Medicine for treating tumors

Abstract

The invention relates to a medicine for treating tumors. The pharmaceutical composition comprises one or more compounds, or a stereoisomer or tautomer thereof, or an optical isomer or raceme thereof, or a solvate thereof, or a pharmaceutically acceptable salt, ester, amide, or a prodrug thereof, or a metabolite thereof, or an analogue thereof, or a derivative thereof, or a crystal compound thereof, or a nitrogen oxide thereof, or a deuterated substance thereof, or a pharmaceutically acceptable salt, ester, amide, or a prodrug thereof, or a metabolite thereof, or an analogue thereof, or a derivative thereof, or a crystal compound thereof, or a nitrogen oxide thereof, or a deuterated substance thereof. Or a combination of these substances. The invention also relates to application of the XIAP E3 ligase as a tumor treatment target and a targeting compound thereof.

Description

Technical Field

[0001] The present application relates to a drug for treating tumors, and specifically to the application of XIAP E3 ligase as a tumor treatment target and its targeting compound, belonging to the field of biochemical medicine technology. Background Art

[0002] X-linked inhibitor of apoptosis protein (XIAP) is a major member of the inhibitor of apoptosis (IAP) family. It contains three N-terminal BIR repeat domains and a C-terminal RING domain. XIAP is widely implicated in numerous physiological and pathological processes, including cell survival, immune regulation, and apoptosis. The XIAP gene is overexpressed in various tumor cell types, including breast cancer, glioblastoma, ovarian cancer, bladder cancer, prostate cancer, renal cancer, liver cancer, colorectal cancer, lung cancer, metastatic melanoma, head and neck squamous cell carcinoma, esophageal cancer, acute and chronic leukemias, and other malignancies. XIAP expression is closely correlated with tumor invasion and metastasis. Therefore, XIAP is widely used as a target for drug development in various tumors and immune diseases. Early research on XIAP inhibitors focused primarily on the BIR domain of XIAP, and several apoptosis-inducing tumor therapeutics targeting the BIR domain of XIAP have been developed. However, since the BIR domain plays an important role in maintaining normal cell function, and these tumor therapeutic drugs target the BIR domain of XIAP while also targeting the BIR domain of other IAPs (such as cIAP1 and cIAP2), resulting in significant side effects, other research methods are urgently needed. Summary of the Invention

[0003] The inventors have discovered that a binding site at the C-terminus of XIAP (particularly the RING domain) that alters E3 ligase activity is an important target for screening anti-tumor or autoimmune disease treatment drugs. Based on this target, multiple compounds with anti-tumor effects have been screened and further functionally validated (see Tables 1, 2, and 8). In this application, anti-tumor effects include not only inhibiting the growth of various tumors, such as bladder cancer, muscle-invasive bladder cancer, breast cancer, pancreatic cancer, colorectal cancer, lung adenocarcinoma, gastric cancer, gastric adenocarcinoma, melanoma, and renal cancer, but also inhibiting the metastasis (such as lung metastasis of bladder cancer cells) and invasion of these tumors. Therefore, this application first provides the use of the C-terminus of XIAP, the RING domain of XIAP, a fragment of XIAP with E3 ubiquitin ligase activity, an amino acid site of XIAP corresponding to the 467H site of human XIAP, or any binding site of the RING domain of XIAP that, when bound, alters E3 ligase activity as a target in the preparation of a drug for treating tumors. Examples of targets include one or more of positions 440, 444, 446, 447, 448, 449, 451, 454, 457, 458, 467, 468, 469, 483, 494, 495, 496, or 497 of human XIAP, and all compounds of the present application are obtained based on these targets. Secondly, the present application also provides the use of inhibitors (such as compounds or formulations) that inhibit the E3 ubiquitin ligase activity of XIAP in the preparation of medicaments for treating tumors. These inhibitors are, for example, all compounds of the present application, including compounds of the general formula, specific compounds, or one or more combinations thereof. In some embodiments, the inhibitor is a substance that binds to the C-terminus of XIAP, or to the RING domain of XIAP, or to an amino acid site of XIAP corresponding to the 467H site of human XIAP, or to the RING domain of human XIAP, thereby inhibiting the E3 ligase activity. In some embodiments, these inhibitors can be used in combination with additional therapeutic agents. In some embodiments, these inhibitors can be in any suitable dosage form or administered in any suitable manner, such as the specific dosage forms and specific modes of administration mentioned in this application. In some embodiments, the tumor can be any tumor defined in the art, such as the specific tumor or cancer type mentioned in this application. In some embodiments, the drug is any pharmaceutical form defined in the art, such as the pharmaceutical composition mentioned in this application, or all compounds of this application, including general compounds, specific compounds, or one or more combinations thereof, together with pharmaceutically acceptable excipients, diluents, carriers, and / or excipients. Detailed description is as follows.

[0004] The present application first provides a compound of the general formula represented by formula (I), or a stereoisomer thereof, a tautomer thereof, an optical isomer thereof, a racemate thereof, a solvate thereof, a prodrug thereof, a metabolite thereof, an analogue thereof, a derivative thereof, a crystalline compound thereof, a nitrogen oxide thereof, a deuterated compound thereof, or a combination of these substances:

[0005] Wherein: Ring A is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Y1 is -(CH2) m -C(=O)-NR a R b 、-(CH2) n -R6 or -

[0006] (CH2) m -C(=O)-NH-C(=O)-R c ; Y2 is -NH-C(=O)-R5, -(CH2) n -R6, -X9-(CH2) t -R 4n or -X9-(CH2) t -C(=O)-NR a R b ; R c is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 aminoalkyl, C1-C6 cycloalkyl, C1-C6 halocycloalkyl, C1-C6 hydroxycycloalkyl, C1-C6 aminocycloalkyl or phenyl; R 4n Each is independently H, halogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b 、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R b or CN, the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl or phenyl is optionally substituted with one or more R9; R5 and R6 are each independently H, halogen, -OH, C1-C6 alkyl, C4-C 10 Cycloalkyl, C4-C6 cycloalkenyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl, the C1-C6 alkyl, C4-C 10Cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl or phenyl is optionally substituted with one or more R7; R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b or CN, the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is optionally substituted with one or more R9; R8 is H or C1-C4 alkyl; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connected atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; R9 is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C (=O)NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl, wherein the C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl is independently optionally substituted by 0, 1 or 2 C1-C6 alkyl or C1-C6 haloalkyl; X9 is S or O; m, n and t are each independently 0, 1, 2 or 3.

[0007] In some embodiments, R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3-10 membered heterocyclyl, 4-10 membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b 、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R bor CN, the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is optionally substituted with one or more R9.

[0008] In some embodiments, Ring A is Wherein: R1, R2, R3 and R4 are each independently H, halogen, -OH, -CN, -NO2, -NR a R b , -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b , C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl; wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is independently optionally substituted by one or more R7; X1, X2, X3, X4, X5, X6 and X7 are each independently N or CH. Preferably, R1, R2, R3 and R4 are each independently H, halogen, -OH, -CN, -NO2, -NR a R b , -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b , C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl or phenyl; wherein the C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl or phenyl is independently optionally substituted by one or more R7. Preferably, R5 and R6 are each independently C4-C6 cycloalkyl, C4-C6 cycloalkenyl, 4 to 6-membered azacycloalkyl, bicyclo[2.2.2]octane or phenyl; wherein the C4-C6 cycloalkyl, C4-C6 cycloalkenyl, 4 to 6-membered azacycloalkyl, bicyclo[2.2.2]octane or phenyl is optionally substituted by one or more R7. Preferably, R7 is H, halogen, C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, 4 to 6-membered azacycloalkyl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2 or CN; wherein the C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, 4 to 6-membered azacycloalkyl or phenyl is independently optionally substituted by one or more R9.

[0009] In some embodiments, the compound of the present application has the structure of the compound of formula (II-A), (II-B) or (II-C):

[0010]

[0011] Among them, R1, R 1a R5 is H or C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 hydroxyalkyl; Ring Ar is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Ring G is a C3-C7 cycloalkyl, C3-C7 cycloalkenyl, a 4-7 membered heterocyclyl, a 4-7 membered heteroaryl, a 4-7 membered aryl or a 5-9 membered bicyclyl; Ring G is optionally substituted with 0, 1 or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or C1-C4 alkoxy; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connecting atoms, they form a 4-, 5-, 6- or 7-membered cyclic amine group, which is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S. R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b 、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R b or CN, the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl is optionally substituted with one or more R9. Preferably, ring G is C4-C6 cycloalkyl, C4-C6 cycloalkenyl, phenyl, or a 5- to 9-membered bicyclic group; preferably cyclohexyl, cyclohexenyl, phenyl, bicyclo[2.2.1]heptane, or bicyclo[2.2.2]octane; the ring G is optionally substituted with 0, 1, or 2 halogens, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, or C1-C4 alkoxy. Preferably, R ais H, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl; R b is H, C1-C4 alkyl, C3-C7 cycloalkyl, phenyl, The C1-C4 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with 0, 1, 2 or 3 halogen, -OH or C1-C4 alkoxy; or R a and R b Together with the atoms connected to it, it forms azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole, triazole, The azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole or triazole is optionally substituted with 0, 1, 2 or 3 halogen, -OH or C1-C4 alkoxy.

[0012] In some embodiments, the compounds of the present application have the structures of the compounds of formula (III-A), (III-B), and (III-C):

[0013]

[0014] Among them, R a and R b are each independently H, C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 hydroxyalkyl; or, R a and R b Together with the connecting atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R 4b substituted, the 4, 5, 6 or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; the 4, 5, 6 or 7-membered cyclic amine group is preferably azetidine, tetrahydropyrrole, piperidine, piperazine or azepane; R4, R 4a 、R 4b 、R 4c Each is independently H, halogen, -OH, -CN, -NO2, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or C1-C4 alkoxy; t and t1 are independently optionally 0, 1, 2, 3, 4 or 5; m is 0, 1, 2 or 3; X7 is N or CH; X9 is S or O; R 4m and R 4n Each is independently H, halogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R b or CN, the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is optionally substituted with one or more R9; R8 is H or C1-C4 alkyl; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connecting atoms, they form a 4-, 5-, 6- or 7-membered cyclic amine group, which is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; R9 is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl, and the C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl is independently optionally substituted by 0, 1 or 2 C1-C6 alkyl or C1-C6 haloalkyl.

[0015] In some embodiments, the compounds of the present application have the structures of the compounds of the general formula (IV-A), (IV-B), (IV-C), and (IV-D):

[0016]

[0017] wherein ring Ar is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; ring P is a 4-10 membered cycloalkyl, a 4-10 membered azacycloalkyl, a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring;

[0018] R 5a 、R 5b is H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or phenyl; R 5cis H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C3-C6 cycloalkyl, -(C=O)-R8, tetrahydropyrrolyl, piperidinyl, piperazinyl; the C3-C6 cycloalkyl, tetrahydropyrrolyl, piperidinyl, piperazinyl are optionally substituted with 0, 1 or 2 halogen, -OH, -NH2 or C1-C4 alkyl; R 5d is H, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl; R 5e is C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C3-C6 cycloalkyl, phenyl or C1-C4 alkylphenyl; the C3-C6 cycloalkyl, phenyl or C1-C4 alkylphenyl is optionally substituted with 0, 1 or 2 halogen, -OH, -NH2 or C1-C4 alkyl; X5a, X5b, X5c, X5d or X5e are each independently N or CH.

