Antibody-drug conjugate carrying pan-ras inhibitor payload

Abstract

Provided is an antibody-drug conjugate carrying a pan-RAS inhibitor payload, which innovatively uses a pan-RAS inhibitor as a bioactive molecule of an antibody-drug conjugate. The antibody-drug conjugate has high anti-tumor activity, can effectively kill tumor cells, and has a bystander killing effect.

Description

An antibody-drug conjugate loaded with a pan-RAS inhibitor Technical Field

[0001] This disclosure relates to pan-RAS inhibitor antibody-drug conjugates comprising a pan-RAS inhibitor and an antibody or antigen-binding fragment thereof that binds to an antigen target (e.g., an antigen expressed on cancer cells). This disclosure further relates to methods for preparing and using antibody-drug conjugates comprising pan-RAS inhibitors. Background Technology

[0002] Antibody-drug conjugates (ADCs) have shown promising therapeutic effects in recent years due to their unique structure and targeted delivery and killing properties. Currently, more than ten ADC drugs have been approved for marketing, covering various types of cancer and providing patients with more treatment options.

[0003] RAS is one of the most frequently mutated genes in human tumors, occurring in approximately 30% of cancer patients, with KRAS accounting for about 85% of RAS mutations. KRAS mutations are found in 88% of pancreatic cancers, 50% of colorectal adenocarcinomas, and 32% of lung adenocarcinomas, making the development of KRAS-targeting inhibitors of great clinical significance and value.

[0004] KRAS is a membrane-bound protein with GTPase activity. It acts as a "molecular switch" by cycling between the GDP-binding inactive conformation and the GTP-binding active conformation through nucleotide exchange. In its GTP-bound state, KRAS can activate multiple downstream signaling pathways, including RAF-MEK-ERK and PI3K-AKT, to regulate life processes such as cell growth, proliferation, differentiation, and apoptosis.

[0005] KRAS mutations (such as G12C, G12D, G12V, G13D, etc.) affect GTP hydrolysis mediated by GTPase activating proteins (GAPs), increasing the number of KRAS in a GTP-bound activated state, overactivating downstream signaling pathways, and ultimately leading to tumorigenesis and development. However, because the KRAS protein lacks a corresponding hydrophobic pocket suitable for drug binding, and its affinity for GTP and GDP is in the picomolar range (~20 pM), the development of inhibitors that competitively bind to KRAS is extremely difficult. For decades, KRAS has been considered an untreatable target.

[0006] In May 2021, AMG510 was approved by the FDA for the treatment of KRAS carriers. G12CLocally advanced or metastatic non-small cell lung cancer with mutations has broken the historical barrier of KRAS being "untreatable." However, G12C mutations represent only a small fraction of KRAS mutations, and for mutations at other KRAS sites, there is currently a lack of satisfactory and effective inhibitory compounds, leaving a significant unmet clinical need. Pan-RAS inhibitors may potentially cause toxicity due to simultaneous inhibition of wild-type RAS in normal tissues, while targeted delivery of ADCs holds promise for addressing this issue. Combining the characteristics of pan-RAS inhibitors and ADCs, ADCs loaded with pan-RAS inhibitors promise to provide safer and more effective treatment options for patients with various RAS mutations. Summary of the Invention

[0007] This invention provides a novel antibody-drug conjugate, characterized by the innovative use of a pan-RAS inhibitor as the bioactive molecule (drug payload) of the antibody-drug conjugate. The antibody-drug conjugate of this invention exhibits high antitumor activity, effectively killing tumor cells and demonstrating a bystander-killing effect.

[0008] On the one hand, this disclosure provides an antibody-pan-RAS inhibitor conjugate of Formula I, or a stereoisomer, tautomer, prodrug thereof, pharmaceutically acceptable salt thereof, or pharmaceutically acceptable solvate thereof.

[0009] in,

[0010] Ab represents an antibody or antigen-binding fragment, or an antigen ligand;

[0011] q is the number of LPs coupled to Ab. q can represent the number of LPs coupled to a single Ab, or the average number of LPs coupled to Ab in a batch of drugs. q is selected from any value between 1.0 and 16.0.

[0012] L is a segment that covalently connects Ab and P. L is -L1-L2-L3-L4-L5-, where L1 is connected to Ab, L4 or L5 can be a direct bond, and L4 or L5 is connected to P.

[0013] P is a pan-RAS inhibitor fragment, which is a structural unit shown in Formula II, and P is connected to L5 or L4 through its oxygen, sulfur, or nitrogen atoms:

[0014] in:

[0015] Cya said or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups;

[0016] A represents a 4- to 6-membered heterocyclic alkylene, phenylene, or a 5- to 6-membered heterocyclic aryl group, wherein each of the 4- to 6-membered heterocyclic alkylene, phenylene, or 5- to 6-membered heterocyclic aryl group can be independently represented by 0, 1, 2, 3, or 4 R's. x replace;

[0017] B represents a 5- to 6-membered heteroaryl group or a phenylene group, wherein the 5- to 6-membered heteroaryl group or phenylene group can be independently represented by 0, 1, 2, 3, or 4 R groups. x replace;

[0018] X and Y independently represent hydrogen, C1-C6 aminoalkyl, C1-C6 hydroxyalkyl, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C 12 Cycloalkyl, 5- to 6-membered heteroaryl or phenyl, wherein the C1-C6 aminoalkyl, C1-C6 hydroxyalkyl, C1-C6 alkyl, 4- to 12-membered heterocycloalkyl, C3-C 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2 or 3 heteroatoms selected from N, O, and S;

[0019] Z represents -OR a -SR a Or -NR a R a ';

[0020] R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 membered heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a 'The methylene group on the C0-C6 alkylene, C1-C6 alkylene, C1-C6 alkyl, or C1-C6 haloalkyl group can be replaced with a carbonyl group or -NR group.' a-, -O-, or -S-, and optionally, each of the C0-C6 alkylene, C1-C6 alkylene, C1-C6 alkyl, and C1-C6 haloalkyl groups can be independently substituted by 0, 1, 2, 3, or 4 substituents selected from halogens or C1-C3 alkyl groups, and the two substituents of the same C atom can form a 3-8 membered ring with the C atom, the 3-8 membered ring optionally also containing 0, 1, 2, or 3 heteroatoms selected from N, O, or S; each of the C3-C8 cycloalkyl or 4-8 membered heterocycloalkyl groups can be independently substituted by 0, 1, 2, 3, or 4 substituents selected from halogens, oxoalkyl, -OR-, -O-, -O-, -S-, -O ... a -SR a -NR a R a ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-(C3-C8 cycloalkyl) or -(C0-C3 alkylene)-(4-8 heterocyclic alkyl), -C(O)R a -C(O)OR a -C(O)NR a R a '、-NR a C(O)R a '、-OC(O)R a '、-OC(O)NR a R a '、-NR a C(O)NR a R a '、-S(O)R a -S(O)2R a -NR a S(O)2R a Substituents of ';

[0021] R2 represents a C1-C6 alkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), or -(C0-C6 alkylene)-(4-8 heterocyclic alkyl), which may optionally be substituted with 0, 1, or 2 substituents selected from the following: -OR a -SR a Or -NR a R a ';

[0022] R3 and R3' each independently represent hydrogen, halogen, C1-C6 alkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl) or -(C0-C6 alkylene)-CN;

[0023] R4 represents hydrogen, C1-C6 alkyl, -(C0-C6 alkylene)-OR a -(C0-C6 alkylene)-SRa -(C0-C6 alkylene)-NR a R a ', -(C0-C6 alkylene)-(C3-C8 cycloalkyl) or -(C0-C6 alkylene)-(4-12 heterocyclic alkyl), -(C0-C6 alkylene)-phenyl or -(C0-C6 alkylene)-(5-6 heteroaryl), wherein any methylene group on the C0-C6 alkylene or C1-C6 alkyl group can be replaced with a carbonyl group, -NR a -, -O-, or -S-, and optionally, the C0-C6 alkylene or C1-C6 alkyl group may be substituted with 0, 1, 2, 3, or 4 substituents selected from halogens or C1-C3 alkyl groups, and the two substituents of the same C atom may form a 3-8 membered ring with the C atom, the 3-8 membered ring optionally also containing 0, 1, 2, or 3 heteroatoms selected from N, O, or S; the C1-C6 alkyl, C3-C8 cycloalkyl, 4-12 membered heterocycloalkyl, phenyl, and 5-6 membered heteroaryl groups may each be independently substituted with 0, 1, 2, 3, or 4 substituents selected from halogens, oxo-, -OR-, -O ... a -SR a -NR a R a ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-(C3-C8 cycloalkyl) or -(C0-C3 alkylene)-(4-8 heterocyclic alkyl), -C(O)R a -C(O)OR a -C(O)NR a R a '、-NR a C(O)R a '、-OC(O)R a '、-OC(O)NR a R a '、-NR a C(O)NR a R a '、-S(O)R a -S(O)2R a -NR a S(O)2R a Substituents of ';

[0024] L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a-, -O- or -S-, and optionally, the methylene groups on the -(C1-C6) alkylene groups can each be independently replaced by 0, 1, 2, 3 or 4 C1-C3 alkyl groups, and the two substituents on the same C atom can form a 3-8 membered ring with the C atom;

[0025] Cy1 represents C3-C 12 Cycloalkyl or 4-12 membered heterocyclic alkyl, wherein the ring can be monocyclic, spirocyclic, bridged, or fused.

[0026] R5 represents hydrogen, halogen, oxometalate, and =NR independently. a -OR a -SR a -NR a R a ', cyano, -C(O)OR a -C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )R a ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a Substitution with ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-C3-C8 cycloalkyl or -(C0-C3 alkylene)-4-8 heterocyclic alkyl groups;

[0027] m represents 0, 1, 2, or 3;

[0028] R x Each independently represents hydrogen, halogen, oxo, =NR a -OR a -SR a -NR a R a ', cyano, -C(O)OR a -C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )Ra ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl;

[0029] R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; when R a R a When connected to the same N atom, the R a and R a 'The N atom and the N atom bonded together can form a 4-8 membered ring, which may optionally contain one, two or three heteroatoms selected from N, O or S;

[0030] The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms.

[0031] In some embodiments, P is connected to L by removing an H atom or a monovalent group attached to an oxygen, sulfur, or nitrogen atom; preferably, P is connected to L by removing Ra from Z.

[0032] In some implementations, -P has the structure shown in Formula IIa:

[0033] Where Za represents O, S, or NR a .

[0034] In some implementations, Cya indicates or It may optionally be substituted with 0, 1, 2, or 3 substituents selected from halogens or C1-C3 alkyl groups; preferably, Cya represents or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups; more preferably, Cya is preferred. or

[0035] In some implementations, A represents a 5-6 member heteroaryl group, which can be substituted with 0, 1, 2, 3, or 4 R groups. x Substitution; preferably, A represents an imidazolyl group, which can be replaced by 0, 1, 2, 3 or 4 R groups. x Substitution; more preferably, A represents an imidazolyl group.

[0036] In some implementations, B represents or The structure can be 0, 1, 2, 3, or 4 Rs. x Replacement; preferably, B indicates or

[0037] In some implementations, X and Y independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C 12 C1-C6 alkyl, 4-12 heterocyclic alkyl, C3-C6 alkyl, 5-6 heterocyclic alkyl, or phenyl, wherein the C1-C6 alkyl, 4-12 heterocyclic alkyl, or C3-C6 alkyl is a cyclic alkyl group. 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further comprise 0, 1, 2, or 3 heteroatoms selected from N, O, and S; preferably, X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C6 alkyl, or C4- to C5-C6 alkyl. 12 C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C 12 Each cycloalkyl group can be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2, or 3 heteroatoms selected from N, O, and S; more preferably, X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, or C3- to C6-membered cycloalkyl, wherein the C1- to C6 alkyl, 4- to 8-membered heterocyclic alkyl, or C3- to C6-membered cycloalkyl may each independently be represented by 0, 1, 2, or 3 R atoms. x Alternatively, X and Y can form a 3-6 element ring, which can be optionally divided by 0, 1, or 2 R elements. x The ring may further include 0, 1, or 2 heteroatoms selected from N, O, and S.

[0038] In some implementations, Z represents -OR a Or -NR a R a ', preferably Z represents -OH or -NHR a More preferably, Z represents -OH.

[0039] In some embodiments, R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a Preferably, R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 heterocyclic alkyl); more preferably, R1 represents C1-C6 alkyl or C1-C6 haloalkyl; even more preferably, R1 represents ethyl or -CH2CF3.

[0040] In some embodiments, R2 represents a C1-C6 alkyl group, which may be substituted with 0 or 1 -ORa; preferably, R2 represents 1-methoxyethyl; more preferably, R2 represents Where * indicates the location where R2 is connected to the part connected to it in the formula.

[0041] In some embodiments, R3 and R3' each independently represent hydrogen, halogen, and C1-C6 alkyl; preferably, R3 and R3' are H.

[0042] In some implementations, R4 represents hydrogen, -OR a -SR a Or -NR a R a Preferably, R4 represents H.

[0043] In some implementations, L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a -, -O-, or -S-; preferably, L D1 L D2 Each can be independently represented as a single bond or -(C1-C6) alkylene-.

[0044] In some embodiments, Cy1 represents a 4-12 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring; preferably, Cy1 represents a 4-8 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring.

[0045] In some implementations, R5 independently represents hydrogen, oxygen, and =NR. a -S(O)2Ra -C(O)R a C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl groups can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a The alkyl group is substituted with a cyano group, a C1-C6 alkyl group, a C3-C8 cycloalkyl group, or a 4-8 membered heterocyclic alkyl group; preferably, each of the R5 groups independently represents a C3-C8 cycloalkyl group or a 4-8 membered heterocyclic alkyl group, and each of the aforementioned C3-C8 cycloalkyl groups or 4-8 membered heterocyclic alkyl groups can be independently substituted with 0, 1, 2, 3, or 4 groups selected from halogen, oxo, -OR a -SR a -NR a R a The substituents are ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 heterocyclic alkyl, and when R5 is a C5-C6 cycloalkyl or 5-6 heterocyclic alkyl, the number of substituents is greater than 0.

[0046] In some implementations, m represents 0, 1, or 2.

[0047] In some implementation schemes, R x Each can be used independently to represent hydrogen, halogen, oxo, -OR a -SR a -NR a R a ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl; preferably, R x Each can independently represent hydrogen, halogen, oxo, -OH, -SH, -NH2, cyano, C1-C6 alkyl; more preferably, R x Each can be independently represented as hydrogen, halogen, -OH, -NH2, cyano, or C1-C3 alkyl.

[0048] In some implementations, P is the structural unit shown in Formula III, and P is connected to L5 or L4 through its contained oxygen, sulfur, or nitrogen atoms:

[0049] in:

[0050] Cya said or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups;

[0051] B indicates or The structure can be 0, 1, 2 or 3 Rs x replace;

[0052] X and Y independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, and C3-C6 alkyl. 12 C1-C6 alkyl, 4-12 heterocyclic alkyl, C3-C6 alkyl, 5-6 heterocyclic alkyl, or phenyl, wherein the C1-C6 alkyl, 4-12 heterocyclic alkyl, or C3-C6 alkyl is a cyclic alkyl group. 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2 or 3 heteroatoms selected from N, O, and S;

[0053] Z represents -OR a Or -NR a R a ';

[0054] R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 membered heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a ';

[0055] L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a -、-O- or -S-;

[0056] Cy1 represents C3-C 12 Cycloalkyl or 4-12 membered heterocyclic alkyl, wherein the ring can be monocyclic, spirocyclic, bridged, or fused.

[0057] R5 represents hydrogen, halogen, oxidative oxidation, and =NR independently. a -OR a -SR a -NR a R a ', cyano, -C(O)OR a-C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )R a ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a Substitution with ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-C3-C8 cycloalkyl or -(C0-C3 alkylene)-4-8 heterocyclic alkyl groups;

[0058] m represents 0, 1, 2, or 3;

[0059] R x Each can be used independently to represent hydrogen, halogen, oxo, -OR a -SR a -NR a R a ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl;

[0060] R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; when R a R a When connected to the same N atom, the R a and R a 'The N atom and the N atom bonded together can form a 4-8 membered ring, which may optionally contain one, two or three heteroatoms selected from N, O or S;

[0061] The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms.

[0062] In some implementations, P is the structural unit shown in Formula III, and P is connected to L5 or L4 through its contained oxygen, sulfur, or nitrogen atoms:

[0063] in:

[0064] Cya said or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups;

[0065] B is selected from or The structure can be 0, 1, 2 or 3 Rs x replace;

[0066] X and Y independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, and C3-C6 alkyl. 12 C1-C6 alkyl, 4-12 heterocyclic alkyl, C3-C6 alkyl, 5-6 heterocyclic alkyl, or phenyl, wherein the C1-C6 alkyl, 4-12 heterocyclic alkyl, or C3-C6 alkyl is a cyclic alkyl group. 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2 or 3 heteroatoms selected from N, O, and S;

[0067] Z represents -OR a Or -NR a R a ';

[0068] R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 membered heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a ';

[0069] L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a -、-O- or -S-;

[0070] Cy1 represents C3-C 12 Cycloalkyl or 4-12 membered heterocyclic alkyl, wherein the ring can be monocyclic, spirocyclic, bridged, or fused.

[0071] R5 represents hydrogen, halogen, oxidative oxidation, and =NR independently. a -ORa -SR a -NR a R a ', cyano, -C(O)OR a -C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )R a ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a Substitution with ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-C3-C8 cycloalkyl or -(C0-C3 alkylene)-4-8 heterocyclic alkyl groups;

[0072] m represents 0, 1, 2, or 3;

[0073] R x Each can be used independently to represent hydrogen, halogen, oxo, -OR a -SR a -NR a R a ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl;

[0074] R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; when R a R a When connected to the same N atom, the R a and R a 'The N atom and the N atom bonded together can form a 4-8 membered ring, which may optionally contain one, two or three heteroatoms selected from N, O or S;

[0075] The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms.

[0076] In some implementations, P is the structural unit shown in Formula III, and P is connected to L5 or L4 through its contained oxygen, sulfur, or nitrogen atoms:

[0077] Cya represents or

[0078] B indicates or

[0079] X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl, wherein the C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl can each be independently represented by 0, 1, 2, or 3 R. x Alternatively, X and Y can form a 3-6 element ring, which can be optionally divided by 0, 1, or 2 R elements. x Alternatively, the ring may further include 0, 1, or 2 heteroatoms selected from N, O, and S;

[0080] Z represents -OH or -NHR a ;

[0081] R1 represents ethyl or trifluoroethyl;

[0082] L D1 L D2 Each can independently represent a single bond or a -(C1-C6) alkylene group;

[0083] Cy1 represents a 4-8 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring.

[0084] R5 can independently represent C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, wherein each of the aforementioned C3-C8 cycloalkyl and 4-8 membered heterocyclic alkyl groups can be independently selected from 0, 1, 2, 3 or 4 ions chosen from halogen, oxo, -OR a -SR a -NR a R a The substituents of ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, and when R5 is a C5-C6 cycloalkyl or 5-6 membered heterocyclic alkyl, the number of substituents is greater than 0;

[0085] m represents 0, 1, or 2;

[0086] R x Each can independently represent hydrogen, halogen, -OH, -NH2, cyano, and C1-C3 alkyl;

[0087] R a R aEach can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl;

[0088] The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms.

[0089] 68. In some embodiments, P is selected from segments of the following structures, wherein P is connected to L5 or L4 via an oxygen, nitrogen, or sulfur atom contained therein:

[0090] In some implementations, -P is selected from the following structures:

[0091] In some implementations, q is selected from any value between 1.0 and 16.0.

[0092] In some implementations, q is selected from any value between 2.0 and 16.0.

[0093] In some implementations, q is selected from any value between 1.0 and 8.0.

[0094] In some implementations, q is selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0095] In some implementations, q is selected from 2, 4, 6, or 8.

[0096] In some implementations, q is selected from any value between 4.0 and 8.0.

[0097] In some implementations, q is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16.

[0098] In some implementations, q is selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0099] In some implementations, q is selected from 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0100] In some implementation schemes, q is selected from 6, 7, 8, 9, 10, 11, and 12.

[0101] In some implementations, q is selected from 4, 5, 6, 7, and 8; preferably, q is selected from 4, 6, and 8.

[0102] In some implementations, L1 is selected from:

[0103] In some implementations, L2 is selected from: direct-connect key,

[0104] Among them, E is independently selected from direct-connect key, The phenyl group, the 5-6-membered heteroaryl group, and the amide group may optionally be substituted with 0, 1, or 2 of the following substituents: F, Cl, methyl, methoxy;

[0105] a1 is independently selected from 1, 2, 3, 4, 5, and 6;

[0106] a2 is independently selected from 0, 1, 2, 3, 4, 5, and 6;

[0107] a3 is independently selected from 1, 2, 3, 4, 5, and 6;

[0108] R x1 Each is independently selected from H, methyl, or -CH2CH2N(Me)2;

[0109] R x2 Each is independently selected from H or methyl;

[0110] R x3 Each is independently selected from -NH2, -OH, -OMe, -NHMe, -N(Me)2 or -NHCH2COOH.

[0111] In some implementations, L3 may or may not exist. When L3 does not exist, it is a direct connection key; when L3 exists, L3 is selected from: -ML 3a -or-L 3a -M-,

[0112] in,

[0113] M is selected independently from: direct connection key,

[0114] L 3a Each is independently selected from: direct-connect key,

[0115] b1 is independently selected from 1, 2, 3, 4 and 5;

[0116] b2 is independently selected from 0, 1, 2, 3, 4 and 5;

[0117] b3 is independently selected from 1, 2, 3, 4 and 5;

[0118] b4 is independently selected from 0, 1, 2, and 3;

[0119] R y1 Each is independently selected from H, methyl, -CH2CH2N(Me)2 or HL;

[0120] R y2 Each is independently selected from H, methyl, -CH2CH2N(Me)2 or HL;

[0121] R y3 Each is independently selected from H, methyl, or -CH2CH2N(Me)2;

[0122] R y4 Each is independently selected from H, methyl, or -CH2CH2N(Me)2;

[0123] R y5 Each is independently selected from H, C1-C4 alkyl, or HL, wherein HL is selected from: Where c1 is selected from 0, 1, or 2; c2 is selected from 2 to 20; c3 is selected from 1 to 20; R y6 Selected from H, methyl, or acetyl groups;

[0124] R y7 Each is independently selected from H, Where c4 is selected from 2-20; c5 is selected from 2-20; c6 is selected from 1, 2, or 3; c7 is selected from 2-20; R y8 Selected from H or methyl.

[0125] In some embodiments, L4 is selected from a straight link, a short peptide consisting of 2-5 amino acid residues, or an amide consisting of amino acid residues and a carboxylic acid / amine.

[0126] In some implementations, L4 is selected from direct-connect key.

[0127] In some embodiments, L4 is selected from amides composed of amino acid residues and carboxylic acid residues, or amides composed of amino acid residues and amine residues.

[0128] In some implementations, L4 is Where p is selected from 2, 3, 4, or 5; R pEach can be independently selected from: H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)Me, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2) p1 N(R z )2、-(CH2)3NHCOMe、-(CH2)3NHCONH2、-(CH2)4NHCONH2、where p1 is selected from 1, 2, 3, 4; R z Each can be independently selected from H, C1-C4 alkyl or HL, where HL is as defined above.

[0129] In some implementations, L4 is Where r is selected from 1 or 2; R p As defined above.

[0130] In some implementations, L4 is selected from:

[0131] Among them, R z Each is independently selected from H, C1-C4 alkyl, or HL, as defined above.

[0132] In some implementations, L4 is selected from:

[0133] In some implementations, L4 is selected from:

[0134] In some implementations, L5 is selected from: direct-connect key,

[0135] Among them, R w1 Each is independently selected from: -CH2NH-HL, -CH2N(Me)-HL, -CH2CH2NH-HL, -CH2CH2N(Me)-HL, Where d1 is selected from 0, 1 or 2, and d2 is selected from 1 to 20;

[0136] R w2 Each is independently selected from: H, Me, -CH2CH2N(Me)2 or -CH2CH2SO2Me;

[0137] R w3 Each is independently selected from: H, Me, -CH2CH2N(Me)2, -CH2CH2SO2Me or -CH2CH2OCH2CH2OH;

[0138] Rw4 Each is independently selected from: H, Me, -CH2CH2N(Me)2, -CH2CH2SO2Me or -CH2CH2OCH2CH2OH;

[0139] R w5 Each is independently selected from: H, Me, or -CH2CH2N(Me)2;

[0140] R w6 Each is independently selected from: H, Me, or -CH2CH2N(Me)2;

[0141] W 1 Each independently selected

[0142] W 2 -L w1 -L w2 or -L w3 -L w4 L w1 It is a short peptide composed of 2-5 amino acid residues; L w2 H, C1-C4 alkyl, (C1-C4 alkyl)-acyl, HL, where HL is as defined above; L w3 An amide composed of a carboxylic acid / amine and 1-3 amino acid residues; L w4 -NH (C1-C4 alkyl), -N (C1-C4 alkyl)2, Where R e It is H or C1-C4 alkyl, and e1 or e3 is each chosen as an integer from 1 to 20;

[0143] Each d is independently selected from 0, 1, or 2.

[0144] In some implementations, L5 is selected from: direct-connect key.

[0145] In some implementations, L5 is selected from

[0146] Where R w6 Each is independently selected from H or methyl;

[0147] Each d is independently selected from 1 or 2;

[0148] W 2 -L w1 -L w2 or -L w3 -L w4 ,

[0149] Among them, L w1 for L w1 N-terminus and Lw2 Connect, p is selected from 2, 3, 4 or 5; R p Each can be independently selected from: H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)Me, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3N(Me)2, -(CH2)3NHCOMe, -(CH2)3NHCONH2, -(CH2)4NH2, -(CH2)4N(Me)2, -(CH2)4N(Et)2, -(CH2)4N(nPr)2, -(CH2)4NHCONH2;

[0150] L w2 For H, methyl, acetyl, HL, where HL is as defined above;

[0151] L w3 for L w3 The left side and L w4 Connect, where r is selected from 1 or 2; R p Each can be independently selected from: H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)Me, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3N(Me)2, -(CH2)3NHCOMe, -(CH2)3NHCONH2, -(CH2)4NH2, -(CH2)4N(Me)2, -(CH2)4N(Et)2, -(CH2)4N(nPr)2, -(CH2)4NHCONH2;

[0152] L w4 -NHMe, -N(Me)2, e1, e2, or e3 can each be chosen from integers between 1 and 20.

[0153] In some implementations, L5 is selected from:

[0154] In some implementations, L5 is selected from:

[0155] In some implementations, -L4-L5- is selected from:

[0156] In some implementations, -L4-L5- is selected from:

[0157] In some implementations, -L1-L2- is selected from:

[0158] In some implementations, -L3- is selected from: direct-connect key,

[0159] In some implementations, -L- is selected from the following structures or the succinimide hydrolysis ring-opening structures of the following structures:

[0160] In some embodiments, the antibody-drug conjugate can be obtained by a coupling reaction between an Ab and the following LP intermediate, wherein the LP intermediate has the following structure:

[0161] L H -L2-L3-L4-L5-P

[0162] Among them, L H -Selected from;

[0163] P, L2, L3, L4, and L5 are as described above.

[0164] In some embodiments, the antibody-drug conjugate can be obtained by a conjugation reaction of Ab and the following LP:

[0165] In some implementations, Ab is an antibody or an antigen ligand.

[0166] In some implementations, Ab is a ligand that binds to a target antigen, wherein the target is highly expressed in tumor cells but lowly expressed or not expressed in normal cells.

[0167] In some embodiments, Ab is an antibody or antigen ligand, and the target of said antibody or antigen is, for example, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ANGPTL4, ASLG659, Axl, B7H3, B7H4, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCL5, CCR5, CCR7, CD123, CD138, CD142, CD147, CD166, CD19. CD22, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD46, CD47, CD49D(ITGA4), CD5 6. CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH3, CDH6, CDH11, CDH17, CD11b, CEA, CEACAM5, CEACAM6, CLDN18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTGF, CTSD, CTSS, CXCL11, CXCL10, CXCR5, DDIT4, DLL3, DLL 4. DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcR H1, FGF2, FGFR2, FGFR3, FLT3, FOLR-α, GD2, GEDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HM OX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KR T33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3C D, PLOD2, PSCA, PSMA, PTK7, RNF43, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STC2, STEAP1, STEAP2, TCF 4. TENB2, TGF, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, etc. .

[0168] In some implementations, Ab is an anti-antigen antibody, and the antigen target is, for example, cMET, CEACAM5, CEACAM6, CDH17, MSLN, HER2, TROP2, EGFR, HER3, B7H3, etc.

[0169] Anti-inflammatory therapies, Ab, Adalimumab, Aducanumab, Alemtuzumab, Altumomab, amivantamab,atezolizumab,anetumab,avelumab,bapineuzumab,basil ximab, bectumomab, bermekimab, besilesomab, bevacizumab, bezlotoximab, brentuximab, brodalumab, catumaxomab, cemiplimab, cetuximab, cin panemab, clivatuzumab, crenezumab, daclizumab, daratumumab, denosumab, dinutuximab, dostarlimab, durvalumab, edrecolomab, elotuzumab, and mapalumab, enfortumab, epcoritamab, epratuzumab, etaracizumab, gemtuzumab, glofitamab, girentuximab, gosuranemab, ibritumomab, inbili zumab, infliximab, inotuzumab, ipilimumab, isatuximab, ixekizumab, J591, labetuzumab, lecanemab, loncastuximab, mirzotamab, mogamulizum ab, mosunetuzumab, necitumumab, nimotuzumab, natalizumab, naratuximab, naxitamab, nivolumab, ocrelizumab, ofatumumab, olaratumab, orego vomab,panitumumab,pembrolizumab,pertuzumab,polatuzumab,prasinezumab,racotumomab,ramucirumab,rituximab,sacituzumab,semorinema b,siltuximab,solanezumab,tacatuzumab,tafasitamab,telesotuzumab,teprotumumab,tilavonemab,tocilizumab,tocitumomab,trastuzumab,tusamitamab, ustekinumab, vedolizumab, votumumab, zagotenemab, zanidatamab, zalutumumab, zanolimumab, or fragments thereof or similar substances.

[0170] In some implementations, the Ab is an anti-cMET antibody, such as Telisotuzumab.

[0171] In some implementations, the Ab is an anti-TROP2 antibody, such as Sacituzumab.

[0172] In some implementations, the Ab is an anti-HER3 antibody, such as Patritumab.

[0173] In some implementations, the Ab is an anti-CEACAM5 antibody, such as Tusamitamab.

[0174] In some implementations, the Ab is an anti-B7H3 antibody, such as Mirzotamab.

[0175] In some implementations, the Ab is an anti-EGFR antibody, such as Cetuximab.

[0176] In some implementations, Ab is a bispecific antibody, such as a bispecific antibody with any combination of the above-mentioned antigen targets.

[0177] In some implementation schemes, the Ab is a bispecific antibody, such as EGFR / c-Met bispecific antibody, EGFR / HER3 bispecific antibody, FOLR1 / TRPV6 bispecific antibody, HER2 / TROP2 bispecific antibody, HER2 / HER2 bispecific antibody, EGFR / MUC1 bispecific antibody, HER3 / TROP2 bispecific antibody, etc.

[0178] In some embodiments, the structure of the antibody-drug conjugate is shown in Formula I-1:

[0179] Among them, Ab, L1, L2, L3, L4, L5, P, and q are as defined above.

[0180] In some embodiments, the antibody-drug conjugate has the structure shown in Formula III-a, Formula III-b, or Formula III-c:

[0181] The groups are as defined above.

[0182] In some embodiments, the antibody-drug conjugate has the structure shown in formula III-d, III-e, III-f, III-g, or III-h:

[0183] in,

[0184] mL is 0, 1, or 2; R 5L Remove one H subunit from R5; R 1L Remove one H subunit from R1; R 4L Remove one H subunit from R4;

[0185] Ab,Cya,A,B,X,Y,Z,R1,R2,R3,R3',R4,R5,L D1 L D2 Cy1, m, q, L1, L2, L3, L4, and L5 are as defined previously.

[0186] In some embodiments, the antibody-drug conjugate structure is shown as in Formula IV-a or Formula IV-b:

[0187] The groups are as defined above.

[0188] In some embodiments, the antibody-drug conjugate structure is shown in Formula IV-e:

[0189] in,

[0190] mL is 0, 1, or 2; R 5L Remove one H subunit from R5;

[0191] Ab,Cya,B,X,Y,Z,R1,R5,L D1 L D2 Cy1, m, q, L1, L2, L3, L4, and L5 are as defined above.

