-87055-9

[1834]

[1835] In a 25 mL reaction flask, intermediate SMP-87055-8 (240.0 mg, 0.18 mmol) and N,N-dimethylformamide (4 mL) were added and stirred at room temperature. Then, 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (54.0 mg, 0.36 mmol) was added, and the reaction was continued for 20 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain compound SMP-87055-9, weighing 173.0 mg, with a yield of 85.2%.

[1836] MS(ESI) m / z: 1129 [M+H] + .

[1837] Step 9: Preparation of compound SMP-87055

[1838]

[1839] Compound SMP-93566-4 (9.2 mg, 0.024 mmol) and N,N-dimethylformamide (1.5 mL) were added to a 10 mL reaction flask and stirred at room temperature. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (9.1 mg, 0.024 mmol) and N,N-diisopropylethylamine (3.9 mg, 0.030 mmol) were added sequentially. After reacting for 5 minutes, compound SMP-87055-9 (22.0 mg, 0.020 mmol) was added, and the reaction was continued for 25 minutes. TLC showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by HPLC to obtain the target product SMP-87055, weighing 12.0 mg, with a yield of 40.1%. MS (ESI) m / z: 1494 [M+H] + .

[1840] Example 87: Preparation of compound SMP-34146

[1841]

[1842] Step 1: Preparation of compound SMP-34146-2

[1843]

[1844] SMP-34146-1 (500.0 mg, 0.83 mmol), p-nitrobenzene chloroformate (251.0 mg, 1.25 mmol), and N,N-dimethylformamide (10 mL) were added to a 50 mL reaction flask. After complete clarification, N,N-diisopropylethylamine (321 mg, 2.49 mmol) was added. The mixture was stirred at room temperature for 3 hours. TLC showed that the reaction was complete. The N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by thin-layer chromatography (dichloromethane:methanol = 15:1) to give compound SMP-34146-2, weighing 600.0 mg, with a yield of 94%.

[1845] MS(ESI) m / z: 767 [M+H] + .

[1846] Step 2: Preparation of compound SMP-34146-3

[1847]

[1848] SMP-34146-2 (300.0 mg, 0.39 mmol), iribulin (285.0 mg, 0.39 mmol), and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask. After complete dissolution, 1-hydroxybenzotriazole (58.1 mg, 0.43 mmol) and N,N-diisopropylethylamine (150.9 mg, 1.17 mmol) were added sequentially. The mixture was stirred at room temperature for 1 hour. TLC showed that the reaction was complete. N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by thin-layer chromatography (dichloromethane:methanol = 12:1) to give compound SMP-34146-3, weighing 180.0 mg, with a yield of 33.9%.

[1849] MS(ESI) m / z: 679 [M / 2+H] + .

[1850] Step 3: Preparation of compound SMP-34146-4

[1851]

[1852] SMP-34146-3 (176.5 mg, 0.13 mmol), 1,8-diazobisspirocyclo[5.4.0]undec-7-ene (29.1 mg, 0.19 mmol), and N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask and stirred at room temperature for 30 minutes. LC-MS analysis showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34146-4, weighing 90.0 mg, with a yield of 61%.

[1853] MS(ESI) m / z: 568 [M / 2+H] + .

[1854] Step 4: Preparation of compound SMP-34146

[1855]

[1856] Intermediate SMP-34146-4 (34.1 mg, 0.03 mmol), SMP-93566-4 (11.5 mg, 0.03 mmol), and N,N-dimethylformamide (2 mL) were added to a 10 mL reaction flask. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (11.4 mg, 0.03 mmol) and N,N-diisopropylethylamine (10.3 mg, 0.08 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. LC-MS analysis showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-34146, weighing 33.3 mg, with a yield of 74%.

[1857] MS(ESI) m / z: 750 [M / 2+H] + .

[1858] Example 88: Preparation of compound SMP-02692

[1859]

[1860] Step 1: Preparation of compound SMP-02692

[1861]

[1862] In a 10 mL reaction flask, SMP-93566 (10 mg, 0.007 mmol), acetone (1 mL), and iodomethane (2.0 mg, 0.014 mmol) were added. The mixture was stirred overnight at room temperature under nitrogen protection. LC-MS analysis confirmed the reaction was complete. A small amount of acetonitrile and water were added directly to the reaction solution, and the mixture was lyophilized to obtain compound SMP-02692, weighing 10.2 mg, with a yield of 93.7%.