[0019] The present application also provides a compound represented by formula (Ii), or a stereoisomer, a tautomer, an optical isomer, a racemate, a solvate, a prodrug, a metabolite, an analog, a derivative, a crystalline compound, a nitrogen oxide, a deuterated compound, or a combination thereof:

[0020] Wherein, Ring A is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Y1 is -(CH2) m -C(=O)-NR a R b , or -(CH2) m -C(=O)-NH-C(=O)-R c ; Y2 is -NH-C(=O)-R5, -

[0021] (CH2) n -R6 or -X9-(CH2) t -C(=O)-NR a R b ; R c is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 aminoalkyl, C1-C6 cycloalkyl, C1-C6 halocycloalkyl, C1-C6 hydroxycycloalkyl, C1-C6 aminocycloalkyl or phenyl; R5 and R6 are each independently C4-C 10 Cycloalkyl, C4-C6 cycloalkenyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl, the C4-C 10Cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl or phenyl is optionally substituted with one or more R7; R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b or CN, the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is optionally substituted with one or more R9; R8 is H or C1-C4 alkyl; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connected atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; R9 is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C (=O)NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl, wherein the C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl is independently optionally substituted by 0, 1 or 2 C1-C6 alkyl or C1-C6 haloalkyl; X9 is S or O; m, n and t are each independently 0, 1, 2 or 3.

[0022] In some embodiments, Ring A is R1, R2, R3 and R4 are each independently H, halogen, -OH, -CN, -NO2, -NR a R b , -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl; wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is independently optionally substituted by one or more R7; X1, X2, X3, X4, X5, X6 and X7 are each independently N or CH. Preferably, R1, R2, R3 and R4 are each independently H, halogen, -OH, -CN, -NO2, -NR a R b , -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b , C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl or phenyl; wherein the C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl or phenyl is independently optionally substituted by one or more R7. Preferably, R5 and R6 are each independently C4-C6 cycloalkyl, C4-C6 cycloalkenyl, 4 to 6-membered azacycloalkyl, bicyclo[2.2.2]octane or phenyl; wherein the C4-C6 cycloalkyl, C4-C6 cycloalkenyl, 4 to 6-membered azacycloalkyl, bicyclo[2.2.2]octane or phenyl is optionally substituted by one or more R7. R7 is H, halogen, C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, 4 to 6-membered azacycloalkyl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2 or CN; wherein the C1-C4 alkyl, C1-C4 alkoxy, C3-C6 cycloalkyl, 4 to 6-membered azacycloalkyl or phenyl is independently optionally substituted by one or more R9.

[0023] In some embodiments, the compound is of the structure described by formula (III-Ai): Among them, R1, R 1a R5 is H or C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 hydroxyalkyl; Ring Ar is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Ring G is a C3-C7 cycloalkyl, C3-C7 cycloalkenyl, a 4-7 membered heterocyclyl, a 4-7 membered heteroaryl, a 4-7 membered aryl or a 5-9 membered bicyclyl; Ring G is optionally substituted with 0, 1 or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or C1-C4 alkoxy; R a and Rb Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connected atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S. Preferably, the compound has a structure described by formula (III-BI):

[0024] In some embodiments, the compound is of the structure described by formula (III-Ai): Among them, R a and R b are each independently H, C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 hydroxyalkyl; or, R a and R b Together with the connecting atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R 4b substituted, the 4, 5, 6 or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; the 4, 5, 6 or 7-membered cyclic amine group is preferably azetidine, tetrahydropyrrole, piperidine, piperazine or azepane; R4, R 4a 、R 4b Each is independently H, halogen, -OH, -CN, -NO2, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or C1-C4 alkoxy; t1 is 0, 1, 2, 3, 4 or 5. Preferably, the compound has the structure described by formula (III-BI):

[0025]

[0026] In some embodiments, the compound is of the structure described by formula (IV-Ai): wherein Ring Ar is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Ring P is a 4-10 membered cycloalkyl, a 4-10 membered azacycloalkyl, a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; R 5a 、R 5b is H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or phenyl; R5c is H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C3-C6 cycloalkyl, -(C=O)-R8, tetrahydropyrrolyl, piperidinyl, piperazinyl; the C3-C6 cycloalkyl, tetrahydropyrrolyl, piperidinyl, piperazinyl are optionally substituted with 0, 1 or 2 halogen, -OH, -NH2 or C1-C4 alkyl; R 5d is H, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl; R 5e is C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C3-C6 cycloalkyl, phenyl or C1-C4 alkylphenyl; the C3-C6 cycloalkyl, phenyl or C1-C4 alkylphenyl is optionally substituted with 0, 1 or 2 halogen, -OH, -NH2 or C1-C4 alkyl. Preferably, the compound is of formula (IV-BI), (IV-CI) or (IV-DI):

[0027] where X 5a 、X 5b 、X 5c 、X 5d or X 5e are each independently N or CH.

[0028] In some embodiments, ring G is C4-C6 cycloalkyl, C4-C6 cycloalkenyl, phenyl, 5-9 membered bicyclic group; preferably cyclohexyl, cyclohexenyl, phenyl, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane; and ring G is optionally substituted with 0, 1 or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy.

[0029] In some embodiments, wherein R a is H, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl; R b is H, C1-C4 alkyl, C3-C7 cycloalkyl, phenyl, The C1-C4 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with 0, 1, 2 or 3 halogen, -OH or C1-C4 alkoxy; or R a and R b Together with the atoms connected to it, it forms azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole, triazole,

[0030] The azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole or triazole is optionally substituted with 0, 1, 2 or 3 halogen, -OH or C1-C4 alkoxy.

[0031] In some embodiments, the specific compounds of the present application have at least one of the following structures:

[0032]

[0033]

[0034]

[0035]

[0036] Table 1 Correspondence between the general formula and compounds 1-65

[0037]

[0038] On the other hand, the present application provides a composition, characterized in that it comprises the aforementioned compound, or its stereoisomer, its tautomer, or its optical isomer or its racemate, or its solvate, or its prodrug, or its metabolite, or its analog or derivative, or its crystalline compound, or its nitrogen oxide, or its deuterated form, or a combination of these substances; optionally, further comprising a pharmaceutically acceptable excipient, diluent, carrier, and / or vehicle.

[0039] The aforementioned composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent.

[0040] In another aspect, the present application provides use of the aforementioned compound, or its stereoisomer, tautomer, optical isomer or racemate, or solvate, or prodrug, or metabolite, or analog or derivative, or crystalline compound, or nitrogen oxide, or deuterated form, or combination of these substances, or the aforementioned composition in the preparation of a medicament for preventing and / or treating XIAP-mediated diseases and / or conditions.

[0041] In some embodiments, the diseases and / or conditions are all types of diseases in which cells cannot undergo apoptosis, such as tumors, cancers, or immune diseases.

[0042] In some embodiments, the aforementioned compound, or its stereoisomer, or its tautomer, or its optical isomer, or its racemate, or its solvate, or its prodrug, or its metabolite, or its analog or derivative, or its crystalline compound, or its nitrogen oxide, or its deuterated form, or a combination of these substances, or the aforementioned composition is used as an active ingredient or therapeutic agent in achieving the aforementioned use, for example, as the sole active ingredient or the main active ingredient. In some embodiments, "as an active ingredient or therapeutic agent" means that its content reaches a therapeutically effective amount; or "as the sole active ingredient or the main active ingredient" means that its content accounts for 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of all active ingredients, and the percentage is a mass ratio or a molar ratio; or "as the sole active ingredient or the main active ingredient" means that its contribution to the efficacy of the disease treatment is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0043] In some embodiments, the aforementioned compound, or its stereoisomer, or its tautomer, or its optical isomer or racemate, or its solvate, or its prodrug, or its metabolite, or its analog or derivative, or its crystalline compound, or its nitrogen oxide, or its deuterated form, or a combination of these substances, or the aforementioned composition and the additional therapeutic agent are formulated or designed for continuous administration, or simultaneous administration, or sequential administration, or alternating administration, or intermittent administration, or separate administration. In some embodiments, systemic administration, or local administration, or parenteral administration (such as through mucosal administration, transdermal administration, microneedle administration), or non-invasive administration, or non-invasive administration.

[0044] In some embodiments, the concentration or proportion of the aforementioned compound, or its stereoisomer, its tautomer, or its optical isomer or racemate, or its solvate, or its prodrug, or its metabolite, or its analog or derivative, or its crystalline compound, or its nitrogen oxide, or its deuterated substance, or a combination of these substances in the aforementioned composition is at least not less than 0.001%, preferably 0.01% to 25%, preferably 0.05% to 5%, and more preferably 0.15%; or the concentration or proportion of these substances or their combination is less than 0.001%, and the percentage is expressed as mass / volume concentration (ratio) or mass ratio or molar (number) ratio.

[0045] In some embodiments, the compound of the present application is prepared into an injection, tablet, lyophilized powder injection, capsule, effervescent tablet, chewable tablet, buccal tablet, granule, ointment, syrup, aerosol, nasal drops, external preparation, ophthalmic dosage form, oral preparation, oil-water mixture, suspension, liniment, lotion, cream, drops, granule, spray, ointment, patch, paste, pill, suppository or emulsion, health product, food, dietary supplement, nutrition or beverage. Solid dosage form can generally contain 1% to 95% (w / w) of active compound. In some embodiments, the range of the active compound is from 5% to 70% (w / w).

[0046] In some embodiments, the patient with the disease is a human, such as an infant, child, teenager, middle-aged adult, or elderly person.

[0047] In some embodiments, the XIAP-mediated disease and / or condition is a neoplastic or autoimmune disease, including but not limited to carcinoma, sarcoma, Kaposi's sarcoma, erythroblastoma, malignant glioma, meningioma, astrocytoma, melanoma, and myoblastoma; brain cancer, skin cancer, adenocarcinoma, malignant epithelial tumors, urological tumors, prostate cancer, urothelial carcinoma, locally advanced or metastatic urothelial carcinoma, bladder urothelial carcinoma, bladder cancer, muscle invasive bladder cancer (MIBC), non-muscle invasive bladder cancer (NMIBC), metastatic bladder cancer, advanced bladder cancer, ovarian cancer, breast cancer, uterine cancer, pancreatic cancer, liver cancer, colon cancer, blood cancer, lung adenocarcinoma, lung cancer, bone cancer, neuroblastoma, intestinal cancer such as colorectal cancer, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary non-polyposis colorectal cancer, esophageal cancer, Lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cell carcinoma, cervical cancer, cervical squamous cell carcinoma and adenocarcinoma, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, invasive breast cancer, urethral cancer, melanoma, brain tumors, gliomas, astrocytomas, meningiomas, medulloblastomas, peripheral neuroectodermal tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, leukemia, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, B-cell lymphoma, polycythemia vera, hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma,

[0048] Head and neck tumors, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, thyroid tumors, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, plasmacytoma, systemic lupus erythematosus, or rheumatoid arthritis.