[0192] In some embodiments, the antibody-drug conjugate is selected from the following structures or the succinimide hydrolysis ring-opening structures of the following structures:

[0193] Where Ab is the antibody or its antigen-binding fragment, and q is any value between 1.0 and 16.0;

[0194] Cya said or

[0195] B indicates or

[0196] X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl, wherein the C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl can each be independently represented by 0, 1, 2, or 3 R. x Alternatively, X and Y can form a 3-6 element ring, which can be optionally divided by 0, 1, or 2 R elements. x Alternatively, the ring may further include 0, 1, or 2 heteroatoms selected from N, O, and S;

[0197] Z represents -OR a Or -NR a R a ';

[0198] R1 represents ethyl or trifluoroethyl;

[0199] L D1 L D2 Each can independently represent a single bond or a -(C1-C6) alkylene group;

[0200] Cy1 represents a 4-8 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring.

[0201] R5 can independently represent C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, wherein each of the aforementioned C3-C8 cycloalkyl and 4-8 membered heterocyclic alkyl groups can be independently selected from 0, 1, 2, 3 or 4 ions chosen from halogen, oxo, -OR a -SR a -NR a R a The substituents of ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, and when R5 is a C5-C6 cycloalkyl or 5-6 membered heterocyclic alkyl, the number of substituents is greater than 0;

[0202] R 5L This means that one H subunit has been removed from R5;

[0203] m represents 0, 1, or 2;

[0204] mL represents 0 or 1;

[0205] R xEach can independently represent hydrogen, halogen, -OH, -NH2, cyano, and C1-C3 alkyl;

[0206] R a R a Each of the following can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 heterocyclic alkyl; the alkyl, alkylene, cycloalkyl, cycloalkylene, heterocyclic alkyl or heterocyclic alkylene can each be independently substituted by 0, 1, 2, 3, 4, 5 or 6 halogen atoms.

[0207] In some implementations, -P in Formula I-1 and -P in Formula III-a In Equation III-b In formula III-c In Equation III-d In Equation III-e In formula III-f, In formula III-g In Equation III-h In formula IV-a In formula IV-b In formula IV-e IV-a-1 to IV-a-24 It has a structure selected from that shown in claim 21.

[0208] In some embodiments, the antibody-drug conjugate is selected from the following structures or the succinimide hydrolysis ring-opening structures of the following structures:

[0209] Where Ab is the antibody or its antigen-binding fragment, and q is any value between 1.0 and 16.0;

[0210] For example, Ab is an anti-cMET antibody, such as Telisotuzumab; Ab is an anti-TROP2 antibody, such as Sacituzumab; Ab is an anti-CEACAM5 antibody, such as Tusamitamab; Ab is an anti-HER3 antibody, such as Patritumab.

[0211] For example, q can be any value from 2.0 to 12.0; preferably, q is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12; preferably, q is about 4, about 6, about 8, or about 10.

[0212] Another aspect of the present invention relates to a pharmaceutical composition comprising a compound or antibody-drug conjugate (ADC) as described in the present invention, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotope derivative, N-oxide, or prodrug, and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

[0213] Another aspect of the present invention relates to the use of the aforementioned compound or antibody-drug conjugate (ADC), or its stereoisomers, tautomers, solvates, pharmaceutically acceptable salts, metabolites, isotope derivatives, N-oxides, or prodrugs, or the use of the pharmaceutical composition of the present invention in the preparation of a medicament for the prevention or treatment of cancer-related diseases or conditions, or for the prevention or treatment of cancer-related diseases or conditions. The cancer is a solid tumor or hematologic malignancy, specifically selected from esophageal cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, liver cancer, kidney cancer, head and neck cancer, brain tumor, urothelial carcinoma, skin cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, leukemia, or lymphoma, etc.

[0214] The present invention also relates to a method for preventing or treating cancer-related diseases or conditions, comprising administering to a patient in need of treatment a therapeutically effective amount of the aforementioned compound or antibody-drug conjugate (ADC) of the present invention, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, metabolite, isotope derivative, N-oxide, or prodrug, or a pharmaceutical composition of the present invention. Attached Figure Description

[0215] Figure 1 illustrates the bystander killing effect of antibody-drug conjugate ADC3-4.

[0216] Figure 2 shows the mouse pharmacokinetic results of the antibody-drug conjugate and its corresponding naked antibody.

[0217] Figure 3A shows the efficacy of intravenous administration of ADC drugs in the CL-40 xenograft model.

[0218] Figure 3B shows the changes in body weight in mice in the CL-40 xenograft model after intravenous administration of ADC drugs.

[0219] Figure 4A shows the efficacy of a single intravenous administration of ADC drugs in the HPAC xenograft model.

[0220] Figure 4B shows the changes in body weight in mice in the HPAC xenograft model after a single intravenous administration of ADC drugs.

[0221] Invention Details

[0222] It is particularly noteworthy that, in this article, when referring to “compounds” or “antibody-drug conjugates” with a specific structural formula, this generally also includes their stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotope derivatives.

[0223] As is known to those skilled in the art, the salt, solvate, and hydrate of a compound or antibody-drug conjugate are alternative forms of the compound, and they can all be converted into the compound under certain conditions. Therefore, it is particularly noteworthy that when a compound is mentioned herein, its pharmaceutically acceptable salt is generally also included, and further included, its solvate and hydrate.

[0224] Similarly, when referring to a compound or antibody-drug conjugate in this article, it generally also includes its prodrug, metabolites, and nitrogen oxides.

[0225] The term "stereoisomer" in this invention refers to the enantiomer produced when the compound of formula (I) contains an asymmetric carbon atom; the cis-trans isomer produced when the compound contains a carbon-carbon double bond or a cyclic structure; and the tautomer produced when the compound contains a ketone or oxime. All enantiomers, diastereomers, racemic isomers, cis-trans isomers, tautomers, geometric isomers, epimers, rotational isomers, and mixtures thereof of the compound of formula (I) are included within the scope of this invention.

[0226] The "pharmaceutically acceptable salts" described in this invention refer to pharmaceutically acceptable addition salts of acids and bases or their solvates. Such pharmaceutically acceptable salts include salts of the following acids: hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, sulfurous acid, formic acid, toluenesulfonic acid, methanesulfonic acid, nitric acid, benzoic acid, citric acid, tartaric acid, maleic acid, hydroiodic acid, and alkanes (such as acetic acid, HOOC-(CH2)n-COOH (where n is 0-4)). Salts of bases include: sodium salts, potassium salts, calcium salts, ammonium salts, etc. Many non-toxic pharmaceutically acceptable addition salts are known to those skilled in the art.

[0227] The precursors or metabolites described in this invention can be any precursors or metabolites known in the art, as long as they are metabolized and transformed in vivo to form a compound. For example, "prodrug" refers to those prodrugs of the compounds of this invention that, within a reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly converted in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism.

[0228] definition

[0229] Unless otherwise specified, the terms used in this application (including the specification and claims) are defined as follows. It should be noted that in the specification and appended claims, unless otherwise clearly indicated, the singular form "a" includes the plural meaning. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0230] Unless the context clearly indicates otherwise, the terms “comprising” or “including” should be understood to encompass the listed components or steps, whether the components or steps are presented alone or in combination with one or more additional components or steps.

[0231] As used herein, endpoints are included when providing a range.

[0232] As used herein, the term "about" is used to refer to a numerical value that includes the standard deviation of the error of the means or method used to determine that value. In some embodiments, the term "about" refers to a range of a numerical value along any direction (greater or less than) within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or a lower percentage of that value, unless otherwise specified or otherwise apparent from the context (e.g., when the number would exceed 100% of the possible value).

[0233] In this invention, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to a target cell-associated antigen or receptor. These ligands include, but are not limited to, antibodies, peptides, fusion proteins, nanobodies, or other molecules that can bind to cells, receptors, or antigens.

[0234] The term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0235] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA). The definitions in (86:9268–9272) are provided. For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0236] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0237] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0238] The term "antigen-binding fragment" of an antibody refers to a fragment of a polypeptide, such as a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety." See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single-domain antibodies (sdAb, nanobodies), and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. A review of engineered antibody variants is available in the literature (Holliger et al., 2005, Nat Biotechnol, 23:1126-1136).

[0239] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0240] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0241] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0242] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113; Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv. In some embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFv containing NH2-VH-VH-COOH and NH2-VL-VL-COOH.

[0243] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, referring to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0244] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0245] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0246] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0247] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0248] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0249] The term "semi-humanized antibody" refers to an antibody, in contrast to a humanized antibody or a fully humanized antibody, in which one antibody chain contains a murine variable region (as in chimeric antibodies) and the other antibody chain contains a humanized variable region.

[0250] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. Chimeric antibodies or fragments thereof according to this application can be prepared using gene recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter and a sequence encoding the variable region of a non-human, particularly mouse, monoclonal antibody according to this application, and a sequence encoding the constant region of a human antibody. The chimeric antibody of this application encoded by such a recombinant gene will be, for example, a mouse-human chimera, whose specificity is determined by the variable region derived from mouse DNA and whose isotype is determined by the constant region derived from human DNA.

[0251] The term "monoclonal antibody" or "monoclonal antibody" refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0252] The term "bispecific antibody" or "biantibody" refers to an antibody that simultaneously binds to two antigenic epitopes. These two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two binding regions fused to them (similar to natural antibodies, except that the two arms bind different antigenic markers or epitopes). The antigen-binding regions can be single-chain antibodies (SCVs) or Fab fragments.

[0253] In this invention, the term "antibody or antigen-binding fragment" generally refers to an immunologically binding agent that extends to all antibodies from all species. Antibodies include monoclonal antibodies, polyclonal antibodies, dimers, multimers, proantibodies, chimeric antibodies, fully human antibodies, humanized antibodies, recombinant antibodies, and fragments thereof. An antigen-binding fragment can refer to one or more fragments of an antibody that maintain its ability to specifically bind antigens.

[0254] In this invention, the term "antibody-drug conjugate" or "ADC" refers to a substance obtained by linking a bioactive compound fragment (drug molecule) to an antibody or antigen-binding fragment. In some embodiments of this invention, the bioactive molecule and the targeting moiety are linked via a linker L. This linker can cleave under specific conditions (e.g., hydrolytic enzymes and / or low pH environments within tumors) or under specific actions (e.g., the action of lysosomal proteases), thereby separating the bioactive molecule from the antibody or antigen-binding fragment. In some embodiments of this invention, the bioactive molecule and the antibody or antigen-binding fragment are directly linked by a covalent bond, which can cleave under specific conditions or actions, thereby separating the bioactive molecule from the antibody or antigen-binding fragment.

[0255] In this invention, the term "drug-antibody ratio" or "DAR" refers to the ratio of drug to antibody in an antibody-drug conjugate. For example, it can represent the number of drug molecules linked to each antibody, or the average number of drug molecules linked to a group of ADCs (i.e., average or mean DAR). The DAR of an ADC can be any value within the range of 1 to 20. For example, DAR can be 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 10.0, 12.0, or 20.0.

[0256] In this invention, L1 is linked to Ab via an S atom. As will be understood by those skilled in the art, the present invention can open the disulfide bond of Ab with a reagent (such as the reducing agent TCEP, etc.), and the resulting thiol group reacts with the linker drug conjugate (i.e., LP) (such as a substitution reaction or an addition reaction, etc.) to obtain the antibody-drug conjugate.

[0257] In this invention, when the connection direction of the listed linking groups is not specified, the connection direction is arbitrary, for example... In this case, L stands for -C(O)NH-. -C(O)NH- can be formed by connecting the phenyl group and the cyclohexyl group in a left-to-right reading order. Alternatively, the phenyl and cyclohexyl groups can be connected in the reverse reading order from left to right to form the structure. The combination of the linking group and the linked group is only permitted if it results in a stable compound. In some preferred embodiments of the invention, the order is read from left to right.

[0258] Unless otherwise defined, the substituents in this invention are independent of each other and not related to each other, for example (enumerating rather than exhaustively listing), in one aspect, for R in the substituents a (or R) a Regarding R, it is independent of the definitions of different substituents. Specifically, for R a (or R) a Choosing a meaning among substituents does not mean that R a (or R) a The same meaning applies to all other substituents. More specifically, for example (not exhaustive list only) for NR... a R a In 'middle, when R a (or R) a When the meaning of ') is taken from hydrogen, it does not mean that in -OR a Or -C(O)-NR a R a R in ' a (or R)a ') must be hydrogen, and they can each independently represent those selected from R a (or R) a Other substituents in the definition of '). In another aspect, when a substituent contains more than one R... a (or R) a When '), these R a (or R) a ') are also independent. For example, in the substituent -(CR a R a’ ) m -O-(CR a R a’ ) n In the case where m+n is greater than or equal to 2, there are m+n R values. a (or R) a ') are independent and can represent selections from R a (or R) a The same or different substituents in the definition of ').

[0259] Unless otherwise defined, the meaning of "substituted by x A substituents or B substituents" as described in this invention is the same as "substituted by x substituents selected from A and B," and when the number of substituents is greater than 1, the x substituents may be the same or different. For example, "R1 may be substituted by 0, 1, or 2 R..." x "Replace" means that R1 can be optionally selected from R1 by 0, 1, or 2. x Substituents are substituted, and when the number of substituents is greater than one, these substituents can be derived from R. x Refers to the same or different substituents. For example, "R1 may be substituted with 0, 1 or 2 H, C1-C3 alkyl or C3-C6 cycloalkyl" means that R1 may optionally be substituted with 0, 1 or 2 substituents selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, and when the number of substituents is greater than 1, these substituents may be the same or different. For example, when the number of substituents is 2, these 2 substituents may be, for example, 2 H, may be, for example, 2 C1-C3 alkyl, may be, for example, one is H and the other is C1-C3 alkyl, may be, for example, one is C1-C3 alkyl and the other is C3-C6 cycloalkyl.

[0260] Unless otherwise defined, the phrase "and the two substituents form a ring" as described in this invention means that two monovalent or polyvalent residues derived from the removal of one or more H atoms from each of the two substituents or any group form one or more covalent bonds, which can be single, double, or triple bonds, thereby forming a cyclic structure together with the same atom attached to them, or together with different atoms attached to them and the atoms between them. This description is a description of the structure, without regard to whether the two substituents can form a ring through a chemical reaction or what kind of chemical reaction is required to form a ring. All stable or chemically feasible cyclic structures formed by the above description are included within the scope of this invention. If further characteristic descriptions are provided (e.g., "forming a 4-12 membered heterocycle", "the ring comprises a monocyclic, bridged, or spirocyclic ring", "the ring may also contain one or two heteroatoms selected from N, O, or S", or "the ring may also be substituted by one or two substituents selected from Rz"), then the above-described cyclic structure may additionally have the features described therein.

[0261] The term “optionally substituted X” (e.g., “optionally substituted alkyl”) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein the alkyl group is optionally substituted”). It is not intended that the characteristic “X” (e.g., alkyl) itself is optional. As described herein, certain compounds may contain one or more “optionally substituted” moieties. Generally, the term “substituted”, whether preceded by the terms “optionally” or “arbitrarily”, means that one or more hydrogens of the specified moiety are replaced by suitable substituents, such as any of the substituents or groups described herein. Unless otherwise indicated, a “optionally substituted” group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. For example, in the term “optionally substituted C1-C6 alkyl-C5-C6 heteroaryl,” the alkyl moiety, the heteroaryl moiety, or both may be optionally substituted. The combinations of substituents contemplated in this disclosure are preferably combinations that form stable or chemically viable compounds. As used herein, the term "stable" means that a compound remains substantially unchanged when subjected to conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for one or more of the purposes disclosed herein. In this document, "any substitution" has the same meaning as "optional substitution." Unless otherwise defined, "optional substitution" can refer to substitution by a monovalent substituent and / or a divalent substituent.For example, the monovalent substituent is selected from the following substituents, such as hydrogen, alkyl, hydroxyalkyl, aminoalkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic, halogen, hydroxyl, alkoxy, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino, disubstituted amino (where the two amino substituents are selected from alkyl, aryl, or arylalkyl), alkanoylamino, arylanoylamino, arylalkylanoylamino, substituted alkanoylamino, substituted arylamino, substituted arylalkylanoylamino, thio, alkylthio, arylthiothio, arylalkylthio, arylthiocarbonyl, arylalkylthiocarbonyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, aminosulfonyl (e.g., -SO2NH2), substituted sulfonylamino, nitro, cyano, carboxyl, ammonia The substituents may be alkyl, such as -CONH2; substituted carbamoyl, such as -CONHalkyl, -CONHaryl, -CONHarylalkyl; or have two substituents selected from alkyl, aryl, or arylalkyl on nitrogen; alkoxycarbonyl; aryl; substituted aryl; guanidine; heterocyclic, such as indolyl; imidazolyl; furanyl; thiophene; thiazolyl; pyrrolyl; pyridyl; pyrimidinyl; pyrrolyl; piperidinyl; morpholinyl; piperazinyl; homopiperazinyl; and substituted heterocyclic groups; for example, divalent substituents may be selected from the following substituents: =O, =S, =NNRr2, =NNHC(O)Rr, =NNHC(O)ORr, =NNHS(O)2Rr, =NRr, =NORr, and alkylene (e.g., -(C(Rr2)). 2-3 -、-(C(Rr2)) 2-3 O-、-O(C(Rr2)) 2-3 -、-O(C(Rr2)) 2-3 O-, -S(C(Rr2))2-3S-), etc., where Rr can represent hydrogen, alkyl, heteroalkyl, aromatic, heteroaryl, etc.

[0262] Unless otherwise defined, the terms “single bond” or “bond” or “direct bond” as used herein refer to two atoms connected by a single saturated covalent bond. For example, when L represents a single bond, “ALB” means that A and B are connected by a single saturated covalent bond, i.e., “AB”; as another example, when L represents a single bond, “-CH2-L-NH-” means that -CH2- and -NH- are connected by a single saturated covalent bond, i.e., “-CH2-NH-”.

[0263] As used herein, the term "alkyl" or "alkylene" is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, "C1-C6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl). Alkyl groups can be unsubstituted or substituted, and when substituted, they can be substituted at any usable link, preferably from one or more of hydrogen, deuterium, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In this document, alkyl is an alkyl group having 1 to 12 carbon atoms, preferably having 1 to 10, 1 to 8, 1 to 6, 1 to 4, and more preferably having 1 to 4 carbon atoms.

[0264] As used herein, the term "alkylene" is intended to include branched, straight-chain, saturated aliphatic hydrocarbon groups having a specified number of carbon atoms, comprising or not comprising cyclic alkyl groups, which are residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms. For example, "C0-C6 alkylene" means an alkylene having 0 (i.e., bond), 1, 2, 3, 4, 5, or 6 carbon atoms. Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (e.g., -(CH2)3-, -(CHCH3)CH2-, -(CHCH2CH)-), butylene (e.g., -(CH2)4-, -CH2CH(CH2CH3)-, -CH2(CHCH2CH)-, etc.), and pentylene (e.g., -(CH2)5-, -CH2CH(CH(CH3)2)-, -CH2(CH... (e.g., CH2CH)CH2-), hexanediol (e.g., -(CH2)6-, -CH2CH2CH(CH(CH3)2)-, -CH2(CHCH(CH3)CH)CH2-, etc.). In this document, alkylene groups are preferably alkylene groups having 0-6, 0-4, 0-3, 0-2, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-3 carbon atoms. In this document, alkylene groups are preferably alkylene groups that do not contain cyclic alkyl groups.

[0265] Similarly, the term "X-subgroup" or "X-subgroup" as used herein is intended to include divalent residues derived by removing two hydrogen atoms from the same atom or two different atoms of the parent compound X. The parent compound X is defined as in other paragraphs herein. For example, X can be alkyl, cycloalkyl, heterocycloalkyl, or phenyl, and correspondingly, X-subgroup represents alkylene, cycloalkylene, heterocycloalkylene, or phenylene.

[0266] The term "cycloalkyl" refers to monocyclic, polycyclic, or branched cycloalkyl groups. For example, C3-C 12 Cyclic alkyl groups, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and norbornel. Branched cycloalkyl groups such as 1-methylcyclopropyl and 2-methylcyclopropyl are included in the definition of "cycloalkyl". In this invention, cycloalkyl groups are preferably saturated carbocyclic. Polycyclic cycloalkyl groups, such as bicyclic and tricyclic cycloalkyl groups, include bridged rings, spirocyclic, or fused ring cycloalkyl groups. In this invention, cycloalkyl groups are preferably C3-C6. 12 Cycloalkyl, C3-C8 cycloalkyl, C8-C 12 Cycloalkyl, C3-C7 cycloalkyl, C8-C 12 Cycloalkyl, C4-C8 cycloalkyl, C5-C 10 Cycloalkyl, C3-C6 cycloalkyl. For example, in some embodiments, the cycloalkyl group in monocyclic form is C3-C8, C3-C6, or C5-C6. In some embodiments, the cycloalkyl group in bicyclic form is C7-C6. 12 In some embodiments, the cycloalkyl group in spirocyclic form is C5-C6. 12 Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Exemplary arrangements of bicyclic cycloalkyl groups having 7 to 12 ring atoms include, but are not limited to, [4,4], [4,5], [5,5], [5,6], or [6,6] ring systems. Exemplary bridging bicyclic cycloalkyl groups include, but are not limited to, bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, and bicyclic [3.2.2]nonane. Examples of spirocycloalkyl groups include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane, and spiro[4.5]decane. The cycloalkyl group can be unsubstituted or substituted. When substituted, it can be substituted at any usable linker. The substituent is preferably one or more of halogen, hydroxyl, amino, cyano, oxo, alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. According to common practice in the art, a cycloalkyl group can be monocyclic without being specifically indicated in the text. For example, unless otherwise specified, "the C3-C8 cycloalkyl group can be spirocyclic, bridged, or fused" is equivalent to "the C3-C8 cycloalkyl group can be monocyclic, spirocyclic, bridged, or fused."

[0267] The term “heteroalkyl” refers to an alkyl group as defined herein, wherein one or more carbon atoms in the chain are replaced by heteroatoms selected from O, S and N.

[0268] Similarly, the term "heterocyclic alkyl" refers to a cyclic structure in which at least one carbon atom in the cycloalkyl ring is replaced by a heteroatom selected from N, O, S, and P. The N atom may optionally be quaternized, and the N and S atoms may optionally be oxidized (i.e., NO, SO, and SO2). It includes monocyclic, bicyclic, and tricyclic heterocyclic systems, wherein bicyclic and tricyclic heterocyclic systems include spirocyclic, fused, and bridged heterocyclic systems. Heterocyclic alkyl groups can be unsubstituted or substituted, and when substituted, they can be substituted at any usable junction. The substituents are preferably one or more selected from halogens, hydroxyl groups, amino groups, cyano groups, oxo groups, alkyl groups, alkoxy groups, haloalkyl groups, cycloalkyl groups, heterocyclic alkyl groups, aryl groups, and heteroaryl groups. In this invention, the heterocyclic alkyl group is preferably a 4-12 membered heterocyclic alkyl group, more preferably a 4-8 membered heterocyclic alkyl group. According to common practice in the art, a heterocyclic alkyl group may be monocyclic without special indication herein. For example, unless otherwise specified, "the C3-C8 heterocyclic alkyl group can be a spirocyclic, bridged, or fused ring" is equivalent to "the C3-C8 heterocyclic alkyl group can be a monocyclic, spirocyclic, bridged, or fused ring".

[0269] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing one or more carbon-carbon double bonds and typically having a length of 2 to 20 carbon atoms, and includes groups having "cis" and "trans" orientations, or optionally "E" and "Z" orientations. For example, "C2-C6 alkenyl" is an alkenyl group containing two to six carbon atoms and having one, two, or three carbon-carbon double bonds. In some instances, the alkenyl group is C2-C... 18 alkenyl, C2-C 16 alkenyl, C2-C 14 alkenyl, C2-C 12 alkenyl, C2-C 10 The alkenyl group can be C2-C8, C2-C6, C2-C4, or C2-C3. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, and 1-methyl-2-buten-1-yl. In this invention, the alkenyl group is preferably C2-C6. In this invention, the alkenyl group preferably contains one or two double bonds, more preferably one double bond.

[0270] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing one or more carbon-carbon triple bonds and typically ranging from 2 to 20 carbon atoms in length. For example, "C2-C6 alkynyl" is an alkynyl group containing two to six carbon atoms and having one, two, or three carbon-carbon triple bonds. In some instances, the alkenyl group is C2-C... 18 alkynyl group, C2-C 16 alkynyl group, C2-C 14 alkynyl group, C2-C 12 alkynyl group, C2-C 10The alkynyl group can be C2-C8, C2-C6, C2-C4, or C2-C3. Representative alkynyl groups include, but are not limited to, ethynyl, propynyl-1-yl (-C≡C-CH2), propynyl-2-yl (propynyl, -CH2-C≡CH), butynyl-1-yl, butynyl-2-yl, and butynyl-3-yl. In this document, the alkynyl group is preferably C2-C6 alkynyl.

[0271] The term "cycloalkenyl" refers to a non-aromatic hydrocarbon cyclic group having at least one carbon-carbon double bond. Cycloalkenyl encompasses monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged cyclic systems. Examples include C3-C8 cyclic alkenyl groups, including but not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and norcamphenyl. Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohexen-3-enyl, and cyclohexadienyl. Exemplary arrangements of bicyclic cycloalkenyl groups having 7 to 12 ring atoms include, but are not limited to, [4,4], [4,5], [5,5], [5,6], or [6,6] cyclic systems. Exemplary bridging bicyclic cycloalkenyl groups include, but are not limited to, bicyclic [2.2.1]heptene, bicyclic [2.2.2]octene, and bicyclic [3.2.2]nonene. Examples of spirocycloalkyl groups include spiro[2.2]pentene, spiro[2.3]hexene, spiro[2.4]heptene, spiro[2.5]octene, and spiro[4.5]decene. Branched cycloalkenyl groups such as 1-methylcyclopropenyl and 2-methylcyclopropenyl are also included in the definition of "cycloalkenyl". In some embodiments of the invention, the cycloalkenyl group is C3-C5 cycloalkenyl, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl, C3-C8 cycloalkenyl, C3-C9 cycloalkenyl, C3-C 10 Cycloalkenyl, C3-C 11 Cycloalkenyl, C3-C 12 Cycloalkenyl.

[0272] Similarly, "cycloalkynyl" refers to a non-aromatic hydrocarbon cyclic group having at least one carbon-carbon triple bond. Cycloalkynyl groups encompass monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged ring systems. In some embodiments of the present invention, the cycloalkynyl group is C3-C5 cycloalkynyl, C3-C6 cycloalkynyl, C3-C7 cycloalkynyl, C3-C8 cycloalkynyl, C3-C9 cycloalkynyl, C3-C... 10 Cycloalkynyl, C3-C 11 Cycloalkynyl, C3-C 12 Cycloacetic group.

[0273] Similarly, the term "heterocyclic alkenyl" refers to a non-aromatic heterocyclic group having at least one carbon-carbon double bond. Heterocyclic alkenyls encompass monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged ring systems.

[0274] Similarly, the term "heterocyclic ynyl" refers to a non-aromatic heterocyclic group having at least one carbon-carbon triple bond. Heterocyclic ynyl encompasses monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged ring systems.

[0275] The term "aryl" or "aromatic ring" refers to a carbocyclic aromatic group having a specified number of carbon atoms. If the number of carbon atoms is not specified, it is at most 14 carbon atoms. The aryl group can be unsubstituted or substituted, and when substituted, it can be substituted at any usable junction. The substituent is preferably one or more of deuterium, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments of the invention, the aryl group includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, 2-naphthyl, etc.

[0276] The term "heteroaryl" refers to an aromatic heterocycle having at least one monocyclic or fused polycyclic ring selected from oxygen, nitrogen, and sulfur. Suitable heteroaryl groups do not include ring systems such as pyranium that must be charged to be aromatic. Heteroaryl groups can be stable 5-, 6-, or 7-membered aromatic monocyclic or bicyclic, or 7-, 8-, 9-, 10-, 11-, or 12-membered aromatic polycyclic heterocycles. A suitable 5-membered heteroaryl ring (as a monocyclic heteroaryl or as part of a polycyclic heteroaryl) has one oxygen, sulfur, or nitrogen ring atom, or one nitrogen plus one oxygen or sulfur, or 2, 3, or 4 nitrogen ring atoms. A suitable 6-membered heteroaryl ring (as a monocyclic heteroaryl or as part of a polycyclic heteroaryl) has 1, 2, or 3 nitrogen ring atoms. The nitrogen in the heterocycle may optionally be quaternized. Preferably, when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. Preferably, the total number of S and O atoms in the heterocycle is not greater than 1. The heteroaryl group can be unsubstituted or substituted, and if the resulting compound is stable, the heterocyclic group described herein can be substituted at any usable connection point, wherein the substituent is preferably one or more of halogen, hydroxyl, amino, cyano, alkyl, alkoxy, haloalkyl, cycloalkyl, heterocyclic alkyl, aryl and heteroaryl. Examples of heteroaryl groups include, but are not limited to, pyridinyl, imidazolyl, imidazopyridinyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrroleyl, quinolinyl, isoquinolinyl, indoleyl, benzimidazolyl, benzofuranyl, cenolinyl, indazolyl, indazinyl, phthalazinyl, pyridazinyl, triazinyl, isoindoleyl, pteridinyl, purine, oxadiazolyl, triazolyl, thiazolyl, furazanyl, benzofuranyl, benzothiopheneyl, benzothiazolyl, benzooxazolyl, quinazolinyl, quinoxolinyl, naphthidyl, and furopyridyl. The term "heteroaryl" may also include biaryl structures formed by an "aryl" as defined above and a monocyclic "heteroaryl", such as, but not limited to, "-phenylbipyridinyl-", "-phenylbipyrimidinyl", "-pyridylbiphenyl", "-pyridylbipyrimidinyl-", and "-pyrimidinylbiphenyl-"; wherein the present invention also includes fused-ring and spirocyclic compounds containing, for example, the rings described above.

[0277] The term "heterocyclic" or "heterocyclic group" refers to any monocyclic, bicyclic, polycyclic, fused, spirocyclic, or bridged non-aromatic ring system that is fully saturated, partially unsaturated, or fully unsaturated, having, for example, 3 to 20 ring atoms, wherein the ring atoms are carbon, and at least one carbon atom is replaced by a heteroatom selected from nitrogen, sulfur, or oxygen. If any ring atom in the cyclic system is a heteroatom, the system is a heterocyclic system, regardless of the connection points between the cyclic system and the rest of the molecule. In one example, a heterocyclic group comprises 3-11 ring atoms ("members") and includes monocyclic, bicyclic, tricyclic, spirocyclic, and bridged ring systems, wherein the ring atoms are carbon, and at least one atom in the ring or cyclic system is a heteroatom selected from nitrogen, sulfur, or oxygen. In other examples, a heterocyclic group comprises 4-10 or 5-10 ring atoms. In one example, a heterocyclic group comprises 1 to 4 heteroatoms. In one example, a heterocyclic group comprises 1 to 3 heteroatoms. In another example, the heterocyclic group comprises a 3- to 7-membered monocyclic ring having 1-2, 1-3, or 1-4 heteroatoms selected from nitrogen, sulfur, or oxygen. In another example, the heterocyclic group comprises a 4- to 6-membered monocyclic ring having 1-2, 1-3, or 1-4 heteroatoms selected from nitrogen, sulfur, or oxygen. In another example, the heterocyclic group comprises a 3-membered monocyclic ring. In another example, the heterocyclic group comprises a 4-membered monocyclic ring. In another example, the heterocyclic group comprises a 5- to 6-membered monocyclic ring. In some embodiments, the heterocyclic alkyl group comprises at least one nitrogen atom. In one example, the heterocyclic group comprises 0 to 3 double bonds. Any nitrogen or sulfur heteroatom may optionally be oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom may optionally be quaternized (e.g., [NR4+]Cl-, [NR4+]OH-).Examples of heterocycles include ethylene oxide, aziridinyl, thiohexacyclopropane, aziridinyl, oxetane, thiohexacyclobutane, 1,2-dithiohexacyclobutane, 1,3-dithiohexacyclobutane, pyrrolyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, imidazoalkyl, piperidinyl, piperazinyl, isoquinolinyl, tetrahydroisoquinolinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinyl, thiazinyl, thioxanyl, homopiperazinyl, and homopiperidinyl. ridinyl), azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazaphene, triazaphene, thiazepanyl, tetrahydrothiaranyl, oxazolyl, thiazolyl, isothiazolyl, 1,1-dioxoisothiazolinyl, 1,1-dioxoisothiazolyl, oxazolidinyl, imidazolinone, 4,5,6,7-tetrahydro[2H]inzolyl, tetrahydrobenzo[2H]-benzyl Imidazolyl, 4,5,6,7-tetrahydrobenzo[d]imidazolyl, thiazinyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxahiazinyl, thiatriazinyl, oxtriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, indololinyl, thiaranyl, 2H-pyranyl, 4H-pyranyl, dioxalyl, 1,3-dioxolanecycloyl, pyrazolinyl, pyrazolylalkyl, dithiopheneyl, dithiohexacyclopentyl, pyrimidinone, pyrimidinedione, pyrimidin-2,4-dicarboxyl, piperazinoneyl, piperazinedioneyl, pyrazolylalkyliminoimidazolinyl, 3-azabicyclo[3.1.0]hexyl, 3,6-diazabicyclo[3.1.0] 1] Heptyl, 6-azabicyclo[3.1.1]heptyl, 3-azabicyclo[3.1.1]heptyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 2-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl, 2-azabicyclo[2.2.2]octyl, 8-azabicyclo[2.2.2]octyl, 7-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonyl, azaspiro[2.5]octyl, azaspiro[4.5]decyl, 1-azaspiro[4.5]dec-2-yl, azaspiro[5.5]undecane, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindolyl, 1,1-dioxahexahydrothiopyranyl.