[1863] MS(ESI) m / z: 1428 [M+H] + .

[1864] II. Preparation of Targeted Connector-Drug Conjugates

[1865] Conjugation Method: Antibody-drug conjugates are generated by drug conjugation using disulfide bonds on the antibody. First, the antibody is prepared as a 20 mg / mL solution. 0.1 mL of antibody is added to a 1.5 mL centrifuge tube, along with 293 μL of PBS (pH 7.4) / DTPA solution. Then, 6.4 μL of 5 mM TCEP aqueous solution (equivalent to 2.4 eq per molecule of antibody) is added, and the mixture is incubated at 25°C for 2 hours to reduce the disulfide bonds in the antibody's hinge region to thiol groups. Next, 10% medical-grade DMSO and 13.3 μL of payload (equivalent to 10 eq per molecule of antibody) are added to the above solution. The mixture is then shaken at 25°C (400 rpm) for 2 hours to connect the drug linker and the antibody, yielding the antibody-drug conjugate.

[1866] Preparation of antibody-drug conjugates corresponding to Examples 13-88 in Examples 89

[1867] The compounds prepared in Examples 13-88 were used as drug linking assembly units and conjugated with Her2 antibodies using the aforementioned conjugation method to obtain the corresponding antibody-drug conjugates.

[1868] Concentration of antibody-drug conjugates: The antibody-drug conjugates were placed in a 5000K (Milipore Co.) ultrafiltration tube and centrifuged at 2°C (3500G for 10 minutes) using a high-speed refrigerated centrifuge (GENESPEED 1580R).

[1869] The following is the preparation method of the comparative sample.

[1870] Comparative section

[1871] I. Preparation of Comparative Targeting Connectors-Drug Conjugates

[1872] Comparative Example 1: Preparation of Compound SMP-81641

[1873]

[1874] Step 1: Preparation of compound SMP-81641

[1875]

[1876] In a 10 mL reaction flask, 6-maleimide hexanoic acid (6.0 mg, 0.020 mmol), intermediate SMP-58054-1 (21.0 mg, 0.018 mmol), N,N-dimethylformamide (3 mL), 1-hydroxybenzotriazole (2.7 mg, 0.021 mmol), and triethylamine (5.1 mg, 0.050 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. The reaction was confirmed to be complete by LC-MS. The reaction solution was purified by preparative HPLC to obtain compound SMP-81641, weighing 8.9 mg, with a yield of 23%.

[1877] MS(ESI) m / z: 1378 [M+H] + .

[1878] Comparative Example 2: Preparation of Compound SMP-31896

[1879]

[1880] Step 1: Preparation of compound SMP-31896

[1881]

[1882] 6-Maleimide hexanoic acid (2.1 mg, 0.006 mmol) and N,N-dimethylformamide (0.5 mL) were added to a 4 mL reaction flask. After the reaction solution became clear, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.8 mg, 0.01 mmol) and N,N-diisopropylethylamine (1.3 mg, 0.01 mmol) were added sequentially. The mixture was reacted at room temperature for 5 minutes, and then intermediate SMP-82444-1 (14.0 mg, 0.006 mmol) was added. The mixture was stirred for 25 minutes. TLC showed that the reaction was complete. The reaction solution was purified by HPLC to give compound SMP-31896, weighing 10.9 mg, with a yield of 72%.

[1883] MS(ESI) m / z: 1280 [M / 2+H] + .

[1884] Comparative Example 3: Preparation of Compound SMP-74544

[1885]

[1886] Step 1: Preparation of compound SMP-74544

[1887]

[1888] Compound 6-maleimide hexanoic acid (13.0 mg, 0.06 mmol) and solvent N,N-dimethylformamide (3 mL) were added to a 10 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (22.8 mg, 0.06 mmol) and N,N-diisopropylethylamine (7.6 mg, 0.06 mmol) were added sequentially. After reacting at room temperature for 5 minutes, intermediate D (131.0 mg, 0.06 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain compound SMP-74544, weighing 115.0 mg, with a yield of 80%. MS (ESI) m / z: 1192 [M / 2+H] + .