[0049] In some embodiments, the tumor includes but is not limited to lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, bronchioalveolar carcinoma), pleural mesothelioma, esophageal cancer (squamous cell carcinoma, adenocarcinoma, neuroendocrine carcinoma), gastric cancer (adenocarcinoma (intestinal type / diffuse type), signet ring cell carcinoma, gastric lymphoma (MALT lymphoma)), colorectal cancer (adenocarcinoma (mucinous adenocarcinoma / signet ring cell carcinoma), anal canal squamous cell carcinoma), hepatobiliary system (hepatocellular carcinoma (HCC), cholangiocarcinoma (CCC), hepatoblastoma, gallbladder cancer, ampullary carcinoma), pancreatic cancer (ductal adenocarcinoma, acinar cell carcinoma, pancreatoblastoma), renal cancer (clear cell carcinoma, papillary renal cell carcinoma, chromophobe cell carcinoma), gastric adenocarcinoma, bladder cancer (urothelial carcinoma (transitional cell carcinoma), squamous cell carcinoma, adenocarcinoma, muscle invasive bladder cancer, non-muscle invasive bladder cancer, primary bladder cancer, invasive bladder cancer, early bladder cancer, intermediate bladder cancer, metastatic bladder cancer or advanced bladder cancer) bladder cancer), prostate cancer (adenocarcinoma (ductal / acinar), neuroendocrine cancer), testicular cancer (seminoma, embryonal carcinoma, teratoma, choriocarcinoma), breast cancer, ovarian cancer, cervical cancer (squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma), endometrial cancer (endometrioid adenocarcinoma, serous carcinoma, clear cell carcinoma), leukemia (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)), bone marrow cancer Proliferative tumors (polycythemia vera, primary myelofibrosis), lymphoma (Hodgkin lymphoma (nodular sclerosis type / mixed cell type, etc.), non-Hodgkin lymphoma (diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, etc.)), multiple myeloma (plasmacytoma), liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), glioma (glioblastoma (GBM), astrocytoma (WHO grade I-IV), oligodendroglioma), medulloblastoma,

[0050] Ependymoma, meningioma, neuroblastoma, neurilemmoma (schwannoma), malignant peripheral nerve sheath tumor (MPNST), teratoma, yolk sac tumor, embryonal carcinoma, choriocarcinoma, thyroid cancer (papillary, follicular, medullary, undifferentiated), adrenocortical carcinoma, pheochromocytoma / paraganglioma, pituitary adenoma, melanoma, basal cell carcinoma, squamous cell carcinoma, cutaneous T-cell lymphoma (mycosis fungoides), osteosarcoma, chondrosarcoma, Ewing sarcoma, chordoma, pulmonary carcinoid, gastroenteropancreatic neuroendocrine tumors (GEP-NETs), small cell neuroendocrine carcinoma, metastatic carcinoma, brain metastasis, bone metastasis, liver metastasis, tumor of unknown primary site, metastatic poorly differentiated carcinoma, metastatic adenocarcinoma, retinoblastoma, Wilms tumor, hepatoblastoma, primitive neuroectodermal tumor (PNET).

[0051] The present application also provides the use of the aforementioned compound, or its stereoisomer, tautomer, optical isomer, racemate, solvate, prodrug, metabolite, analog, derivative, crystalline compound, nitrogen oxide, deuterated form, or combination thereof, or the aforementioned composition, in the preparation of a medicament or formulation for binding to XIAP. In some embodiments, binding to XIAP involves binding to the C-terminus of XIAP, or to the RING domain of XIAP, or to an amino acid site of XIAP corresponding to site 467H of human XIAP (e.g., XIAP protein sequence, Uniprot ID: P98170), or to the RING domain of human XIAP, thereby altering (e.g., inhibiting) E3 ligase activity.

[0052] The present application further provides the use of the C-terminus of XIAP, or the RING domain of XIAP, or a fragment of XIAP having E3 ubiquitin ligase activity, or an amino acid site of XIAP corresponding to the 467H site of human XIAP, or a binding site in the RING domain of XIAP that is bound and thereby alters E3 ligase activity as a target for tumor treatment.

[0053] The present application further provides the use of the C-terminus of XIAP, or the RING domain of XIAP, or a fragment of XIAP having E3 ubiquitin ligase activity, or an amino acid site of XIAP corresponding to the 467H site of human XIAP, or a binding site of the RING domain of XIAP that is bound and thereby alters E3 ligase activity as a target in the preparation of a drug for treating tumors.

[0054] The present application further provides the use of a compound or formulation that inhibits the E3 ubiquitin ligase activity of XIAP in the preparation of a drug for treating tumors. In some embodiments, the compound or formulation that inhibits the E3 ubiquitin ligase activity of XIAP is a substance that binds to the C-terminus of XIAP, or to the RING domain of XIAP, or to the amino acid site of XIAP corresponding to the 467H site of human XIAP, or to the RING domain of human XIAP, thereby altering (e.g., inhibiting) the E3 ligase activity. In some preferred embodiments, the compound or formulation that inhibits the E3 ubiquitin ligase activity of XIAP is a compound of the present application, or a stereoisomer thereof, a tautomer thereof, an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, or an analog thereof, or a derivative thereof, or a crystalline compound thereof, or a nitrogen oxide thereof, or a deuterated form thereof, or a combination of these substances, or a composition of the present application.

[0055] In some embodiments, a tumor treatment or tumor therapeutic drug inhibits tumor development or progression by inhibiting the E3 ubiquitin ligase activity of XIAP. In some embodiments, inhibition of the E3 ubiquitin ligase activity of XIAP can be achieved by any technique known in the art, such as mutating key enzyme active sites, knocking out key fragments, or conjugating small molecules or macromolecules. In one embodiment, the above embodiments can be implemented individually or in combination with other examples or embodiments disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Western blot experiment after candidate compounds acted on human bladder cancer T24T cells.

[0057] Figure 2 The results of testing the migration and invasion abilities of T24T cells under the action of candidate compounds.

[0058] Figure 3 Effects of compound 1 on primary basal-type muscle-invasive bladder cancer in mice.

[0059] Figure 4 Results of Compound 1 administration in a BBN-induced mouse model of primary basal muscle invasive bladder cancer. (A) After treatment, mice were sacrificed and bladders were removed; (B) Bladder weights of mice in each group; (C) Bladder / body weight ratio of mice in each group; (D) Body weight changes of mice during the treatment period.

[0060] Figure 5Compound 1 inhibits subcutaneous tumorigenesis of human BMIBC cells in nude mice. (A) Tumors were removed from nude mice after sacrifice 33 days after treatment. (B) Weights of subcutaneous tumors in nude mice and statistical analysis. (C) Tumor volumes were calculated using the formula V = π / 6 × (longest diameter) × (short diameter)² and statistical analysis was performed.

[0061] Figure 6 Effects of compound 2 on primary basal-type muscle-invasive bladder cancer in mice.

[0062] Figure 7 Differential analysis of XIAP gene expression in various tumor tissues.

[0063] Figure 8 Western blot experiments after compound 1 acted on different tumor cells.

[0064] Figure 9 Compound 1 and its modified compounds inhibit the malignant proliferation of human melanoma cells. (A) Soft agar colony formation assay of A375 cells treated with different compounds to assess their malignant proliferation. (B) Corresponding colony formation numbers and data analysis results.

[0065] Figure 10 Compound 1 inhibits the malignant proliferation of human lung cancer and prostate cancer cells. (A-B) Soft agar colony formation assay of human prostate cancer cells PC-3 treated with Compound 1, along with the corresponding number of colonies formed and data analysis results. (B-C) Soft agar colony formation assay of human lung adenocarcinoma cells HCC827 treated with Compound 1, along with the corresponding number of colonies formed and data analysis results.

[0066] Figure 11 Compound 1 and its modifications and analogs inhibit the invasion and metastasis of human bladder cancer cells, T24T cells. (A)-(D) are the compound structures. (E)-(H) show the changes in T24T cell migration and invasion abilities after treatment with different compounds, compared to the effects of treatment with Compound 1. (I)-(L) show the statistical results of the corresponding Transwell assays.

[0067] Figure 12 The XIAP RING domain plays an important role in BBN-induced basal-type muscle-invasive bladder cancer.

[0068] Figure 13 The XIAP RING domain uses DNMT3B protein as a substrate and plays an important role in basal-type muscle-invasive bladder cancer.

[0069] Figure 14Figure 2 shows the inhibitory effect of Compound 2 (Com2) on T24T bladder cancer subcutaneous xenografts. (A) Timeline of nude mouse subcutaneous tumor growth; (B) Comparison of tumor size and morphology between the control and drug-treated groups; (C) Dynamic curves of tumor volume changes in the control and drug-treated groups; (D) Weighing and statistical analysis of nude mouse subcutaneous tumors.

[0070] Figure 15 Figure 1 shows the inhibitory effect of Compound 1 (Com1) on lung metastasis of human bladder cancer T24T cells. (A) Lung tissue fixed with a saturated picric acid solution was photographed, showing lung metastases; (B) Paraffin-embedded tissue was sectioned and stained with HE; (C) The number of metastatic foci in nude mouse lung tissue was counted.

[0071] Figure 16 Figure 1 shows the target-dependent effects of Compound 1 (Com1) on human bladder cancer T24T cells. (A) Nude mice in the T24T (KOXIAP / XIAP) vehicle control group, the T24T (KOXIAP / XIAP) drug-treated group (10 mg / kg / day), the T24T (KOXIAP / H467A) vehicle control group, and the T24T (KOXIAP / H467A) drug-treated group (10 mg / kg / day) were sacrificed and their subcutaneous tumors were removed for comparison of tumor size and morphology. (B) Subcutaneous tumors were weighed and statistically analyzed. (C) Tumor volume curves were plotted for mice. (D) Percentage of tumor inhibition in the drug-treated groups was calculated based on tumor volume.

[0072] Figure 17 Figure 1 shows the inhibitory effect of different concentrations of Compound 1 (Com1) on subcutaneous tumor growth in human gastric cancer AGS cells. (A) Photos of mice in the control and drug-treated groups (5 mg / kg / day, 20 mg / kg / day, and 100 mg / kg / day) on day 24; (B, C) Tumor volumes and growth curves for the control and drug-treated groups; (D) Tumor weights for the control and drug-treated groups; (E) Percentage of tumor inhibition in the drug-treated groups calculated based on tumor volume; (F) Weight curves for mice in the control and drug-treated groups; (G) Photos of tumors in the control and drug-treated groups.

[0073] Figure 18 Figure 1 shows the inhibitory effect of different concentrations of Compound 1 (Com1) on subcutaneous tumor growth in mouse gastric cancer MFC cells. (A) Photos of mice in the control and drug-treated groups (5 mg / kg / day, 10 mg / kg / day, and 40 mg / kg / day) on day 24; (B, C) Tumor volumes and growth curves for the control and drug-treated groups; (D) Tumor weights for the control and drug-treated groups; (E) Percentage of tumor inhibition in the drug-treated groups calculated based on tumor volume; (F) Body weight curves for mice in the control and drug-treated groups; (G) Photos of tumors in the control and drug-treated groups.

[0074] Figure 19 Figure 1 shows the inhibitory effect of different concentrations of Compound 1 (Com1) on subcutaneous tumor growth in mouse pancreatic cancer KPC cells. (A) Comparison of tumor size and morphology between the control group and the drug-treated groups (10 mg / kg / day, 40 mg / kg / day, and 100 mg / kg / day, intraperitoneal injection); (B, C) Quantitative comparison of tumor size and volume between the control group and each drug-treated group; (D) Calculation of the percentage of tumor inhibition in the drug-treated group based on tumor volume; (E) Tumor volume change curves for the control and drug-treated groups; (F) Body weight change curves for the control and drug-treated groups.

[0075] Figure 20 Figures showing the inhibitory effect of Compound 1 (Com1) on subcutaneous tumor growth in human melanoma A375 cells. (A) Photos of mice in the control and drug-treated groups (Com1, 50 mg / kg / day) on day 25; (B) Comparison of tumor size and morphology between the control and drug-treated groups; (C, D) Quantitative statistics of tumor volume and weight after treatment in the control and drug-treated groups; (E) Calculation of the percentage of tumor inhibition in the drug-treated group based on tumor volume; (F) Tumor volume change curves for mice in the control and drug-treated groups.

[0076] Figure 21 Figures showing the inhibitory effect of different concentrations of compound 9 (X-10) on subcutaneous tumor growth of mouse renal cell carcinoma RENCA cells. (A) Comparison of tumor size and morphology after treatment in the control group, intraperitoneal injection groups at different concentrations (5 mg / kg / day, 20 mg / kg / day, 40 mg / kg / day), and the drinking water group (40 mg / kg / day); (B, C) Comparison of tumor weight and volume after treatment in each group; (D) Calculation of the percentage of tumor inhibition after treatment in each group based on tumor weight; (E) Tumor volume change curves for each group; (F) Mouse body weight change curves for each group.