[0278] Similarly, the terms "carbocyclic" or "carbocyclic group" or "cyclic hydrocarbon group" refer to any monocyclic, bicyclic, polycyclic, fused, spirocyclic, or bridged non-aromatic ring system that is fully saturated, partially unsaturated, or fully unsaturated, having, for example, 3 to 20 ring atoms, wherein said ring atoms are carbon.

[0279] In a specific embodiment, the heterocyclic group or the heteroaryl group is attached at a carbon atom of the heterocyclic group or the heteroaryl group. By way of example, carbon-bonded heterocyclic groups include the following bonding arrangements: at positions 2, 3, 4, 5, or 6 of the pyridine ring; at positions 3, 4, 5, or 6 of the pyridazine ring; at positions 2, 4, 5, or 6 of the pyrazine ring; at positions 2, 3, 5, or 6 of the furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole rings; at positions 2, 4, or 5 of the oxazole, imidazole, or thiazole rings; at positions 3, 4, or 5 of the isoxazole, pyrazole, or isothiazole rings; at positions 2 or 3 of the aziridine ring; at positions 2, 3, or 4 of the azacyclic butane ring; at positions 2, 3, 4, 5, 6, 7, or 8 of the quinoline ring; or at positions 1, 3, 4, 5, 6, 7, or 8 of the isoquinoline ring.

[0280] In some embodiments, the heterocyclic or heteroaryl group is N-linked. By way of example, nitrogen-bonded heterocyclic or heteroaryl groups include the following bonding arrangements: at the 1 position of aziridine, aziridine, pyrrole, pyrrolidine, 2-pyrrololine, 3-pyrrololine, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindole, 1H-indazole, at the 2 position of isoindole or isodihydroindole, at the 4 position of morpholine, and at the 9 position of carbazole or β-carboline.

[0281] In this invention, the term "fused ring" or "fused ring" refers to a polycyclic group formed by two or more ring structures sharing two adjacent atoms.

[0282] In this invention, the term "bridged ring" refers to a polycyclic group in which two rings in the system share two or more ring atoms.

[0283] In this invention, the term "spirocyclic" refers to a polycyclic group in which single rings share a single carbon atom (called a spiro atom).

[0284] The term "alkoxy" or "alkyloxy" refers to -O-alkyl. For example, "C1-C6 alkoxy" (or alkyloxy) is intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and tert-butoxy. In this document, alkoxy groups are preferably alkoxy groups having 1 to 6, more preferably 1 to 4, carbon atoms. Similarly, "alkylthio" or "thioalkoxy" refers to an alkyl group as defined above that is bridging a sulfur group and has a specified number of carbon atoms; for example, -S-methyl and -S-ethyl. Alkoxy groups can be unsubstituted or substituted, and when substituted, they can be substituted at any usable connection point, wherein the substituent is preferably one or more of deuterium, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0285] In this invention, "halogenated" or "halogen" includes fluorine, chlorine, bromine, and iodine. "Haloalkyl" / "halogenated alkylene" is intended to include branched and straight-chain saturated alkyl / alkylene groups having a specified number of carbon atoms and substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl. Similarly, "halogenated cycloalkyl" / "halogenated heterocycloalkyl" is intended to include cycloalkyl / heterocycloalkyl groups having a specified number of carbon atoms and substituted with one or more halogens. In this invention, the halogen atom is preferably fluorine or chlorine, more preferably fluorine. In this document, unless specifically stated that a certain alkyl, cycloalkyl, heterocycloalkyl, or alkylene group cannot be substituted with a halogen, or can be inferred from the context that the group cannot be halogenated, or is considered unsuitable for halogenation based on common knowledge in the art, then these groups are considered to be halogenated, for example, substituted with one, two, three, or four halogens; for example, substituted with one, two, or three halogens; for example, substituted with one or two halogens; for example, substituted with one halogen; in some other preferred embodiments of the invention, these groups are not halogenated.

[0286] "Haloalkoxy" or "haloalkyloxy" means a haloalkyl group as defined above that is oxygen-bridged and has a specified number of carbon atoms. For example, "haloC1-C6 alkoxy" is intended to include C1, C2, C3, C4, C5, and C6 haloalkoxy groups. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluoroethoxy. Similarly, "haloalkylthio" or "thiohaloalkoxy" means a haloalkyl group as defined above that is sulfur-bridged and has a specified number of carbon atoms; for example, trifluoromethyl-S- and pentafluoroethyl-S-.

[0287] In this text, "oxo" means that at least two hydrogen atoms or bonding electrons on at least one atom of a specified group are replaced by the =O atom. Oxidation can occur on carbon atoms and / or heteroatoms. For example, oxoation on a C atom can oxidize -CH2- to -C(=O)-; oxoation on a S atom can oxidize -S- to -S(=O)- or -S(=O)2-. A specified group can have 0, 1, 2, 3, 4, or even more atoms oxidized.

[0288] In this paper, the lines drawn from the ring system indicate that the bond can be attached to any suitable ring atom. If the ring is a bicyclic fused ring system, the substituent can be attached to any position on either ring in the bicyclic system.

[0289] In this document, wavy lines intersecting with bonds in a chemical structure represent the connection points between atoms or groups connected to wavy bonds in the chemical structure and the remainder of the molecule or molecular segment. When a chemical structure contains two wavy lines intersecting with bonds, the structure can be connected to the remainder of the molecule or molecular segment in either orientation.

[0290] As used herein, in the context of describing adjacent atoms, the term "adjacent" refers to two atoms directly connected by a covalent bond.

[0291] In some embodiments, the divalent group generally described does not have a specific bonding configuration. It should be understood that, unless otherwise stated, the general description is intended to include two bonding structures. For example, in the group R1-R2-R3, if group R2 is described as -CH2C(O)-, it should be understood that the group can be bonded as R1-CH2C(O)-R3 and R1-C(O)CH2-R3, unless otherwise stated.

[0292] As used herein, the term "substitution" means the replacement of at least one hydrogen atom with a non-hydrogen group, provided that the normal valence is maintained and the substitution results in a stable compound. The cyclic double bond used herein refers to a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N).

[0293] In this disclosure, C is used when referring to certain substituent groups. x1 -C x2The expression indicates that the number of carbon atoms in the substituent group can be x1 to x2. For example, C0-C8 indicates that the group contains 0, 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms; C1-C8 indicates that the group contains 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms; C2-C8 indicates that the group contains 2, 3, 4, 5, 6, 7, or 8 carbon atoms; C3-C8 indicates that the group contains 3, 4, 5, 6, 7, or 8 carbon atoms; C4-C8 indicates that the group contains 4, 5, 6, 7, or 8 carbon atoms; C0-C6 indicates that the group contains 0, 1, 2, 3, 4, 5, or 6 carbon atoms; C1-C6 indicates that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms; C2-C6 indicates that the group contains 2, 3, 4, 5, or 6 carbon atoms; and C3-C6 indicates that the group contains 3, 4, 5, or 6 carbon atoms.

[0294] In this disclosure, when referring to cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, and heterocycloalkyl), the expression "x1-x2 membered ring" is used, indicating that the number of ring atoms in the group can be x1 to x2. For example, the 3-12 membered cyclic group can be a 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 membered ring, and its number of ring atoms can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; a 3-6 membered ring indicates that the cyclic group can be a 3, 4, 5, or 6 membered ring, and its number of ring atoms can be 3, 4, 5, or 6; a 3-8 membered ring indicates that the cyclic group can be a 3, 4, 5, 6, 7, or 8 membered ring, and its number of ring atoms can be 3, 4, 5, 6, 7, or 8; a 3-9 membered ring indicates that the cyclic group can be a 3, 4, 5, 6, 7, 8, or 9 membered ring, and its number of ring atoms can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; 8 or 9; 4-7 membered ring indicates that the cyclic group can be a 4, 5, 6, or 7 membered ring, and its number of ring atoms can be 4, 5, 6, or 7; 5-8 membered ring indicates that the cyclic group can be a 5, 6, 7, or 8 membered ring, and its number of ring atoms can be 5, 6, 7, or 8; 5-12 membered ring indicates that the cyclic group can be a 5, 6, 7, 8, 9, 10, 11, or 12 membered ring, and its number of ring atoms can be 5, 6, 7, 8, 9, 10, 11, or 12; 6-12 membered ring indicates that the cyclic group can be a 6, 7, 8, 9, 10, 11, or 12 membered ring, and its number of ring atoms can be 6, 7, 8, 9, 10, 11, or 12. The ring atoms can be carbon atoms or heteroatoms, for example, heteroatoms selected from N, O, and S. When the ring is a heterocycle, the heterocycle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cyclic heteroatoms, for example heteroatoms selected from N, O and S.

[0295] In cases where nitrogen atoms (e.g., amines) are present on the compounds of the present invention, these nitrogen atoms can be converted into N-oxides by treatment with an oxidizing agent (e.g., mCPBA and / or hydrogen peroxide) to obtain other compounds of the present invention. Therefore, the nitrogen atoms shown and claimed are considered to encompass both the shown nitrogen and its N-oxides to obtain derivatives of the present invention.

[0296] When any variable appears more than once in any composition or formula of a compound, its definition for each occurrence is independent of its definition for each other occurrence. Thus, for example, if a substituent group is shown to have 0-3 R groups, the substituent group may optionally be substituted with up to three R groups, and each occurrence of R is independently selected from the definition of R. Furthermore, combinations of substituents and / or variables are only permitted if such combinations produce a stable compound.

[0297] Unless otherwise stated, the terms “compound(s) of the invention” and “compound(s) of the present invention” include compounds of general formula and compounds listed in the specific list, including their stereoisomers, tautomers, solvates, precursors, metabolites, isotope derivatives and salts (e.g., pharmaceutically acceptable salts).

[0298] The "stereoisomerism" described in this invention is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism (i.e., geometric isomerism) and optical isomerism (also called enantiomerism). Conformational isomerism refers to the phenomenon where organic molecules with a certain configuration exhibit different spatial arrangements of atoms or atomic groups due to the rotation or twisting of carbon atoms or carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair and boat conformations in cyclohexane. Cis-trans isomers are isomers caused by the presence of C=C double bonds, C=N double bonds, or ring systems, which make rotation difficult, and are usually represented by Z and E. Optical isomers, also called enantiomers, refer to two stereoisomers of a compound that are non-overlapping mirror images of each other. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center. The prefixes d and l, or (+) and (-), are used to indicate the rotational sign of a compound with respect to plane-polarized light, where (-) or 1 indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Mixtures of enantiomers are generally referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur in a chemical reaction or process without stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two optically inactive enantiomer species. The compounds of the present invention may contain one or more asymmetric carbon atoms. Therefore, the compounds may exist in the form of diastereomers, enantiomers, or mixtures thereof.

[0299] The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that interconvert through low-barrier transformations. For example, proton tautomers (also known as proton-transformed tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. Valence tautomers include interconversions that occur through the recombination of some bonded electrons. The compounds described in this invention can exist in tautomer forms, having different hydrogen bonding sites through one or more double bond shifts.

[0300] The term "chirality" refers to a molecule that does not overlap with its mirror-image partner, while the term "chirality" refers to a molecule that can overlap with its mirror-image partner.

[0301] The term "diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral properties, or biological activity. Mixtures of diastereomers can be separated using high-resolution analytical procedures (such as electrophoresis) and chromatographic methods (such as HPLC).

[0302] The stereochemical definitions and conventions used in this article generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

[0303] All enantiomers, diastereomers, racemates, mesoomers, cis-trans isomers, trans-blocked isomers, tautomers, and mixtures thereof are included within the scope of this invention. All methods for preparing the compounds of this invention and the intermediates therein are considered part of this invention. When preparing enantiomers or diastereomers, they can be separated by conventional methods (e.g., by chromatography or fractional crystallization). The free forms and salts of these end products are within the scope of this invention. If desired, one form of the compound can be converted to another. Free bases or acids can be converted to salts; salts can be converted to free compounds or another salt; mixtures of isomers of this invention can be separated into individual isomers. The compounds of this invention, their free forms, and salts can exist in various tautomer forms, wherein hydrogen atoms are transposed to other parts of the molecule and thus the chemical bonds between the atoms of the molecule are rearranged. It should be understood that all possible tautomer forms are included within this invention.

[0304] In the structures shown herein, where the stereochemistry of any specific chiral atom is not specified, all stereoisomers are considered as and included in the compound of the invention. When the stereochemistry is specified by a solid wedge or dashed line indicating a specific configuration, the stereoisomer is specified and defined. Unless otherwise stated, the use of a solid wedge or dashed line signifies relative stereochemistry.

[0305] In this invention, the term "pharmaceutical-acceptable salt" or "pharmaceutically acceptable salt" means that, within a reasonable medical judgment, it is suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. The salt can be prepared in situ during the final separation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent, as outlined below. For example, the free base functional group can react with a suitable acid, which can be an organic or inorganic acid; the free acid functional group can react with a suitable base, which can be an organic or inorganic base. Examples of pharmaceutically acceptable inorganic acid addition salts are salts formed by amino groups with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed using other methods in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, sodium alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, disaccharide, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-enolate, glyceryl phosphate, gluconate, hernisulfate, heptaate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pyrate, pectinate, persulfate, 3-phenylpropionate, phosphate, bitter salts, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically usable salts include (where appropriate) non-toxic ammonium salts, quaternary ammonium salts, and amine cations formed by counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0306] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example by dissolving the compounds of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol, and acetonitrile), adding an excess of an aqueous solution of an organic or inorganic acid to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid, and then separating the precipitated salt.

[0307] The precursors or metabolites described in this invention can be precursors or metabolites known in the art, as long as they are metabolized and transformed in vivo to form compounds. For example, "prodrug" refers to those prodrugs of the compounds of this invention that, within a reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism, or through N-demethylation of the compounds of this invention.

[0308] The term "solvate" as used in this invention refers to the physical association of the compound of this invention with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0309] The term "isotope derivative" in this invention refers to molecules in which the compounds described herein are isotopically labeled. Commonly used isotopes for isotopic labeling are hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 11 C, 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Tritium, in particular. 3 H and carbon 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2Substitution with H can enhance metabolic stability and prolong the half-life, thereby reducing the dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0310] In this invention, the term "patient" refers to an organism treated by the method of this invention. Such organisms preferably include, but are not limited to, mammals (e.g., rodents, apes / monkeys, horses, cattle, pigs, dogs, cats, etc.), and most preferably, humans.

[0311] In this invention, the term "effective amount" means the amount of a drug or agent (i.e., the compound of this invention) that will elicit a biological or medical response in a tissue, system, animal, or human, as sought by, for example, a researcher or clinician. Furthermore, the term "therapeutic effective amount" means an amount that, compared to a corresponding subject who has not received the aforementioned amount, results in improved treatment, cure, prevention, or reduction of a disease, symptom, or side effect, or a slower rate of progression of a disease or symptom. Effective amounts may be administered, applied, or dosed in one or more administrations and are not intended to be limited to a specific formulation or route of administration. The term also includes effective amounts within its scope that enhance normal physiological function.

[0312] In this invention, the term "treatment" has a broad meaning, encompassing therapeutic and / or preventative treatment of an object. Specifically, "treatment" includes any treatment that results in the mitigation, suppression, elimination, improvement, and / or prevention of symptoms, diseases, disorders, etc., such as alleviating, reducing, regulating, improving, eliminating, preventing, or improving their symptoms. Therapeutic treatment includes alleviating, suppressing, or improving symptoms or conditions of a disease; suppressing the development of complications; improving underlying metabolic syndrome; suppressing the development of a disease or symptom, such as controlling the progression of a disease or condition; alleviating a disease or symptom; reducing a disease or symptom; alleviating complications caused by a disease or symptom; or treating signs caused by a disease or symptom. Preventative treatment includes pre-treatment to prevent, block, delay, slow the occurrence or development of a disease or condition, or reduce its severity.

[0313] Similarly, "therapeutic agents" also include medicines or reagents that provide therapeutic and / or preventative treatment to a subject.

[0314] In this invention, the terms "pharmaceutical" or "pharmaceutical acceptable" are used herein to refer to compounds, substances, compositions, and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, and / or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0315] In this invention, the term "pharmaceutical carrier" means a pharmaceutical substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, zinc stearate, or stearic acid), or solvent encapsulation substance, relating to carrying or delivering a subject compound from one part of an organ or body to another part of an organ or body. Each carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmlessness to the patient.

[0316] In this invention, the term "pharmaceutical composition" means a composition comprising the compounds of this invention and at least one other pharmaceutical carrier. "Pharmaceutical carrier" refers to a medium commonly accepted in the art for delivering a bioactive agent to an animal (specifically a mammal), including (i.e.) adjuvants, excipients, or mediators such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants, depending on the mode of administration and the nature of the dosage form.

[0317] Specific pharmaceutical and medical terminology

[0318] In this invention, the term "acceptable," as used herein, means that a prescription component or active ingredient does not have an excessively harmful effect on the health of a general therapeutic target.

[0319] In this invention, the term "cancer," as used herein, refers to an uncontrolled abnormal growth of cells that, under certain conditions, is capable of metastasis (spread). This type of cancer includes, but is not limited to, solid tumors (such as those of the bladder, intestines, brain, chest, uterus, heart, kidneys, lungs, lymphoid tissue (lymphoma), ovaries, pancreas or other endocrine organs (such as the thyroid), prostate, skin (melanoma), or hematologic malignancies (such as non-leukemic leukemia).

[0320] In this invention, the term "combined administration" or similar terms, as used herein, refers to administering several selected therapeutic agents to a patient in the same or different manners of administration at the same or different times.

[0321] In this invention, the terms "enhancement" or "capability to enhance," as used herein, refer to the expected increase or prolongation of either potency or duration of effect. Therefore, in terms of enhancing the therapeutic effect of a drug, the term "capability to enhance" refers to the ability of a drug in a system to increase or prolong its potency or duration of effect. The term "synergistic value," as used herein, refers to the ability of an ideal system to maximize the enhancement of another therapeutic agent.

[0322] In this invention, the term "immune disease" refers to a disease or symptom that results from an adverse or harmful reaction to endogenous or exogenous antigens. This typically results in cellular dysfunction, or damage leading to functional impairment, or damage to organs or tissues that may produce immune symptoms.

[0323] In this invention, the terms "reagent kit" and "product packaging" are synonyms.

[0324] In this invention, the terms "subject," "subject," or "patient" include both mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as orangutans, apes, and monkeys; agricultural animals such as cattle, horses, goats, sheep, and pigs; livestock such as rabbits and dogs; and laboratory animals including rodents such as rats, mice, and guinea pigs. Non-mammals include, but are not limited to, birds and fish. In a preferred embodiment, the selected mammal is a human.

[0325] As used herein, a compound or pharmaceutical composition, when administered, can improve a disease, symptom, or condition, particularly by improving its severity, delaying its onset, slowing its progression, or reducing its duration. This may be attributable to or related to the administration, whether the administration is fixed or intermittent, continuous or discontinuous. Detailed Implementation

[0326] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or alterations based on the teachings of the present invention without departing from the basic ideas and scope of the invention. In this invention, when no preparation method is mentioned, the relevant raw materials and intermediates are known products that can be synthesized according to methods known in the art or purchased from commercial reagents (e.g., from Bioderm, Pharmaron, etc.).

[0327] The abbreviations used in this invention have the following meanings:

[0328] In the following examples, unless otherwise specified, the reaction temperature is room temperature (10-30°C).

[0329] Unless otherwise specified in the reaction examples, all reactions were carried out under a nitrogen atmosphere. A nitrogen atmosphere refers to a reaction flask connected to a nitrogen balloon of approximately 1L.

[0330] Hydrogenation reactions are typically carried out under vacuum, filled with hydrogen gas, and repeated three times. A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon of approximately 1L.

[0331] Microwave reaction use Initiator + Microwave Reactor.

[0332] The compounds of the present invention were separated and purified by preparative TLC, silica gel column chromatography, Prep-HPLC and / or silica gel fast column chromatography (Flash column chromatography), and their structures were determined by... 1 Confirmation was performed using 1H NMR and / or MS. Reaction monitoring was performed using TLC or LC-MS.

[0333] 1 H-NMR spectra were recorded at 500 MHz on a Bruker instrument. Chemical shift values ​​are expressed in parts per million (ppm), i.e., δ values. The following abbreviations are used for the multiplicity of NMR signals: s = singlet, brs = broad peak, d = doublet, t = triplet, m = multiplet. Coupling constants are listed in J values ​​and measured in Hz. LC-MS experimental conditions were as follows: Instrument: Thermo U3000, ALLtech ELSD, MSQ, UV detector combined with ELSD and MSD (elution ratio 4:1). Column: Waters X-Bridge C-18, 3.5 μm, 4.6 x 50 mm; column temperature: 30 °C. Gradient [time (min) / solvent B in A (%)]: 0.00 / 5.0, 1.40 / 95, 2.80 / 95, 2.82 / 5, 3.00 / 5. (Solvent A = 0.01% trifluoroacetic acid in water; Solvent B = 0.01% trifluoroacetic acid in acetonitrile). UV detection: 214 / 254 / 280 / 300nm; DAD detection: 210-350nm; Flow rate: 2mL / min; MS: ESI, 100-1500m / z.

[0334] Preparative HPLC typically uses either an alkaline or acidic method (alkaline method mobile phase: acetonitrile / 0.05% ammonium bicarbonate aqueous solution; acidic method mobile phase: acetonitrile / 0.05% formic acid aqueous solution); the instrument is a Thermo U3000 AFC-3000; column: Globalsil C-18 12nm, 250x20mm, 10μm, or equivalent; flow rate: 20mL / min, using gradient elution separation.

[0335] The synthesis methods of some intermediates in the invention are as follows:

[0336] Intermediate 1

[0337] Intermediate 1 is prepared by the following steps:

[0338] Step 1: 100 g (756.67 mmol) of methyl 2,2-dimethyl-3-hydroxypropionate (INT-1a) was dissolved in 1 L of N,N-dimethylformamide. Imidazole (128.79 g, 1.89 mol) was added and stirred until dissolved. Tert-butyldiphenylchlorosilane (228.78 g, 832.34 mmol) was added dropwise at 20 °C. After the addition was complete, stirring was continued for 4 hours. After the reaction was complete, the reaction solution was poured into 3 L of ice water. The suspension was extracted with ethyl acetate (1 L * 2). The organic phase was washed three times with water (1 L * 2) and concentrated under reduced pressure to obtain a colorless oily substance, INT-1b. No purification was required; it was used directly in the next step. ESI-MS (m / z): 371.2 [M+H] + ;

[0339] Step 2: Add the residual INT-1b obtained in the previous step to methanol (2L), then add 360g of a prepared 33% sodium hydroxide aqueous solution, and stir at 20°C for 17 hours. After the reaction is complete, add 1L of water, remove methanol under reduced pressure, and extract the residual liquid with petroleum ether (1L*5). After extraction, adjust the pH of the aqueous phase to 4-5 with hydrochloric acid, and a large amount of white solid precipitates. Continue stirring for 30 minutes, filter, and dry to obtain white solid INT-1c (269g, yield 90%). ESI-MS (m / z): 357.8 [M+H] + ;

[0340] Step 3: Dissolve INT-1c (130g, 364.63mmol) in dichloromethane (500mL), add thionyl chloride (130.14g, 1.09mol, 79.35mL) at room temperature, and add N,N-dimethylformamide (0.05mL) dropwise. Stir at 60℃ for 3 hours. After the reaction is complete, remove dichloromethane and the remaining thionyl chloride under reduced pressure. Add petroleum ether (300mL) to the residue and continue distilling until no fraction is distilled off, to obtain a pale yellow oily substance INT-1d, which is used directly in the next step of the reaction without purification.

[0341] Step 4: Dissolve 5-bromoindole INT-1e (64.8 g, 331 mmol) in dichloromethane (400 mL), and add diethylaluminum chloride solution (198 mL, 2 mol / L in hexanes) at 0 °C. After the addition is complete, stir for 30 minutes. Add the dichloromethane solution of INT-1d obtained in the previous step to the reaction flask. After the addition is complete, continue stirring for 2 hours. After the reaction is complete, slowly pour the reaction solution into an ice-cold potassium sodium tartrate aqueous solution (1 L) and stir for 16 hours. After the system stabilizes, concentrate under reduced pressure to remove dichloromethane. Extract the residue with ethyl acetate (1 L * 2), wash with water (1 L * 2), and rotary evaporate the organic phase to obtain a brown oil. Add the oil to a mixed solution of petroleum ether / ethyl acetate = 10 / 1 (2 L), stir at 20 °C to precipitate a solid, filter, and obtain a yellow solid INT-1f (139 g, yield 78%). ESI-MS (m / z): 534.8 [M+H] + ;

[0342] Step 5: Dissolve INT-1f (100g, 187.07mmol) in tetrahydrofuran (500mL), add lithium borohydride (12.23g, 561.21mmol) under ice bath conditions, stir for 20 minutes after the addition is complete, and after the system stabilizes, heat to 60℃ and stir overnight. After the starting material disappears, slowly add the reaction solution to ice water (200mL) to quench the reaction, extract with ethyl acetate (500mL*3), wash the organic phase with water, dry it, concentrate under reduced pressure, and dissolve the residue in dichloromethane (500mL). Diethyl 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate (28.43 g, 112.24 mmol) and p-toluenesulfonic acid (21.35 g, 112.24 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to remove dichloromethane. The residue was dissolved in methanol (500 mL), and a pre-prepared 14% lithium hydroxide aqueous solution (100 mL) was added. The mixture was stirred at room temperature for 3 hours, filtered, and dried at room temperature to give a yellow solid INT-1 g (84 g, yield 86.26%). ESI-MS (m / z): 520.2 [M+H] + ;

[0343] Step 6: Dissolve INT-1 g (50 g, 96 mmol) in tetrahydrofuran (250 mL), add tetrabutylammonium fluoride (197 mL, 1 mol / L in THF), stir overnight at 60 °C. After the reaction is complete, add the reaction solution to water (300 mL), extract with ethyl acetate (200 mL * 3), wash with water, concentrate under reduced pressure to obtain a brown oil. Dissolve the residue in methanol (40 mL), add water (20 mL), wash the mixture with petroleum ether (40 mL * 5), concentrate under reduced pressure to remove methanol, extract the residue with ethyl acetate (50 mL * 2), wash the organic phase with water (50 mL), and dry to obtain a pale yellow oil INT-1h (25 g, yield 90.40%). ESI-MS (m / z): 282.8 [M+H] + ;

[0344] Step 7: Dissolve compound INT-1h (25 g, 88.7 mmol) in dioxane (250 mL), add potassium acetate (21.7 g, 221.8 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (3.24 g, 4.4 mmol), and neopentyl glycol diboronate (24.1 g, 106.4 mmol). React at 90 °C for 4 hours under nitrogen protection. Monitor the reaction of the starting material by LCMS until complete. Use diatomaceous earth as a preservative. The filtrate was filtered, concentrated, and dichloromethane (200 mL) was added to the concentrate. A 15% sodium hydroxide aqueous solution (17.7 g, 3548 mmol) was added, and concentration under reduced pressure continued until the aqueous phase was clear. The solution was filtered, and the aqueous phase was extracted once with dichloromethane (300 mL). The pH of the aqueous phase was adjusted to 3-4 with hydrochloric acid under ice bath conditions, resulting in the precipitation of a large amount of yellow solid. The mixture was stirred for 30 minutes, and then filtered to obtain the yellow solid compound INT-1i (17.96 g, yield 82.1%). ESI-MS (m / z): 248.4 [M+H] + ;

[0345] Step 8: Compound INT-1i (35 g, 142 mmol) and compound INT-1k (51.8 g, 142 mmol) were dissolved in dioxane (350 mL) and water (17.5 mL). Potassium carbonate (39.2 g, 284 mmol) and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (5.2 g, 7.1 mmol) were added. The reaction was carried out at 90 °C for 17 hours under nitrogen protection. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (300 mL). After washing with water (100 mL), the residue was evaporated to dryness to obtain a brown oily compound INT-1j, which was used directly in the next step without further treatment. ESI-MS (m / z): 488.4 [M+H] + ;

[0346] Step 9: Dissolve the crude compound INT-1j in dichloromethane (700 mL). Add 4-dimethylaminopyridine (866 mg, 7.1 mmol) and triethylamine (43.0 g, 426 mmol). Add acetic anhydride (14.5 g, 142 mmol) dropwise at 0 °C. After the addition is complete, remove the ice bath and allow the temperature to rise naturally. Stir for 1-2 hours until the reaction is complete. Wash the reaction solution with water (300 mL * 2), dry, and concentrate to obtain a brown oil. Purify by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1) to obtain a pale yellow oil INT-1l (62.3 g, yield 83.2%). ESI-MS (m / z): 530.5 [M + H] + ;

[0347] Step 10: Compound INT-1l (62.3 g, 117.9 mmol) was dissolved in N,N-dimethylformamide (620 mL), and N-iodosuccinimide (26.5 g, 117.9 mmol) was added. The mixture was reacted overnight at 10 °C. LCMS was used to monitor the reaction until complete. The reaction solution was slowly poured into ice water (3000 mL), and a solid precipitated upon stirring. The solid was filtered, washed with water (100 mL), and dried to obtain a yellow solid compound INT-1m (64.2 g, 87% yield). ESI-MS (m / z): 656.3 [M+H] + ;

[0348] Step 11: Compound INT-1m (64 g, 99.1 mmol) was dissolved in tetrahydrofuran (640 mL) and water (128 mL). Lithium hydroxide monohydrate (11.86 g, 282.4 mmol) was added, and the mixture was stirred at 70 °C for 1 hour. The reaction mixture was monitored by LCMS until the starting material was completely reacted. Water (300 mL) was added to the reaction mixture, and the solution was concentrated under reduced pressure. Then, methyltetrahydrofuran (200 mL) was added, and the pH was adjusted to 4–5 with 4 M hydrochloric acid. The mixture was then extracted with methyltetrahydrofuran (200 mL * 3). The organic phases were combined, washed with brine (100 mL), and evaporated thoroughly to dryness to obtain a yellow solid compound INT-1n (56.5 g, 95% yield). ESI-MS (m / z): 600.5 [M + H] + ;

[0349] Step 12: INT-1n (57 g, 95.0 mmol), 1-methylimidazole (38.9 g, 475 mmol), and (S)-hexahydropyridazine-3-carboxylic acid methyl ester trifluoroacetate (52.7 g, 142.5 mmol) were dissolved in acetonitrile (800 mL). A solution of N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (40.0 g, 142.5 mmol) in acetonitrile (400 mL) was added at 0 °C. After the addition was complete, the mixture was stirred for 1 hour. LC-MS was used to monitor the reaction until complete. Water (1000 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (1000 mL * 3). The extract was evaporated to dryness to give a yellow solid, INT-1o (56.5 g, 95% yield). ESI-MS (m / z): 726.3 [M+H] + ;

[0350] Step 13: Compound INT-1o (56.5 g, 77.8 mmol) was dissolved in tetrahydrofuran (560 mL) and water (112 mL). Lithium hydroxide (4.66 g, 194.7 mmol) was added, and the mixture was reacted at 10 °C for 2 hours. LCMS was used to monitor the reaction until complete. Water (300 mL) was added, and the pH was adjusted to 5-6 with 4 mol / L hydrochloric acid. The mixture was concentrated, extracted with methyltetrahydrofuran (200 mL * 3), washed with brine (100 mL), and after thorough separation, the solvent was completely removed by rotary evaporation to obtain a yellow solid compound INT-1p (55.4 g, yield 88.16%). ESI-MS (m / z): 712.6 [M+H] + ;

[0351] Step 14: N,N,N',N'-Tetramethylchloromethanesulfonamide hexafluorophosphate (59.1 g, 210.8 mmol) and 1-methylimidazole (26.5 g, 323.2 mmol) were added to acetonitrile (2000 mL), stirred until dissolved, and a THF solution of compound INT-1p (100 g / 1000 mL, 140.5 mmol) was added dropwise at 10-20 °C. After the addition was complete, the mixture was stirred for 1-2 hours. The reaction mixture was monitored by LCMS to ensure complete reaction of the starting material. The solvent was removed by rotary evaporation, and the residue was extracted with water (1000 mL) and dichloromethane (1000 mL * 3). The pH was adjusted to 3-4 with hydrochloric acid, and the organic phase was evaporated to dryness to give a yellow solid. Recrystallization from isopropanol gave compound INT-1q (59 g, 60% yield). ESI-MS (m / z): 694.6 [M+H] + ;

[0352] Step 15: Compound INT-1q (37 g, 53.35 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (6.6 g, 16.0 mmol), tris(dibenzylacetone)dipalladium (5.86 g, 6.40 mmol), and potassium acetate (18.3 g, 186.7 mmol) were dissolved in toluene (370 mL). Pinara-borane (34.1 g, 266.7 mmol, 38.7 mL) was added under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. The reaction mixture was monitored by LCMS to ensure complete reaction. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-1 (31 g, yield 82%). ESI-MS (m / z): 694.8 [M+H] + .