[1889] Comparative Example 4: Preparation of Compound SMP-79786

[1890]

[1891] Step 1: Preparation of compound SMP-79786

[1892]

[1893] Compound 6-maleimide hexanoic acid (13.0 mg, 0.06 mmol) and solvent N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (22.8 mg, 0.06 mmol) and N,N-diisopropylethylamine (7.7 mg, 0.06 mmol) were added sequentially. After reacting at room temperature for 5 minutes, intermediate A (62.0 mg, 0.06 mmol) was added, and the reaction was continued at room temperature for 25 minutes. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain compound SMP-79786, weighing 48.0 mg, with a yield of 65%.

[1894] MS(ESI) m / z: 1241 [M+H] + .

[1895] Comparative Example 5: Preparation of Compound SMP-50206

[1896]

[1897] Step 1: Preparation of compound SMP-50206

[1898]

[1899] In a 4 mL reaction flask, maleimide-tetraethylene glycol-propionic acid (2.0 mg, 0.006 mmol) and N,N-dimethylformamide (0.5 mL) were added sequentially. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.1 mg, 0.06 mmol) and N,N-diisopropylethylamine (10.3 mg, 0.08 mmol) were added. After reacting at room temperature for 5 minutes, intermediate D (13 mg, 0.006 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-50206, weighing 11.1 mg, with a yield of 72%.

[1900] MS(ESI) m / z: 1259 [M / 2+H] + .

[1901] Comparative Example 6: Preparation of Compound SMP-77169

[1902]

[1903] Step 1: Preparation of compound SMP-77169-1

[1904]

[1905] In a 10 mL reaction flask, SMP-34048-5 (50.0 mg, 0.099 mmol), intermediate B-1 (76.5 mg, 0.119 mmol), N,N-diisopropylethylamine (19.1 mg, 0.148 mmol), and N,N-dimethylformamide (4 mL) were added sequentially. The mixture was stirred at room temperature, and then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (45.0 mg, 0.119 mmol) was added. The mixture was reacted for 20 minutes under these conditions. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-77169-1, weighing 45.0 mg, with a yield of 40.1%. MS (ESI) m / z: 1134 [M+H] + .

[1906] Step 2: Preparation of compound SMP-77169-2

[1907]

[1908] In a 10 mL reaction flask, SMP-77169-1 (45.0 mg, 0.040 mmol), N,N-dimethylformamide (3 mL), and 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (9.0 mg, 0.060 mmol) were added sequentially. The mixture was stirred at room temperature for 30 minutes. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by HPLC to obtain compound SMP-77169-2, weighing 25.0 mg, with a yield of 68.5%.

[1909] MS(ESI) m / z: 913 [M+H] + .

[1910] Step 3: Preparation of compound SMP-77169

[1911]

[1912] In a 10 mL reaction flask, SMP-77169-2 (25.0 mg, 0.027 mmol), maleimide propionic acid (5.6 mg, 0.033 mmol), N,N-diisopropylethylamine (5.3 mg, 0.041 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. The mixture was stirred at room temperature, and then N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (12.5 mg, 0.033 mmol) was added. The mixture was reacted for 20 minutes under these conditions. LC-MS showed that most of the molecules were of the target compound. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain compound SMP-77169, weighing 15.0 mg, with a yield of 52.3%. MS (ESI) m / z: 1063 [M+H] + .

[1913] Comparative Example 7: Preparation of Compound SMP-32666

[1914]

[1915] Step 1: Preparation of compound SMP-32666

[1916]

[1917] In a 10 mL reaction flask, intermediate C (20 mg, 0.008 mmol), maleimide-tetraethylene glycol-carboxylic acid (2.8 mg, 0.008 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.0 mg, 0.008 mmol) and N,N-diisopropylethylamine (3.0 mg, 0.024 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-32666, weighing 10.0 mg, with a yield of 43.7%.