[0077] Figure 22 Figures showing the inhibitory effect of compound 9 (X-10) on subcutaneous tumor growth in human lung adenocarcinoma H1299 cells. (A) Photos of mice in the control and drug-treated groups (intraperitoneal injection, 40 mg / kg / day) on day 32; (C) Comparison of tumor size and morphology in the control and drug-treated groups; (C, D) Quantitative statistics of tumor size and volume after treatment in the control and drug-treated groups; (E) Tumor volume curves for mice in the control and drug-treated groups; (F) Calculation of the percentage of tumor inhibition in the drug-treated group based on tumor volume.

[0078] Figure 23 The results of the inhibitory effect of compound 9 (X-10) on subcutaneous tumor growth of mouse colorectal cancer MC38 cells are shown in Figure 1. (A) Control group and drug-treated group (X-10, 40 mg / kg / day);

[0079] (B) Comparison of tumor size and morphology between the control group and the drug-treated group; (C, E) Quantitative statistics of tumor size and volume between the control group and the drug-treated group; (E) Tumor volume change curve between the control group and the drug-treated group; (F) Calculation of the tumor inhibition percentage of the drug-treated group based on tumor volume.

[0080] Figure 24 .H NMR spectrum of compound 3 (T4489).

[0081] Figure 25 Compound 1 and its modified analogs inhibited the invasion and metastasis of human bladder cancer cells, T24T cells. (A)-(D) are the compound structures. (E)-(H) are Transwell assays of T24T cells treated with different compounds, examining changes in cell migration and invasion, and comparing the effects with those after treatment with Compound 1. (I)-(L) are the statistical results of the Transwell assays.

[0082] Figure 26 .Amino acid sites involved in binding to small molecule compounds in the TargetMol database and the frequency of binding to small molecule compounds.

[0083] Figure 27 .Amino acid sites involved in binding to small molecule compounds in the Chemdiv database and the frequency of binding to small molecule compounds. DETAILED DESCRIPTION

[0084] The following describes embodiments of the present application with reference to the accompanying drawings. Elements and features described in one drawing or one embodiment of the present application may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that, for the sake of clarity, representations and descriptions not relevant to the present application and known to those of ordinary skill in the art have been omitted from the drawings and descriptions. The present application is further described below with reference to the accompanying drawings.

[0085] Table 2 Functional verification of representative compounds

[0086]

[0087]

[0088] Definitions of Terms. Abbreviations used herein have their conventional meanings in chemistry and biology. The chemical structures and formulas described herein are constructed according to standard chemical valence rules known in the chemical art. For terms not specifically defined herein, they should be given the meanings that would be given to them by those skilled in the art based on the disclosure and context.

[0089] "XIAP inhibitors" or "XIAP antagonists" refer to substances that can target and inhibit the XIAP protein RING structure or E3 ligase biological function to exert anti-tumor effects. In some embodiments, these inhibitors are selected from one or more of nucleic acid molecules, small molecules, antibody drugs, polypeptides, proteins, nucleic acid constructs, interfering lentiviruses, interfering adeno-associated viruses, and gene editing systems. In some embodiments, the inhibitor is a drug, compound, composition, or formulation, such as a compound or composition (such as a pharmaceutical composition) of the present application. Therefore, the compounds and related compositions of the present application can bind to XIAP. Further, the compounds and related compositions of the present application can bind to the C-terminus of XIAP or the RING domain of XIAP. Furthermore, the compounds and related compositions of the present application can bind to the 467H site of human XIAP, or amino acid sites of XIAP in other species corresponding to the 467H site of human XIAP, such as the 466H site of mouse XIAP. Furthermore, the compounds and related compositions of the present application bind to the human XIAP RING domain and thereby alter E3 ligase activity.

[0090] The term "treat" refers to alleviating, preventing, ameliorating or delaying the onset of one or more unwanted conditions or symptoms of a disease in a patient; or to controlling the progression of a disease or maintaining disease stability.

[0091] As used herein, "subject" or "patient" refers to a human or non-human animal (eg, a mammal), including but not limited to dogs, cats, horses, cows, pigs, sheep, goats, chickens, monkeys, rabbits, rats, and mice.

[0092] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a compound or a pharmaceutically acceptable salt thereof (typically as part of a pharmaceutical composition) sufficient to inhibit, halt, ameliorate, alleviate, delay the onset of, or produce an improvement in the symptoms of one or more of the conditions being treated when administered alone or in combination with another drug for the treatment of a specific subject or population of subjects.

[0093] "Pharmaceutical composition" refers to a compound suitable for administration in pharmaceutical or veterinary settings. A pharmaceutical composition comprises a therapeutically effective amount of a compound as described above, or a pharmaceutically acceptable salt or other form thereof, together with one or more pharmaceutically acceptable excipients. It will be understood that determining the appropriate dosage form, dose, and route of administration for a particular patient is within the level of ordinary skill in the pharmaceutical and medical arts.

[0094] The compounds and related compositions of the present application can be administered alone or in combination with other pharmaceutically active substances, for example, to patients who have received unsatisfactory traditional treatments, such as unsatisfactory radiotherapy or chemotherapy, and who have developed drug resistance. Other pharmaceutically active substances can be used to treat diseases or conditions that are the same as or different from the diseases or conditions of the present application. If a patient has received or is receiving multiple pharmaceutically active substances, the substances can be administered simultaneously or sequentially. Common methods for treating tumors include surgery, chemotherapy, radiotherapy, immunotherapy, photodynamic therapy and targeted therapy, etc. Drugs for treating tumors include chemotherapeutic drugs, immunotherapy drugs and targeted therapy drugs, etc. The compounds and compositions of the present application can be used in combination with these drugs or methods. The administration of the compounds and compositions of the present application can be carried out before, simultaneously with or after the combined treatment. A variety of administration routes can be used. A variety of non-limiting methods of administering the compounds and related compositions to a patient include oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) infusion, intracranial, intradermal, inhalation, extra-digestive route, intravaginal, intraperitoneal, intravesical, topical (powders, ointments or drops) or as a buccal or nasal spray. In addition, the substance or composition comprising the active substance can be administered once, for example by bolus injection, multiple times, for example, by a series of tablets or delivered substantially uniformly over a period of time, for example using transdermal delivery. It should be noted that the dosage of the substance can change over time. The compounds and compositions of the present application can also benefit from a variety of drug delivery systems, including timed release, delayed release or sustained release drug delivery systems, or novel drug delivery systems (e.g., using nanocarriers).

[0095] The present application also includes isotopically enriched compounds, which are equivalent to those compounds listed herein, except that one or more of their atoms are replaced by atoms having an atomic weight or mass number different from that found in nature. The compounds of the present application may exist in the form of non-solvates or solvates (such as hydrates). The compounds of the present application may be administered as pharmaceutically acceptable salts, esters, amides or prodrugs. Among them, salts include but are not limited to inorganic or organic salts, acid addition salts and / or base addition salts of the compounds of the present application. The compounds and salts of the present application may also exist in the form of tautomers, such as in the form of enols or imines, and their corresponding ketone and enamine forms, and their geometric isomers and compounds. Tautomers exist as mixtures of tautomer groups in solution. The compounds of the present application may contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereoisomeric forms of the compounds and mixtures thereof, including racemic mixtures, constitute part of the present application. In addition, the present application includes all stereo and positional isomers. For example, if a compound contains a double bond, both the cis and trans forms (designated Z and E, respectively), as well as mixtures, are included.

[0096] The compounds of the present invention can be administered to patients alone or as part of a pharmaceutical composition in a therapeutically effective amount. Typically, the dose of the active compound is from about 0.01 mg / kg to

[0097] 1000mg / kg. It is expected that a dosage range of 50-500mg / kg is suitable, preferably administered intravenously, intramuscularly or intradermally, and administered once or several times a day. In some embodiments, the dosage regimen of the compound or composition of the present application can be oral 1mg to 2000mg / day, preferably 1 to 1000mg / day, more preferably 50 to 600mg / day, divided into two to four (preferably two) separate doses; cyclical treatment (for example, one week out of three weeks, or three weeks out of four weeks) can also be used.

[0098] Preparation Example 1 Preparation of Compound Y205-0880

[0099] Reaction roadmap

[0100]

[0101] Bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride (1.0 g, 5.6 mmol) was dissolved in ethanol, and 10 mL of 20% NaOH solution was added. The reaction was stirred under reflux at 70°C for 5 h. After removing the ethanol by distillation under reduced pressure, the pH was adjusted to 2-3 with 6N-HCl. The crystals were washed out by cooling in an ice-water bath, filtered under reduced pressure, and dried to obtain bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic acid (intermediate 2) with a yield of 50%.

[0102] p-Aminobenzoic acid (500 mg, 3.65 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (1.899 g, 3.65 mmol), and N,N-diisopropylethylamine (707 mg, 5.47 mmol) were dissolved in N,N-dimethylformamide and stirred at room temperature for 10 minutes. Pyrrolidine (307 mg, 4.38 mmol) was then added and the mixture was stirred overnight. After the reaction was complete, the solvent was removed by distillation under reduced pressure, and the mixture was extracted with ethyl acetate / water. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and dried by suction. The crude product was purified by silica gel column to obtain the off-white intermediate 4-pyrrolidinoylaniline (Intermediate 3) in an 81% yield.

[0103] Intermediate 2 (500 mg, 2.80 mmol), benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate (524 mg, 4.9 mmol), and N,N-diisopropylethylamine (200 mg, 1.52 mmol) were dissolved in N,N-dimethylformamide and stirred at room temperature for 10 minutes. 4-Pyrrolidinoylaniline (0.57 g, 2.8 mmol) was then added. The reaction was heated to 70°C for 5 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and the mixture was extracted with ethyl acetate / water. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and dried. The crude product was purified on a silica gel column to obtain Intermediate 4 in a 71% yield.

[0104] Intermediate 4 (500 mg, 2.80 mmol) was dissolved in methanol, and after adding a catalytic amount of Pd / C and triethylamine, hydrogen was introduced and reacted for 30 minutes. After the reaction was completed, water was added to terminate the reaction. Methanol was removed by distillation under reduced pressure, and the mixture was extracted with an ethyl acetate / water system. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and spin-dried. The crude product was purified by silica gel column to obtain the final product Y205-0880 (669734-34-3) with a yield of 47%. The nuclear magnetic resonance data of product Y205-0880 are shown in Table 8. The retention time in LC-MS is 0.861 min, and m / z (ESI) = 371.1 (M+H) + .

[0105] For compounds with similar structures and synthetic routes, reference may be made to the preparation methods of the specific compounds in this application or according to conventional methods in the art. The NMR data of compound E587-0499 are shown in Table 8. The retention time in LC-MS is 0.595 min, and m / z (ESI) = 450.3 (M+H) + .

[0106] The H NMR spectrum of compound T4489 is shown in Figure 24 .

[0107] Preparation Example 2 Preparation of Compounds X-1, X-4, X-3 and X-12

[0108]

[0109] Reagents and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90°C; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0110] Different substituted aminobenzoic acids (X-1: R1 is H, p-; X-4: R1 is H, m-; X-3: R1 is CH3, m-; X-12: R1 is H, o-; X-2: R1 is CH3, R1 and The following were dissolved in 6 mL of DMF, stirred at room temperature, and reacted overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound a was obtained by separation and purification by column chromatography.

[0111] Compound a (2.8 mmol), monomethyl phthalate (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.8 mmol), and N,N-diisopropylethylamine (4.2 mmol) were dissolved in 6 mL of DMF. The reaction system was stirred at 90°C. The reaction progress was monitored by TLC. After approximately 4 h of reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound b was isolated and purified by column chromatography.