[0353] Intermediate 2

[0354] Intermediate 2 is prepared by the following steps:

[0355] Step 1: Compound INT-2a (43 g, 199 mmol), pinacol diborate (55.6 g, 219 mmol), methoxy(cyclooctadiene)iridium dimer (1.30 g, 1.99 mmol), and 4,4-di-tert-butylbipyridine (2.67 g, 9.95 mmol) were added to tetrahydrofuran (500 mL). The mixture was heated to 75 °C under nitrogen atmosphere and stirred for 16 hours. LCMS monitoring showed complete conversion of the starting material. Excess tetrahydrofuran was removed by rotary evaporation to obtain a brown residue, INT-2b, which was used directly in the next reaction without purification. ESI-MS (m / z): 342.4 [M+H] + .

[0356] Step 2: The residual liquid INT-2b obtained in the previous step was added to methanol (200 mL), followed by concentrated hydrochloric acid (100 mL). The reaction solution was refluxed for 3 hours. LCMS monitoring showed the starting material had disappeared. Methanol was removed by rotary evaporation. The residual liquid was added to water (200 mL), and the pH was adjusted to 13 with 30% sodium hydroxide solution. Extraction was performed with dichloromethane (400 mL * 2) to remove impurities. The aqueous phase was cooled to 0-5℃, and the pH was adjusted to 6-7 with hydrochloric acid. Stirring continued to wash out the solid, which was then filtered and dried to obtain a white solid compound INT-2c (41.3 g, yield 80%). ESI-MS (m / z): 260.2 [M+H] + .

[0357] Step 3: Compound INT-2c (41.3 g, 159 mmol) was dissolved in acetonitrile (400 mL), and N-iodosuccinimide (35.8 g, 239 mmol) was added. The mixture was heated to 80 °C and stirred overnight. LC-MS showed that the starting material disappeared. Acetonitrile was removed by rotary evaporation of the reaction solution. The residue was added to ethyl acetate (300 mL), washed with water (100 mL), dried, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give a white solid compound INT-2d (45.6 g, 85% yield). ESI-MS (m / z): 342.5 [M+H] + .

[0358] Step 4: Compound INT-2d (5 g, 14.6 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of zinc cyanide (1.03 g, 8.8 mmol) and tetrakis(triphenylphosphine)palladium (1.69 g, 1.46 mmol). The mixture was stirred overnight at 100 °C under nitrogen protection. The reaction was monitored by LCMS until complete. The reaction was quenched with ammonia (5 mL), and extracted with ethyl acetate (200 mL x 2). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 10:1) to give a colorless oily compound INT-2e (2.1 g, yield 59.6%). ESI-MS (m / z): 241.2 [M+H] + .

[0359] Step 5: Compound INT-2e (2.1 g, 8.7 mmol) was dissolved in a mixed solution of ethanol (20 mL) and water (4 mL). Potassium hydroxide (0.54 g, 9.6 mmol) was added, and the reaction mixture was refluxed for 16 hours. LC-MS monitoring showed the starting material had disappeared. The reaction mixture was concentrated to give a white solid compound INT-2f (2.26 g, 100% yield). ESI-MS (m / z): 258.0 [MH] - .

[0360] Step 6: Dissolve compound INT-2f (770 mg, 2.96 mmol) in methanol (10 mL), and add thionyl chloride (1.06 g, 8.9 mmol). Stir the reaction mixture at 70 °C for 3 hours. LCMS monitoring showed complete reaction of the starting material. Concentrate the reaction mixture to give a pale yellow solid compound INT-2g (800 mg, yield 98.6%). ESI-MS (m / z): 274.1 [M+H] + .

[0361] Step 7: Dissolve compound INT-2 g (600 mg, 2.19 mmol) in ethanol (6 mL), and add hydrazine hydrate (329 mg, 6.57 mmol). Stir the reaction mixture at 90 °C for 16 hours. Monitor the reaction progress using LC-MS until complete. Concentrate the reaction mixture. Purify the residue by column chromatography (dichloromethane / methanol = 20:1) to obtain a colorless oily compound INT-2h (550 mg, yield 91.7%). ESI-MS (m / z): 274.2 [M+H] + .

[0362] Step 8: Compound INT-2h (300 mg, 1.09 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by the addition of INT-2i (376 mg, 1.64 mmol), 1-hydroxybenzotriazole (222 mg, 1.64 mmol), N,N-diisopropylethylamine (424 mg, 3.28 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (315 mg, 1.64 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by LCMS until the starting material was completely reacted. Water (40 mL) was added to the system, and the mixture was extracted with dichloromethane (60 mL * 3). The organic phase was dried, dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was purified by column chromatography (dichloromethane / methanol = 10:1) to give a pale yellow oily compound INT-2j (480 mg, yield 90.4%). ESI-MS (m / z): 485.3 [M+H] + .

[0363] Step 9: Compound INT-2j (480 mg, 0.99 mmol) was dissolved in tetrahydrofuran (5 mL), and Burgess reagent (354 mg, 1.48 mmol) was added. The reaction mixture was stirred at 80 °C for 16 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted, and the reaction mixture was concentrated. The residue was purified by column chromatography (dichloromethane / methanol = 20:1) to give a colorless oily compound INT-2k (320 mg, yield 69.2%). ESI-MS (m / z): 467.2 [M+H] + .

[0364] Step 10: Trifluoroacetic acid (2 mL) was added dropwise to a solution of compound INT-2k (390 mg, 0.83 mmol) in dichloromethane (6 mL). The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by LCMS until completion. The reaction mixture was concentrated by vacuum distillation to obtain the crude product INT-2l. ESI-MS (m / z): 366.8 [M+H] + .

[0365] Step 11: The crude product INT-2 was dissolved in tetrahydrofuran (4 mL), and N,N-diisopropylethylamine (539 mg, 4.17 mmol) was added. Then, benzyl chloroformate (214 mg, 1.25 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed as monitored by LCMS, the reaction mixture was concentrated by vacuum distillation and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to obtain a colorless oily compound INT-2 (350 mg, yield 83.7%). ESI-MS (m / z): 501.0 [M+H] + .

[0366] Intermediate 3

[0367] Intermediate 3 is prepared by the following steps:

[0368] Step 1: Dissolve INT-3a (663 mg, 2.92 mmol) in ethanol (8 mL), add sodium ethoxide (199 mg, 2.92 mmol) at room temperature, and stir for 30 minutes at room temperature. Filter to remove the solid, then add an ethanol solution of INT-2h (400 mg, 1.46 mmol) to the filtrate. Stir the reaction mixture at 85 °C for 16 hours. After the reaction is complete, concentrate under reduced pressure. Purify the residue by silica gel column chromatography (dichloromethane / methanol = 20:1) to give a pale yellow solid compound INT-3b (516 mg, yield 75.8%). ESI-MS (m / z): 466.3 [M+H] + .

[0369] Step 2: Under a nitrogen atmosphere and ice bath conditions, sodium hydride (48 mg, 1.2 mmol, 60% dispersion in oil) was added to a tetrahydrofuran solution of compound INT-3b (160 mg, 0.343 mmol). The reaction mixture was stirred at room temperature for 2 hours, then cooled to 0°C, and 2-(trimethylsilyl)ethoxymethyl chloride (114 mg, 0.686 mmol) was added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for another 16 hours. The reaction was monitored by LCMS until complete. Water (40 mL) was added, followed by extraction with ethyl acetate (40 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated by filtration. The residue was purified by reversed-phase silica gel column chromatography (acetonitrile = 100%) to give compound INT-3 (134 mg, yield 65.6%). ESI-MS (m / z): 596.4 [M+H] + .

[0370] Intermediate 4

[0371] Intermediate 4 is prepared by the following steps:

[0372] Step 1: Compound INT-4a (5.0 g, 39.0 mmol) was dissolved in dichloromethane (50 mL) and methanol (10 mL). Trimethylsilyldiazomethane (2 mol / L, 29.3 mL) was added dropwise at 0 °C. After the addition was complete, the mixture was stirred for 1 hour, and the reaction was monitored by TLC until complete. The solution was concentrated to obtain a colorless oily compound INT-4b (5.6 g, 100% yield). ESI-MS (m / z): 143.4 [M+H] + . 1 H NMR(500MHz,Chloroform-d)δ3.70(s,3H),3.32–3.20(m,2H),2.87–2.77(m,3H),2.68–2.60(m,2H).

[0373] Step 2: Compound INT-4b (5.6 g, 39.4 mmol) was dissolved in n-heptane (60 mL), and tert-butyl hydrazide formate (5.5 g, 41.4 mmol) was added. The reaction mixture was heated to 70 °C and stirred for 16 hours. LC-MS was used to monitor the reaction until complete. The reaction solution was concentrated, and the residue was recrystallized from (n-heptane / isopropanol = 30 / 1) to give a white solid compound INT-4c (9.0 g, yield 89.1%). ESI-MS (m / z): 257.3 [M+H] + .

[0374] Step 3: Compound INT-4c (3.0 g, 11.7 mmol) was dissolved in methanol (30 mL), and platinum dioxide (300 mg, 10% wt) was added. The reaction mixture was stirred for 16 hours under a hydrogen atmosphere, and the reaction was monitored by LC-MS to ensure complete reaction of the starting materials. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain a colorless oily compound INT-4d (3.0 g, yield 99.2%). ESI-MS (m / z): 259.3 [M+H] + .

[0375] Step 4: Compound INT-4d (3.0 g, 11.6 mmol) was dissolved in tetrahydrofuran (30 mL). Di-tert-butyl dicarbonate (3.0 g, 14.0 mmol), triethylamine (3.5 g, 34.8 mmol), and 4-dimethylaminopyridine (0.14 g, 1.2 mmol) were added sequentially at 0 °C. The reaction was stirred at room temperature for 2 hours, and the reaction was monitored by LCMS until complete. The reaction was quenched with water (60 mL), and the mixture was extracted with ethyl acetate (70 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give a colorless oily compound INT-4e (3.2 g, yield 75.7%). ESI-MS (m / z): 359.2 [M+H] + .

[0376] Step 5: Compound INT-4e (2.0 g, 5.6 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL). Lithium bis(trimethylsilylamino)ene (1 mol / L, 16.7 mL) was added dropwise at -70 °C under a nitrogen atmosphere. After the addition was complete, the mixture was stirred at this temperature for 30 minutes. Then, trimethylchlorosilane (1.8 g, 16.7 mmol) was added, and stirring continued for 1 hour. N-bromosuccinimide (3.0 g, 16.7 mmol) was then added, and the reaction mixture was heated to room temperature and stirred for 16 hours. LC-MS was used to monitor the reaction until complete. The reaction was quenched with water (60 mL), and the mixture was extracted with ethyl acetate (70 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product INT-4f was used directly in the next step of the reaction. ESI-MS (m / z): 509.3 [M+H] + .

[0377] Step 6: The crude compound INT-4f was dissolved in methanol (20 mL), and potassium carbonate (1.5 g, 11.2 mmol) was added at 0 °C. The reaction was stirred at room temperature for 2 hours, and the reaction was monitored by LCMS until the starting material was completely reacted. The reaction was quenched by adding water (60 mL), and the mixture was extracted with ethyl acetate (70 mL * 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give a colorless oily compound INT-4g (1.4 g, two-step yield 57.4%). ESI-MS (m / z): 437.2 [M+H] + .

[0378] Step 7: Compound INT-4 g (1.4 g, 3.2 mmol) was dissolved in acetonitrile (140 mL), and cesium carbonate (3.1 g, 9.6 mmol) was added. The reaction was heated to 60 °C and stirred for 16 hours. The reaction was monitored by LCMS to ensure complete reaction of the starting material. The reaction was quenched by adding water (100 mL) to the system, and extracted with ethyl acetate (100 mL * 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative liquid chromatography and SFC to obtain a white solid compound INT-4h (0.21 g, yield 18.4%). ESI-MS (m / z): 357.2 [M+H] + . 1 H NMR(500MHz,Chloroform-d)δ5.24–4.93(m,1H),4.52–4.42(m,1H),3.78–3.71(m,3H),2.93 –2.84(m,1H),2.42–2.35(m,1H),2.17–2.09(m,1H),1.61–1.55(m,2H),1.55–1.46(m,18H).

[0379] Step 8: Dissolve compound INT-4h (100 mg, 0.24 mmol) in dichloromethane (1 mL), and add trifluoroacetic acid (1 mL) to the solution under ice bath conditions. Stir the reaction mixture at room temperature for 2 hours. After the reaction is complete, concentrate the reaction solution to give a colorless oily compound INT-4 (91 mg, 100% yield). ESI-MS (m / z): 157.3 [M+H] + .

[0380] Intermediate 5

[0381] Intermediate 5 is prepared by the following steps:

[0382] Step 1: Compound INT-1k (1.0 g, 2.7 mmol) was dissolved in tetrahydrofuran (5 mL) and water (5 mL). Lithium hydroxide monohydrate (230 mg, 5.5 mmol) was added at 0 °C, and the mixture was stirred for 2 hours. LC-MS was used to monitor the reaction until complete. The solution was diluted with water (60 mL), adjusted to pH 5 with dilute hydrochloric acid, and extracted with ethyl acetate (100 mL * 3). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to give a yellow solid compound INT-5a (900 mg, yield 93.6%). ESI-MS (m / z): 350.8 [M+H] + .

[0383] Step 2: Compound INT-4 (91 mg, 0.24 mmol) was dissolved in dichloromethane (5 mL). Diisopropylethylamine (118 mg, 0.91 mmol), compound INT-5a (80 mg, 0.23 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (104 mg, 0.27 mmol) were added sequentially at room temperature. The reaction mixture was stirred at room temperature for 2 hours, and the reaction proceeded to completion as monitored by LC-MS. The reaction was quenched with water (40 mL), and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to obtain a colorless oily compound INT-5 (100 mg, yield 89.7%). ESI-MS (m / z): 489.2 [M+H] + .

[0384] Intermediate 6

[0385] Intermediate 6 is prepared by the following steps:

[0386] By replacing compound INT-4 in the synthesis of compound INT-5 with compound INT-6a, and using a similar method and reaction steps, compound INT-6 can be obtained. ESI-MS (m / z): 477.3 [M+H] + .

[0387] Intermediate 7

[0388] Intermediate 7 is prepared by the following steps:

[0389] Step 1: Compound INT-2k (10 g, 21.4 mmol) was dissolved in 1,4-dioxane (300 mL), followed by the addition of potassium acetate (4.2 g, 42.8 mmol), neopentyl glycol diboronate (7.3 g, 32.1 mmol), and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride (1.4 g, 2.14 mmol). The reaction was stirred at 85 °C for 16 hours under a nitrogen atmosphere, and the reaction was monitored for completeness by LC-MS. Water (100 mL) was added to the system, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to obtain crude INT-7a, a brown oily substance. This crude product was used directly in the next reaction without purification. ESI-MS (m / z): 501.2 [M+H] + .

[0390] Step 2: The crude compound INT-7a obtained above was dissolved in 1,4-dioxane (150 mL) and water (15 mL). INT-7b (8.5 g, 13.2 mmol), potassium carbonate (5.5 g, 39.4 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (0.96 g, 1.3 mmol) were added sequentially. The reaction mixture was stirred at 85 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored to be complete by LC-MS. Water (100 mL) was added to the system, and the mixture was extracted with ethyl acetate (200 mL * 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give a colorless oily compound INT-7c (4.1 g, two-step yield 20.5%). ESI-MS (m / z): 906.7 [M+H] + .

[0391] Step 3: Compound INT-7c (1.4 g, 1.5 mmol) was dissolved in N,N-dimethylformamide (15 mL), and cesium carbonate (1.5 g, 4.5 mmol) and 2,2,2-trifluoroethyltrifluoromethanesulfonate (1.0 g, 4.5 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (150 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (100 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give a pale yellow solid compound INT-7d (770 mg, yield 51.3%). ESI-MS (m / z): 988.7 [M+H] + .

[0392] Step 4: Compound INT-7d (770 mg, 0.78 mmol) was dissolved in tetrahydrofuran (3 mL), and tetrabutylammonium fluoride (1 M, 3.9 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (40 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 3) to give a pale yellow solid compound INT-7e (526 mg, yield 90.0%). ESI-MS (m / z): 750.6 [M + H] + .

[0393] Step 5: Compound INT-7e (400 mg, 0.54 mmol) was dissolved in acetonitrile (4 mL), and trimethyliodosilane (190 mg, 0.8 mmol) was added to it under ice bath conditions. The reaction mixture was stirred under ice bath conditions for 1 hour. After the reaction was complete, sodium bicarbonate aqueous solution (40 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (40 mL * 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give a pale yellow solid compound INT-7f (325 mg, yield 93.8%). ESI-MS (m / z): 650.6 [M + H] + .

[0394] Step 6: Compound INT-7f (325 mg, 0.5 mmol) was dissolved in dichloromethane (5 mL), and 3-oxetane (72 mg, 1.0 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 10 minutes, and then sodium triacetoxyborohydride (318 mg, 1.5 mmol) was added, followed by stirring for 2 hours. After the reaction was complete, sodium bicarbonate aqueous solution (20 mL) was added to the reaction system, and the mixture was extracted with dichloromethane (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid compound INT-7 (225 mg, yield 77.9%). ESI-MS (m / z): 706.6 [M+H] + .

[0395] Intermediate 8

[0396] Intermediate 8 is prepared by the following steps:

[0397] Step 1: Compound INT-7 (220 mg, 0.31 mmol) was dissolved in 1,4-dioxane (5 mL), followed by the addition of potassium acetate (92 mg, 0.94 mmol), pinacol diborate (118 mg, 0.47 mmol), and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride (20 mg, 0.031 mmol). The mixture was stirred at 70 °C for 16 hours under a nitrogen atmosphere, and the reaction was monitored for completeness by LC-MS. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid compound INT-8a (173 mg, yield 73.9%). ESI-MS (m / z): 754.6 [M+H] + .

[0398] Step 2: Compound INT-8a (101 mg, 0.13 mmol) was dissolved in 1,4-dioxane (3 mL) and water (0.3 mL). INT-5 (66 mg, 0.13 mmol), potassium carbonate (46 mg, 0.34 mmol), and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride (8.7 mg, 0.013 mmol) were added sequentially. The reaction mixture was stirred at 70 °C under a nitrogen atmosphere for 16 hours, and the reaction was monitored for completeness by LC-MS. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid compound INT-8b (118 mg, yield 85.0%). ESI-MS (m / z): 1036.7 [M+H] + .

[0399] Step 3: Compound INT-8b (118 mg, 0.11 mmol) was dissolved in tetrahydrofuran (2 mL) and water (1 mL). Lithium hydroxide monohydrate (14 mg, 0.33 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. LC-MS was used to monitor the reaction until complete. The mixture was diluted with water (40 mL), adjusted to pH 5 with dilute hydrochloric acid, and extracted with ethyl acetate (40 mL * 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow solid compound INT-8c (101 mg, 90.0% yield). ESI-MS (m / z): 1022.7 [M+H] + .

[0400] Step 4: N,N,N',N'-Tetramethylchloromethanesulfonamide hexafluorophosphate (56 mg, 0.2 mmol) and 1-methylimidazole (41 mg, 0.5 mmol) were added to acetonitrile (3 mL), stirred until dissolved, and a THF solution of compound INT-8c (101 mg, 0.1 mmol) in 1 mL was added dropwise at room temperature. After the addition was complete, the mixture was stirred for 1 hour, and the reaction was monitored by LCMS to ensure complete reaction of the starting material. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid compound INT-8d (72 mg, yield 65.0%). ESI-MS (m / z): 1004.7 [M+H] + .

[0401] Step 5: Compound INT-8d (30 mg, 0.03 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (40 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-8 (25 mg, yield 92.6%). ESI-MS (m / z): 904.7 [M + H] + .

[0402] Intermediate 9

[0403] By replacing compound INT-5 in the synthesis step of compound INT-8 with compound INT-6, and using a similar method and reaction steps, compound INT-9 can be obtained. ESI-MS (m / z): 892.6 [M+H] + .

[0404] Intermediate 10

[0405] Intermediate 10 is prepared by the following steps:

[0406] Step 1: Compound INT-2 (200 mg, 0.40 mmol) was dissolved in a mixed solution of 1,4-dioxane (5 mL) and water (0.5 mL). Then, INT-1 (332 mg, 0.48 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (29 mg, 0.040 mmol), and potassium phosphate (251 mg, 1.20 mmol) were added sequentially. The reaction mixture was stirred at 70 °C for 16 hours under nitrogen protection. After the reaction was complete, water (40 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography (ethyl acetate) to give a pale yellow solid compound INT-10a (300 mg, yield 76.1%). ESI-MS (m / z): 988.0 [M+H] + .

[0407] Step 2: Compound INT-10a (300 mg, 0.30 mmol) was dissolved in N,N-dimethylformamide (3 mL), and cesium carbonate (198 mg, 0.61 mmol) and iodoethane (71 mg, 0.46 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (50 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (60 mL * 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography (petroleum ether / ethyl acetate = 1:2) to give a pale yellow solid compound INT-10b (110 mg, yield 35.7%). ESI-MS (m / z): 1016.9 [M+H] + .

[0408] Step 3: Compound INT-10b (110 mg, 0.108 mmol), 10% palladium hydroxide (22 mg), 10% palladium on carbon (22 mg), and isopropanol (5 mL) were added to a reaction flask. The reaction mixture was stirred at 60 °C for 16 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound INT-10 (85 mg, yield 89.0%). ESI-MS (m / z): 882.2 [M+H] + .

[0409] Intermediate 11

[0410] Intermediate 11 is prepared by the following steps:

[0411] Step 1: Compound INT-10 (140 mg, 0.16 mmol) was dissolved in 1,2-dichloroethane (2 mL) / methanol (2 mL), and 1-methylazacyclobutane-3-one hydrochloride (41 mg, 0.48 mmol) was added at room temperature. After 20 minutes, sodium cyanoborohydride (50 mg, 0.79 mmol) was slowly added to the reaction solution, and the reaction solution was stirred at 40 °C for 16 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL), extracted with ethyl acetate (40 mL * 3), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and the concentrated organic phase was purified by preparative thin-layer chromatography (dichloromethane / methanol = 15:1) to obtain a grayish-white solid compound INT-11a (60 mg, yield 39%). ESI-MS (m / z): 951.1 [M+H] + .

[0412] Step 2: Compound INT-11a (44 mg, 0.081 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath to adjust the pH to 8. The mixture was extracted with ethyl acetate (40 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a grayish-white solid compound INT-11 (29 mg, yield 81.0%). ESI-MS (m / z): 850.2 [M + H] + .

[0413] Intermediate 12

[0414] By replacing the 1-methylazacyclobutane-3-one hydrochloride in the synthesis of compound INT-11 with 3-oxetane, compound INT-12 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 837.8 [M+H] + LC-MS retention time RT = 1.55 min.

[0415] Intermediate 13

[0416] By replacing the 1-methylazacyclobutane-3-one hydrochloride in the synthesis of compound INT-11 with an aqueous formaldehyde solution, compound INT-13 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 796.0 [M+H] + LC-MS retention time RT = 1.58 min.

[0417] Intermediate 14

[0418] Compound INT-14 was prepared by the following steps:

[0419] Step 1: Compound INT-14a (5 g, 42.33 mmol), potassium carbonate (17.55 g, 126.98 mmol), and tetrabutylammonium iodide (782 mg, 2.12 mmol) were dissolved in acetonitrile (50 mL). Benzyl bromide (7.24 g, 42.33 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction was quenched by adding water (50 mL). The mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7 / 3) to give a colorless oily compound INT-14b (8.8 g, 100% yield). ESI-MS (m / z): 226.0 [M + NH4] + .

[0420] Step 2: Compound INT-14b (1.92 g, 9.20 mmol), [[2-(FMOC-amino)acetamido]methyl acetate (1.13 g, 3.07 mmol), and pyridine 4-methylbenzenesulfonic acid (77 mg, 0.31 mmol) were dissolved in dichloromethane (15 mL). The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was then concentrated, and the residue was purified by reversed-phase column chromatography (acetonitrile / water = 8 / 2) to give a pale yellow solid compound INT-14c (912 mg, yield 57%). ESI-MS (m / z): 517.0 [M+H] + .

[0421] Step 3: Compound INT-14c (450 mg, 0.87 mmol) was dissolved in N,N,-dimethylformamide (5 mL), and diethylamine (0.5 mL) was added. The reaction mixture was stirred at room temperature for 1 h, and then concentrated to obtain a pale yellow oily compound INT-14d (256 mg, 100% yield). ESI-MS (m / z): 295.0 [M+H] + .

[0422] Step 4: Compound INT-14d (256 mg, 0.87 mmol), (((9H-fluoro-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine (524 mg, 1.05 mmol), and N,N-diisopropylethylamine (337 mg, 2.61 mmol) were dissolved in N,N,-dimethylformamide (5 mL). HATU (430 mg, 1.13 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction was quenched by adding water (10 mL). The mixture was extracted with ethyl acetate (20 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a white solid compound INT-14e (645 mg, 95% yield). ESI-MS (m / z): 795.4 [M + NH4] + .

[0423] Step 5: Compound INT-14e (195 mg, 0.25 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated to obtain a pale yellow oily compound INT-14 (139 mg, 100% yield). ESI-MS (m / z): 556.0 [M+H] + .

[0424] Intermediate 15

[0425] Compound INT-15 was prepared by the following steps:

[0426] Step 1: Compound INT-15a (250 mg, 0.55 mmol), N-acetylpoly-10-sarcosine Ac-pSar10-COOH (384 mg, 0.5 mmol), and HATU (285 mg, 0.75 mmol) were dissolved in N,N,-dimethylformamide (2 mL). N,N-diisopropylethylamine (322 mg, 2.5 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated, and the residue was purified by reversed-phase column chromatography (acetonitrile / water = 1 / 1) to obtain a white solid compound INT-15b (532 mg, 90% yield). ESI-MS (m / z): 1194.0 [M+NH4] + .

[0427] Step 2: Compound INT-15b (242 mg, 0.21 mmol) was dissolved in dichloromethane (5 mL), and 4 M hydrochloric acid-dioxane solution (1 mL) was added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated to obtain a white solid compound INT-15c (235 mg, 100% yield). ESI-MS (m / z): 1137.7 [M+NH4] + .

[0428] Step 3: Compound INT-15c (235 mg, 0.21 mmol), compound INT-14 (139 mg, 0.25 mmol), and N,N-diisopropylethylamine (80 mg, 0.62 mmol) were dissolved in N,N,-dimethylformamide (2 mL). TSTU (93 mg, 0.31 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated, and the residue was purified by reversed-phase column chromatography (acetonitrile / water = 4 / 6) to give a white solid compound INT-15d (172 mg, yield 50%). ESI-MS (m / z): 1675.3 [M+NH4] + .

[0429] Step 4: Compound INT-15d (53 mg, 0.032 mmol), 10% palladium on carbon (5 mg), and ethanol (2 mL) were added to a reaction flask. The reaction mixture was stirred at room temperature for 2 h under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give compound INT-15 (48 mg, 95% yield). ESI-MS (m / z): 793.5 [(M+NH4+H) / 2] + .

[0430] Intermediate 16

[0431] Compound INT-16 was prepared by the following steps:

[0432] Step 1: Compound INT-16a (2.5 g, 12.24 mmol) and maleic anhydride (1.2 g, 12.24 mmol) were dissolved in acetic acid (3 mL). The reaction mixture was stirred at room temperature for 3 h, and then concentrated. The residue was slurried using hexane / dichloromethane (10 mL / 10 mL), and the solid was collected and dried under reduced pressure. The solid was added to toluene (9 mL), followed by N,N,-dimethylacetamide (0.5 mL) and triethylamine (3.72 g, 36.72 mmol). The reaction mixture was heated to 60 °C until the solid was completely dissolved. The temperature was then increased to 110 °C and stirred for 16 h. The reaction mixture was concentrated, and the residue was dissolved in ethyl acetate (20 mL) and washed with an aqueous solution containing 10% citric acid (2 × 30 mL) and saturated saline solution (2 × 30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid compound INT-16b (3.03 g, yield 69.7%, purity 80%). ESI-MS (m / z): 285.5 [M+H] + .

[0433] Step 2: Compound INT-16b (2.44 g, 8.85 mmol) was dissolved in N,N,-dimethylformamide (10 mL), and N-hydroxysuccinimide (1.09 g, 9.44 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.81 g, 9.44 mmol) were added. The reaction mixture was stirred at room temperature for 3 h, and then water (50 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (100 mL * 3), and the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 7) to give a pale yellow solid compound INT-16 (426 mg, yield 13%). ESI-MS (m / z): 382.6 [M+H] + .

[0434] Intermediate 17

[0435] Compound INT-17 was prepared by the following steps:

[0436] Step 1: Compound INT-7d (2.0 g, 2.02 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (5 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (40 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-17a (1.3 g, yield 72.3%). ESI-MS (m / z): 890.4 [M + H] + .

[0437] Step 2: Compound INT-17a (1.0 g, 1.12 mmol) was dissolved in tetrahydrofuran (10 mL). N,N-diisopropylethylamine (436 mg, 3.37 mmol) and benzyl chloroformate (288 mg, 1.69 mmol) were added to the solution under ice bath conditions. The reaction mixture was stirred at room temperature for 16 hours. A saturated sodium bicarbonate solution (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid, compound INT-17b (1.15 g, 100% yield). ESI-MS (m / z): 1022.6 [M+H] + .

[0438] Step 3: Compound INT-17b (1.15 g, 1.12 mmol) was dissolved in tetrahydrofuran (6 mL), and tetrabutylammonium fluoride (1 M, 3.4 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (40 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to give a pale yellow solid compound INT-17c (551 mg, yield 62.5%). ESI-MS (m / z): 785.2 [M + H] + .

[0439] Step 4: Compound INT-17c (551 mg, 0.70 mmol) was dissolved in 1,4-dioxane (10 mL), followed by the addition of potassium acetate (207 mg, 2.11 mmol), pinacol diboronate (267 mg, 1.05 mmol), and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride (51 mg, 0.070 mmol). The mixture was stirred at 80 °C for 16 hours under a nitrogen atmosphere. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to give a pale yellow solid compound INT-17d (467 mg, 80% yield). ESI-MS (m / z): 832.2 [M+H] + .

[0440] Step 5: Compound INT-17d (467 mg, 0.56 mmol) was dissolved in 1,4-dioxane (6 mL) and water (0.6 mL). INT-5 (261 mg, 0.53 mmol), potassium carbonate (233 mg, 1.68 mmol), and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride (36 mg, 0.056 mmol) were added sequentially. The reaction mixture was stirred at 50 °C under a nitrogen atmosphere for 20 hours. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:9) to give a pale yellow solid compound INT-17e (529 mg, yield 84.5%). ESI-MS (m / z): 1114.0 [M+H] + .

[0441] Step 6: Compound INT-17e (519 mg, 0.46 mmol) was dissolved in tetrahydrofuran (5 mL) and water (2.5 mL). Lithium hydroxide monohydrate (14 mg, 0.33 mmol) was added at 0 °C, and stirring was continued for 1 hour. The mixture was diluted with water (40 mL), adjusted to pH 5 with dilute hydrochloric acid, and extracted with ethyl acetate (40 mL * 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow solid compound INT-17f (512 mg, 100% yield). ESI-MS (m / z): 1100.7 [M+H] + .

[0442] Step 7: N,N,N',N'-Tetramethylchloromethanemid hexafluorophosphate (261 mg, 0.93 mmol) and 1-methylimidazole (191 mg, 2.33 mmol) were added to acetonitrile (10 mL), stirred until dissolved, and a THF solution of compound INT-17f (512 mg, 0.46 mmol) was added dropwise at room temperature. After the addition was complete, the mixture was stirred for 1 hour. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:9) to give a pale yellow solid compound INT-17g (348 mg, yield 69.1%). ESI-MS (m / z): 1081.6 [M+H] + .

[0443] Step 8: Compound INT-17 g (230 mg, 0.212 mmol), 10% palladium hydroxide (23 mg), 10% palladium on carbon (23 mg), and isopropanol (5 mL) were added to a reaction flask. The reaction mixture was stirred at 70 °C for 16 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give compound INT-17 (180 mg, yield 89.0%). ESI-MS (m / z): 948.8 [M+H] + .