[1918] MS(ESI) m / z: 1307 [M / 2+H] + .

[1919] Comparative Example 8: Preparation of Compound SMP-88394

[1920]

[1921] Step 1: Preparation of compound SMP-88394

[1922]

[1923] Intermediate SMP-58054-2 (34.0 mg, 0.03 mmol), 3-maleimide propionic acid (45.1 mg, 0.03 mmol), and N,N-dimethylformamide (2 mL) were added to a 10 mL reaction flask. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (11.4 mg, 0.03 mmol) and N,N-diisopropylethylamine (10.3 mg, 0.081 mmol) were added sequentially. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-88394, weighing 25.0 mg, with a yield of 79%.

[1924] MS(ESI) m / z: 668 [M / 2+H] + .

[1925] Comparative Example 9: Preparation of Compound SMP-49994

[1926]

[1927] Step 1: Preparation of compound SMP-49994

[1928]

[1929] In a 10 mL reaction flask, intermediate C (20 mg, 0.008 mmol), 6-maleimide hexanoic acid (1.7 mg, 0.008 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.0 mg, 0.008 mmol) and N,N-diisopropylethylamine (3.0 mg, 0.024 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-49994, weighing 10.0 mg, with a yield of 46.1%.

[1930] MS(ESI) m / z: 1340 [M / 2+H] + .

[1931] Comparative Example 10: Preparation of Compound SMP-54731

[1932]

[1933] Step 1: Preparation of compound SMP-54731

[1934]

[1935] In a 10 mL reaction flask, intermediate C (20 mg, 0.008 mmol), maleimide-octaethylene glycol-carboxylic acid (4.2 mg, 0.008 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (3.0 mg, 0.008 mmol) and N,N-diisopropylethylamine (3.0 mg, 0.024 mmol) were added. The mixture was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-54731, weighing 6.0 mg, with a yield of 24.6%.

[1936] MS(ESI) m / z: 1395 [M / 2+H] + .

[1937] Comparative Example 11: Preparation of Compound SMP-02571

[1938]

[1939] Step 1: Preparation of compound SMP-02571

[1940]

[1941] In a 25 mL reaction flask, maleimide-diethylene glycol-carboxylic acid (20.0 mg, 0.078 mmol) and N,N-dimethylformamide (5 mL) were added sequentially. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (30.0 mg, 0.079 mmol) and N,N-diisopropylethylamine (15.0 mg, 0.12 mmol) were added. After reacting at room temperature for 5 minutes, compound H (200.0 mg, 0.081 mmol) was added and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by HPLC to obtain the target product SMP-02571, weighing 160.0 mg, with a yield of 74%.

[1942] MS(ESI) m / z: 1352 [M / 2+H] + .

[1943] Comparative Example 12: Preparation of Compound SMP-39835

[1944]

[1945] Step 1: Preparation of compound SMP-39835

[1946]

[1947] Maleimide propionic acid (17.0 mg, 0.10 mmol) and N,N-dimethylformamide (5 mL) were added to a 25 mL reaction flask. Then, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (38.0 mg, 0.10 mmol) and N,N-diisopropylethylamine (15.0 mg, 0.12 mmol) were added sequentially. After reacting at room temperature for 5 minutes, compound A (84.0 mg, 0.08 mmol) was added, and the mixture was stirred for 25 minutes. TLC showed that the reaction was complete. Excess N,N-dimethylformamide was removed by concentration under reduced pressure. The residue was purified by preparative HPLC to obtain the target product SMP-39835, weighing 80.0 mg, with a yield of 83%. MS (ESI) m / z: 1199 [M+H] + .

[1948] Comparative Example 13: Preparation of Compound SMP-02524

[1949]

[1950] Step 1: Preparation of compound SMP-02524

[1951]

[1952] In a 10 mL reaction flask, intermediate D (50.0 mg, 0.023 mmol), maleimide-diethylene glycol-carboxylic acid (6.0 mg, 0.023 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete clarification, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (9.0 mg, 0.023 mmol) and N,N-diisopropylethylamine (9.0 mg, 0.069 mmol) were added. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound SMP-02524, weighing 35 mg, with a yield of 63.1%.