[0112] Compound b (1.4 mmol) and lithium hydroxide hydrate (2.8 mmol) were dissolved in 6 mL of a 1:1 tetrahydrofuran / water mixture. The reaction was stirred at room temperature and monitored by TLC. The reaction was complete after approximately 2 h. The solvent was removed by distillation under reduced pressure. The reaction solution was acidified with 1 mol / L dilute hydrochloric acid and extracted with ethyl acetate / water. The organic layers were combined, dried, and concentrated under reduced pressure. A small amount of ethyl acetate was added to dissolve the product, and the product was allowed to stand to precipitate. The product was filtered to obtain 2-((4-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-1, white powder, 45% yield), 2-((3-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-4, flesh-colored powder, 40% yield), 2-((2-methyl-5-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-3, white powder, 41% yield), and 2-((2-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-12, white powder, 43% yield). The structural formulas and NMR characterization results of compounds X-1, X-4, X-3, and X-12 are shown in Table 8.

[0113] Preparation Example 3 Preparation of Compounds X-5, X-6, X-7, X-8 and X-10

[0114]

[0115] Reagents and conditions: (IV) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90°C; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0116] Differently substituted p-aminobenzoic acids (X-5: R1 is H, R2 is CH; X-6: R1 is H, R2 is Cl; X-7: R1 is H, R2 is OH; X-8: R1 is -OCH3, R2 is H; X-10: R1 is OH, R2 is H) (3.6 mmol), pyrrolidine (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (3.6 mmol), and N,N-diisopropylethylamine (5.5 mmol) were dissolved in 6 mL of DMF and stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound c was isolated and purified by column chromatography.

[0117] Synthesis Route II: Compound d was synthesized by referring to Synthesis Route II in Preparation Example 2.

[0118] Synthesis Route III was prepared by referring to Synthesis Route III in Preparation Example 2 to obtain the products 2-((3-methyl-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-5, white powder, 35% yield), 2-((3-chloro-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-6, white powder, 19% yield), 2-((3-hydroxy-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-7, brown powder, 10% yield), 2-((2-methoxy-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-8, white powder, 40% yield), and 2-((2-hydroxy-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)benzoic acid (X-10, white powder, 15% yield). The structural formulas and NMR characterization results of compounds X-5, X-6, X-7, X-8 and X-10 are shown in Table 8.

[0119] Preparation Example 4 Preparation of Compounds LYS10-LYS25

[0120]

[0121] Reagents and conditions: (V) PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90°C; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0122] Table 3 Amines in Synthesis Route V

[0123]

[0124]

[0125] 4-Amino-2-chlorobenzoic acid (3.6 mmol), various amines (see Table 3) (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.6 mmol), and N,N-diisopropylethylamine (5.5 mmol) were dissolved in 6 mL of DMF, stirred at room temperature, and reacted overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound e was isolated and purified by column chromatography.

[0126] Synthesis Route II: Compound f was synthesized by referring to Synthesis Route II in Preparation Example 2.

[0127] Synthesis Route III was prepared by referring to Synthesis Route III in Preparation Example 2, and the following was finally synthesized:

[0128] 2-((3-chloro-4-(cyclobutylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-10, white powder, 35%),

[0129] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0130] 2-((3-chloro-4-(piperidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (LYS-12, white powder, 19%),

[0131] 2-((3-chloro-4-(4-methylpiperazine-1-carbonyl)phenyl)carbamoyl)benzoic acid (LYS-13, white powder, 10%),

[0132] 2-((4-(azetidine-1-carbonyl)-3-chlorophenyl)carbamoyl)benzoic acid (LYS-14, white powder, 38%),

[0133] 2-((3-chloro-4-(morpholine-4-carbonyl)phenyl)carbamoyl)benzoic acid (LYS-15, white powder, 32%),

[0134] 2-((3-chloro-4-((thiophen-2-ylmethyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-16, white powder, 41%),

[0135] 2-((4-(Azacyclohexane-1-carbonyl)-3-chlorophenyl)carbamoyl)benzoic acid (LYS-17, white powder, 30%),

[0136] 2-((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-18, white powder, 33%),

[0137] 2-((3-chloro-4-(ethylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-19, white powder, 40%),

[0138] 2-((3-chloro-4-(4-(4-methoxybenzoyl)piperidin-1-carboxyl)phenyl)carbamoyl)benzoic acid (LYS-20, white powder, 20%),

[0139] 2-((3-chloro-4-(diethylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-21, white powder, 28%),

[0140] 2-((3-chloro-4-((3,5-dimethoxyphenyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-22, white powder, 45%),

[0141] 2-((3-chloro-4-((3-chlorophenyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-23, white powder, 35%),

[0142] 2-((3-chloro-4-((4-morpholinophenyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-24, white powder, 24%),

[0143] 2-((3-chloro-4-((thiophen-2-ylmethyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-24, white powder, 15%). The structural formula and NMR characterization results of compound LYS10-25 are shown in Table 8.

[0144] Preparation Example 5 Preparation of Compounds Y-9-2 and Y-9-3

[0145]

[0146] Reagents and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (VI) PyBOP, DIPEA, DMF, 70°C; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0147] Synthesis Route I Referring to the synthesis route I in Preparation Example 2, compound g was synthesized.

[0148] Compound g (2.6 mmol), (1R,2R)-2-(methoxycarbonyl)cyclohexanecarboxylic acid (2.94 mmol), benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate (2.45 mmol), and N,N-diisopropylethylamine (3.67 mmol) were dissolved in 8 mL of N,N-dimethylformamide and stirred at room temperature for 10 minutes. (4-amino-3-methylphenyl)(pyrrolidin-1-yl)methanone (2.45 mmol) was then added. The reaction temperature was increased to 70°C and monitored by TLC. After approximately 4 hours, the reaction was complete and evaporated under reduced pressure. The organic layers were extracted with ethyl acetate / saturated brine, and the combined organic layers were dried and concentrated under reduced pressure. Column chromatography was used to separate and purify the product, methyl (1R,2R)-2-((3-methyl-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)cyclohexane-1-carboxylate (Y-9-2), a white powder in a 60% yield.

[0149] Synthesis Route III was followed by that described in Preparation Example 2 to yield (1R,2R)-2-((3-methyl-4-(pyrrolidine-1-formyl)phenyl)carbamoyl)cyclohexane-1-carboxylic acid (Y-9-3, flesh-colored solid powder, 30% yield). The structures and NMR characterization results of compounds Y-9-2 and Y-9-3 are shown in Table 8.

[0150] Preparation Example 6 Preparation of Compounds X-15, X-16, X-17, X-18 and X-19

[0151]

[0152] Reagents and conditions: (I) PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90℃; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0153] Table 4 Amines in Synthesis Route I and 2-(Methoxycarbonyl)cyclocarboxylic acids in Synthesis Route II

[0154]

[0155] p-Aminobenzoic acid (3.6 mmol), various amines (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.6 mmol), and N,N-diisopropylethylamine (5.5 mmol) were dissolved in 6 mL of DMF and stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound a was isolated and purified by column chromatography.

[0156] Compound a (2.8 mmol), various 2-(methoxycarbonyl)cycloalkanecarboxylic acids (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.8 mmol), and N,N-diisopropylethylamine (4.2 mmol) were dissolved in 6 mL of DMF. The reaction system was stirred at 90°C. The reaction progress was monitored by TLC. After approximately 4 h of reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound b was isolated and purified by column chromatography.

[0157] Compound b (1.4 mmol) and lithium hydroxide hydrate (2.8 mmol) were dissolved in 6 mL of a tetrahydrofuran / water (1:1) mixed solution, stirred at room temperature, and the reaction was monitored by TLC. The reaction was completed after about 2 h. The solvent was removed by distillation under reduced pressure, and the reaction solution was acidified with 1 mol / L dilute hydrochloric acid, then extracted with ethyl acetate / water, the organic layers were combined, dried, and concentrated under reduced pressure. A small amount of ethyl acetate was added to dissolve, and the product was allowed to stand and precipitate. It was filtered to obtain the product 2-((4-(diethylcarbamoyl)phenyl)carbamoyl)cyclohexane-1-carboxylic acid (X-15, white powder, yield 21%), 2-((4-(4-methylpiperidin-1-formyl)phenyl)carbamoyl)cyclohexane-1-carboxylic acid (X-16, white powder, yield 20%), 2-((4-(isobutylcarbamoyl)phenyl) Carbamoyl) cyclohexane-1-carboxylic acid (X-17, gray powder, yield 30%), 3-((4-(piperidin-1-carboxyl)phenyl)carbamoyl)bicyclo[2.2.1]heptane-2-carboxylic acid (X-18, white powder, yield 15%) and (1R,6S)-6-((4-(pyrrolidine-1-carboxyl)phenyl)carbamoyl)cyclohex-3-ene-1-carboxylic acid (X-19, white powder, yield 10%). Compounds X-15, X-16, X-

[0158] The structural formulas and NMR characterization results of 17, X-18 and X-19 are shown in Table 8.

[0159] Preparation Example 7 Preparation of Compound X-20

[0160]

[0161] Reagents and conditions: (IV) 4-Methylpiperazine, STAB, CH2Cl2, CH3COOH, rt; (V) Pd(DPPF)Cl2.CH2Cl2, K2CO3, dioxane / H2O 5:1, N2, 100°C;

[0162] 6-Bromopyridine-3-carboxaldehyde (2.2 mmol), 4-methylpiperazine (2.2 mmol), and sodium triacetoxyborohydride (4.3 mmol) were dissolved in 6 mL of 1,2-dichloroethane. Two drops of acetic acid were added dropwise with stirring and the mixture was allowed to react at room temperature for 4 hours. The reaction was monitored by TLC. Upon completion, the solvent was removed by vacuum distillation, and the mixture was extracted with a water / methylene chloride:methanol system. The organic layers were combined, dried, and concentrated under reduced pressure. Purification by column chromatography afforded a colorless, transparent oil, c.

[0163] Compound c (0.83 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (0.99 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (0.04 mmol), and potassium carbonate (2.49 mmol) were dissolved in 6 mL of a 5:1 mixture of dioxane and water. The mixture was reacted at 100°C for 2 h under nitrogen. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / water. The organic layers were combined, dried, and concentrated under reduced pressure. An appropriate amount of dichloromethane was added to produce a precipitate, which was allowed to settle and filtered to obtain the final product, 4-(5-((4-methylpiperazin-1-yl)methyl)pyridin-2-yl)benzamide (X-20), a white solid powder in a 60% yield. The structural formula and NMR characterization results of compound X-20 are shown in Table 8.

[0164] Preparation Example 8 Preparation of Compound X-21

[0165]

[0166] Reagents and conditions: (I) 4-Methylbenzylamine, PyBOP, DIPEA, DMF, rt; (VI) Pd(PPh3)4, Cs2CO3, dioxane / H2O 5:1, N2, 100°C;

[0167] 6-Bromonicotinic acid (2.5 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (2.5 mmol), and N,N-diisopropylethylamine (3.7 mmol) were dissolved in 6 mL of N,N-dimethylformamide. After stirring at room temperature for 10 minutes, p-methylbenzylamine (3.0 mmol) was added and the mixture was allowed to react for 5 hours. After the reaction was complete, the solvent was removed by distillation under reduced pressure, resulting in the precipitation of a solid. An appropriate amount of ethyl acetate was added to the reaction solution, which was allowed to stand and then filtered to obtain a yellow powder d.

[0168] Compound d (0.66 mmol), 2-(4-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (0.79 mmol), tetrakis(triphenylphosphine)palladium (0.06 mmol), and cesium carbonate (1.3 mmol) were dissolved in 6 mL of dioxane:water (5:1) and reacted at 100°C under nitrogen for 2 h. After completion of the reaction, the solvent was distilled off under reduced pressure, and the mixture was extracted with ethyl acetate / water. The organic layers were combined, dried, and concentrated under reduced pressure. The crude product was purified on a silica gel column to yield the final product, 6-(4-fluorophenyl)-N-(4-methylbenzyl)nicotinamide (X-21, white solid powder, 62%). The structure and NMR characterization results of compound X-21 are shown in Table 8.