[0444] Intermediate 18

[0445] Compound INT-18 was prepared by the following steps:

[0446] Step 1: Compound INT-5 (137 mg, 0.28 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (20 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-18a (108 mg, yield 94.1%). ESI-MS (m / z): 389.5 [M + H] + .

[0447] Step 2: Under ice bath conditions, (R)-2-hydroxy-3-methylbutyric acid (65 mg, 0.554 mmol), N,N-diisopropylethylamine (107 mg, 0.832 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (200 mg, 0.527 mmol) were added sequentially to a solution of compound INT-18a (135 mg, 0.277 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred under these conditions for 2 hours. After the reaction was complete, water (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phase was dried, dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 95:5) to give a pale yellow oily compound INT-18 (130 mg, yield 95.7%). ESI-MS (m / z): 489.5 [M+H] + .

[0448] Intermediate 22

[0449] By replacing the N-acetyl-10-sarcosine Ac-pSar10-COOH in the synthesis of compound INT-15 with N-acetyl-poly-14-sarcosine Ac-pSar14-COOH, compound INT-22 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 934.5 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.21 min.

[0450] Intermediate 23

[0451] Compound INT-23 was prepared by the following steps:

[0452] Step 1: Compound INT-14b (686 mg, 3.29 mmol), (S)-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propamido)methyl acetate (420 mg, 1.10 mmol), and pyridine 4-methylbenzenesulfonic acid (28 mg, 0.11 mmol) were dissolved in dichloromethane (15 mL). The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was then concentrated, and the residue was purified by reversed-phase column chromatography (acetonitrile / water = 8 / 2) to give a pale yellow solid compound INT-23a (185 mg, yield 31%). ESI-MS (m / z): 531.5 [M+H] + .

[0453] Step 2: Compound INT-23a (185 mg, 0.348 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 1 h, and then concentrated to obtain a pale yellow oily compound INT-23b (107 mg, 100% yield). ESI-MS (m / z): 309.5 [M+H] + .

[0454] Step 3: Compound INT-23b (107 mg, 0.348 mmol), (((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-alanine (200 mg, 0.523 mmol), and N,N-diisopropylethylamine (135 mg, 1.05 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (238 mg, 0.627 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then purified by reversed-phase column chromatography (acetonitrile / water = 70 / 30) to obtain a white solid compound INT-23c (148 mg, yield 63%). ESI-MS (m / z): 673.7 [M+H] + .

[0455] Step 4: Compound INT-23c (148 mg, 0.22 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated to obtain a pale yellow oily compound INT-23 (99 mg, 100% yield). ESI-MS (m / z): 451.7 [M+H] + .

[0456] Intermediate 24

[0457] By replacing INT-14 in the synthesis of compound INT-15 with INT-23, and using a similar method and reaction steps, compound INT-24 can be obtained. ESI-MS (m / z): 741.3 [(M+NH4) / 2] + LC-MS retention time RT = 1.18 min.

[0458] Intermediate 25

[0459] By replacing INT-14a in the synthesis of compound INT-14 with (S)-2-hydroxy-3-methylbutyric acid, compound INT-25 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 556.0 [M+H] + LC-MS retention time RT = 1.39 min.

[0460] Intermediate 26

[0461] By replacing INT-14 in the synthesis of compound INT-15 with INT-25, and using a similar method and reaction steps, compound INT-26 can be obtained. ESI-MS (m / z): 793.1 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.28 min.

[0462] Intermediate 27

[0463] Compound INT-27 was prepared by the following steps:

[0464] Compounds INT-27a (150 mg, 0.219 mmol) and INT-15a (111 mg, 0.241 mmol) were dissolved in N,N,-dimethylformamide (2 mL), and N,N-diisopropylethylamine (85 mg, 0.656 mmol) was added. The reaction mixture was stirred at room temperature for 2 h, and then purified by reversed-phase column chromatography (acetonitrile / water = 70 / 30) to give anhydrous oily compound INT-27 (160 mg, yield 73%). ESI-MS (m / z): 1014.3 [M+NH4] + LC-MS retention time RT = 1.73 min.

[0465] Intermediate 28

[0466] By replacing INT-15b in the synthesis of compound INT-15 with INT-27, and using a similar method and reaction steps, compound INT-28 can be obtained. ESI-MS (m / z): 702.6 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.33 min.

[0467] Intermediate 29

[0468] Compound INT-29 was prepared by the following steps:

[0469] Step 1: Methylamine hydrochloride (82.92 g, 1.23 mol) was dissolved in methanol (200 mL), and triethylamine (136.71 g, 1.35 mol) was added. The reaction mixture was stirred at room temperature for 1 hour. Then, compound INT-29a (22 g, 122.82 mmol) was added, and the reaction mixture was stirred at room temperature for another 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a white solid compound INT-29b (25.81 g, 95% yield). ESI-MS (m / z): 211.1 [M+H] + .

[0470] Step 2: Compound INT-29b (24.7 g, 117.5 mmol) was dissolved in tetrahydrofuran (200 mL). Borane dimethyl sulfide complex (147 mL, 294 mmol, 2 M in THF) was slowly added dropwise under ice bath conditions. The reaction mixture was heated to 70 °C and stirred for 5 h, then cooled to room temperature. Hydrochloric acid-dioxane solution (5 mL, 4 M in 1,4-dioxane) was slowly added dropwise to the reaction system under ice bath conditions. After the addition was complete, the reaction mixture was heated to 65 °C and stirred for 8 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a white solid compound INT-29c (27.3 g, 99% yield). ESI-MS (m / z): 197.1 [M+H] + .

[0471] Step 3: Compound INT-29c (15 g, 64.47 mmol) and imidazole (21.92 g, 322.35 mmol) were dissolved in dichloromethane (200 mL), and tert-butyldiphenylchlorosilane (26.58 g, 96.71 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 8 h, then poured into ice water (1 L), extracted with ethyl acetate (400 mL * 2), and the organic phases were combined. The mixture was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give a pale yellow oily compound INT-29d (18.95 g, yield 67%). ESI-MS (m / z): 435.3 [M+H] + .

[0472] Step 4: Compound INT-29d (15 g, 34.51 mmol) was dissolved in tetrahydrofuran (200 mL), and 9-fluorenylmethyl-N-succinimide carbonate (12.22 g, 36.24 mmol) and triethylamine (6.99 g, 69 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. Water (160 mL) was added, and the mixture was extracted with ethyl acetate (150 mL * 2). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 1) to give a pale yellow solid compound INT-29e (18.95 g, yield 83%). ESI-MS (m / z): 657.1 [M+H] + .

[0473] Step 5: Compound INT-29e (15.14 g, 23.05 mmol), 10% palladium on carbon (1.5 g), methanol (100 mL), and ethyl acetate (200 mL) were added to a reaction flask. The reaction mixture was stirred at room temperature for 5 h under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 7 / 3) to give compound INT-29f (12.85 g, yield 88.9%). ESI-MS (m / z): 649.2 [M+Na] + .

[0474] Step 6: Compound INT-29f (1.86 g, 2.92 mmol), (tert-butoxycarbonyl)-L-valine-L-alanine (1.03 g, 3.56 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.47 g, 5.93 mmol) were dissolved in dichloromethane (20 mL) and methanol (10 mL). The mixture was stirred at room temperature for 16 h. The reaction solution was then concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 55 / 45) to give a white solid compound INT-29g (2.0 g, yield 75%). ESI-MS (m / z): 914.2 [M+NH4] + LC-MS retention time RT = 2.09 min.

[0475] Step 7: Dissolve compound INT-29g (287mg, 0.32mmol) in tetrahydrofuran (2mL), 0 oTetrabutylammonium fluoride (0.96 mL, 0.96 mmol, 1 M in THF) was added at C, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 80 / 20) to give a white solid compound INT-29h (108 mg, yield 77%). ESI-MS (m / z): 436.2 [M+H] + LC-MS retention time RT = 1.20 min.

[0476] Step 8: Compound INT-29h (108 mg, 0.247 mmol), N-acetylpoly-10-sarcosine Ac-pSar10-COOH (191 mg, 0.247 mmol), and HATU (141 mg, 0.371 mmol) were dissolved in N,N,-dimethylformamide (2 mL). N,N-diisopropylethylamine (96 mg, 0.742 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then purified by reversed-phase column chromatography (acetonitrile / water = 1 / 1) to obtain a white solid compound INT-29i (184 mg, yield 62%). ESI-MS (m / z): 1206.3 [M+NH4] + LC-MS retention time RT = 1.15 min.

[0477] Step 9: INT-29i (100 mg, 0.084 mmol) and di(p-nitrobenzene) carbonate (51 mg, 0.168 mmol) were dissolved in N,N,-dimethylformamide (2 mL). N,N-diisopropylethylamine (33 mg, 0.252 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then purified by reversed-phase column chromatography (acetonitrile / water = 1 / 1) to obtain a pale yellow solid, INT-29i (95 mg, yield 83%). ESI-MS (m / z): 1372.4 [M+NH4] + LC-MS retention time RT = 1.43 min.

[0478] Intermediate 30

[0479] Compound INT-30 was prepared by the following steps:

[0480] Compound INT-30a (1 g, 3.91 mmol) and 7-aminoheptanoic acid (567 mg, 3.91 mmol) were dissolved in acetic acid (20 mL). The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was then concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate / dichloromethane = 20 / 80) to give a white solid compound INT-30 (1.1 g, yield 73%). ESI-MS (m / z): 401.0 [M+NH4] + LC-MS retention time RT = 1.16 min.

[0481] Intermediate 31

[0482] Replacing INT-5 in the synthesis step of compound INT-17g with INT-6, and using a similar method and reaction steps, yields compound INT-31. ESI-MS (m / z): 1071.6 [M+H] + LC-MS retention time RT = 2.02 min.

[0483] Intermediate 32

[0484] Compound INT-32 was prepared by the following steps:

[0485] Step 1: Compound INT-31 (114 mg, 0.106 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (40 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a pale yellow solid compound INT-32a (100 mg, 96% yield). ESI-MS (m / z): 970.9 [M + H] + LC-MS retention time RT = 1.83 min.

[0486] Step 2: Compound INT-32a (100 mg, 0.103 mmol), (2R)-3-methyl-2-hydroxybutyric acid (18 mg, 0.154 mmol), and N,N-diisopropylethylamine (40 mg, 0.309 mmol) were dissolved in N,N,-dimethylformamide (2 mL), and HATU (70 mg, 0.185 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. Water (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give a pale yellow oily compound INT-32b (90 mg, yield 81%). ESI-MS (m / z): 1071.4 [M+H] + LC-MS retention time RT = 1.87 min.

[0487] Step 3: Compound INT-32b (90 mg, 0.084 mmol), 10% palladium hydroxide (5 mg), 10% palladium on carbon (5 mg), and isopropanol (5 mL) were added to a reaction flask. The reaction mixture was stirred at 65 °C for 16 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound INT-32c (70 mg, yield 88%). ESI-MS (m / z): 936.5 [M+H] + LC-MS retention time RT = 1.53 min.

[0488] Step 4: Compound INT-32c (70 mg, 0.075 mmol) and N-tert-butoxycarbonyl-4-piperidinone (45 mg, 0.244 mmol) were dissolved in 1,2,-dichloroethane (5 mL), and the reaction mixture was stirred at room temperature for 0.5 hours. Sodium borohydride acetate (79 mg, 0.374 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. Water (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give a pale yellow solid compound INT-32d (34 mg, yield 40%). ESI-MS (m / z): 1119.8 [M+H] + LC-MS retention time RT = 1.90 min.

[0489] Step 5: Compound INT-32d (34 mg, 0.030 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (40 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow oily compound INT-32 (26 mg, yield 85%). ESI-MS (m / z): 1019.4 [M+H] + LC-MS retention time RT = 1.47 min.

[0490] Intermediate 33

[0491] Compound INT-33 was prepared by the following steps:

[0492] Step 1: Under a nitrogen atmosphere, 5-hexyneic acid succinimide ester (500 mg, 2.39 mmol), 5-bromo-2-methylmercaptopyrimidine (539 mg, 2.63 mmol), bis(triphenylphosphine) palladium dichloride (49.3 mg, 0.239 mmol), cuprous iodide (45.5 mg, 0.239 mmol), and triethylamine (725 mg, 7.17 mmol) were dissolved in N,N,-dimethylformamide (5 mL). The reaction mixture was stirred at 95 °C for 3 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50 / 50) to give a pale yellow solid compound INT-33a (650 mg, yield 81%). ESI-MS (m / z): 334.1 [M+H] + LC-MS retention time RT = 1.71 min.

[0493] Step 2: Compound INT-33a (650 mg, 1.95 mmol) was dissolved in dichloromethane (20 mL), and m-chloroperoxybenzoic acid (675 mg, 3.91 mmol) was added. The reaction solution was reacted at room temperature for 2 h. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 90) to give a white solid compound INT-33 (545 mg, yield 76%). ESI-MS (m / z): 366.0 [M+H] + LC-MS retention time RT = 1.46 min.

[0494] Compound INT-33 can also be prepared by the following method:

[0495] Step 1: 5-Bromo-2-methylmercaptopyrimidine is oxidized by m-chloroperoxybenzoic acid in a solution such as DCM at room temperature to give intermediate INT-33b.

[0496] Step 2: Compound INT-33b, 5-hexynyl succinimide, bis(triphenylphosphine) palladium dichloride, cuprous iodide and triethylamine are dissolved in N,N,-dimethylformamide and stirred for about 3 hours under nitrogen protection and heating at 95°C to obtain intermediate INT-33 by Sonogashira coupling reaction.

[0497] Example 1: Synthesis of drug loading P

[0498] Example 1.1: Synthesis of Compound 1

[0499] Compound 1 was prepared by the following steps:

[0500] Under ice bath conditions, a solution of compound INT-13 (30 mg, 0.038 mmol) in N,N-dimethylformamide (3 mL) was successively added with (S)-2-hydroxy-3-methylbutyric acid (4.5 mg, 0.038 mmol), N,N-diisopropylethylamine (14.6 mg, 0.113 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (14 mg, 0.038 mmol). The reaction mixture was stirred under these conditions for 2 hours. After the reaction was complete, water (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phase was dried, dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was purified by preparative liquid chromatography to give compound 1 (13 mg, yield 38.5%) as a white solid. ESI-MS (m / z): 896.4 [M+H] + LC-MS retention time RT = 1.70 min.

[0501] 1H NMR (500MHz, DMSO-d6) δ9.32(d,J=2.0Hz,1H),8.54–8.50(m,1H),8.29(d,J=2.0Hz,1H),8.00(d,J=9.5Hz,1H),7.86(s,1H),7.78(dd,J=8.5 ,1.6Hz,1H),7.62(d,J=8.5Hz,1H),5.62–5.55(m,2H),5.18–5.13(m,1H),4.42–4.35(m,2H),4.27–4.20(m,2H),4.15–4.07(m,1H),3.73–3. 70(m,1H),3.61–3.57(m,2H),3.30(s,3H),3.03–2.98(m,2H),2.81–2 .75(m,3H),2.45–2.42(m,1H),2.18(s,3H),2.13–2.00(m,7H),1.86–1 .77(m,4H),1.55–1.48(m,1H),1.41(d,J=6.0Hz,3H),1.25–1.23(m,1H ),0.96(d,J=7.0Hz,3H),0.92(s,3H),0.91–0.86(m,6H),0.36(s,3H).

[0502] Example 1.2: Synthesis of Compound 2

[0503] By replacing (S)-2-hydroxy-3-methylbutyric acid in the synthesis of compound 1 with (R)-2-hydroxy-3-methylbutyric acid, compound 2 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 896.7 [M+H] + LC-MS retention time RT = 1.66 min.

[0504] 1H NMR (500MHz, DMSO-d6) δ9.32(d,J=2.0Hz,1H),8.53(d,J=1.5Hz,1H),8.29(d,J=2.0Hz,1H),8.04(d,J=9.0Hz,1H),7.85(s,1H),7.78(dd,J=8.5,1.5Hz ,1H),7.62(d,J=8.5Hz,1H),5.62–5.55(m,1H),5.37–5.31(m,1H),5.17–5. 13(m,1H),4.41–4.35(m,2H),4.27–4.20(m,2H),4.15–4.08(m,1H),3.82–3 .78(m,1H),3.61–3.56(m,2H),3.30(s,3H),3.04–2.98(m,2H),2.82–2.75( m,3H),2.46–2.42(m,1H),2.18(s,3H),2.13–1.98(m,7H),1.87–1.76(m,4H ),1.58–1.46(m,1H),1.41(d,J=6.0Hz,3H),1.23(s,1H),0.94(d,J=7.0Hz, 3H), 0.92 (s, 3H), 0.88 (t, J = 7.0Hz, 3H), 0.78 (d, J = 6.5Hz, 3H), 0.37 (s, 3H).

[0505] Example 1.3: Synthesis of Compound 3

[0506] Compound 3 was prepared by the following steps:

[0507] Under ice bath conditions, a solution of compound INT-11 (31 mg, 0.036 mmol) in N,N-dimethylformamide (3 mL) was successively added with (S)-2-hydroxy-3-methylbutyric acid (4.3 mg, 0.036 mmol), N,N-diisopropylethylamine (14 mg, 0.109 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (13.9 mg, 0.036 mmol). The reaction mixture was stirred under these conditions for 2 hours. After the reaction was complete, water (20 mL) was added to the system, followed by extraction with ethyl acetate (40 mL * 3). The organic phase was dried, dried over anhydrous sodium sulfate, and concentrated by filtration. The residue was purified by preparative liquid chromatography to give compound 3 (12 mg, yield 34.6%) as a white solid. ESI-MS (m / z): 951.9 [M+H] + LC-MS retention time RT = 1.65 min.

[0508] 1 H NMR (500MHz, DMSO-d6) δ9.32(d,J=2.0Hz,1H),8.54–8.49(m,1H),8.30(d,J=2.0Hz,1H),8.00(d,J=9.5Hz,1H),7.86(s,1H),7.78(dd,J=8.5,1. 5Hz,1H),7.62(d,J=8.5Hz,1H),5.62–5.56(m,2H),5.19–5.13(m,1H),4 .42–4.35(m,2H),4.27–4.20(m,2H),4.15–4.08(m,1H),3.73–3.70(m,1H ),3.60–3.57(m,2H),3.30(s,3H),3.06–3.00(m,2H),2.83–2.68(m,7H) ,2.45–2.41(m,1H),2.22(s,3H),2.13–2.09(m,1H),2.08–1.89(m,7H), 1.83–1.74(m,4H),1.55–1.49(m,1H),1.41(d,J=6.0Hz,3H),1.32–1.26 (m,1H),0.98–0.95(m,3H),0.92(s,3H),0.90–0.86(m,6H),0.36(s,3H).

[0509] Example 1.4: Synthesis of Compound 4

[0510] By replacing (S)-2-hydroxy-3-methylbutyric acid in the synthesis of compound 3 with (R)-2-hydroxy-3-methylbutyric acid, compound 4 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 951.2 [M+H] + LC-MS retention time RT = 1.61 min.

[0511] 1H NMR(500MHz,DMSO-d6)δ9.32(d,J=2.0Hz,1H),8.55–8.51(m,1H),8.30(d,J=2.0 Hz,1H),8.04(d,J=9.0Hz,1H),7.85(s,1H),7.78(dd,J=8.5,1.5Hz,1H),7.62(d ,J=8.5Hz,1H),5.61–5.55(m,1H),5.38–5.31(m,1H),5.17–5.12(m,1H),4.41–4 .35(m,2H),4.26–4.20(m,2H),4.15–4.08(m,1H),3.82–3.79(m,1H),3.60–3.57( m,2H),3.30(s,3H),3.07–2.99(m,2H),2.85–2.80(m,1H),2.79–2.66(m,6H),2. 47–2.42(m,1H),2.22(s,3H),2.13–1.97(m,5H),1.96–1.89(m,2H),1.85–1.75(m ,4H),1.55–1.48(m,1H),1.41(d,J=6.0Hz,3H),1.32–1.25(m,1H),0.94(d,J=7. 0Hz,3H),0.92(s,3H),0.88(t,J=7.0Hz,3H),0.78(d,J=7.0Hz,3H),0.37(s,3H).

[0512] Example 1.5: Synthesis of Compound 5

[0513] By replacing INT-13 in the synthesis of compound 1 with INT-12, and replacing (S)-2-hydroxy-3-methylbutyric acid with (R)-2-hydroxy-3-methylbutyric acid, compound 5 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 938.9 [M+H] + LC-MS retention time RT = 1.63 min.

[0514] 1H NMR(500MHz,DMSO-d6)δ9.33(d,J=2.0Hz,1H),8.53(s,1H),8.34–8.27(m,1H),8 .04(d,J=9.0Hz,1H),7.85(s,1H),7.79(d,J=8.5Hz,1H),7.62(d,J=8.5Hz,1H), 5.61–5.55(m,1H),5.40–5.29(m,1H),5.19–5.11(m,1H),4.53(t,J=6.5Hz,2H), 4.43(t,J=6.0Hz,2H),4.41–4.35(m,2H),4.27–4.19(m,2H),4.15–4.07(m,1H),3 .82–3.78(m,1H),3.61–3.56(m,2H),3.43–3.41(m,2H),3.30(s,3H),3.11–3.06 (m,1H),3.03–2.98(m,1H),2.80–2.70(m,3H),2.46–2.42(m,1H),2.13–2.06(m,3 H),2.05–1.91(m,4H),1.88–1.76(m,4H),1.56–1.48(m,1H),1.41(d,J=6.0Hz,3 H),0.97–0.90(m,6H),0.88(t,J=7.0Hz,3H),0.78(d,J=6.5Hz,3H),0.37(s,3H).

[0515] Example 1.6: Synthesis of Compound 6

[0516] By replacing compound INT-13 in the synthesis step of compound 1 with compound INT-9, and replacing (S)-2-hydroxy-3-methylbutyric acid with (R)-2-hydroxy-3-methylbutyric acid, compound 6 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 992.9 [M+H] + LC-MS retention time RT = 1.68 min.

[0517] 1H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.0Hz,1H),8.56–8.51(m,1H),8.29–8.26(m ,1H),8.06(d,J=9.5Hz,1H),7.91(s,1H),7.87(dd,J=9.0,1.5Hz,1H),7.80(d, J=8.5Hz,1H),5.71–5.61(m,1H),5.59–5.53(m,1H),5.37–5.31(m,1H),5.15–5 .11(m,1H),4.91–4.81(m,1H),4.54(t,J=6.5Hz,2H),4.43(t,J=6.0Hz,2H),4.3 7–4.32(m,1H),4.29–4.21(m,2H),3.83–3.79(m,1H),3.61–3.56(m,2H),3.43– 3.41(m,2H),3.33(s,3H),3.10–3.03(m,2H),2.79–2.70(m,3H),2.46–2.42(m, 1H),2.13–2.06(m,3H),2.04–1.93(m,4H),1.87–1.78(m,4H),1.55–1.49(m,1H ),1.42(d,J=6.0Hz,3H),0.96–0.91(m,6H),0.78(d,J=7.0Hz,3H),0.33(s,3H).

[0518] Example 1.7: Synthesis of Compound 7

[0519] Compound 7 was prepared by the following steps:

[0520] Step 1: Compound INT-1 (18.5 g, 26.67 mmol) was dissolved in dichloromethane (80 mL), and trifluoroacetic acid (40 mL) was added. The reaction was carried out at room temperature for 4 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in dichloromethane (200 mL), washed twice with saturated sodium bicarbonate aqueous solution, washed with water (80 mL) in the organic phase, dried over sodium sulfate, filtered, and concentrated to give a yellow solid compound 7a (15.5 g, yield 97.9%). ESI-MS (m / z): 594.3 [M+H] + .

[0521] Step 2: Under ice bath conditions, (S)-2-hydroxy-3-methylbutyric acid (199 mg, 1.68 mmol), N,N-diisopropylethylamine (653 mg, 5.05 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (641 mg, 1.68 mmol) were added sequentially to a solution of compound 7a (1.0 g, 1.68 mmol) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred under these conditions for 2 hours. After the reaction was complete, water (50 mL) was added to the system, followed by extraction with ethyl acetate (60 mL x 3), washing with water (50 mL x 3) of the organic phase, drying over anhydrous sodium sulfate, and filtration to concentrate. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:8) to give a yellow solid compound 7b (853 mg, yield 73.0%). ESI-MS (m / z): 694.7 [M+H] + .

[0522] Step 3: Compound 7b (108 mg, 0.155 mmol) was dissolved in a mixed solution of 1,4-dioxane (4 mL) and water (0.4 mL). INT-3 (84 mg, 0.141 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (10 mg, 0.014 mmol), and potassium phosphate (90 mg, 0.423 mmol) were added sequentially. The reaction mixture was stirred at 70 °C for 16 hours under nitrogen protection. After the reaction was complete, water (40 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 7c. ESI-MS (m / z): 1083.5 [M + H] + .

[0523] Step 4: The crude compound 7c was dissolved in N,N-dimethylformamide (4 mL), and cesium carbonate (138 mg, 0.423 mmol) and iodoethane (66 mg, 0.423 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (40 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined and washed with water (30 mL * 2) and saturated brine (20 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography (dichloromethane / methanol = 20:1) to give compound 7d (83 mg, yield 53.0%). ESI-MS (m / z): 1111.3 [M+H] + .

[0524] Step 5: Compound 7d (83 mg, 0.075 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath to adjust the pH to 8. The mixture was extracted with dichloromethane (40 mL * 3), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude compound 7e. ESI-MS (m / z): 881.3 [M + H] + .

[0525] Step 6: The crude product 7e was dissolved in 1,2-dichloroethane (3 mL), and formaldehyde aqueous solution (19 mg, 37%) was added at room temperature. After 10 minutes, sodium borohydride acetate (79 mg, 0.373 mmol) was slowly added to the reaction solution, and the reaction solution was stirred at room temperature for 3 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution (5 mL), extracted with ethyl acetate (40 mL * 3), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and the organic phase was concentrated and purified by preparative thin-layer chromatography (dichloromethane / methanol = 12:1) and preparative liquid chromatography to obtain a white solid compound 7 (13 mg, two-step yield 19.5%). ESI-MS (m / z): 895.1 [M+H] + LC-MS retention time RT = 1.58 min.

[0526] 1H NMR(500MHz,DMSO-d6)δ9.29(d,J=2.0Hz,1H),8.53–8.50(m,1H),8.17(d,J=2.0Hz,1H),8.00(d,J=9.5Hz,1H),7.84(s,1H),7.78–7.7 4(m,1H),7.60(d,J=8.5Hz,1H),5.62–5.52(m,2H),5.17–5.12(m,1H),4.39–4.31(m,2H),4.27–4.22(m,2H),4.17–4.11(m,1H),3.73–3 .71(m,1H),3.60–3.57(m,2H),3.29(s,3H),3.03–2.99(m,1H),2.84–2.74(m,5H),2.43–2.41(m,1H),2.18(s,3H),2.13–2.08(m,1H), 2.05–1.91(m,7H),1.83–1.76(m,4H),1.54–1.49(m,1H),1.40(d,J=6.0Hz,3H),0.96(d,J=7.0Hz,3H),0.92–0.85(m,9H),0.37(s,3H).

[0527] Example 1.8: Synthesis of Compound 8

[0528] By replacing compound INT-13 in the synthesis step of compound 1 with compound INT-8, and replacing (S)-2-hydroxy-3-methylbutyric acid with (R)-2-hydroxy-3-methylbutyric acid, compound 8 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 1004.5 [M+H] + LC-MS retention time RT = 1.76 min.

[0529] Example 1.9: Synthesis of Compound 9

[0530] Compound 9 was prepared by the following steps:

[0531] Step 1: Compound INT-12 (15 mg, 0.018 mmol), BOC-N-methyl-L-valine (6 mg, 0.027 mmol), and N,N-diisopropylethylamine (7 mg, 0.054 mmol) were dissolved in N,N-dimethylformamide (2 mL), and HATU (13 mg, 0.033 mmol) was added. The mixture was reacted at room temperature for 1 hour. The reaction solution was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give a yellow oily compound 9a (13 mg, yield 69%). ESI-MS (m / z): 1050.7 [M+H] + .

[0532] Step 2: Compound 9a (13 mg, 0.012 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath to adjust the pH to 8. The mixture was extracted with dichloromethane (40 mL * 3), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and the residue was purified by preparative liquid chromatography to give a white solid compound 9 (6 mg, yield 35%). ESI-MS (m / z): 950.5 [M + H] + LC-MS retention time RT = 1.58 min.

[0533] Example 1.10: Synthesis of Compound 10

[0534] By replacing compound BOC-N-methyl-L-valine in the synthesis of compound 9 with compound Boc-N-methyl-D-valine, and using a similar method and reaction steps, compound 10 can be obtained. ESI-MS (m / z): 950.5 [M+H] + LC-MS retention time RT = 1.57 min.

[0535] Example 1.11: Synthesis of Compound 11

[0536] By replacing INT-13 in the synthesis of compound 1 with INT-12, and replacing (S)-2-hydroxy-3-methylbutyric acid with 1-hydroxycyclopropanecarboxylic acid, compound 11 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 922.2 [M+H] + LC-MS retention time RT = 1.54 min.

[0537] Example 1.12: Synthesis of Compound 12

[0538] By replacing INT-13 in the synthesis of compound 1 with INT-12, and (R)-2-cyclopentyl-2-hydroxyacetic acid with (S)-2-hydroxy-3-methylbutyric acid, compound 12 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 964.8 [M+H] + LC-MS retention time RT = 1.66 min.

[0539] Example 1.13: Synthesis of Compound 13

[0540] By replacing INT-13 in the synthesis step of compound 1 with INT-12, and replacing (S)-2-hydroxy-3-methylbutyric acid with 1-hydroxycyclobutylcarboxylic acid, compound 13 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 936.1 [M+H] + LC-MS retention time RT = 1.59 min.

[0541] Example 1.14: Synthesis of Compound 14

[0542] By replacing INT-13 in the synthesis step of compound 1 with INT-12, and replacing (S)-2-hydroxy-3-methylbutyric acid with 1-hydroxy-cyclopentanoic acid, compound 14 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 950.2 [M+H] + LC-MS retention time RT = 1.63 min.

[0543] Example 1.15: Synthesis of Compound 15

[0544] Compound 15 was prepared by the following steps:

[0545] Step 1: Compound INT-7e (217 mg, 0.318 mmol) was dissolved in 1,4-dioxane (5 mL) and water (0.5 mL). INT-18 (130 mg, 0.265 mmol), potassium carbonate (110 mg, 0.796 mmol), and [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium dichloride (17 mg, 0.026 mmol) were added sequentially. The reaction mixture was stirred at 50 °C under a nitrogen atmosphere for 16 hours. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 95:5) to give a pale yellow solid compound 15a (153 mg, yield 53.3%). ESI-MS (m / z): 1081.3 [M+H] + .

[0546] Step 2: Compound 15a (64 mg, 0.059 mmol) was dissolved in tetrahydrofuran (5 mL) and water (2.5 mL). Lithium hydroxide monohydrate (4.26 mg, 0.177 mmol) was added at 0 °C, and stirring was continued for 1 hour. The mixture was diluted with water (20 mL), adjusted to pH 5 with dilute hydrochloric acid, and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow solid, compound 15b (51 mg, yield 80.7%). ESI-MS (m / z): 1067.1 [M+H] + .

[0547] Step 3: N,N,N',N'-Tetramethylchloromethanesulfonamide hexafluorophosphate (26.8 mg, 0.096 mmol) and 1-methylimidazole (19.6 mg, 0.026 mmol) were added to acetonitrile (10 mL), stirred until dissolved, and a THF solution of compound 15b (51 mg, 0.047 mmol) in 5 mL was added dropwise at room temperature. After the addition was complete, the mixture was stirred for 1 hour. Water (40 mL) was added to the system, and the mixture was extracted with ethyl acetate (40 mL * 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 95:5) to give a pale yellow solid compound 15c (28 mg, yield 55.8%). ESI-MS (m / z): 1049.1 [M+H] + .

[0548] Step 4: Compound 15c (28 mg, 0.026 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (20 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative liquid chromatography to give a white solid compound 15 (10 mg, yield 39.5%). ESI-MS (m / z): 948.4 [M+H] + LC-MS retention time RT = 1.53 min.

[0549] 1 H NMR(500MHz,DMSO-d6)δ9.31(s,1H),8.27(d,J=26.9Hz,1H),8.15–8.00(m,1H),7.97–7.66(m,4H),6.00(d,J=11.2Hz,1H),5.71–5.56(m,1H ),5.35(d,J=27.0Hz,2H),4.94–4.80(m,1H),4.74(d,J=11.0Hz,1H),4.57–4.31(m,3H),4.05–3.99(m,1H),3.97–3.90(m,1H),3.82(s,1H),3 .31(s,3H),3.05–3.00(m,1H),2.89(s,2H),2.68–2.61(m,1H),2.45– 2.41(m,2H),2.38–2.29(m,3H),2.17–2.09(m,1H),2.04–1.98(m,1H), 1.91–1.80(m,1H),1.68–1.58(m,1H),1.45–1.32(m,4H),1.31–1.20(m ,1H),1.17–1.08(m,1H),0.98–0.87(m,6H),0.80(s,3H),0.29(s,3H).