[1953] MS(ESI) m / z: 1215 [M / 2+H] + .

[1954] Comparative Example 14: Preparation of Compound DS-8201

[1955]

[1956] Step 1: Preparation of compound DS-8201

[1957]

[1958] In a 10 mL reaction flask, intermediate B (50.0 mg, 0.06 mmol), maleimide hexanoic acid (12.6 mg, 0.06 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete dissolution, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (22.2 mg, 0.06 mmol) and N,N-diisopropylethylamine (23.2 mg, 0.18 mmol) were added sequentially. The reaction was stirred at room temperature for 20 minutes. TLC showed that the reaction was complete. The reaction solution was purified by preparative HPLC to obtain compound DS-8201, weighing 35 mg, with a yield of 56.4%.

[1959] MS(ESI) m / z: 1034 [M+H] + .

[1960] II. Preparation of Comparative Targeted Connector-Drug Conjugates

[1961] Referring to the method for preparing target linker-drug conjugates in the Examples section, the compounds prepared in Comparative Examples 1-14 were used as drug linking assembly units and conjugated with Her2 antibody using the conjugation method of Example 89 to obtain the corresponding comparative antibody-drug conjugates.

[1962] The following uses antibody-drug conjugates (ADCs) as an example to illustrate the effects of introducing the structure shown in Formula A into the linker of the target adapter-drug conjugate in this invention:

[1963] Experimental Example 1: Detection of Antibody-Drug Conjugates

[1964] 1. Detection methods for antibody-drug conjugates

[1965] Test samples: the ADC of the present invention prepared in Example 89 above and the comparative ADC prepared in the comparative example.

[1966] Size exclusion high-performance liquid chromatography (SE-HPLC) was used to detect antibody-drug conjugate polymers (polymer components). A Biocore SEC column (7.8 × 300 mm, 5 μm) was used. The mobile phase was 50 mM phosphate containing 300 mM sodium chloride (pH 6.8). The flow rate was 0.5 mL / min, the sample injection volume was 10 μL (50 μg), and the UV absorbance at 280 nm was observed for at least 30 min. The polymer content of the antibody-drug conjugate was calculated using the area normalization method.

[1967] The antibody-drug conjugate ratio (DAR) of antibody-drug conjugates was determined using reversed-phase high-performance liquid chromatography-mass spectrometry (RP-HPLC-MS). The ADC sample was diluted to 1 mg / mL with 50 mmol / L Tris buffer (pH 8.0), and freshly prepared dithiothreitol (DTT) stock solution was added to a final DTT concentration of 50 mmol / L. The mixture was incubated at 37°C for 30 min. A PLRP-S column was used. Porosity (2.1 × 50 mm, 5 μm), column temperature 70℃. Flow rate 0.25 mL / min. Inject 10–20 μg of sample. Mobile phase A was an aqueous solution of 0.1% formic acid (V / V) and 0.025% trifluoroacetic acid (V / V), and mobile phase B was an acetonitrile solution of 0.1% formic acid (V / V) and 0.025% trifluoroacetic acid (V / V). The flow rate was 0–3 min, with mobile phase B at 27% for 3 min; 3–25 min, mobile phase B increased from 27% to 49%; 25–26 min, mobile phase B increased from 49% to 95%; 26–31 min, mobile phase B increased from 95% to 27%; 31.5–45 min, mobile phase B increased to 27%. UV absorbance at 280 nm was observed. The mass spectrometry drying gas temperature was 350℃, the drying gas flow rate was 7.0 L / min, the nebulizer pressure was 40 psi, and the capillary voltage was ±3000V -3000V. The mass range was 500–1600 m / z. The molecular weight of each mass spectrometry peak (range 20000–70000) was calculated by deconvolution and compared with the molecular weight of the light and heavy chains of the unconjugated antibody to calculate the drug loading of each peak and determine the loading of the corresponding UV peak. The percentage of UV peak area for each heavy and light chain was calculated by integration, and the weighted average DAR of the antibody-drug conjugate was calculated based on the drug loading distribution of each peak.