[0169] Preparation Example 9 Preparation of Compounds C1-2-C3-5

[0170]

[0171] Reagents and conditions: (VI) Pyrrolidine (Cyclopropylamine), PyBOP, DIPEA, DMF, 70°C; (II) PyBOP, DIPEA, DMF, 90°C; (III)

[0172] LiOH·H2O,THF / H2O 1:1,rt; (V)SOCl2,DMF,DCM,rt; (VI)Amines,DCM,rt.

[0173] 4-Amino-2-chlorobenzoic acid A (2.9 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.9 mmol), and N,N-diisopropylethylamine (4.4 mmol) were dissolved in 6 mL of anhydrous DMF. After stirring at room temperature for 5 minutes, cyclopropylamine (3.5 mmol) was added and the mixture was heated in an oil bath to 70°C for 4 hours. The reaction was monitored by TLC. After completion, most of the anhydrous DMF was removed by vacuum distillation. The mixture was then extracted with ethyl acetate / saturated brine. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 90:1) to obtain Compound B.

[0174] Monomethyl phthalate (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.8 mmol), and N,N-diisopropylethylamine (4.2 mmol) were dissolved in 10 mL of anhydrous DMF and stirred at room temperature for 5 minutes. Compound B (2.8 mmol) was then added, and the reaction was heated in an oil bath to 90°C and stirred for 4 hours. TLC was used to monitor the reaction progress. Upon completion, the anhydrous DMF was removed by vacuum distillation. The mixture was then extracted with ethyl acetate and saturated brine. The organic layers were combined, dried over anhydrous MgSO₄, filtered, and the solvent removed by vacuum distillation to yield the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 75:1) to obtain compound C1-2. By replacing monomethyl phthalate with monomethyl isophthalate or monomethyl terephthalate, while maintaining all other conditions, compounds C2-2 and C3-2, respectively, were obtained.

[0175] Dissolve C2-2 (1.4 mmol) in 6 mL of a 1:1 tetrahydrofuran:water solution, add lithium hydroxide hydrate (2.8 mmol), and stir at room temperature for 2 h. Monitor by TLC. After the reaction is complete, remove the solvent by distillation under reduced pressure. Adjust the pH to a weakly acidic state with 1 M dilute hydrochloric acid solution, extract with ethyl acetate / saturated brine, and remove the solvent by distillation under reduced pressure. Add an appropriate amount of ethyl acetate and allow to stand until stable crystals precipitate. Filter to obtain compound C2-3 and dry in an oven at low temperature. Replace C2-2 with C3-2, while keeping other conditions unchanged, to obtain C3-3.

[0176] Replace different positions Dissolve 1.4 mmol of thionyl chloride (1.4 mmol) in 6 mL of anhydrous dichloromethane, add 2.8 mmol of thionyl chloride dropwise, stopper the bottle with a rubber stopper, and add a catalytic amount of DMF dropwise via syringe. Stir at room temperature and react for 4 h. Monitor the reaction by TLC. Once the reaction is complete, remove the solvent by distillation under reduced pressure, seal the bottle, and proceed to the next step.

[0177] The product from the previous step was dissolved in 6 mL of anhydrous dichloromethane and placed in a low-temperature reaction chamber, stirring at -20°C. Using a pipette, various amine compounds (1.7 mmol) were quickly transferred to the reaction flask. After 5 minutes, the mixture was stirred at room temperature and allowed to react for 5 hours. TLC was used to monitor the reaction progress. Upon completion, the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate and saturated brine. The organic layers were combined and dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. The crude product was isolated and purified by column chromatography (dichloromethane:methanol = 25:1) to afford compounds C1-4, C1-5, C2-4, C2-5, C3-4, and C3-5. The corresponding relationships between the substitution of the product and the raw material in step V and the different amine compounds in step VI are: (C1-4: o-, diethylamine; C1-5: o-, azetidine; C2-4: m-, diethylamine; C2-5: m-, azetidine; C3-4: p-, diethylamine; C3-5: p-, azetidine)

[0178] Preparation Example 10 Preparation of Compounds C5-2-1, C5-2-2, and C6-2-1

[0179]

[0180] Reagents and conditions: (V) SOCl2, DMF, DCM, rt; (VI) B, DCM, rt.

[0181] Will Dissolve 1.5 mmol of thionyl chloride (1.5 mmol) in 5 mL of anhydrous dichloromethane, add 3.0 mmol of thionyl chloride dropwise, stopper the bottle with a rubber stopper, and add a catalytic amount of DMF dropwise via syringe. Stir at room temperature and react for 5 h. Monitor the reaction by TLC. Once the reaction is complete, remove the solvent by distillation under reduced pressure, seal the bottle, and proceed to the next step.

[0182] The product of the previous step and 4-amino-2-chloro-N-cyclopropylbenzamide B (1.8 mmol) were dissolved in 5 mL of anhydrous dichloromethane, stirred at room temperature, and reacted overnight. TLC monitored the reaction progress. After the reaction was complete, the solvent was distilled off under reduced pressure, extracted with ethyl acetate / saturated brine, the organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 30:1) to obtain compound C5-2-1. Replace with With other conditions unchanged, C5-2-2 and C6-2-1 can be prepared respectively.

[0183] Preparation Example 11 Preparation of Compounds C7-3, C8-3, and C9-3

[0184]

[0185] Reagents and conditions: (VI) Pyrrolidine, (Cyclopropylamine) PyBOP, DIPEA, DMF, 70°C; (VII) PyBOP, DIPEA, DMF, 100°C; (III) LiOH·H2O, THF / H2O 1:1, rt.

[0186] Will (2.7mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.2mmol) and N,N-diisopropylethylamine (3.4mmol) were dissolved in 10mL of anhydrous DMF and stirred at room temperature for 5min. Compound B (2.2mmol) was added and the reaction was heated to 100℃ in an oil bath and stirred for 4h. The reaction progress was monitored by TLC. After the reaction was complete, anhydrous DMF was removed by vacuum distillation. The mixture was extracted with ethyl acetate / saturated brine. The organic layers were combined, dried over anhydrous MgSO4, filtered, and the solvent was removed by vacuum distillation to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane: methanol = 75:1) to obtain compound

[0187] The product of the previous step (1.4 mmol) was dissolved in 6 mL of tetrahydrofuran: water (1:1) solution, lithium hydroxide hydrate (2.8 mmol) was added and stirred at room temperature for 2 h, monitored by TLC. After the reaction was completed, the solvent was removed by distillation under reduced pressure, the pH was adjusted to weak acidity with 1 M dilute hydrochloric acid solution, and the mixture was extracted with ethyl acetate / saturated brine, and the solvent was removed by distillation under reduced pressure. An appropriate amount of ethyl acetate was added and the mixture was allowed to stand until the crystals were stably precipitated. Compound C7-3 was obtained by filtration and placed in an oven for low-temperature drying. Replace with With other conditions unchanged, C8-3 and C9-3 can be prepared respectively.

[0188] General synthetic route of compound C1-2-C3-5:

[0189]

[0190] Reagent and conditions: (VI) Pyrrolidine, PyBOP, DIPEA, DMF, 70℃; (II) PyBOP, DIPEA, DMF, 90℃; (III) LiOH·H2O, THF / H2O 1:1, rt; (V) SOCl2, DMF, DCM, rt; (VI) Amines, DCM, rt.

[0191] 4-Amino-2-chlorobenzoic acid A (2.9 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.9 mmol), and N,N-diisopropylethylamine (4.4 mmol) were dissolved in 6 mL of anhydrous DMF. After stirring at room temperature for 5 minutes, cyclopropylamine (3.5 mmol) was added and the mixture was heated in an oil bath to 70°C for 4 hours. The reaction was monitored by TLC. After completion, most of the anhydrous DMF was removed by vacuum distillation. The mixture was then extracted with ethyl acetate / saturated brine. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 90:1) to obtain Compound B.

[0192] Monomethyl phthalate (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.8 mmol), and N,N-diisopropylethylamine (4.2 mmol) were dissolved in 10 mL of anhydrous DMF. After stirring at room temperature for 5 minutes, compound B (2.8 mmol) was added, and the reaction was heated in an oil bath to 90°C and stirred for 4 hours. TLC was used to monitor the reaction progress. After completion, the anhydrous DMF was removed by vacuum distillation, and the mixture was extracted with ethyl acetate and saturated brine. The organic layers were combined, dried over anhydrous MgSO₄, filtered, and the solvent was removed by vacuum distillation to yield the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 75:1) to afford compounds C1-2, C2-2, and C3-2.

[0193] Methyl ((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)benzoate with different substitution positions (1.4 mmol) was dissolved in 6 mL of a tetrahydrofuran:water (1:1) solution. Lithium hydroxide hydrate (2.8 mmol) was added and stirred at room temperature for 2 h. TLC monitoring was performed. After the reaction was completed, the solvent was removed by distillation under reduced pressure. The pH was adjusted to a weak acidic state with 1 M dilute hydrochloric acid solution. Extraction was performed with ethyl acetate / saturated brine, and the solvent was removed by distillation under reduced pressure. An appropriate amount of ethyl acetate was added and the mixture was allowed to stand until stable crystals precipitated. Compounds C2-3 and C3-3 were obtained by filtration and dried in an oven at low temperature.

[0194] Dissolve variously substituted ((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)benzoic acid (1.4 mmol) in 6 mL of anhydrous dichloromethane. Add thionyl chloride (2.8 mmol) dropwise. Seal the vial with a rubber stopper and add a catalytic amount of DMF dropwise via syringe. Stir at room temperature for 4 h. Monitor the reaction by TLC. Upon completion, remove the solvent by distillation under reduced pressure. Seal the vial with a lid and proceed to the next step.

[0195] The product from the previous step was dissolved in 6 mL of anhydrous dichloromethane and placed in a low-temperature reaction chamber, stirring at -20°C. Using a pipette, various amine compounds (1.7 mmol) were quickly transferred to the reaction flask. After 5 minutes, the mixture was stirred at room temperature and allowed to react for 5 hours. TLC was used to monitor the reaction progress. Upon completion, the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate and saturated brine. The organic layers were combined and dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. The crude product was isolated and purified by column chromatography (dichloromethane:methanol = 25:1) to afford compounds C1-4, C1-5, C2-4, C2-5, C3-4, and C3-5.

[0196] General synthetic route of compounds C5-2-1–C6-2-1:

[0197]

[0198] Reagent and conditions: (V)SOCl2,DMF,DCM,rt; (VI)B,DCM,rt.

[0199] Dissolve sulfamoyl-substituted benzoic acid (1.5 mmol) in 5 mL of anhydrous dichloromethane. Add thionyl chloride (3.0 mmol) dropwise. Seal the vial with a rubber stopper and add a catalytic amount of DMF dropwise via syringe. Stir at room temperature for 5 h. Monitor the reaction by TLC. Once the reaction is complete, remove the solvent by distillation under reduced pressure. Seal the vial and proceed to the next step.

[0200] The product from the previous step and 4-amino-2-chloro-N-cyclopropylbenzamide B (1.8 mmol) were dissolved in 5 mL of anhydrous dichloromethane and stirred at room temperature overnight. The reaction progress was monitored by TLC. Upon completion, the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate and saturated brine. The organic layers were combined and dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 30:1) to obtain compounds C5-2-1, C5-2-2, and C6-2-1.

[0201] General synthetic route of compounds C7-3–C9-3:

[0202]

[0203] Reagents and conditions: (VI) Pyrrolidine, PyBOP, DIPEA, DMF, 70℃; (VII) PyBOP, DIPEA, DMF, 100℃; (III) LiOH·H2O, THF / H2O 1:1, rt.