[0550] Example 1.16: Synthesis of Compound 16

[0551] Compound 16 was prepared by the following steps:

[0552] Step 1: Compound INT-17 (35 mg, 0.037 mmol) was dissolved in 1,2-dichloroethane (6 mL), and N-methyl-4-piperidinone (12.5 mg, 0.11 mmol) was added at room temperature. After 20 minutes, sodium borohydride acetate (39 mg, 0.184 mmol) was slowly added to the reaction solution, and the reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL), extracted with ethyl acetate (40 mL * 3), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and the concentrated organic phase was purified by preparative thin-layer chromatography (dichloromethane / methanol = 8:1) to obtain a colorless oily compound 16a (22 mg, yield 57%). ESI-MS (m / z): 1043.8 [M+H] + .

[0553] Step 2: Compound 16a (22 mg, 0.021 mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (2 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (20 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid, compound 16b (19 mg, 96% yield). ESI-MS (m / z): 944.8 [M + H] + .

[0554] Step 3: Under ice bath conditions, (R)-2-hydroxy-3-methylbutyric acid (3.7 mg, 0.031 mmol), N,N-diisopropylethylamine (8.2 mg, 0.063 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (14.4 mg, 0.038 mmol) were added sequentially to a solution of compound 16b (19 mg, 0.20 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred under these conditions for 2 hours. The reaction mixture was purified by preparative liquid chromatography to give a white solid compound 16 (4 mg, yield 18.1%). ESI-MS (m / z): 1044.5 [M+H] + LC-MS retention time RT = 1.56 min.

[0555] 1H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.1Hz,1H),8.46(s,1H),8.27(s,1H),8.14(d,J= 8.9Hz,1H),7.92(s,1H),7.88(d,J=8.7Hz,1H),7.81(d,J=8.7Hz,1H),6.02(d,J=11 .0Hz,1H),5.70–5.61(m,1H),5.46–5.39(m,1H),5.37–5.30(m,1H),4.94–4.81(m,1 H),4.76(d,J=11.1Hz,1H),4.55–4.48(m,1H),4.39–4.32(m,1H),3.83(d,J=3.6Hz, 1H),3.58–3.51(m,5H),3.33(s,3H),3.09–3.02(m,3H),2.94–2.88(m,2H),2.83–2. 79(m,2H),2.69–2.63(m,2H),2.35–2.27(m,3H),2.15(s,3H),2.10–2.00(m,4H),1. 92–1.84(m,2H),1.81–1.76(m,2H),1.71–1.63(m,3H),1.51–1.46(m,1H),1.43(d,J =6.0Hz,3H),0.96(d,J=6.8Hz,3H),0.92(s,3H),0.81(d,J=6.8Hz,3H),0.31(s,3H).

[0556] Example 1.17: Synthesis of Compound 17

[0557] Compound 17 was prepared by the following steps:

[0558] Step 1: Compound INT-17 (171 mg, 0.18 mmol) was dissolved in 1,2-dichloroethane (6 mL), and 1-benzyloxycarbonyl-3-ketoazacyclobutane (111 mg, 0.54 mmol) was added at room temperature. After 20 minutes, sodium borohydride acetate (191 mg, 0.901 mmol) was slowly added to the reaction solution, and the reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL), extracted with ethyl acetate (40 mL * 3), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (dichloromethane / methanol = 93:7) to obtain a colorless oily compound 17a (180 mg, yield 87%). ESI-MS (m / z): 1137.4 [M+H] +.

[0559] Step 2: Compound 17a (180 mg, 0.158 mmol), 10% palladium hydroxide (18 mg), 10% palladium on carbon (18 mg), and methanol (5 mL) were added to a reaction flask. The reaction mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give compound 17b (150 mg, yield 94.5%). ESI-MS (m / z): 1003.3 [M+H] + .

[0560] Step 3: Compound 17b (150 mg, 0.119 mmol) was dissolved in methanol (5 mL), and formaldehyde aqueous solution (0.5 mL) was added at room temperature. After 20 minutes, sodium borohydride acetate (127 mg, 0.598 mmol) was slowly added to the reaction solution, and the reaction solution was stirred at room temperature for 16 hours. The reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL), extracted with ethyl acetate (40 mL * 3), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and the organic phase was concentrated and purified by preparative thin-layer chromatography (dichloromethane / methanol = 8:1) to obtain a colorless oily compound 17c (60 mg, yield 49.3%). ESI-MS (m / z): 1016.8 [M+H] + .

[0561] Step 4: Compound 17c (10 mg, 0.009 mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added to the reaction system under ice bath conditions to adjust the pH to 8. The mixture was extracted with dichloromethane (20 mL * 2), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a pale yellow solid compound 17d (ESI-MS (m / z): 917.3 [M + H)). + .

[0562] Step 5: Under ice bath conditions, (R)-2-hydroxy-3-methylbutyric acid (1.74 mg, 0.015 mmol), N,N-diisopropylethylamine (3.81 mg, 0.029 mmol), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6.73 mg, 0.017 mmol) were added sequentially to a solution of compound 17d (9 mg, 0.009 mmol) in N,N-dimethylformamide (2 mL). The reaction mixture was stirred under these conditions for 2 hours. The reaction mixture was purified by preparative liquid chromatography to give a white solid compound 17 (5 mg, 50% yield). ESI-MS (m / z): 1016.8 [M+H] + LC-MS retention time RT = 1.53 min.

[0563] 1 H NMR(500MHz,DMSO-d6)δ9.35(d,J=2.1Hz,1H),8.46(s,1H),8.28(s,1H),8.14(d,J=9.0H z,1H),7.92(s,1H),7.88(d,J=8.7Hz,1H),7.82(d,J=8.7Hz,1H),6.02(d,J=11.1Hz,1H) ,5.71–5.61(m,1H),5.46–5.39(m,1H),5.37–5.29(m,1H),4.93–4.82(m,1H),4.76(d,J= 11.0Hz,1H),4.54–4.49(m,1H),4.40–4.33(m,1H),3.83(d,J=3.6Hz,1H),3.57–3.55(m, 4H),3.33(s,3H),3.09–3.01(m,2H),2.87–2.82(m,2H),2.79–2.76(m,2H),2.74–2.69(m ,2H),2.68–2.64(m,2H),2.36–2.34(m,1H),2.23(s,3H),2.18–2.12(m,1H),2.09–2.02( m,3H),1.97–1.91(m,2H),1.84–1.76(m,2H),1.67–1.63(m,1H),1.43(d,J=6.0Hz,3H),0 .96(d,J=6.8Hz,3H),0.92(s,3H),0.89–0.86(m,1H),0.81(d,J=6.8Hz,3H),0.31(s,3H).

[0564] Example 1.18: Synthesis of Compound 18

[0565] By replacing compound 1e in the synthesis of compound 1 with compound INT-8, and using a similar method and reaction steps, compound 18 can be obtained. ESI-MS (m / z): 1004.5 [M+H] + LC-MS retention time RT = 1.64 min.

[0566] 1 H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.2Hz,1H),8.44(s,1H),8.28(s,1H),8.10(d,J=9 .0Hz,1H),7.93(s,1H),7.87(d,J=8.7Hz,1H),7.81(d,J=8.6Hz,1H),6.03(d,J=11.0H z,1H),5.71–5.65(m,1H),5.62(d,J=6.0Hz,1H),5.39–5.31(m,1H),4.92–4.81(m,1H) ,4.76(d,J=11.0Hz,1H),4.56–4.47(m,3H),4.43(t,J=6.1Hz,2H),4.35(d,J=6.1Hz,1 H),3.78–3.74(m,1H),3.55(s,2H),3.43–3.38(m,3H),3.33(s,3H),3.12–3.03(m,2H) ,2.75–2.70(m,2H),2.68–2.64(m,1H),2.48–2.44(m,2H),2.37–2.32(m,1H),2.14(t, J=9.8Hz,1H),2.10–2.06(m,3H),2.00–1.93(m,2H),1.87–1.79(m,2H),1.64(t,J=9.3 Hz,1H),1.42(d,J=6.0Hz,3H),0.98(d,J=6.8Hz,3H),0.96–0.90(m,6H),0.30(s,3H).

[0567] Example 1.19: Synthesis of Compound 19

[0568] Replace compound INT-12 in the synthesis step of compound 9 with compound INT-8; replace compound BOC-N-methyl-L-valine in the synthesis step of compound 9 with compound Boc-N-methyl-L-valine; compound 19 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 1118.0 [M+H] + LC-MS retention time RT = 1.75 min.

[0569] Example 2: Synthesis of linker-drug conjugate LP

[0570] Example 2.1: Synthesis of compound LP-1

[0571] Compound LP-1 was prepared by the following steps:

[0572] Step 1: Compound INT-15 (48 mg, 0.032 mmol), intermediate INT-13 (25 mg, 0.032 mmol), and N,N-diisopropylethylamine (12 mg, 0.094 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (18 mg, 0.047 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated, and the residue was purified by preparative liquid chromatography to obtain a white solid compound LP-1a (20 mg, yield 27%). ESI-MS (m / z): 1173.5 1 / 2 [M+H] + LC-MS retention time RT = 1.74 min.

[0573] Step 2: Compound LP-1a (10 mg, 0.004 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated. The residue was dissolved in N,N,-dimethylformamide (2 mL), and 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolyl) ester (3 mg, 0.008 mmol) and N,N-diisopropylethylamine (2 mg, 0.015 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 h. The reaction mixture was purified by preparative liquid chromatography to obtain a white solid compound LP-1 (8 mg, yield 79%). ESI-MS (m / z): 1195.4 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.45 min.

[0574] 1H NMR(500MHz,DMSO-d6)δ9.32(d,J=2.1Hz,1H),9.11(s,2H),8.81(d,J=9.3Hz,1H),8.53(s,1H) ,8.31–8.04(m,8H),7.84(s,1H),7.78(d,J=8.7Hz,1H),7.61(d,J=8.7Hz,1H),7.27–7.21(m,5H ),7.21–7.14(m,1H),5.56(t,J=9.2Hz,1H),5.14(d,J=12.0Hz,1H),4.94–4.87(m,1H),4.57–4. 48(m,2H),4.40–4.37(m,2H),4.35–4.31(m,3H),4.26–4.19(m,7H),4.11–3.90(m,10H),3.79–3 .70(m,7H),3.62–3.56(m,3H),3.40(s,3H),3.31(s,3H),3.08–3.01(m,3H),2.95–2.72(m,44H) ,2.56–2.54(m,2H),2.46–2.40(m,2H),2.34–2.30(m,2H),2.18(s,3H),2.08–2.03(m,4H),2.01 –1.96(m,3H),1.86–1.78(m,6H),1.70–1.61(m,1H),1.55–1.48(m,2H),1.40(d,J=6.0Hz,3H),1 .38–1.35(m,1H),0.94–0.90(m,6H),0.87(t,J=7.1Hz,3H),0.81(d,J=6.8Hz,3H),0.35(s,3H).

[0575] Example 2.2: Synthesis of compound LP-2

[0576] By replacing INT-13 in the synthesis of compound LP-1 with INT-12, and using a similar method and reaction steps, compound LP-2 can be obtained. ESI-MS (m / z): 1208.7 [M / 2+H] + LC-MS retention time RT = 1.60 min.

[0577] 1H NMR(500MHz,DMSO-d6)δ9.32(d,J=2.2Hz,1H),9.11(s,2H),8.79(s,1H),8.53(s,1H),8.42–8.36( m,2H),8.30(d,J=2.2Hz,1H),8.29–8.23(m,1H),8.24–8.18(m,1H),8.18–8.12(m,1H),8.11–7.99( m,2H),7.84(s,1H),7.78(d,J=8.6Hz,1H),7.61(d,J=8.8Hz,1H),7.24(d,J=4.5Hz,5H),7.21(s,1 H),6.72(s,1H),6.66(s,1H),5.56(s,1H),5.32(dd,J=5.5,4.2Hz,3H),5.13(d,J=12.5Hz,1H),4.9 1(s,1H),4.53(t,J=6.4Hz,3H),4.43(t,J=6.1Hz,2H),4.41–4.31(m,5H),4.22(d,J=17.6Hz,8H), 4.13–3.85(m,8H),3.80–3.69(m,3H),3.63–3.54(m,4H),3.40(s,3H),3.24(s,3H),2.97–2.72(m,5 0H),2.33(s,4H),2.08(d,J=11.9Hz,4H),1.83–1.77(m,4H),1.69–1.61(m,3H),1.51(s,2H),1.46 (d,J=7.2Hz,4H),1.41(d,J=5.9Hz,3H),0.85(t,J=7.0Hz,9H),0.81(d,J=6.8Hz,3H),0.35(s,3H).

[0578] Example 2.3: Synthesis of compound LP-3

[0579] By replacing INT-13 in the synthesis of compound LP-1 with INT-9, and using a similar method and reaction steps, compound LP-3 can be obtained. ESI-MS (m / z): 1244.1 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.45 min.

[0580] Example 2.4: Synthesis of compound LP-4

[0581] By replacing INT-13 in the synthesis of compound LP-1 with INT-8, and using a similar method and reaction steps, compound LP-4 can be obtained. ESI-MS (m / z): 1240.4 [M / 2+H] + LC-MS retention time RT = 1.48 min.

[0582] 1 H NMR(500MHz,DMSO-d6)δ9.36–9.32(m,1H),9.11(s,2H),8.84–8.78(m,1H),8.48(s,1H),8.31(d,J =9.1Hz,1H),8.30–8.25(m,1H),8.24(s,1H),8.17(d,J=8.0Hz,1H),8.11–8.01(m,3H),7.92(s,1H) ,7.91–7.76(m,3H),7.28–7.20(m,5H),7.21–7.15(m,1H),6.01(d,J=11.0Hz,1H),5.71–5.59(m,1H ),5.38–5.30(m,1H),4.91–4.84(m,2H),4.78(d,J=11.0Hz,1H),4.56–4.50(m,4H),4.46–4.40(m,3 H),4.38–4.31(m,4H),4.27–4.19(m,4H),4.06–3.90(m,12H),3.80–3.76(m,4H),3.75–3.67(m,6H) ,3.41(s,3H),3.33(s,3H),3.11–3.02(m,4H),2.97–2.72(m,44H),2.35–2.31(m,2H),2.20–2.14(m ,1H),2.12–2.06(m,2H),2.02–1.93(m,5H),1.86–1.75(m,6H),1.68–1.61(m,2H),1.42(d,J=5.9Hz ,3H),1.39–1.35(m,1H),1.31–1.26(m,2H),0.95–0.90(m,6H),0.82(d,J=6.8Hz,3H),0.30(s,3H).

[0583] Example 2.5: Synthesis of compound LP-5

[0584] Compound LP-5 was prepared by the following steps:

[0585] Step 1: Compound INT-14e (22 mg, 0.028 mmol), 10% palladium on carbon (5 mg), and ethanol (2 mL) were added to a reaction flask. The reaction mixture was stirred at room temperature for 2 h under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give compound LP-5a (18 mg, 93% yield). ESI-MS (m / z): 703.9 [M+NH4] + .

[0586] Step 2: Compound LP-5a (18 mg, 0.028 mmol), intermediate INT-11 (15 mg, 0.016 mmol), and N,N-diisopropylethylamine (6 mg, 0.047 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (9 mg, 0.023 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated, and the residue was purified by preparative liquid chromatography to obtain a white solid compound LP-5b (5 mg, 20% yield). ESI-MS (m / z): 1519.2 [M+H] + LC-MS retention time RT = 1.71 min.

[0587] Step 3: Compound LP-5b (5 mg, 0.003 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated to obtain a pale yellow oily compound LP-5c (4 mg, 100% yield). ESI-MS (m / z): 649.5 [M / 2+H] + .

[0588] Step 4: LP-5c (4 mg, 0.003 mmol), succinimide 6-(maleimide)hexanoate (2 mg, 0.006 mmol), and N,N-diisopropylethylamine (2 mg, 0.015 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then purified by preparative liquid chromatography to obtain a white solid compound LP-5 (2 mg, 40% yield). ESI-MS (m / z): 745.2 [M / 2+H] + LC-MS retention time RT = 1.53 min.

[0589] Example 2.6: Synthesis of compound LP-6

[0590] By replacing INT-11 in the synthesis of compound LP-5 with INT-12, and using a similar method and reaction steps, compound LP-6 can be obtained. ESI-MS (m / z): 1478.3 [M+H]+ LC-MS retention time RT = 1.77 min.

[0591] Example 2.7: Synthesis of compound LP-7

[0592] By replacing INT-13 in the synthesis of compound LP-1 with INT-11 and INT-15 with INT-26, compound LP-7 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 1215.2 [M / 2+H] + LC-MS retention time RT = 1.47 min.

[0593] Example 2.8: Synthesis of compound LP-8

[0594] Replacing INT-13 in the synthesis of compound LP-1 with INT-11, and using a similar method and reaction steps, yields compound LP-8. ESI-MS (m / z): 1214.3 [M / 2+H] + LC-MS retention time RT = 1.40 min.

[0595] 1H NMR(500MHz,DMSO-d6)δ9.32(d,J=2.1Hz,1H),9.11(s,2H),8.82–8.76(m,1H),8.53(s,1H),8.40 –8.36(m,1H),8.29(d,J=2.2Hz,1H),8.24(s,1H),8.20(s,1H),8.14(s,1H),8.09–7.99(m,3H),7 .84(s,1H),7.78(d,J=8.7Hz,1H),7.61(d,J=8.9Hz,1H),7.27–7.22(m,5H),5.61–5.54(m,1H),5 .13(d,J=11.8Hz,1H),4.91(s,1H),4.56–4.49(m,2H),4.41–4.31(m,7H),4.29–4.18(m,9H),4.1 2–3.90(m,14H),3.81–3.69(m,8H),3.40(s,3H),3.31(s,3H),3.07–3.02(m,4H),2.96–2.84(m,2 5H),2.79–2.72(m,16H),2.46–2.42(m,4H),2.35–2.30(m,4H),2.21(s,3H),2.10–2.04(m,1H),1 .97–1.89(m,4H),1.83–1.76(m,6H),1.70–1.63(m,2H),1.54–1.49(m,2H),1.40(d,J=5.9Hz,3H) ,1.30–1.26(m,2H),0.94–0.90(m,6H),0.87(t,J=7.1Hz,3H),0.81(d,J=6.8Hz,3H),0.35(s,3H).

[0596] Example 2.9: Synthesis of compound LP-9

[0597] Compound LP-9 was prepared by the following steps:

[0598] Step 1: Compound INT-15 (78 mg, 0.050 mmol), intermediate INT-8 (45 mg, 0.050 mmol), and N,N-diisopropylethylamine (20 mg, 0.15 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (28 mg, 0.075 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated, and the residue was purified by preparative liquid chromatography to obtain a white solid compound LP-9a (25 mg, 20% yield). ESI-MS (m / z): 1226.8 [M / 2+H]+ LC-MS retention time RT = 1.59 min.

[0599] Step 2: Compound LP-9a (25 mg, 0.010 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated to remove residual diethylamine and N,N,-dimethylformamide, yielding a pale yellow oily compound LP-9b. ESI-MS (m / z): 1116.0 [M / 2+H] + LC-MS retention time RT = 1.44 min.

[0600] Step 3: Compound LP-9b (5 mg, 0.002 mmol) and succinimide 6-(maleimide)hexanoate (1 mg, 0.003 mmol) were dissolved in N,N,-dimethylformamide (2 mL), and N,N-diisopropylethylamine (1 mg, 0.006 mmol) was added. The reaction mixture was stirred at room temperature for 1 h, and then purified by preparative liquid chromatography to obtain a white solid compound LP-9 (3 mg, 60% yield). ESI-MS (m / z): 1212.3 [M / 2+H] + LC-MS retention time RT = 1.49 min.

[0601] 1H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.2Hz,1H),8.79(s,1H),8.48(s,1H),8.40–8.32(m,3H),8.32–8.28(m,1H) ,8.17–8.12(m,2H),8.04–8.00(m,1H),7.97–7.92(m,1H),7.92(s,1H),7.88(d,J=8.6Hz,1H),7.81(d,J=8.7H z,1H),7.27–7.20(m,4H),7.20–7.15(m,1H),6.99(s,2H),6.00(d,J=11.2Hz,1H),5.72–5.60(m,1H),5.38–5. 31(m,1H),4.89–4.84(m,1H),4.78(d,J=10.9Hz,1H),4.57–4.49(m,5H),4.43(t,J=6.1Hz,2H),4.39–4.30(m, 5H),4.30–4.16(m,7H),4.10–3.99(m,6H),3.95–3.87(m,5H),3.80–3.75(m,3H),3.74–3.68(m,3H),3.67–3.6 1(m,4H),3.55(s,3H),3.10–3.03(m,4H),2.96–2.70(m,40H),2.47–2.42(m,2H),2.19–2.15(m,1H),2.12–2.0 8(m,4H),2.02–1.94(m,5H),1.86–1.77(m,4H),1.68–1.61(m,2H),1.49–1.44(m,5H),1.42(d,J=6.0Hz,3H),1 .39–1.35(m,1H),1.30–1.26(m,1H),1.20–1.14(m,3H),0.95–0.90(m,6H),0.82(d,J=6.8Hz,3H),0.30(s,3H).

[0602] Example 2.10: Synthesis of compound LP-10

[0603] Compound LP-10 was prepared by the following steps:

[0604] Step 1: Compound LP-9b (5 mg, 0.002 mmol), intermediate INT-16 (2.3 mg, 0.006 mmol), and N,N-diisopropylethylamine (1 mg, 0.006 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then purified by preparative liquid chromatography to obtain a white solid compound LP-10a (3.5 mg, yield 68%). ESI-MS (m / z): 1249.3 [M / 2+H] + LC-MS retention time RT = 1.53 min.

[0605] Step 2: Under ice bath conditions, compound LP-10a (3.5 mg, 0.01 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.1 mL) was added. The reaction mixture was stirred at room temperature for 3 h, then concentrated. The residue was purified by preparative liquid chromatography to obtain a white solid compound LP-10 (1.0 mg, yield 21%). ESI-MS (m / z): 1198.8 [M / 2+H] + LC-MS retention time RT = 1.41 min.

[0606] Example 2.11: Synthesis of compound LP-11

[0607] Compound LP-11 was prepared by the following steps:

[0608] Bromoacetic acid (11 mg, 0.079 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (19 mg, 0.076 mmol) were dissolved in N,N,-dimethylformamide (2 mL). After stirring the reaction solution at room temperature for 10 min, compound LP-9b (10 mg, 0.005 mmol) and 2,6-dimethylpyridine (8 mg, 0.076 mmol) were added. After stirring the reaction solution at room temperature for 2 h, the reaction solution was purified by preparative liquid chromatography to give a white solid compound LP-11 (4 mg, yield 38%). ESI-MS (m / z): 1177.1 [M / 2+H] + LC-MS retention time RT = 1.47 min.

[0609] 1H NMR(500MHz,DMSO-d6)δ9.35(d,J=2.1Hz,1H),8.82–8.75(m,1H),8.49(s,1H),8.45(d,J=7.7Hz,1H),8. 41–8.36(m,1H),8.34–8.26(m,3H),8.17–8.12(m,2H),8.01(s,1H),7.92(s,1H),7.88(d,J=87.0Hz,1H) ,7.81(d,J=8.7Hz,1H),7.30–7.16(m,5H),6.01(d,J=11.1Hz,1H),5.71–5.60(m,1H),5.39–5.32(m,1H) ,4.93–4.84(m,2H),4.79(d,J=11.1Hz,1H),4.59–4.50(m,5H),4.44(t,J=6.1Hz,2H),4.39–4.32(m,5H), 4.31–4.17(m,7H),4.12–3.98(m,7H),3.97–3.86(m,7H),3.81–3.76(m,3H),3.75–3.71(m,3H),3.67–3. 61(m,2H),3.56(s,3H),3.10–3.04(m,4H),2.98–2.72(m,38H),2.47–2.42(m,2H),2.21–2.14(m,1H),2. 12–2.07(m,2H),2.03–1.96(m,4H),1.90–1.74(m,5H),1.71–1.62(m,2H),1.55–1.49(m,1H),1.43(d,J= 5.9Hz,3H),1.40–1.35(m,1H),1.33–1.23(m,2H),0.96–0.90(m,6H),0.83(d,J=6.8Hz,3H),0.31(s,3H).

[0610] Example 2.15: Synthesis of compound LP-15

[0611] Compound LP-15 was prepared by the following steps:

[0612] Step 1: Compound LP-15a (69 mg, 0.095 mmol), 9-fluorenemethyl-N-succinimide carbonate (48 mg, 0.142 mmol), and N,N-diisopropylethylamine (37 mg, 0.284 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by reversed-phase column chromatography (acetonitrile / water = 40:60) to give a white solid compound LP-15b (83 mg, 92% yield). ESI-MS (m / z): 967.3 [M+NH4] + LC-MS retention time RT = 1.22 min

[0613] Step 2: Compound LP-15b (83 mg, 0.87 mmol) was dissolved in methanol (5 mL), and sulfoxide (21 mg, 0.175 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 3 h, and then concentrated. The residue was purified by reversed-phase column chromatography (acetonitrile / water = 50:50) to give a white solid compound LP-15c (68 mg, 80% yield). ESI-MS (m / z): 966.6 [M+H] + LC-MS retention time RT = 1.31 min

[0614] Step 3: Compound LP-15c (263 mg, 0.273 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated. The residue was dissolved in N,N,-dimethylformamide (2 mL), and fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (174 mg, 0.409 mmol), N,N-diisopropylethylamine (106 mg, 0.817 mmol), and HATU (186 mg, 0.490 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was purified by reversed-phase column chromatography (acetonitrile / water = 50:50) to give a white solid compound LP-15d (214 mg, yield 68%). ESI-MS (m / z): 1166.4 [M+NH4] + LC-MS retention time RT = 1.50 min.

[0615] Step 4: LP-15d (214 mg, 0.186 mmol) was dissolved in dichloromethane (5 mL), and 4M hydrochloric acid-dioxane solution (1 mL) was added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated. The residue was dissolved in N,N,-dimethylformamide (2 mL), and INT-14 (206 mg, 0.372 mmol), N,N-diisopropylethylamine (72 mg, 0.558 mmol), and TSTU (112 mg, 0.372 mmol) were added. The reaction mixture was stirred at room temperature for 1 h, and then purified by reversed-phase column chromatography (acetonitrile / water = 50:50) to obtain a white solid compound LP-15e (150 mg, yield 92%). ESI-MS (m / z): 1648.0 [M+NH4] + LC-MS retention time RT = 1.52 min.

[0616] Step 5: Compound LP-15e (150 mg, 0.032 mmol), 10% palladium on carbon (15 mg), and ethanol (2 mL) were added to the reaction flask. The reaction mixture was stirred at room temperature for 2 h under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated. The residue was purified by reversed-phase column chromatography (acetonitrile / water = 40:60) to give compound LP-15f (80 mg, yield 56%). ESI-MS (m / z): 779.1 [(M+NH4+H) / 2] + .

[0617] Step 6: Compound INT-15f (80 mg, 0.054 mmol), intermediate INT-8 (40 mg, 0.045 mmol), and N,N-diisopropylethylamine (17 mg, 0.134 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (26 mg, 0.068 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then purified by preparative liquid chromatography to obtain a white solid compound LP-15g (60 mg, yield 55%). ESI-MS (m / z): 1207.6 [M / 2+H] + LC-MS retention time RT = 1.53 min.

[0618] Step 7: Dissolve compound LP-15 g (20 mg, 0.008 mmol) in tetrahydrofuran (2 mL), add lithium hydroxide monohydrate aqueous solution (0.1 mL, 1 M) at 0 °C, and stir the reaction solution at 0 °C for 1 hour. Add diethylamine (10 μL) to the reaction solution and continue stirring at 0 °C for 1 hour. Quench the reaction solution with acetic acid (10 μL). Concentrate the reaction solution, and purify the residue by preparative liquid chromatography to obtain a white solid compound LP-15 h (10 mg, yield 55%). ESI-MS (m / z): 1089.5 [M / 2+H] + LC-MS retention time RT = 1.35 min.

[0619] Step 8: Compound LP-15h (8 mg, 0.004 mmol) and succinimide 6-(maleimide)hexanoate (2 mg, 0.005 mmol) were dissolved in N,N,-dimethylformamide (2 mL), and N,N-diisopropylethylamine (1 mg, 0.006 mmol) was added. The reaction mixture was stirred at room temperature for 1 h, and then purified by preparative liquid chromatography to obtain a white solid compound LP-15 (6 mg, yield 67%). ESI-MS (m / z): 1215.0 [M / 2+H] + LC-MS retention time RT = 1.34 min.

[0620] 1H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.1Hz,1H),9.11(s,1H),9.00–8.87(m,1H),8.55(s,1H) ),8.42(s,2H),8.38–8.29(m,3H),8.27(s,1H),8.24–8.13(m,1H),7.90(s,1H),7.87(d,J= 8.7Hz,1H),7.80(d,J=8.7Hz,1H),7.28–7.21(m,5H),7.19–7.14(m,1H),5.71–5.62(m,1H ),5.58–5.50(m,1H),5.13(d,J=12.0Hz,1H),4.94–4.82(m,2H),4.57–4.48(m,4H),4.43(t ,J=6.1Hz,2H),4.36–4.18(m,12H),4.11–3.94(m,9H),3.79–3.70(m,6H),3.63–3.52(m,7 H),3.42–3.38(m,6H),3.10–3.05(m,4H),2.93–2.81(m,22H),2.79–2.73(m,16H),2.43–2. 41(m,2H),2.11–2.05(m,4H),1.99–1.93(m,4H),1.86–1.75(m,8H),1.55–1.47(m,1H),1.4 2(d,J=5.9Hz,3H),0.94(s,3H),0.91(d,J=6.8Hz,3H),0.80(d,J=6.8Hz,3H),0.31(s,3H).

[0621] Example 2.16: Synthesis of compound LP-16

[0622] Compound LP-16 was prepared by the following steps:

[0623] Step 1: Compound INT-15 (21 mg, 0.013 mmol), compound 17d (11 mg, 0.012 mmol), and N,N-diisopropylethylamine (4.6 mg, 0.036 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (7 mg, 0.018 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then purified by preparative liquid chromatography to obtain a white solid compound LP-16a (10 mg, 33% yield). ESI-MS (m / z): 1233.5 [M / 2+H] + LC-MS retention time RT = 1.49 min.

[0624] Step 2: Compound LP-16a (10 mg, 0.004 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated to remove residual diethylamine and N,N,-dimethylformamide, yielding a pale yellow oily compound LP-16b. ESI-MS (m / z): 1122.9 [M / 2+H] + LC-MS retention time RT = 1.35 min.

[0625] Step 3: Compound LP-16b (10 mg, 0.004 mmol) and succinimide 6-(maleimide)hexanoate (1 mg, 0.003 mmol) were dissolved in N,N,-dimethylformamide (2 mL), and N,N-diisopropylethylamine (3 mg, 0.007 mmol) was added. The reaction mixture was stirred at room temperature for 1 h, and then purified by preparative liquid chromatography to obtain a white solid compound LP-16 (5 mg, 45% yield). ESI-MS (m / z): 1248.0 [M / 2+H] + LC-MS retention time RT = 1.39 min.

[0626] 1H NMR(500MHz,DMSO-d6)δ9.35(d,J=2.2Hz,1H),9.12(s,2H),8.81(s,1H),8.49(s,1H),8.28 (s,1H),8.24–8.20(m,1H),8.17–8.13(m,1H),8.10–8.02(m,2H),7.93(s,1H),7.88(d,J=9. 0Hz,1H),7.82(d,J=6.8Hz,1H),7.27–7.19(m,7H),6.67(s,1H),6.01(d,J=11.0Hz,1H),5.6 6(s,1H),5.36–5.30(m,2H),4.91–4.84(m,2H),4.79(d,J=11.0Hz,1H),4.56–4.51(m,3H),4 .38–4.31(m,6H),4.26–4.19(m,8H),4.09–4.03(m,5H),3.95–3.88(m,6H),3.81–3.77(m,4H ),3.73–3.70(m,4H),3.58–3.55(m,5H),3.41(s,3H),3.09–3.05(m,4H),2.95–2.77(m,44H) ,2.35–2.32(m,2H),2.22(s,3H),2.03–1.97(m,8H),1.83–1.76(m,6H),1.68–1.63(m,3H),1 .49–1.45(m,1H),1.43(d,J=5.7Hz,3H),0.95–0.91(m,3H),0.86–0.82(m,6H),0.31(s,3H).