[1968] The content of naked antibody in antibody-drug conjugates (ADCs) was determined using hydrophobic interaction chromatography (HIC). A Biocore HIC-Butyl column (4.6 × 150 mm, 5 μm) was used. Mobile phase A was a 100 mM phosphate aqueous solution containing 2 M ammonium sulfate (pH 7.0), and mobile phase B was a 100 mM phosphate aqueous solution (pH 7.0):isopropanol = 80:20 (V / V). 50 μg of sample was injected, followed by 10 μL. Mobile phase A was run at 100% for 3 min at a flow rate of 1 mL / min. From 3 to 25 min, mobile phase B was increased from 0% to 100%, and at 25.1 min, mobile phase B decreased to 0%, running for 30 min. UV absorbance at 280 nm was observed. The content of naked antibody in the ADC was calculated using the area normalization method based on the retention time of the antibody-targeted naked antibody peak.

[1969] 2. Detection results of antibody-drug conjugates

[1970] Table 1. Detection results of antibody-drug conjugates

[1971]

[1972]

[1973]

[1974] In Table 1 above, the polymer content represents the percentage of polymers (high molecular weight components) in the ADC detected by size exclusion high-performance liquid chromatography. Excessively high polymer content may induce immunogenic reactions. The naked antibody content represents the percentage of naked antibody in the ADC detected by hydrophobic interaction chromatography. Excessively high naked antibody content may cause the naked antibody to become a competitive inhibitor of the ADC. Both excessively high polymer and naked antibody contents can affect the binding of the ADC to its target, thus impacting its therapeutic efficacy.

[1975] The DAR value represents the average number of drug-linked assembly units conjugated to an antibody. A DAR value that is too low cannot achieve effective cell killing, resulting in insufficient ADC efficacy. Under the condition that the ADC allows, a higher DAR value can achieve more efficient drug delivery. However, as the DAR value increases, the content of hydrophobic payload increases, which leads to increased hydrophobicity of the ADC, thereby accelerating the clearance of the ADC in the body, resulting in a shortened ADC half-life and increased toxicity.

[1976] The difference between the ADC structure of this embodiment and the comparative ADC structure lies in whether the connector contains the structure shown in Formula A.

[1977] Meanwhile, the increase in hydrophobicity can be manifested in the polymer content during the coupling step. When the hydrophobicity of the small molecule increases, the hydrophobic amino acids inside the antibody may be exposed due to the influence of the small molecule, thereby increasing the overall hydrophobicity of the ADC and causing the ADC to aggregate more rapidly, thus increasing the number of polymers. The exposure of hydrophobic amino acids inside the antibody may also change the higher-order structure of the antibody, resulting in a decrease in antibody binding activity and thus reducing the targeting ability of the ADC. By introducing the structure of formula A on the linker, the hydrophobicity of the small molecule can be reduced, preventing the non-covalent binding of the hydrophobic payload to the antibody and preventing the exposure of hydrophobic amino acids inside the antibody, thereby reducing the overall hydrophobicity of the ADC. Comparing the data of polymer content, naked antibody content, and DAR value in Table 1, it can be seen that the ADCs of Comparative Examples 1-13 have high polymer content and high naked antibody content, while the corresponding ADCs of the present invention have low polymer content and low naked antibody content; although the polymer content and naked antibody content of Comparative Example 14 are not high, its plasma stability is not as good as that of 88480 which introduced the structure of formula A, which also proves this point.

[1978] Experimental results show that by introducing the structure shown in Formula A onto the linker of the ADC, the content of ADC polymer and naked antibody can be reduced, so that the DAR value of the prepared ADC is maintained within a suitable range, thereby improving the stability of the ADC, reducing its immunogenicity, reducing competitive inhibition, and improving the efficacy of the drug.

[1979] The ADC prepared by this invention has low polymer content and naked antibody content, suitable DAR value, good stability, low immunogenicity, and high purity.

[1980] Experimental Example 2: Stability Test of Antibody-Drug Conjugates

[1981] 1. Experimental Methods

[1982] Test samples: the ADCs of the present invention prepared in the examples and the comparative ADCs prepared in the comparative examples.