[0204] 4-Amino-2-chlorobenzoic acid A (2.9 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.9 mmol), and N,N-diisopropylethylamine (4.4 mmol) were dissolved in 6 mL of anhydrous DMF. After stirring at room temperature for 5 minutes, cyclopropylamine (3.5 mmol) was added and the mixture was heated in an oil bath to 70°C for 4 hours. The reaction was monitored by TLC. After completion, most of the anhydrous DMF was removed by vacuum distillation. The mixture was then extracted with ethyl acetate / saturated brine. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 90:1) to obtain Compound B.

[0205] Different substituted carboxylic acids (2.7 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.2 mmol), and N,N-diisopropylethylamine (3.4 mmol) were dissolved in 10 mL of anhydrous DMF. After stirring at room temperature for 5 minutes, compound B (2.2 mmol) was added, and the reaction was heated to 100°C in an oil bath and stirred for 4 hours. TLC was used to monitor the reaction progress. After completion, the anhydrous DMF was removed by vacuum distillation, and the mixture was extracted with ethyl acetate and saturated brine. The organic layers were combined, dried over anhydrous MgSO₄, filtered, and the solvent was removed by vacuum distillation to yield the crude product. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 75:1) to afford compounds E1, E2, and E3.

[0206] Dissolve different carboxylic acid methyl ester compounds (1.4 mmol) in 6 mL of a tetrahydrofuran:water (1:1) solution, add lithium hydroxide hydrate (2.8 mmol), and stir at room temperature for 2 h. Monitor by TLC. After the reaction is complete, remove the solvent by distillation under reduced pressure. Adjust the pH to a weakly acidic state with 1 M dilute hydrochloric acid solution, extract with ethyl acetate / saturated brine, and remove the solvent by distillation under reduced pressure. Add an appropriate amount of ethyl acetate and allow to stand until stable crystals precipitate. Filter to obtain compounds C7-3, C8-3, and C9-3, and dry them in an oven at low temperature.

[0207] X-2 solvent: deuterated DMSO

[0208] Methyl 2-((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)benzoate C1-2

[0209] The preparation method is the same as compound C1, except that B18 is used to replace B1 to prepare compound LYS26.

[0210] General synthetic route of series A compounds

[0211]

[0212] Reagent and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90℃; (III) LiOH·H2O, THF / H2O1:1, rt;

[0213] Differently substituted aminobenzoic acids (3.6 mmol), pyrrolidine (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.6 mmol), and N,N-diisopropylethylamine (5.5 mmol) were dissolved in 6 mL of DMF and stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound a was isolated and purified by column chromatography.

[0214] Compound a (2.8 mmol), monomethyl phthalate (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (2.8 mmol), and N,N-diisopropylethylamine (4.2 mmol) were dissolved in 6 mL of DMF. The reaction system was stirred at 90°C. The reaction progress was monitored by TLC. After approximately 4 h of reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound b was isolated and purified by column chromatography.

[0215] Compound b (1.4 mmol) and lithium hydroxide hydrate (2.8 mmol) were dissolved in 6 mL of a 1:1 tetrahydrofuran / water mixture. The reaction was stirred at room temperature and monitored by TLC. The reaction was complete after approximately 2 hours. The solvent was removed by distillation under reduced pressure. The reaction solution was acidified with 1 mol / L dilute hydrochloric acid and extracted with ethyl acetate / water. The organic layers were combined, dried, and concentrated under reduced pressure. A small amount of ethyl acetate was added to dissolve the solution. The product precipitated after standing and was filtered to obtain series A compounds LYS1-4.

[0216]

[0217] Table 5A series compounds

[0218]

[0219] General synthetic route of B series compounds

[0220] Reagent and conditions: (IV) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90℃; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0221] Differently substituted p-aminobenzoic acids (3.6 mmol), pyrrolidine (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.6 mmol), and N,N-diisopropylethylamine (5.5 mmol) were dissolved in 6 mL of DMF and stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound c was isolated and purified by column chromatography.

[0222] Synthesis route II is the same as above, and compound d is synthesized.

[0223] Synthesis route III is the same as above, and compound LYS 5-9 is finally synthesized.

[0224]

[0225] Table 6

[0226]

[0227] General synthetic route of C series compounds

[0228]

[0229] Reagent and conditions: (V) PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90℃; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0230] 4-Amino-2-chlorobenzoic acid (3.6 mmol), various amines (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (3.6 mmol), and N,N-diisopropylethylamine (5.5 mmol) were dissolved in 6 mL of DMF and stirred at room temperature overnight. The reaction was monitored by TLC. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound e was isolated and purified by column chromatography.

[0231] Synthesis route II is the same as above, and compound f is synthesized.

[0232] Synthesis route III is the same as above, and compound LYS10-25 is finally synthesized.

[0233]

[0234] Table 7

[0235]

[0236]

[0237] General synthetic route of D series compounds

[0238]

[0239] Reagent and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (VI) PyBOP, DIPEA, DMF, 70℃; (III) LiOH·H2O, THF / H2O1:1, rt;

[0240] Synthesis route I is the same as above, and compound g is synthesized.

[0241] Compound g (2.6 mmol), (1R,2R)-2-(methoxycarbonyl)cyclohexanecarboxylic acid (2.94 mmol), benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate (2.45 mmol), and N,N-diisopropylethylamine (3.67 mmol) were dissolved in 8 mL of N,N-dimethylformamide and stirred at room temperature for 10 minutes. (4-amino-3-methylphenyl)(pyrrolidin-1-yl)methanone (2.45 mmol) was then added. The reaction temperature was increased to 70°C and monitored by TLC. The reaction was complete after approximately 4 hours. The reaction was then evaporated under reduced pressure and extracted with ethyl acetate / saturated brine. The organic layers were combined, dried, and concentrated under reduced pressure. Compound LYS26 (Y-9-2, i.e., 11) was isolated and purified by column chromatography to obtain a white powder in a 60% yield.

[0242] Synthesis route III was the same as above, and compound LYS27 (Y-9-3) was finally synthesized as a flesh-colored solid powder with a yield of 30%.

[0243] Example 1 Screening of small molecule drugs targeting the XIAP RING domain

[0244] The Site Finder module of MOE software (version 2022) was used to analyze the possible binding sites on the surface of human XIAP protein (PDBID: 4IC2, 4IC3 and 5O6T), and the binding pockets predicted by the co-crystal structure near amino acids 467 and 495 in 4IC3 were found: Site 1 (LEU444 GLU446 GLU447 LYS448 LEU449 LYS451 MET454ASN457ILE458 HIS467 LEU468 VAL469 MET483 ILE494 MET496) and Site 2: (LEU444GLU447 LYS448LEU468 LEU495 MET496 SER497). Among them, the PLB of Site1 is>1, so the Site1 site of the cocrystal structure 4IC3 was selected for virtual screening, and the XIAP protein sequence reference Uniprot ID: P98170. This application selected a TargetMol compound library containing 36,861 biologically active compounds and a Chemdiv compound library containing 1,583,358 structurally diverse compounds as screening databases, including a total of 1.62 million molecules. Affinity, druggability properties, structural diversity and binding specificity were used as screening indicators to select compounds that can directly interact with the XIAP RING domain (E3 ligase). Among them, the high-frequency interaction amino acid sites near amino acid 467 and amino acid 495 (key sites for E3 ligase activity) are Asn457, Ile458, Lys448, Met496 and Ser497 (see Figure 26-Figure 27 ), for example, amino acid Ile458 participated in the interaction patterns of 68 compounds. Ultimately, 65 compounds with relatively strong affinity scores, good druggability properties, and strong binding specificity (e.g., Compound 1 had an affinity score of -8.40 kcal / mol for XIAP E3 ligase) were screened for subsequent biological activity testing. When those skilled in the art need to use methods similar to those of the present application to screen for XIAP inhibitors from human XIAP with other amino acid sequences or from XIAP from organisms other than humans, they can screen for amino acid sites corresponding to the aforementioned human XIAP protein without inventive steps, for example, H467 of human XIAP corresponds to H466 of mouse XIAP.

[0245] Example 2 Functional validation of small molecule drugs targeting the XIAP RING domain

[0246] 1.1 In vitro assays to detect the antitumor activity of small molecule compounds

[0247] 1.1.1 Screening and identification of small molecule compounds

[0248] The small molecule compounds obtained from the multiple screenings were treated with bladder cancer cells and tested for anti-tumor activity using the CCK8 assay. In the initial screening experiment, the compounds were treated at a concentration of 1 μM for 72 hours. IC50 values ​​were then determined and further experiments were performed. The results are shown in Table 8.

[0249] Table 8

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] 1.1.2 Detection of targeting specificity and ability to inhibit cancer cell invasion of small molecule compounds

[0261] Reported direct substrate proteins of XIAP E3 ligase include p62, Bcl-2, PTEN and DNMT3B discovered in the team's previous studies. The compounds obtained in the above preliminary screening were applied to T24T cancer cells at a concentration verified to be safe and non-cytotoxic by CCk8 experiments for 24 hours, and the proteins were collected. By immunoblotting (Western blotting), the changes in DNMT3B and other reported direct substrate proteins of XIAP E3 ligase after the action of the compounds, as well as the changes in marker proteins related to cell invasion (such as MMP2, MMP9 and N-cadherin) were detected, and compounds with consistent upregulation of substrate proteins and consistent decrease in invasion-related protein indicators were screened out. We finally screened out lead small molecule compounds targeting the active site of XIAP RING domain E3 ubiquitin ligase with potential anti-tumor activity, including compound 1 and compound 2 (see Figure 1Western blot experiments in human bladder cancer T24T cells following treatment with candidate compounds examined protein changes in DNMT3B and other reported direct substrates of the XIAPE3 ligase, as well as changes in marker proteins associated with cell invasion. Compounds 1 and 2 consistently upregulated substrate protein expression and decreased invasion-related protein expression.

[0262] Given that cell invasion is a key characteristic of cancer progression, and cancer cell metastasis is a major lethal factor in bladder tumors, the search for small molecule compounds with significant inhibitory effects on tumor cell invasion and metastasis is a key focus of this research. The team previously successfully established a stable XIAP knockout cell line, T24T (KOXIAP), and a control cell line, T24T (vector). Furthermore, they constructed corresponding cell models, T24T (KOXIAP / XIAP) and T24T (KOXIAP / H467A), by transfecting the XIAP knockout cells with an HA-tagged XIAP plasmid and an HA-tagged XIAP RING H467 mutant plasmid. Transwell assays were then used to examine the effects of these compounds on the migration and invasion abilities of T24T (vector), T24T (KOXIAP), T24T (KOXIAP / XIAP), and T24T (KOXIAP / H467A) cells. 5% FBS medium containing the test compound was added to the lower chamber, and cells were diluted with 0.1% FBS medium containing the same concentration of compound to form a uniform single-cell suspension (20,000 cells / well) and added to the upper chamber for 24 hours. Medium containing the corresponding DMSO working concentration was used as a control. Figure 2 The results of the migration and invasion of T24T cells under the action of small molecule inhibitors are shown. (A&F) Photos of the migration and invasion abilities of T24T (vector), T24T (KOXIAP), T24T (KOXIAP / XIAP), and T24T (KOXIAP / H467A) cells under the action of DMSO and two small molecule inhibitors (1, 2) were detected by Transwell assay; (B&G) Histograms of the migration abilities corresponding to panels A and F; (C&H) Histograms of the invasion abilities corresponding to panels A and F; The histograms represent the mean ± standard deviation of the counts from five independent fields of view.

[0263] (D) Photographs showing the migration and invasion abilities of T24T cells in the presence of DMSO and small molecule inhibitor A at different concentrations as assessed by a Transwell assay. (E) Line graph of the corresponding migration and invasion abilities in Figure D. Asterisks (*) indicate statistically significant results compared with the control group (P < 0.05).