[0627] Example 2.17: Synthesis of compound LP-17

[0628] Compound LP-17 was prepared by the following steps:

[0629] Step 1: Compound LP-17a (1.09 g, 2.49 mmol) and compound N6-BOC-L-lysine tert-butyl hydrochloride (902 mg, 2.98 mmol) were dissolved in tetrahydrofuran (10 mL) and water (2 mL). Sodium bicarbonate (418 mg, 4.98 mmol) was added under ice bath conditions. The reaction mixture was stirred under ice bath conditions for 3 h. Water (20 mL) was added to the system, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 60 / 40) to give a pale yellow solid compound LP-17b (1.1 g, 71% yield). ESI-MS (m / z): 624.7 [M+H] + LC-MS retention time RT = 2.09 min.

[0630] Step 2: Compound INT-17b (1.1 g, 1.76 mmol) was dissolved in dichloromethane (10 mL), and 4 M hydrochloric acid-dioxane solution (2 mL) was added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated to obtain a pale yellow solid compound INT-17c (800 mg, yield 97%). ESI-MS (m / z): 468.5 [M+H] + LC-MS retention time RT = 1.36 min.

[0631] Step 3: Compound LP-17c (416 mg, 0.889 mmol) and n-propionaldehyde (258 mg, 4.45 mmol) were dissolved in dichloromethane (10 mL). After stirring the reaction solution at room temperature for 0.5 h, sodium borohydride acetate (754 mg, 3.56 mmol) was added to the system, and the reaction solution was stirred at room temperature for another 16 h. The reaction was quenched by adding saturated ammonium chloride aqueous solution (20 mL), and extracted with dichloromethane (30 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 70 / 30) to give a pale yellow solid compound LP-17d (400 mg, yield 81%). ESI-MS (m / z): 551.9 [M+H] + LC-MS retention time RT = 1.43 min.

[0632] Step 4: Compound LP-17d (270 mg, 0.489 mmol), compound INT-14d (216 mg, 0.734 mmol), and N,N-diisopropylethylamine (189 mg, 1.47 mmol) were dissolved in N,N,-dimethylformamide (2 mL), and HATU (279 mg, 0.734 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL * 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give a pale yellow solid compound LP-17e (228 mg, yield 56%). ESI-MS (m / z): 827.9 [M+H] + LC-MS retention time RT = 1.98 min.

[0633] Step 5: LP-17e (61 mg, 0.074 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction mixture was stirred at room temperature for 0.5 h, and then concentrated. The residue was dissolved in N,N,-dimethylformamide (2 mL), and INT-15c (55 mg, 0.049 mmol), N,N-diisopropylethylamine (19 mg, 0.147 mmol), and HATU (28 mg, 0.074 mmol) were added. The reaction mixture was stirred at room temperature for 2 h, and then purified by reversed-phase column chromatography (acetonitrile / water = 40:60) to obtain a white solid compound LP-17f (72 mg, yield 85%). ESI-MS (m / z): 855.7 [M / 2+H] + LC-MS retention time RT = 1.68 min.

[0634] Step 6: Compound LP-17f (72 mg, 0.042 mmol), 10% palladium on carbon (10 mg), tetrahydrofuran (2 mL), and water (2 mL) were added to a reaction flask. The reaction mixture was stirred at room temperature for 2 h under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give compound LP-17g (50 mg, yield 73%). ESI-MS (m / z): 818.8 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.40 min.

[0635] Step 7: Compound INT-17 g (50 mg, 0.03 mmol), intermediate INT-8 (21 mg, 0.024 mmol), and N,N-diisopropylethylamine (9 mg, 0.071 mmol) were dissolved in N,N,-dimethylformamide (2 mL). HATU (14 mg, 0.036 mmol) was added. After stirring the reaction solution at room temperature for 1 h, the reaction solution was rapidly purified by reversed-phase column chromatography (acetonitrile / water = 40:60) to remove impurities related to the condensing agent. The crude product was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. After stirring the reaction solution at room temperature for 0.5 h, the reaction solution was purified by preparative liquid chromatography to obtain a white solid compound LP-17 h (10 mg, yield 18%). ESI-MS (m / z): 1141.1 [M / 2+H] + LC-MS retention time RT = 1.60 min.

[0636] Step 8: Compound LP-17h (10 mg, 0.004 mmol) and succinimide 6-(maleimide)hexanoate (2 mg, 0.005 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then purified by preparative liquid chromatography to obtain a white solid compound LP-17 (4 mg, yield 39%). ESI-MS (m / z): 1266.6 [M / 2+H] + LC-MS retention time RT = 1.52 min.

[0637] 1H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.2Hz,1H),9.11(s,2H),8.89(s,1H),8.48(s,1H),8 .32–8.28(m,1H),8.26(s,1H),8.23–8.18(m,1H),8.11–8.06(m,1H),8.06–8.00(m,1H), 7.90(s,1H),7.87(d,J=8.6Hz,1H),7.83–7.77(m,2H),7.25–7.16(m,1H),6.72–6.60(m ,1H),6.06–5.97(m,1H),5.73–5.62(m,1H),5.36–5.27(m,2H),4.91–4.86(m,1H),4.81– 4.75(m,1H),4.57–4.50(m,2H),4.43(t,J=6.0Hz,2H),4.38–4.17(m,15H),4.11–3.87( m,12H),3.80–3.76(m,2H),3.57–3.53(m,5H),3.40(s,3H),2.97–2.72(m,50H),2.26–2. 22(m,4H),2.10–2.06(m,2H),2.02–1.97(m,8H),1.85–1.79(m,4H),1.66–1.60(m,1H),1 .42(d,J=5.9Hz,3H),1.33–1.28(m,10H),0.92(s,3H),0.86–0.76(m,22H),0.29(s,3H).

[0638] Example 2.18: Synthesis of compound LP-18

[0639] Replacing INT-13 in the synthesis of compound LP-1 with INT-8 and INT-15 with INT-22, compound LP-18 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 1392.7 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.42 min.

[0640] Example 2.19: Synthesis of compound LP-19

[0641] Compound LP-19 was prepared by the following steps:

[0642] Step 1: Compound 19 (37 mg, 0.036 mmol), compound ((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)carbamate tert-butyl ester (40 mg, 0.72 mmol), 2-hydroxypyridine-N-oxide (4 mg, 0.036 mmol), and N,N-diisopropylethylamine (11 mg, 0.109 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 94 / 6) to give a pale yellow oily compound LP-19a (20 mg, yield 38%). ESI-MS (m / z): 1437.8 [M+H] + LC-MS retention time RT = 1.94 min.

[0643] Step 2: Compound LP-19a (20 mg, 0.014 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred under ice bath conditions for 3 h, and then concentrated. The residue was dissolved in N,N,-dimethylformamide (2 mL), and compound INT-15c (19 mg, 0.017 mmol), N,N-diisopropylethylamine (5 mg, 0.042 mmol), and HATU (7 mg, 0.018 mmol) were added. The reaction mixture was stirred at room temperature for 2 h, and then purified by reversed-phase column chromatography (acetonitrile / water = 40:60) to obtain a white solid compound LP-19b (8 mg, yield 23%). ESI-MS (m / z): 1221.5 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.70 min.

[0644] Step 3: Compound LP-19b (8 mg, 0.003 mmol) was dissolved in N,N,-dimethylformamide (2 mL), and diethylamine (0.1 mL) was added. The reaction solution was stirred at room temperature for 0.5 h, and then concentrated to obtain a yellow oily compound LP-19c (6 mg, 100% yield); ESI-MS (m / z): 1110.8 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.54 min.

[0645] Step 4: Compound LP-19c (6 mg, 0.003 mmol) and succinimide 6-(maleimide)hexanoate (2 mg, 0.005 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was then purified by preparative liquid chromatography to obtain a white solid compound LP-19 (4 mg, yield 49%). ESI-MS (m / z): 1234.1 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.58 min.

[0646] Example 2.20: Synthesis of compound LP-20

[0647] Replacing INT-13 in the synthesis of compound LP-1 with INT-8 and INT-15 with INT-24, compound LP-20 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 1190.5 [(M+NH4+H) / 2] + LC-MS retention time RT = 1.44 min.

[0648] Example 2.21: Synthesis of compound LP-21

[0649] By replacing 6-(maleimino)hexanoic acid succinimide in the synthesis of compound LP-9 with maleiminoacetic acid succinimide, compound LP-21 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 1184.8 [M / 2+H] + LC-MS retention time RT = 1.47 min.

[0650] 1H NMR(500MHz, DMSO-d6)δ9.34(d,J=2.1Hz,1H),8.82–8.76(m,1H),8.48(s,1H),8.42–8.34(m,2H),8.28–8.23( m,4H),8.15(d,J=8.1Hz,1H),8.00(s,1H),7.92(s,1H),7.88(d,J=8.7Hz,1H),7.81(d,J=8.7Hz,1H),7.27–7.2 2(m,4H),7.20–7.15(m,1H),7.07(s,2H),6.00(d,J=11.0Hz,1H),5.71–5.60(m,1H),5.38–5.31(m,1H),4.90– 4.83(m,1H),4.78(d,J=11.1Hz,1H),4.57–4.49(m,5H),4.43(t,J=6.1Hz,2H),4.37–4.31(m,4H),4.29–4.18(m ,6H),4.13–4.04(m,6H),4.00–3.86(m,7H),3.82–3.77(m,6H),3.73–3.70(m,4H),3.66–3.62(m,2H),3.56(s, 3H),3.09–3.04(m,4H),2.96–2.86(m,18H),2.83–2.81(m,4H),2.78–2.71(m,16H),2.46–2.43(m,2H),2.20–2. 14(m,1H),2.11–2.07(m,2H),2.01–1.93(m,5H),1.85–1.75(m,4H),1.68–1.62(m,2H),1.52–1.47(m,1H),1.42 (d,J=6.0Hz,3H),1.39–1.35(m,1H),1.31–1.22(m,2H),0.96–0.90(m,6H),0.82(d,J=6.8Hz,3H),0.30(s,3H).

[0651] Example 2.22: Synthesis of compound LP-22

[0652] By replacing INT15 in the synthesis of compound LP-1 with INT28, and using a similar method and reaction steps, compound LP-22 can be obtained. ESI-MS (m / z): 1151.6 [M / 2+H] + LC-MS retention time RT = 1.55 min.

[0653] 1H NMR(500MHz,DMSO-d6)δ9.34(d,J=2.1Hz,1H),9.11(s,2H),8.80(s,1H),8.48–8.43(m,1H),8.44–8.38(m,1H),8.33–8 .27(m,1H),8.27(s,1H),8.25–8.20(m,1H),8.19–8.14(m,1H),8.11–8.01(m,2H),7.92(s,1H),7.87(d,J=8.7Hz,1H),7 .83–7.78(m,2H),7.27–7.19(m,4H),7.19–7.12(m,1H),6.00(d,J=11.0Hz,1H),5.70–5.61(m,1H),5.38–5.30(m,1H),4 .90–4.83(m,2H),4.78(d,J=11.1Hz,1H),4.57–4.49(m,5H),4.43(t,J=6.1Hz,2H),4.38–4.33(m,1H),4.26–4.19(m,1H ),3.80–3.75(m,5H),3.72–3.69(m,4H),3.66–3.62(m,4H),3.58–3.54(m,7H),3.51–3.48(m,33H),3.47–3.45(m,7H), 3.42–3.39(m,9H),3.23(s,3H),3.09–3.04(m,4H),3.00–2.97(m,3H),2.75–2.69(m,3H),2.35–2.31(m,4H),2.30–2.26 (m,2H),2.18–2.15(m,1H),2.11–2.06(m,2H),1.99–1.93(m,3H),1.84–1.78(m,3H),1.66–1.61(m,2H),1.52–1.48(m,1 H),1.42(d,J=5.7Hz,3H),1.37–1.32(m,1H),1.25–1.22(m,1H),0.95–0.89(m,6H),0.82(d,J=6.8Hz,3H),0.30(s,3H).

[0654] Example 2.23: Synthesis of compound LP-23

[0655] Compound LP-23 was prepared by the following steps:

[0656] Step 1: Compound INT-32 (20 mg, 0.019 mmol), compound INT-29 (26 mg, 0.023 mmol), and N,N-diisopropylethylamine (40 mg, 0.309 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was purified by preparative liquid chromatography to give a white solid compound LP-23a (15 mg, 35% yield). ESI-MS (m / z): 1118.4 [M / 2+H] + LC-MS retention time RT = 1.58 min.

[0657] Step 2: LP-23a (15 mg, 0.006 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added under ice bath conditions. The reaction mixture was stirred under ice bath conditions for 2 hours. The reaction mixture was concentrated, and the residue was dissolved in N,N,-dimethylformamide (2 mL), and 6-[2-(methylsulfonyl)-5-pyrimidinyl]-5-hexynic acid (2,5-dioxo-1-pyrrolyl) ester (5 mg, 0.013 mmol) and N,N-diisopropylethylamine (3 mg, 0.023 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by preparative liquid chromatography to give a white solid compound LP-23 (4 mg, 24% yield). ESI-MS (m / z): 1193.5 [M / 2+H] + LC-MS retention time RT = 1.46 min.

[0658] Example 2.24: Synthesis of compound LP-24

[0659] Compound LP-24 was prepared by the following steps:

[0660] Compound LP-9b (5 mg, 0.002 mmol), compound INT-30 (2 mg, 0.005 mmol), and 2,6-dimethylpyridine (1 mg, 0.001 mmol) were dissolved in N,N,-dimethylformamide (2 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was purified by preparative liquid chromatography to give a yellow solid compound LP-24 (2 mg, 37% yield). ESI-MS (m / z): 1298.8 [M / 2+H] + LC-MS retention time RT = 1.50 min.

[0661] Example 3: Preparation of antibody-drug conjugates

[0662] The following are the antibody sequences used.

[0663] Sacituzumab(TROP2)

[0664] (Heavy chain)

[0665] (Light chain)

[0666] Telisotuzumab(cMET)

[0667] (Heavy chain)

[0668] (Light chain)

[0669] Tusamitamab (CEACAM5)

[0670] (Heavy chain)

[0671] (Light chain)

[0672] MAB20(PD-L1)

[0673] (Heavy chain)

[0674] (Light chain)

[0675] Gemtuzumab (CD33)

[0676] (Heavy chain)

[0677] (Light chain)

[0678] Example 3.1: A general method for preparing TROP2 antibody-drug conjugates

[0679] Take 200 μL of 10 mg / mL Sacituzumab antibody and add 40 μL of EDTA solution (1 mM) and mix well. Then add 5.4 μL of TCEP (tris(2-carboxyethyl)phosphine) solution (50 mM) and mix well. Add 154.6 μL of DPBS (VivaCell, 2214082) solution to make the total reaction volume 400 μL. React at 37 °C for 90 min. Add 13.3 μL of dimethyl sulfoxide to the above solution system and mix well. Then add 26.7 μL of dimethyl sulfoxide solution (10 mM) of the linker drug conjugate LP (20 times antibody equivalent) to make the final concentration of dimethyl sulfoxide in the system 10%. Mix by rotation at room temperature for 3 h. After completion, add 8 μL of 100 mM cysteine ​​and mix by rotation at room temperature for 30 min to terminate the reaction. Centrifuge the reaction system at 12000g for 10 min at room temperature, collect the supernatant, and finally use Zeba... TM A desalting centrifuge column (Thermo Fisher, A57761) was used to replace the buffer with a 20 mM histidine and 240 mM sucrose buffer at pH 5.5. The conjugate product of Sacituzumab antibody and the linker drug conjugate LP was obtained. The ADC was characterized by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC), revealing that it was primarily a DAR8 conjugate.

[0680] The general steps for conjugating the antibody and linker drug conjugate of the present invention can also be described as follows: First, the disulfide bonds between the antibody chains are opened using a reducing agent such as TCEP; second, the obtained thiol group reacts with the linker drug conjugate LP of the present invention (if the linker of LP is maleimide, a Michael addition reaction occurs (after conjugation, succinimide can hydrolyze and open the ring of some maleimide linkers LP); if the linker of LP is methanesulfonylpyrimidine, a substitution reaction occurs; if the linker of LP is bromoacetyl, a substitution reaction occurs) to obtain the antibody-drug conjugate.

[0681] Example 3.2: A general method for preparing cMET antibody-drug conjugates

[0682] Referring to the preparation method in Example 3.1, Sacituzumab was replaced with Telisotuzumab to obtain the cMET antibody-drug conjugate, which is mainly a DAR10 conjugate product.

[0683] Example 3.3: A General Method for Preparing CEACAM5 Antibody-Drug Conjugates

[0684] Referring to the preparation method in Example 3.1, Sacituzumab was replaced with Tusamitamab to obtain the CEACAM5 antibody-drug conjugate, which is mainly a DAR8 conjugate product.

[0685] Example 3.4: A general method for preparing PD-L1 antibody-drug conjugates

[0686] Referring to the preparation method in Example 3.1, Sacituzumab was replaced with MAB20 to obtain the PD-L1 antibody-drug conjugate, which is mainly a DAR8 conjugate product.

[0687] Example 3.5: A General Method for Preparing CD33 Antibody-Drug Conjugates

[0688] Referring to the preparation method in Example 3.1, Sacituzumab was replaced with Gemtuzumab to obtain the CD33 antibody-drug conjugate, which is mainly a DAR8 conjugate product.

[0689] HIC-HPLC Method Conditions

[0690] Instrument Model: DIONEX UltiMate 3000

[0691] Chromatographic column: HIC Ethyl, 4.6×35mm, 5μm

[0692] Mobile phase:

[0693] Mobile phase A: 0.1 mol / L NaH₂PO₄, 2 mol / L (NH₄)₂SO₄, pH = 7

[0694] Mobile phase B: 0.1 mol / L NaH2PO4, pH = 7

[0695] Mobile phase C: Isopropanol

[0696] Column temperature: 30℃, sample chamber temperature: room temperature

[0697] The flow rate was 0.8 mL / min, and gradient elution was performed for 40 minutes.

[0698] The mobile phase parameters are:

[0699] Injection volume: 10 μL, Detection wavelength: 280 nm

[0700] Sample preparation: Dilute the sample to 1 mg / mL with DPBS.

[0701] SEC-HPLC method conditions

[0702] Instrument Model: DIONEX UltiMate 3000

[0703] Column: TSKgel G3000SWXL, 7.8mm ID*30cm, 5μm

[0704] Mobile phase: 90% (40 mmol / L NaH₂PO₄, 400 mmol / L NaClO₄), 10% (v / v) acetonitrile, pH = 6.8

[0705] Column temperature: room temperature; Sample chamber temperature: room temperature; Flow rate: 0.8 mL / min

[0706] Injection volume: 10 μL; Detection wavelength: 280 nm

[0707] Sample preparation: Dilute the sample to 1 mg / mL with DPBS.

[0708] The following example ADCs (Sacituzumab, Tusamitamab, MAB20, and Gemtuzumab antibody conjugates) were prepared using common conjugation methods:

[0709] The following example ADCs (Telisotuzumab antibody-conjugated) were prepared using a common conjugation method:

[0710] Experimental Example 1: Test of the inhibitory activity of the compound on the proliferation of tumor cells in vitro

[0711] Assay method for the inhibitory activity of the compound on tumor cells: The compound disclosed herein was co-cultured with tumor cells, and then CTG (CellTiter-Glo) reagent (Promega, G7573) was added. The chemiluminescence value was read using an ELISA reader (manufacturer: Molecular Devices, model: SpectraMax i3x) to evaluate the inhibitory effect of the compound on cell proliferation.

[0712] The tumor cell information used is shown in the table below:

[0713] Universal in vitro cell viability assay: Dilute the compound (3-fold dilution, 9 concentration gradients) with the corresponding assay medium (containing 10% FBS). Digest tumor cells with trypsin, collect and count the cells, and resuspend them in the corresponding assay medium (containing 10% FBS) to a cell density of 3 x 10⁻⁶ cells / mL. 4Cells / mL were collected and 100 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated overnight at 37°C with 5% CO2. After overnight cell adhesion, 100 μL of diluted bioactive molecule was added to each well of the plate, using the detection medium as a control. Cells were then cultured for either 3 days (payload) or 6 days (ADC). Next, 50 μL of CTG reagent was added to each well, and the plates were incubated at room temperature in the dark for 10 min. 100 μL of the solution from each well was then transferred to an opaque white plate, and the chemiluminescence value was read. The inhibition rate was calculated.

[0714] Inhibition rate (100%) = (1 - (sample signal value - average value of cell-free well samples) / (average signal value of non-killing well samples - average value of cell-free well samples)) * 100

[0715] Experimental Example 1.1: The results of the in vitro inhibitory activity test of the bioactive molecule (drug payload) on tumor cell proliferation in this application are shown in the table below:

[0716] As can be seen from the data in the table above, the bioactive molecules (drug payload) used in the ADC of this application have strong inhibitory activity against the proliferation of multiple KRAS-mutant tumor cells.

[0717] Experimental Example 1.2: The results of the in vitro proliferation inhibition activity test of the anti-TROP2 ADC in this application against TROP2-expressing CAPAN2, NCI-H358 and HPAC cells are shown in the table below:

[0718] As can be seen from the data in the table above, the TROP2 ADC compound of this application has strong inhibitory activity against the proliferation of TROP2-expressing CAPAN2, NCI-H358 and HPAC tumor cells.

[0719] Experimental Example 1.3: The results of the in vitro proliferation inhibition activity test of the anti-cMET ADC in this application against multiple cell lines, including Capan-2, are shown in the table below:

[0720] As can be seen from the data in the table above, the cMET ADC compound of this application has strong cell proliferation inhibitory activity against cMET-positive KRAS mutant tumor cells.

[0721] Experimental Example 1.4: The results of the in vitro proliferation inhibitory activity test of the anti-CEACAM5 ADC in this application against HPAC and other cells are shown in the table below:

[0722] As can be seen from the data in the table above, the CEACAM5 ADC compound of this application has strong cell proliferation inhibitory activity against CEACAM5-positive KRAS-mutant tumor cells.

[0723] Experiment Example 2: Thermostability Experiment of Antibody-Drug Conjugates

[0724] Sample preparation: The antibody-drug conjugate of this application was diluted to a working concentration of 4 mg / mL using buffer (20 mM histidine, 240 nM sucrose, pH 5.5), with a total volume of 200 μL. The solution was placed in a 1.5 mL centrifuge tube and heated in a 40 °C water bath. After heating for 0 and 72 hours respectively, the centrifuge tube was removed, mixed thoroughly, and 30 μL of the sample was taken out and stored at -80 °C for later analysis.

[0725] SEC-HPLC detection: Take the incubated sample, dilute the sample to 1 mg / mL with DPBS, inject 10 μL, and detect the antibody aggregation after different incubation times.

[0726] Table 1. Results of the thermal stability of antibody-drug conjugates

[0727] As can be seen from the results in Table 1, the ADCs of different antibodies conjugated with LP in this invention all have good thermal stability.

[0728] Experimental Example 3: Stability Experiment of Antibody-Drug Conjugate in Human Plasma

[0729] The stability of the antibody-drug conjugate of this invention in human plasma was evaluated by measuring the change in its DAR value. The specific steps are as follows:

[0730] 1. Dilute the antibody-drug conjugate to 1 mg / mL using human plasma containing 0.05% sodium azide, and incubate at 37°C for 7 days.

[0731] 2. Resuspend the avidin magnetic beads (ACRO, Cat: SMB-B01-5 mg) in ultrapure water to 1 mg / mL. Take 200 μL of the magnetic beads, adsorb them on a magnetic rack for 2 min, and discard the supernatant.

[0732] 3. Wash the magnetic beads three times with Assay buffer (PBS, pH 7.3, with 0.05% Tween-20).

[0733] 4. Take Human CEACAM5-biotin protein (ACRO, Cat: CE5-H82E0) and dilute it to 100ug / mL using Assay buffer. Discard the supernatant after washing the avidin beads, add 200uL of Human CEACAM5-biotin protein solution, vortex and incubate at room temperature for 2h.

[0734] 5. After incubation, place the magnetic beads on a magnetic rack for 2 minutes to attract them, then discard the supernatant. Wash three times with Assay buffer, discarding the supernatant after the last wash.

[0735] 6. Take 100 μL of human plasma sample containing the antibody-drug conjugate and add it to the magnetic beads. After vortexing, place the mixture on a rotary mixer and incubate at room temperature for 2 hours.

[0736] 7. After incubation, place the magnetic beads on a magnetic rack to attract them for 2 minutes, then discard the supernatant. Wash three times with Assay buffer, discarding the supernatant after the last wash.

[0737] 8. Add 100 μL of 10% acetonitrile aqueous solution, shake for 5 seconds, and discard the supernatant.

[0738] 9. Add 50 μL of Elution Buffer (30% acetonitrile, 1% formic acid aqueous solution), vortex and shake, then place on a rotary mixer and elute by rotation at room temperature for 10 min.

[0739] 10. Place the magnetic beads on a magnetic rack for 2 minutes to attract them, collect the supernatant, and use LC-MS to detect the DAR value.

[0740] Table 2. Results of human plasma stability experiments of the antibody-drug conjugate of the present invention.

[0741] The results shown in Table 2 indicate that the DAR value of the antibody-drug conjugate ADC3-4 of the present invention remained at 7.85 after 7 days of incubation in human plasma, demonstrating good plasma stability.

[0742] Experimental Example 4: Bystander Killing Effect of Antibody-Drug Conjugates

[0743] To detect the bystander effect of antibody-drug conjugates, SK-CO-1 cells were used as CEACAM5-positive cells, and SW480_Luciferase cells (constructed by screening monoclonal strains after infecting SW480 cells with pLV[Exp]-Puro-EF1A>Luciferase lentivirus) were used as CEACAM5-negative cells. Detection was performed using a chemiluminescence method. SK-CO-1 and SW480_Luciferase cells in logarithmic growth phase were harvested, and the cell densities of the two cell types were adjusted to 4 x 102. 5 cells / mL, 4x10 4Cells / mL. Add 50 μL of each of the two cell types to each well of a 96-well plate and incubate overnight at 37°C in a 5% CO2 cell culture incubator. Dilute the antibody-drug conjugate (ADC3-4, 3-fold dilution, 9 concentration gradients) with the corresponding detection medium (containing 10% FBS). After overnight incubation, add 100 μL of the antibody-drug conjugate to each well and incubate for 6 days at 37°C in a 5% CO2 cell culture incubator. After incubation, add 50 μL of One-Glo reagent (Promega, E6130) to each well, incubate at room temperature in the dark for 3 min, and then transfer 100 μL from each well to an opaque white plate. Read the chemiluminescence value using a microplate reader (manufacturer: Molecular Devices, model: SpectraMax i3x).

[0744] As can be seen from the data in Figure 1, the antibody-drug conjugate ADC3-4 of the present invention has an excellent bystander killing effect.

[0745] Experiment Example 5: Pharmacokinetics of Antibody-Drug Conjugates in Non-tumor-bearing Mice

[0746] Non-tumor-bearing mice (CB-17 / SCID) were administered a single intravenous injection of 3 mg / kg of the antibody-drug conjugate ADC3-4 and its corresponding naked anti-tumor tusamitamab. Blood samples were collected intravenously at 0.0035, 1, 2, 4, 7, 14, 21, and 28 days post-administration to evaluate its pharmacokinetics. The concentration of total antibodies in mouse plasma was detected using ELISA. Specific experiments are as follows:

[0747] 1. Plate coating: Take CEACAM5 protein (Essential Biotech, cat: 11077-H08H) diluted to 1ug / mL with 1XDPBS, coat 100uL per well, and incubate overnight at 4℃.

[0748] 2. Blocking: Take a 96-well plate, discard the liquid, wash 4 times with PBST, and then add 300uL of 2% BSA in PBS to each well for blocking at room temperature for 2h.

[0749] 3. Standard curve preparation: Prepare a standard curve according to the analyte, starting at a concentration of 100 ng / mL, diluted 2-fold, with a mouse plasma concentration of 5%.

[0750] 4. After blocking, wash the plate four times with PBST and blot dry. Take the ADC and dilute the standard curve with DPBS containing 5% mouse plasma.

[0751] 5. Sample loading: Take mouse plasma samples and dilute them a certain factor. Add 95 μL of DPBS and 5 μL of diluted plasma sample to each sample well. Add 100 μL of prepared standard curve sample directly to each standard curve well. Gently tap to mix and incubate at room temperature for 2 hours.

[0752] 6. Add secondary antibody: After incubation, wash the plate 4 times with PBST and pat the plate dry. Take the secondary antibody, dilute it 1:2000 with blocking buffer, add 100 μL of secondary antibody to each well, and incubate at room temperature for 1 hour.

[0753] 7. Color development: After secondary antibody incubation, wash the plate 4 times with PBST, then add 100 μL of TMB and develop color at room temperature for 5 min.

[0754] Add 50 μL of stop solution to stop the color development, and read the absorbance at OD450.

[0755] Table 3. Pharmacokinetics of ADC3-4 and its corresponding mouse pharmacokinetic results

[0756] As shown in Table 3 and Figure 2, the antibody-drug conjugate ADC3-4 of the present invention and its corresponding naked anti-Tusamitamab have similar PK performance when administered intravenously, and their pharmacokinetic properties in mice are good.

[0757] Experiment Example 6: Intra-in vivo efficacy test of antibody-drug conjugates

[0758] Experimental Example 6.1: Efficacy Test of the ADC of this Application on a Human Colorectal Cancer Xenograft Model of CL-40 (KRAS G12D)

[0759] The antitumor effect of ADCs in a B-NDG mouse model of subcutaneous xenografted CL-40 human colorectal cancer cells was evaluated. The specific methods are as follows: Human colon cancer CL-40 cells were purchased from ATCC (American College of Medicine) and cultured at 37°C in a 5% CO2 incubator. The culture medium consisted of Dulbecco's Modified Eagle Medium containing 20% ​​inactivated fetal bovine serum. After the cells reached a certain number, they were harvested, and the CL-40 cells, resuspended in PBS and matrix gel at a 1:1 ratio, were incubated at 2 × 10⁻⁶ cells / mL. 6 0.1 mL / mouse was injected subcutaneously into the right back of female B-NDG mice until the average tumor volume reached 150-200 mm. 3 At that time, mice were randomly divided into groups according to tumor volume and body weight. ADC was administered intravenously once a week at a dose of 10 mg / kg for a total of 3 weeks.

[0760] The main observation indicators in this experiment were: 1) Tumor inhibition rate (TGI%), calculated using the formula: TGI TV(%) = [1-(Ti-T0) / (Ci-C0)]×100%, Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; Ci: mean tumor volume of the control group on day i of administration, C0: mean tumor volume of the control group on day 0 of administration; 2) Changes in body weight of mice after administration; 3) Photographs of tumor volume and weight at the endpoint of the experiment.

[0761] Table 4A. Efficacy of ADC in B-NDG mouse CL-40 xenograft model

[0762] As shown in Table 4A and Figure 3A, compounds ADC2-4 and ADC3-4 of the present invention exhibited good antitumor effects in the CL-40 model at a weekly dose of 10 mg / kg. Figure 3B shows that the ADCs of the present invention significantly improved the weight loss in mice caused by tumor burden during administration, demonstrating good safety.

[0763] Experimental Example 6.2: Efficacy Test of the ADC of this Application on HPAC (KRAS G12D) Human Pancreatic Cancer Xenograft Model

[0764] The antitumor effect of the test drug in a female BALB / c-nude mouse model of subcutaneous HPAC xenograft was evaluated. The specific methods are as follows: Human pancreatic cancer HPAC cells were purchased from Nanjing Kebai. Cell culture was conducted at 37℃ in a 5% CO2 incubator. HPAC-specific culture medium was purchased from Pro-Cell (CAT: CM-0361). After the cells reached a certain number, they were harvested, and HPAC cells resuspended in blank culture medium and matrix gel at a 1:1 ratio were cultured at 2×10⁻⁶ cells / year. 6 0.1 mL / mouse was injected subcutaneously into the right back of BALB / C-nude mice until the average tumor volume reached approximately 150 mm. 3 At that time, mice were randomly divided into groups according to tumor volume and body weight, and administered intravenously as a single dose of 10 mg / kg or 3 mg / kg.

[0765] The main observation indicators in this experiment were: 1) Tumor inhibition rate (TGI%), calculated using the formula: TGI TV (%) = [1-(Ti-T0) / (Ci-C0)]×100%, Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; Ci: mean tumor volume of the control group on day i of administration, C0: mean tumor volume of the control group on day 0 of administration; 2) Changes in body weight of mice after administration; 3) Photographs of tumor volume and weight at the endpoint of the experiment.

[0766] Table 4B. Efficacy of ADC in BALB / C-nude mouse HPAC xenograft model

[0767] As shown in Table 4B and Figure 4A, compounds ADC2-4 and ADC3-4 of the present invention, when administered as a single dose at doses of 3 mg / kg and 10 mg / kg, both exhibited good tumor-suppressing effects in the HPAC model. Figure 4B shows that compounds ADC2-4 and ADC3-4 of the present invention have good safety profiles, and no decrease in mouse weight was observed during the experiment.