[1983] Stability testing method: The obtained ADC was subjected to a 14-day stability study. The storage conditions were 40℃ and protected from light. Sampling points were on days 0, 7, and 14. The polymer content and DAR value of the samples at different sampling points were tested according to the method in Experiment Example 1.

[1984] 2. Experimental Results

[1985] Table 2. Stability Test Results

[1986]

[1987]

[1988]

[1989] Table 2 above uses the polymer content and DAR value of the ADC after 0, 7, and 14 days of storage at 40℃ in the dark to evaluate its storage stability. If the ADC still maintains low polymer content and low DAR value after 14 days of storage, it indicates that the ADC has excellent storage stability.

[1990] As can be seen from the data in Table 2, the ADC of t...

Claims

1. A drug linking assembly unit or a salt thereof, characterized in that: The drug linking assembly unit consists of a connector and a drug. The connector end can connect to a target connector, which is a substance capable of targeting and binding to the lesion site. The structure of the connector is selected from: -PEG9 indicates -PEG 16 express 2. The drug linking assembly unit or its salt according to claim 1, characterized in that: The drug is selected from cytotoxic drugs, drugs for treating autoimmune toxicity, or anti-inflammatory drugs.

3. The drug linking assembly unit or its salt according to claim 2, characterized in that: The drug is selected from drugs that target DNA or drugs that target tubulin.

4. The drug linking assembly unit or a salt thereof according to claim 1, characterized in that: The drug is selected from one of the following compounds:

5. The drug linking assembly unit or a salt thereof according to claim 1, characterized in that: The structure of the drug linking assembly unit is selected from: -PEG9 indicates -PEG 16 express 6. A targeted adapter-drug conjugate or a salt thereof, characterized in that: The target connector-drug conjugate is obtained by connecting a target connector and a drug linking assembly unit as described in any one of claims 1 to 5, and its structure is shown in formula J-1. Ab is a target adapter, which is an antibody; q is an integer from 1 to 20; The target connector is a substance that can target and bind to the lesion site.

7. The targeting adapter-drug conjugate or its salt according to claim 6, characterized in that: The DAR value of the target connector-drug conjugate is 1.00 to 20.

00.

8. The targeting adapter-drug conjugate or its salt according to claim 7, characterized in that: The DAR value of the target connector-drug conjugate is 2.0 to 8.

0.

9. The target connector-drug conjugate or its salt according to claim 8, characterized in that: The DAR value of the target connector-drug conjugate is 2.0 to 5.

0.

10. The targeting adapter-drug conjugate or its salt according to claim 6, characterized in that: The structure of the target connector-drug conjugate is selected from: q is an integer from 1 to 20; -PEG9 indicates -PEG 16 express 11. The targeted adapter-drug conjugate or its salt according to any one of claims 6-10, characterized in that: The antibody is an antibody that targets cell surface receptors and tumor-associated antigens.

12. A linker for a targeted connector-drug conjugate or a salt thereof, said linker having a structure selected from: R0” is a leaving group; R0' is a leaving group; -PEG9 indicates -PEG 16 express 13. The linker or its salt according to claim 12, characterized in that: The leaving group is a hydroxyl group.

14. A pharmaceutical preparation for the prevention and / or treatment of tumors, characterized in that: It is a formulation made with the target linker-drug conjugate or its salt as the active ingredient, plus pharmaceutically acceptable excipients, as described in any one of claims 6-11.

15. Use of the targeted adapter-drug conjugate or its salt according to any one of claims 6-11 in the preparation of a pharmaceutical formulation for the prevention and / or treatment of tumors, wherein the tumor is selected from ovarian cancer, breast cancer or gastric cancer.

16. A method for preparing the targeted linker-drug conjugate or a salt thereof according to any one of claims 6-11, characterized in that: The method includes the following steps: (1) Drugs are coupled with linkers to obtain drug linker assembly units; (2) The drug linking assembly unit is coupled with the target adapter to obtain a target adapter-drug conjugate; or, the method includes the following steps: (1') The target connector and the connector are coupled to obtain the target connector-connector unit; (2') The target connector-connector subunit is coupled with the drug to obtain the target connector-drug conjugate.

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