Claims

The antibody-drug conjugate of Formula I, or its stereoisomers, tautomers, racemates, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, or its pharmaceutically acceptable salts or solvates: in, Ab is an antibody or its antigen-binding fragment, or an antigen ligand; L is a segment that covalently connects Ab and P; q is the number of LPs coupled on Ab, and q is any value between 1.0 and 16.0; P is a pan RAS inhibitor fragment, which is a structural unit shown in Formula II, and P is connected to L through an oxygen atom, sulfur atom, or nitrogen atom it contains: in: Cya said or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups; A represents a 4- to 6-membered heterocyclic alkylene, phenylene, or a 5- to 6-membered heterocyclic aryl group, wherein each of the 4- to 6-membered heterocyclic alkylene, phenylene, or 5- to 6-membered heterocyclic aryl group can be independently represented by 0, 1, 2, 3, or 4 R's. x replace; B represents a 5- to 6-membered heteroaryl group or a phenylene group, wherein the 5- to 6-membered heteroaryl group or phenylene group can be independently represented by 0, 1, 2, 3, or 4 R groups. x replace; X and Y independently represent hydrogen, C1-C6 aminoalkyl, C1-C6 hydroxyalkyl, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C 12 Cycloalkyl, 5- to 6-membered heteroaryl or phenyl, wherein the C1-C6 aminoalkyl, C1-C6 hydroxyalkyl, C1-C6 alkyl, 4- to 12-membered heterocycloalkyl, C3-C 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2 or 3 heteroatoms selected from N, O, and S; Z represents -OR a -SR a Or -NR a R a '; R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 membered heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a 'The methylene group on the C0-C6 alkylene, C1-C6 alkylene, C1-C6 alkyl, or C1-C6 haloalkyl group can be replaced with a carbonyl group or -NR group.' a -, -O-, or -S-, and optionally, each of the C0-C6 alkylene, C1-C6 alkylene, C1-C6 alkyl, and C1-C6 haloalkyl groups can be independently substituted by 0, 1, 2, 3, or 4 substituents selected from halogens or C1-C3 alkyl groups, and the two substituents of the same C atom can form a 3-8 membered ring with the C atom, the 3-8 membered ring optionally also containing 0, 1, 2, or 3 heteroatoms selected from N, O, or S; each of the C3-C8 cycloalkyl or 4-8 membered heterocycloalkyl groups can be independently substituted by 0, 1, 2, 3, or 4 substituents selected from halogens, oxoalkyl, -OR-, -O-, -O-, -S-, -O ... a -SR a -NR a R a ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-(C3-C8 cycloalkyl) or -(C0-C3 alkylene)-(4-8 heterocyclic alkyl), -C(O)R a -C(O)OR a -C(O)NR a R a '、-NR a C(O)R a '、-OC(O)R a '、-OC(O)NR a R a '、-NR a C(O)NR a R a '、-S(O)R a -S(O)2R a -NR a S(O)2R a Substituents of '; R2 represents a C1-C6 alkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), or -(C0-C6 alkylene)-(4-8 heterocyclic alkyl), which may optionally be substituted with 0, 1, or 2 substituents selected from the following: -OR a -SR a Or -NR a R a '; R3 and R3' each independently represent hydrogen, halogen, C1-C6 alkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl) or -(C0-C6 alkylene)-CN; R4 represents hydrogen, C1-C6 alkyl, -(C0-C6 alkylene)-OR a -(C0-C6 alkylene)-SR a -(C0-C6 alkylene)-NR a R a ', -(C0-C6 alkylene)-(C3-C8 cycloalkyl) or -(C0-C6 alkylene)-(4-12 heterocyclic alkyl), -(C0-C6 alkylene)-phenyl or -(C0-C6 alkylene)-(5-6 heteroaryl), wherein any methylene group on the C0-C6 alkylene or C1-C6 alkyl group can be replaced with a carbonyl group, -NR a -, -O-, or -S-, and optionally, the C0-C6 alkylene or C1-C6 alkyl group may be substituted with 0, 1, 2, 3, or 4 substituents selected from halogens or C1-C3 alkyl groups, and the two substituents of the same C atom may form a 3-8 membered ring with the C atom, the 3-8 membered ring optionally also containing 0, 1, 2, or 3 heteroatoms selected from N, O, or S; the C1-C6 alkyl, C3-C8 cycloalkyl, 4-12 membered heterocycloalkyl, phenyl, and 5-6 membered heteroaryl groups may each be independently substituted with 0, 1, 2, 3, or 4 substituents selected from halogens, oxo-, -OR-, -O ... a -SR a -NR a R a ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-(C3-C8 cycloalkyl) or -(C0-C3 alkylene)-(4-8 heterocyclic alkyl), -C(O)R a -C(O)OR a -C(O)NR a R a '、-NR a C(O)R a '、-OC(O)R a '、-OC(O)NR a R a '、-NR a C(O)NR a R a '、-S(O)R a -S(O)2R a -NR a S(O)2R a Substituents of '; L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a -, -O- or -S-, and optionally, the methylene groups on the -(C1-C6) alkylene groups can each be independently replaced by 0, 1, 2, 3 or 4 C1-C3 alkyl groups, and the two substituents on the same C atom can form a 3-8 membered ring with the C atom; Cy1 represents C3-C 12 Cycloalkyl or 4-12 membered heterocyclic alkyl, wherein the ring can be monocyclic, spirocyclic, bridged, or fused. R5 represents hydrogen, halogen, oxidative oxidation, and =NR independently. a -OR a -SR a -NR a R a ', cyano, -C(O)OR a -C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )R a ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a Substitution with ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-C3-C8 cycloalkyl or -(C0-C3 alkylene)-4-8 heterocyclic alkyl groups; Where m represents 0, 1, 2 or 3; R x Each independently represents hydrogen, halogen, oxo, =NR a -OR a -SR a -NR a R a ', cyano, -C(O)OR a -C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )R a ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; when R a R a When connected to the same N atom, the R a and R a 'The N atom and the N atom bonded together can form a 4-8 membered ring, which may optionally contain one, two or three heteroatoms selected from N, O or S; The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms. The antibody-drug conjugate as claimed in claim 1, or its stereoisomers, tautomers, racemates, or mixtures thereof, or its pharmaceutically acceptable salts or solvates, wherein, P is connected to L by removing an H atom or a monovalent group attached to an oxygen, sulfur, or nitrogen atom; preferably, P is connected to L by removing Ra from Z. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -P has the structure shown in equation IIa: Where Za represents O, S, or NR a . The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Cya said or It may optionally be substituted with 0, 1, 2, or 3 substituents selected from halogens or C1-C3 alkyl groups; preferably, Cya represents or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups; more preferably, Cya is preferred. or The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... A represents a 5-6 membered heteroaryl group, which can be represented by 0, 1, 2, 3, or 4 R groups. x Substitution; preferably, A represents an imidazolyl group, which can be replaced by 0, 1, 2, 3 or 4 R groups. x Substitution; more preferably, A represents an imidazolyl group. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... B indicates or The structure can be 0, 1, 2, 3, or 4 Rs. x Replacement; preferably, B indicates or The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... X and Y independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, and C3-C6 alkyl. 12 C1-C6 alkyl, 4-12 heterocyclic alkyl, C3-C6 alkyl, 5-6 heterocyclic alkyl, or phenyl, wherein the C1-C6 alkyl, 4-12 heterocyclic alkyl, or C3-C6 alkyl is a cyclic alkyl group. 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further comprise 0, 1, 2, or 3 heteroatoms selected from N, O, and S; preferably, X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C6 alkyl, or C4- to C5-C6 alkyl. 12 C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, C3-C 12 Each cycloalkyl group can be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2, or 3 heteroatoms selected from N, O, and S; more preferably, X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, or C3- to C6-membered cycloalkyl, wherein the C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, or C3- to C6-membered cycloalkyl may each independently be represented by 0, 1, 2, or 3 R atoms. x Alternatively, X and Y can form a 3-6 element ring, which can be optionally divided by 0, 1, or 2 R elements. x The ring may further include 0, 1, or 2 heteroatoms selected from N, O, and S. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Z represents -OR a Or -NR a R a ', preferably Z represents -OH or -NHR a More preferably, Z represents -OH. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 membered heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a Preferably, R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 heterocyclic alkyl); more preferably, R1 represents C1-C6 alkyl or C1-C6 haloalkyl; even more preferably, R1 represents ethyl or -CH2CF3. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... R2 represents a C1-C6 alkyl group, wherein the C1-C6 alkyl group may be substituted with 0 or 1 -ORa; preferably, R2 represents 1-methoxyethyl; more preferably, R2 represents Where * indicates the location where R2 is connected to the part connected to it in the formula. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... R3 and R3' each independently represent hydrogen, halogen, and C1-C6 alkyl; preferably, R3 and R3' are H. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... R4 represents hydrogen, -OR a -SR a Or -NR a R a Preferably, R4 represents H. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a -, -O-, or -S-; preferably, L D1 L D2 Each can be independently represented as a single bond or -(C1-C6) alkylene-. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Cy1 represents a 4-12 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring; preferably, Cy1 represents a 4-8 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... R5 represents hydrogen, oxygen, and =NR independently. a -S(O)2R a -C(O)R a C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl groups can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a The alkyl group is substituted with a cyano group, a C1-C6 alkyl group, a C3-C8 cycloalkyl group, or a 4-8 membered heterocyclic alkyl group; preferably, each of the R5 groups independently represents a C3-C8 cycloalkyl group or a 4-8 membered heterocyclic alkyl group, and each of the aforementioned C3-C8 cycloalkyl groups or 4-8 membered heterocyclic alkyl groups can be independently substituted with 0, 1, 2, 3, or 4 groups selected from halogen, oxo, -OR a -SR a -NR a R a The substituents are ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 heterocyclic alkyl, and when R5 is a C5-C6 cycloalkyl or 5-6 heterocyclic alkyl, the number of substituents is greater than 0. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... m represents 0, 1, or 2. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... R x Each can be used independently to represent hydrogen, halogen, oxo, and -OR. a -SR a -NR a R a ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl; preferably, R x Each can independently represent hydrogen, halogen, oxo, -OH, -SH, -NH2, cyano, C1-C6 alkyl; more preferably, R x Each can be independently represented as hydrogen, halogen, -OH, -NH2, cyano, or C1-C3 alkyl. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... P is selected from the following fragment of the structure III, where P is connected to L through an oxygen or nitrogen atom: in: Cya said or It may optionally be substituted with 0, 1, 2 or 3 substituents selected from halogens or C1-C3 alkyl groups; B is selected from or The structure can be 0, 1, 2 or 3 Rs x replace; X and Y independently represent hydrogen, C1-C6 alkyl, 4- to 12-membered heterocyclic alkyl, and C3-C6 alkyl. 12 C1-C6 alkyl, 4-12 heterocyclic alkyl, C3-C6 alkyl, 5-6 heterocyclic alkyl, or phenyl, wherein the C1-C6 alkyl, 4-12 heterocyclic alkyl, or C3-C6 alkyl is a cyclic alkyl group. 12 The cycloalkyl, 5- to 6-membered heteroaryl, and phenyl groups can each be independently represented by 0, 1, 2, 3, or 4 R groups. x Alternatively, X and Y can form a 3-8 element ring, which can be 0, 1, 2, 3, or 4 R elements. x The ring may further include 0, 1, 2 or 3 heteroatoms selected from N, O, and S; Z represents -OR a Or -NR a R a '; R1 represents C1-C6 alkyl, C1-C6 haloalkyl, -(C0-C6 alkylene)-(C3-C8 cycloalkyl), -(C0-C6 alkylene)-(4-8 membered heterocyclic alkyl), -(C1-C6 alkylene)-OR a -(C1-C6 alkylene)-SR a Or -(C1-C6 alkylene)-NR a R a '; L D1 L D2 Each can independently represent a single bond or a -(C1-C6)alkylene group, wherein any methylene group on the -(C1-C6)alkylene group can be replaced by a carbonyl group or -NR. a -、-O- or -S-; Cy1 represents C3-C 12 Cycloalkyl or 4-12 membered heterocyclic alkyl, wherein the ring can be monocyclic, spirocyclic, bridged, or fused. R5 represents hydrogen, halogen, oxidative oxidation, and =NR independently. a -OR a -SR a -NR a R a ', cyano, -C(O)OR a -C(O)R a -C(O)NR a R a '、-S(O)2R a -S(O)R a -S(O)(NR) a )R a ', C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; each of the above-mentioned C1-C6 alkyl, C3-C8 cycloalkyl, and 4-8 membered heterocyclic alkyl can be independently selected from 0, 1, 2, 3, or 4 alkyl groups selected from halogen, oxo, -OR a -SR a -NR a R a Substitution with ', cyano, C1-C6 alkyl, -(C0-C3 alkylene)-C3-C8 cycloalkyl or -(C0-C3 alkylene)-4-8 heterocyclic alkyl groups; m represents 0, 1, 2, or 3; R x Each can be used independently to represent hydrogen, halogen, oxo, and -OR. a -SR a -NR a R a ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; when R a R a When connected to the same N atom, the R a and R a 'The N atom and the N atom bonded together can form a 4-8 membered ring, which may optionally contain one, two or three heteroatoms selected from N, O or S; The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Cya said or B indicates or X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl, wherein the C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl can each be independently represented by 0, 1, 2, or 3 R. x Alternatively, X and Y can form a 3-6 element ring, which can be optionally divided by 0, 1, or 2 R elements. x Alternatively, the ring may further include 0, 1, or 2 heteroatoms selected from N, O, and S; Z represents -OH or -NHR a ; R1 represents ethyl or trifluoroethyl; L D1 L D2 Each can independently represent a single bond or a -(C1-C6) alkylene group; Cy1 represents a 4-8 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring. R5 can independently represent C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, wherein each of the aforementioned C3-C8 cycloalkyl and 4-8 membered heterocyclic alkyl groups can be independently selected from 0, 1, 2, 3 or 4 ions chosen from halogen, oxo, -OR a -SR a -NR a R a The substituents of ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, and when R5 is a C5-C6 cycloalkyl or 5-6 membered heterocyclic alkyl, the number of substituents is greater than 0; m represents 0, 1, or 2; R x Each can independently represent hydrogen, halogen, -OH, -NH2, cyano, and C1-C3 alkyl; R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... P is selected from fragments of the following structures, their isotopic derivatives, or their stereoisomers, wherein P is linked to L through an oxygen, nitrogen, or sulfur atom: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -P is selected from the following structures, their isotopic derivatives, or stereoisomers: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L is -L1-L2-L3-L4-L5-, where L1 connects to Ab and L5 connects to P. However, when L5 is a direct-connect key, L4 connects to P. L1 is selected from: Preferably, L1 is selected from: or L2 is selected from: direct connection key, Among them, E is independently selected from direct-connect key, The phenyl group, 5-6-membered heteroaryl group, and amide group may optionally be substituted with 0, 1, or 2 of the following substituents: F, Cl, methyl, methoxy. a1 is independently selected from 1, 2, 3, 4, 5, and 6. a2 is independently selected from 0, 1, 2, 3, 4, 5, and 6. a3 is independently selected from 1, 2, 3, 4, 5, and 6. R x1 Each is independently selected from H, methyl, or -CH2CH2N(Me)2. R x2 Each is independently selected from H or methyl. R x3 Each is independently selected from -NH2, -OH, -OMe, -NHMe, -N(Me)2 or -NHCH2COOH; L3 may or may not exist. When L3 does not exist, it is a direct key; when L3 exists, L3 is selected from: -ML 3a -or-L 3a -M-, Among them, M is independently selected from: direct-connect key, Among them, L 3a Each is independently selected from: direct-connect key, b1 is independently selected from 1, 2, 3, 4, and 5. b2 is independently selected from 0, 1, 2, 3, 4, and 5. b3 is independently selected from 1, 2, 3, 4, and 5. b4 is independently selected from 0, 1, 2, and 3. R y1 Each is independently selected from H, methyl, -CH2CH2N(Me)2 or HL. R y2 Each is independently selected from H, methyl, -CH2CH2N(Me)2 or HL. R y3 Each is independently selected from H, methyl, or -CH2CH2N(Me)2. R y4 Each is independently selected from H, methyl, or -CH2CH2N(Me)2. R y5 Each is independently selected from H, C1-C4 alkyl, or HL, wherein HL is selected from: Where c1 is selected from 0, 1, or 2; c2 is selected from 2 to 20; c3 is selected from 1 to 20; R y6 Selected from H, methyl or acetyl, R y7 Each is independently selected from H, Where c4 is selected from 2-20; c5 is selected from 2-20; c6 is selected from 1, 2, or 3; c7 is selected from 2-20; R y8 Selected from H or methyl; L4 is selected from short peptides consisting of 2-5 amino acid residues with a straight link, or amides consisting of amino acid residues and carboxylic acid / amine; L5 is selected from: direct connection key, R w1 Each is independently selected from: -CH2NH-HL, -CH2N(Me)-HL, -CH2CH2NH-HL, -CH2CH2N(Me)-HL, Where d1 is selected from 0, 1 or 2, and d2 is selected from 1 to 20; R w2 Each is independently selected from: H, Me, -CH2CH2N(Me)2 or -CH2CH2SO2Me; R w3 Each is independently selected from: H, Me, -CH2CH2N(Me)2, -CH2CH2SO2Me or -CH2CH2OCH2CH2OH; R w4 Each is independently selected from: H, Me, -CH2CH2N(Me)2, -CH2CH2SO2Me or -CH2CH2OCH2CH2OH; R w5 Each is independently selected from: H, Me, or -CH2CH2N(Me)2; R w6 Each is independently selected from: H, Me, or -CH2CH2N(Me)2; W 1 Each independently selected W 2 -L w1 -L w2 or -L w3 -L w4 L w1 It is a short peptide composed of 2-5 amino acid residues; L w2 H, C1-C4 alkyl, (C1-C4 alkyl)-acyl, HL, where HL is as defined above; L w3 An amide composed of a carboxylic acid / amine and 1-3 amino acid residues; L w4 -NH (C1-C4 alkyl), -N (C1-C4 alkyl)2, Where R e It is H or C1-C4 alkyl, and e1 or e3 is each chosen as an integer from 1 to 20; Each d is independently selected from 0, 1, or 2. The antibody-drug conjugate as described in claim 22, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein, L4 is selected from direct-connect key. The antibody-drug conjugate as described in claim 22, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein, L4 is selected from amides composed of amino acid residues and carboxylic acid residues, or amides composed of amino acid residues and amine residues. The antibody-drug conjugate as described in claim 22, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein, L4 is Where p is selected from 2, 3, 4, or 5; R p Each can be independently selected from: H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)Me, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2) p1 N(R z )2、-(CH2)3NHCOMe、-(CH2)3NHCONH2、-(CH2)4NHCONH2、where p1 is selected from 1, 2, 3, 4; R z Each can be independently selected from H, C1-C4 alkyl or HL. The antibody-drug conjugate as described in claim 22, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein, L4 is Where r is selected from 1 or 2; R p As defined in claim 25. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L4 is selected from: R z Each is independently selected from H, C1-C4 alkyl, or HL. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L4 is selected from: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L4 is selected from: In the antibody-drug conjugate of claim 22, or its stereoisomers, tautomers, racemates, or mixtures thereof, L5 is selected from: direct bonds. The antibody-drug conjugate as described in claim 22, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein L5 is selected from: Where R w6 Each is independently selected from H or methyl; Each d is independently selected from 1 or 2; W 2 -L w1 -L w2 or -L w3 -L w4 , Where L w1 for L w1 N-terminus and L w2 Connect, p is selected from 2, 3, 4 or 5; R p Each can be independently selected from: H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)Me, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3N(Me)2, -(CH2)3NHCOMe, -(CH2)3NHCONH2, -(CH2)4NH2, -(CH2)4N(Me)2, -(CH2)4N(Et)2, -(CH2)4N(nPr)2, -(CH2)4NHCONH2; L w2 For H, methyl, acetyl, HL; L w3 for L w3 The left side and L w4 Connect, where r is selected from 1 or 2; R p Each can be independently selected from: H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)Me, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3N(Me)2, -(CH2)3NHCOMe, -(CH2)3NHCONH2, -(CH2)4NH2, -(CH2)4N(Me)2, -(CH2)4N(Et)2, -(CH2)4N(nPr)2, -(CH2)4NHCONH2; L w4 -NHMe, -N(Me)2, e1, e2, or e3 can each be independently chosen from integers from 1 to 20. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L5 is selected from: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... L5 is selected from: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -L4-L5- Selected from: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -L4-L5- Selected from: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -L1-L2- Selected from: The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -L3- Selected from: Direct Connector The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -L- is selected from the following structures or the succinimide hydrolysis ring-opening structures of the following structures: The antibody-drug conjugate or its stereoisomers, tautomers, racemates or mixtures thereof as described in any of the preceding claims have the structure shown in Formula I-1: in, Ab, P, q are as described in any one of claims 1-21; L1, L2, L3, L4, and L5 are as described in any one of claims 22-38. The antibody-drug conjugate or its stereoisomers, tautomers, racemates or mixtures thereof as described in any of the preceding claims have the structures shown in Formula III-a, Formula III-b or Formula III-c: in, Ab,Cya,A,B,X,Y,R1,R2,R3,R3',R4,R5,L D1 L D2 Cy1, Ra, m, q as described in any one of claims 1-19; L1, L2, L3, L4, and L5 are as described in any one of claims 22-38. The antibody-drug conjugate or its stereoisomers, tautomers, racemates or mixtures thereof as described in any of the preceding claims have the structures shown in Formula III-d, III-e, III-f, III-g or III-h: in, Ab,Cya,A,B,X,Y,Z,R1,R2,R3,R3',R4,R5,L D1 L D2 Cy1, m, q as described in any one of claims 1-19; mL is 0, 1, or 2; R 5L Remove one H subunit from R5; R 1L Remove one H subunit from R1; R 4L Remove one H subunit from R4; L1, L2, L3, L4, and L5 are as described in any one of claims 22-38. The antibody-drug conjugate or its stereoisomers, tautomers, racemates or mixtures thereof as described in any of the preceding claims have the structure shown in Formula IV-a or Formula IV-b: in, Ab,Cya,B,X,Y,R1,R5,L D1 L D2 Cy1, Ra, m, q as described in any one of claims 1-19; L1, L2, L3, L4, and L5 are as described in any one of claims 22-38. The antibody-drug conjugate or its stereoisomers, tautomers, racemates or mixtures thereof as described in any of the preceding claims have the structure shown in Formula IV-e: in, Ab,Cya,B,X,Y,Z,R1,R5,L D1 L D2 Cy1, m, q as described in any one of claims 1-19; mL is 0, 1, or 2; R 5L Remove one H subunit from R5; L1, L2, L3, L4, and L5 are as described in any one of claims 22-38. The antibody-drug conjugate or its stereoisomers, tautomers, racemates or mixtures thereof as described in any of the preceding claims have structures as shown in Formulas IV-a-1 to IV-a-24: ,in, Ab represents the antibody or its antigen-binding fragment, and q is selected from any value between 1.0 and 16.0; Cya said or B indicates or X and Y each independently represent hydrogen, C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl, wherein the C1-C6 alkyl, 4- to 8-membered heterocyclic alkyl, and C3- to C6-membered cycloalkyl can each be independently represented by 0, 1, 2, or 3 R. x Alternatively, X and Y can form a 3-6 element ring, which can be optionally divided by 0, 1, or 2 R elements. x Alternatively, the ring may further include 0, 1, or 2 heteroatoms selected from N, O, and S; Z represents -OR a Or -NR a R a '; R1 represents ethyl or trifluoroethyl; L D1 L D2 Each can independently represent a single bond or a -(C1-C6) alkylene group; Cy1 represents a 4-8 membered heterocyclic alkyl group, wherein the ring can be a monocyclic, spirocyclic, bridged, or fused ring. R5 can independently represent C3-C8 cycloalkyl or 4-8 membered heterocyclic alkyl, wherein each of the aforementioned C3-C8 cycloalkyl and 4-8 membered heterocyclic alkyl groups can be independently selected from 0, 1, 2, 3 or 4 ions chosen from halogen, oxo, -OR a -SR a -NR a R a The substituents are ', cyano, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl, and when R5 is a C5-C6 cycloalkyl or a 5-6 membered heterocyclic alkyl, the number of substituents is greater than 0; R 5L This means that one H subunit has been removed from R5; m represents 0, 1, or 2; mL represents 0 or 1; R x Each can independently represent hydrogen, halogen, -OH, -NH2, cyano, and C1-C3 alkyl; R a R a Each can independently represent hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, or 4-8 membered heterocyclic alkyl; The alkyl, alkylene, cycloalkyl, cycloalkylene, heterocycloalkyl, and heteroalkylene can each be independently substituted with 0, 1, 2, 3, 4, 5, or 6 halogen atoms. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... -P in claim 39, and -P in claim 40 In claim 41 In claim 42 In claim 43 In claim 44 It has a structure selected from that shown in claim 21. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, is selected from the following structures or the succinimide hydrolysis ring-opening structures of the following structures: ,in, Ab represents the antibody or its antigen-binding fragment, and q is selected from any value between 1.0 and 16.

0. The antibody-drug conjugate as described in any of the preceding claims, or in the form of its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... q is selected from any value between 1.0 and 16.0; preferably, q is selected from any value between 2.0 and 16.0; preferably, q is selected from any value between 1.0 and 8.0; preferably, q is selected from any value between 4.0 and 8.0; preferably, q is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16; preferably, q is selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; preferably, q is selected from 6, 7, 8, 9, 10, 11, 12; preferably, q is about 10; preferably, q is selected from 1, 2, 3, 4, 5, 6, 7 or 8; preferably, q is selected from 2, 4, 6 or 8; preferably, q is selected from 4, 5, 6, 7, 8; preferably, q is selected from 4, 6, 8. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is a ligand that binds to a target antigen, which is highly expressed in tumor cells but lowly expressed or not expressed in normal cells. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab refers to an antibody or antigen ligand, and the target sites of said antibody or antigen ligand are, for example, 5T4, ACTA2, ADGRE1, AG-7, AIF1, AKR1C1, AKR1C2, ANGPTL4, ASLG659, Axl, B7H3, B7H4, BAFF-R, BCMA, BMPR1B, BNIP3, C1QA, C1QB, CA6, CADM1, CCL5, CCR5, CCR7, CD123, CD138, CD142, CD147, CD166, CD19, CD2 2. CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD46, CD47, CD49D(ITGA4), CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CDCP1, CDH3, CDH6, CDH11, CDH17, CD11b, CEA, CEACAM5, CEACAM6, CLDN18.2,c-Met,COL6A3,COL7A1,CRIPTO,CSF1R,CTGF,CTSD,CTSS,CXCL11,CXCL10,CXCR5,DDIT4,DLL3,DLL 4,DR5,E16,EFNA4,EGFR,EGFRvIII,EGLN,EGLN3,EMR2,ENPP3,EpCAM,EphA2,EphB2R,ETBR,FcRH2,FcR H1,FGF2,FGFR2,FGFR3,FLT3,FOLR-α,GD2,GEDA,GPC-1,GPNMB,GPR20,GZMB,HER2,HER3,HLA-DOB,HM OX1,IFI6,IFNG,IGF-1R,IGFBP3,IL10RA1,IL-13R,IL-2,IL20Ra,IL-3,IL-4,IL-6,IRTA2,KISS1R,KR T33A,LIV-1,LOX,LRP-1,LRRC15,LUM,LY64,LY6E,Ly86,LYPD3,MDP,MMP10,MMP14,MMP16,MPF,MSLN, MUC-1,NaPi2b,Napi3b,Nectin-4,NOG,P2X5,PDGFRA,PDK1,PD-L1,PFKFB3,PGF,PGK1,PIK3AP1,PIK3C D,PLOD2,PSCA,PSMA,PTK7,RNF43,ROR1,ROR2,SERPINE1,SLC39A6,SLTRK6,STC2,STEAP1,STEAP2,TCF 4,TENB2,TGF,TGFB1,TGFB2,TGFBR1,TNFRSF21,TNFSF9,Trop-2,TrpM4,Tyro7,UPK1B,VEGFA,WNT5A。. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-antigen antibody or its antigen-binding fragment, wherein the antigen target is selected from cMET, CEACAM5, CEACAM6, CDH17, MSLN, HER2, TROP2, EGFR, HER3, and B7H3. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an antibody or antigen-binding fragment thereof, said antibody selected from adalimumab, aducanumab, alemtuzumab, altumomab, amivantamab, atezolizumab, anetumab, avelumab, bapineuzumab, basiliximab, bectumomab, bermekimab, besilesomab, bevacizumab, bezlotoxumab, brentuximab, brodalumab, catumaxomab, cemiplimab, cetuximab, cinpanemab, clivatuzumab, crenezumab, daclizumab, daratumumab, denosumab, dinutuximab, dostarlimab, durvalumab, edrecolomab, elotuzumab, emapalumab, enfortumab, epcoritamab, epratuzumab, etaracizumab, gemtuzumab, glofitamab, girentuximab, gosuranemab, ibritumomab, inebil izumab, infliximab, inotuzumab, ipilimumab, isatuximab, ixekizumab, J591, labetuzumab, lecanemab, loncastuximab, mirzotamab, mogamulizumab, mosunetuzumab, necitumumab, nimotuzumab, natalizumab, naratuximab, naxitamab, nivolumab, ocrelizumab, ofatumumab, olaratumab, oreg ovomab, panitumumab, pembrolizumab, pertuzumab, polatuzumab, prasinezumab, racotumomab, ramucirumab, rituximab, sacituzumab, semorinemab, siltuximab, solanezumab, tacatuzumab, tafasitamab, telisotuzumab, teprotumumab, tilavonemab, tocilizumab, tositumumab, trastuzumab,tusamitamab, ustekinumab, vedolizumab, votumumab, zagotenemab, zanidatamab, zalutumumab, zanolimumab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-cMET antibody or its antigen-binding fragment, preferably Telisotuzumab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-TROP2 antibody or its antigen-binding fragment, preferably Sacituzumab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-HER3 antibody or its antigen-binding fragment, preferably Patritumab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-CEACAM5 antibody or its antigen-binding fragment, preferably Tusamitamab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-B7H3 antibody or its antigen-binding fragment, preferably Mirzotamab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is an anti-EGFR antibody or its antigen-binding fragment, preferably Cetuximab. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is a multifunctional antibody or its antigen-binding fragment, and the antigen target of the bifunctional antibody includes any combination of two or more of the above targets. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein... Ab is a bifunctional antibody or its antigen-binding fragment, wherein the combination of antigen targets of the bifunctional antibody is selected from: EGFR / c-Met, EGFR / HER3, HER2 / TROP2, HER2 / HER2, EGFR / MUC1, HER3 / TROP2. The antibody-drug conjugate as described in any of the preceding claims, or its stereoisomers, tautomers, racemates, or mixtures thereof, is selected from the following structures or the succinimide hydrolysis ring-opening structures of the following structures: in, Ab is Telisotuzumab, Sacituzumab, or Tusamitamab; q is any value from 2.0 to 12.0; preferably, q is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12; preferably, q is about 4, about 6, about 8, about 10, about 10. The pharmaceutical composition comprises an antibody-drug conjugate as described in any of the preceding claims, or a stereoisomer, tautomer, racemate, or mixture thereof, and optionally a pharmaceutically acceptable salt, carrier, diluent, or excipient. The use of the antibody-drug conjugate as described in claims 1-60 or the pharmaceutical composition as described in claim 61 in the preparation of a medicament for the prevention or treatment of cancer-related diseases or conditions. The use of the antibody-drug conjugates of claims 1-60 or the pharmaceutical composition of claim 61 for the prevention or treatment of cancer-related diseases or conditions. Methods for the prevention or treatment of cancer-related diseases or conditions include administering to a patient in need of treatment a therapeutically effective amount of the antibody-drug conjugate as claimed in claims 1-60 or the pharmaceutical composition as claimed in claim 61. In the application as described in claim 62 or 63, or in the method as described in claim 63, the cancer is a solid tumor or a hematologic malignancy, specifically selected from esophageal cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, liver cancer, kidney cancer, head and neck cancer, brain tumor, urothelial carcinoma, skin cancer, prostate cancer, thyroid cancer, neuroblastoma, glioma, leukemia, or lymphoma, etc. The LP intermediate, or its stereoisomers, tautomers, racemates, or mixtures thereof, can be coupled with Ab to prepare antibody-drug conjugates as described in any one of claims 1-60 (e.g., thiol-substituted methyl sulfone, thiol-substituted bromine, or thiol-added maleimide double bond), wherein the LP intermediate has the following structure: L H -L2-L3-L4-L5-P in, L H -Selected from; P is as described in any one of claims 1-21; L2, L3, L4, and L5 are as described in any one of claims 22-38. The LP intermediate as claimed in claim 66, or its stereoisomers, tautomers, racemates, or mixtures thereof, wherein the LP intermediate has a structure selected from the following:

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