[0264] It can be seen that when compound 1 and compound 2 were treated at concentrations of 0.5μM and 2μM, respectively, the invasive ability of T24T (Vector) cells and T24T (KOXIAP / XIAP) cells was significantly reduced compared with the cells in the DMSO-treated group at the same dose, indicating that the compound can effectively inhibit cell invasion. At the same time, T24T (KOXIAP) cells and T24T (KOXIAP / H467A) cells did not show significant inhibition of cell invasion under the same grouping treatment. In addition, compound 1 was used in a concentration gradient (0.0625μM, 0.125μM, 0.25μM, 0.5μM) to act on T24T cells, showing that the cell invasion ability was concentration-dependent. The results suggest that the compound inhibits the invasive ability of bladder cancer cells by specifically targeting and inhibiting the E3 ubiquitin ligase site of XIAP.

[0265] 1.2 In vivo experiments to detect the anti-tumor effects and pharmacokinetic studies of small molecule compounds

[0266] 1.2.1 In vivo testing of antitumor activity of small molecule compounds

[0267] 1.2.1.1 Detection of the antitumor activity of small molecule compounds against primary basal-type muscle-invasive bladder cancer in mice

[0268] Wild-type mice aged 5 to 6 weeks were randomly divided into three groups of 10 mice each: a vehicle control group, a group receiving drinking water containing 0.05% BBN, and a group receiving drinking water containing 0.05% BBN plus the small molecule compound to be tested (100 mg / kg / day). Physiological parameters (such as body weight and respiratory rate) were monitored every three days after drug administration, and the mice were closely observed. After drug treatment, the mice were sacrificed, and the bladders were removed for observation of bladder changes, bladder weight measurement, and calculation of the bladder / mouse weight ratio. The bladders were then divided into two parts: one part was used for pathological staining analysis such as H&E and immunohistochemistry (IHC), and the other part was frozen for future RNA and protein analysis.

[0269] To evaluate the therapeutic effects of targeted small molecule compounds on primary basal-type muscle-invasive bladder cancer.

[0270] Experiment 1 involved 5-6-week-old wild-type C57 / B6 male mice randomly divided into three groups (n=5 per group): ① a control group receiving drinking water containing vehicle fluid, ② a group receiving drinking water containing 0.05% BBN, and ③ a group receiving drinking water containing 0.05% BBN plus a small molecule compound. Group ① received drinking water containing vehicle fluid until 25 weeks, group ② received drinking water containing 0.05% BBN until 25 weeks, and group ③, after 16 weeks of BBN exposure, received drinking water containing BBN plus the test small molecule compound (100 mg / kg / day) until 25 weeks. Both the BBN drinking water and the test small molecule compound were replaced every other day. After treatment, mice were sacrificed, and their bladders were removed. Bladder morphology and volume changes were observed, and bladder weight was measured. The bladder / mouse weight ratio was calculated. Figure 3 Figures show the inhibitory effect of candidate small molecule compound 1 on primary basal-type muscle-invasive bladder cancer in mice. (AD) Comparison of mouse bladder tissue morphology (A), tumor weight (B), tumor size to mouse weight ratio (C), and mouse weight (D) in the drinking water control group, the drinking water containing 0.05% BBN, and the drinking water containing 0.05% BBN plus small molecule compound 1 groups. (E) HE staining observed the inhibitory effect of small molecule compound 1 on the pathological changes of BMIBC induced by BBN exposure.

[0271] The results showed that 80% (4 / 5) of the mice in Group ② developed basal-type muscle-invasive bladder cancer. In Group ③, when treated with Compound 1 at a dose of 100 mg / kg / day, there were no significant differences in bladder morphology, volume, or bladder / mouse weight ratio compared to Group ①, and BBN-induced weight loss in mice was also alleviated. HE staining showed that BBN induced BMIBC pathological changes, while Compound 1 significantly alleviated BMIBC pathological damage after intervention. These results indicate that Compound 1 has a significant therapeutic effect on BBN-induced primary basal-type muscle-invasive bladder cancer in mice.

[0272] Experiment 2 specifically involved 5-6 week old wild-type C57 / B6 male mice randomly divided into three groups, each with 10 mice: ① a control group receiving drinking water containing vehicle, ② a group receiving drinking water containing 0.05% BBN, and ③ a group receiving drinking water containing 0.05% BBN plus drug. Group ① received drinking water containing vehicle for 25 weeks, group ② received drinking water containing 0.05% BBN for 25 weeks, and group ③, after 5 weeks of BBN exposure, received drinking water containing BBN plus compound 1 (Com1, 100 mg / kg / day) for 25 weeks. Both BBN drinking water and Com1 were changed every other day. Results are shown in the table. Figure 4 .

[0273] The results showed that compound 1 in group ③, at a dose of 100 mg / kg / day, significantly inhibited bladder morphology, volume, and bladder / mouse weight ratio in mice compared to group ①, suggesting that compound 1 significantly inhibited the development of BMIBC. These results demonstrate that compound 1 can effectively inhibit the development and progression of primary basal-type muscle-invasive bladder cancer in mice.

[0274] Experiment 3 involved 5-6 week-old wild-type C57 / B6 male mice randomly divided into three groups, each containing five mice: ① a control group receiving vehicle-containing drinking water, ② a group receiving drinking water containing 0.05% BBN, and ③ a group receiving 0.05% BBN drinking water plus drug. Group ① received vehicle-containing drinking water until 25 weeks, group ② received 0.05% BBN drinking water until 25 weeks, and group ③, after 17 weeks of BBN exposure, received BBN-containing drinking water plus Compound 2 (Com2 100 mg / kg / day) until 25 weeks. Both BBN drinking water and Com2 were replaced every other day.

[0275] The results showed (see Figure 6 ) demonstrated the efficacy of Compound 2 in a BBN-induced mouse model of primary basal muscle-invasive bladder cancer. In Group ③, at a dose of 100 mg / kg / day, Compound 2 reduced bladder morphology, volume, and bladder / mouse weight ratio compared to Group ②, demonstrating improvement in BBN-induced BMIBC in mice. These results demonstrate the therapeutic activity of Compound 2 against BBN-induced primary basal muscle-invasive bladder cancer in mice.

[0276] 1.2.1.2 Detection of the inhibitory effect of small molecule compounds on subcutaneous tumor formation of BMIBC cells in nude mice

[0277] 3-4 week old BALB / C-nu nude mice were selected. T24T bladder cancer cells were amplified, digested with trypsin, counted, and resuspended in PBS to adjust the cell density to 2×10 7 / ml. 100μl of cell suspension was drawn up using a 1ml sterile syringe and inoculated subcutaneously into nude mice, generally within half an hour. During the inoculation, the cell suspension was placed on ice to reduce cell metabolism and maintain cell activity. The mice were randomly divided into four groups (nine per group): ① a vehicle control group; ② a group receiving intraperitoneal injection of 10mg / kg compound 1; ③ a group receiving 40mg / kg compound 1 via drinking water; and ④ a group receiving intraperitoneal injection of 10mg / kg compound 1 after 5 days of subcutaneous injection of tumor cells, when significant tumor growth was observed. After 4 weeks of drug treatment, the mice were sacrificed and the changes in subcutaneous tumors were observed to evaluate the inhibitory effect of the small molecule compound on subcutaneous tumor formation of BMIBC cells in nude mice.

[0278] The results showed (see Figure 5): Compared with the control group, the weight and size of the subcutaneous tumors in nude mice treated with compound 1 were significantly reduced, indicating that compound 1 has a good inhibitory effect and therapeutic effect on the subcutaneous tumor formation of BMIBC cells in nude mice.

[0279] 1.2.1.3 Detecting the Antitumor Activity of Small Molecule Compounds in Mice Using a Human PDX Model

[0280] Fresh tumor tissue samples from bladder cancer patients are removed, the capsule and necrotic tissue removed, washed three times with sterile PBS, and cut into fragments approximately 2-3 mm in diameter on a sterile dish. A small incision is made in 4-8 week old nude mice to locate the kidney. Using forceps, the tumor tissue sample is inserted into the tip of a custom-made trocar and transplanted beneath the mouse's renal capsule. Three to four nude mice are inoculated with each tumor, and this inoculation must be completed within 2 hours of tumor removal. This model retains the microenvironment and basic cellular characteristics of the primary tumor, forming the first generation of xenografts (F1). Once the xenografts reach a certain size, the xenografts are removed and aseptically transplanted into a new batch of mice, forming the second generation of xenografts (F2). This procedure is repeated to form the third generation (F3), fourth generation (F4), and so on. F3 and higher generation mice can be used for drug therapy studies.

[0281] F4 generation xenografts were implanted into 40 BALB / C-nu nude mice to establish a bladder cancer PDX model for drug efficacy evaluation. After 18 days of normal maintenance, tumor formation was assessed by CT. Tumor-bearing mice were randomly divided into four groups: a vehicle control group and a group treated with the small molecule compound to be tested. Three dose groups were set: 50 mg / kg / day, 100 mg / kg / day, and 200 mg / kg / day. Drugs were administered by gavage for 30 days. Physiological parameters (such as body weight, heart rate, and respiratory rate) and biochemical parameters (such as blood glucose, lipid profile, and liver function) were monitored every three days after drug administration, and the mice were closely observed. After drug treatment, the mice were sacrificed, bladder changes were observed, bladder weights were measured, and the bladder / mouse weight ratio was calculated. The mice were then divided into two parts: one was used for pathological analysis using H&E and immunohistochemistry (IHC), and the other was frozen for future RNA and protein analysis. This was done to evaluate the therapeutic efficacy of the small molecule compound on bladder cancer PDX.

[0282] Example 3 Screening and identification of small molecule compounds with anti-tumor spectrum

[0283] Based on the fact that XIAP is overexpressed in a variety of epithelial tumors and may play an important biological function, we plan to use the screened small molecule inhibitors targeting human XIAP E3 ligase with anti-bladder cancer activity in different tumor cells to test whether they have the same anti-tumor activity against other tumors with high XIAP expression. Figure 7), XIAP expression in tumors such as breast cancer, colorectal cancer and lung adenocarcinoma is significantly higher than that in normal tissues. The corresponding tumor cells have been collected, and the active small molecule compounds that have been screened are intended to act on each tumor cell to detect their anti-tumor activity.

[0284] Experiment 1: Compound 1 obtained in the initial screening was subjected to a concentration gradient to act on AGS (human gastric adenocarcinoma cells), HGC-27 gastric cancer cells, U2-OS human osteosarcoma cells and MB-231 breast cancer cells for 24 h, and the protein was collected. Figure 8 Western blot analysis of candidate small molecule inhibitor 1 in different tumor cells was performed. Changes in proteins of reported direct substrates of the XIAP E3 ligase and markers of cell invasion were detected. (A & B) Compound 1 treatment of gastric cancer cells AGS and HGC-27 showed upregulation of substrate proteins and decreased invasion markers. (C) Compound 1 treatment of human osteosarcoma cells U2-OS showed upregulation of substrate proteins and decreased invasion markers. (D) Compound 1 treatment of breast cancer cells MB-231 showed upregulation of substrate proteins and decreased invasion markers.

[0285] Western blotting results showed that the expression of DNMT3B and other reported direct substrate proteins of XIAP E3 ligase increased after the action of the compound, and the expression of marker proteins related to cell invasion (such as MMP2 and MMP9) was significantly decreased.

[0286] Experiment 2: Prepare 1.25% agarose solution and BME cell culture medium (2X). Mix 42°C preheated culture medium with 1.25% agarose solution in a 3:2 ratio. Add the solution to a six-well plate (3 mL / well) as quickly as possible, taking care not to create bubbles when plating. Allow the solution to solidify at room temperature for approximately 2 hours. Count the prepared cells and add 8,000 cells per well to the prepared culture medium. Add 1.25% agarose solution proportionally to the cell suspension in the culture medium. Pipette thoroughly to mix thoroughly, then immediately add the solution to the corresponding lower gel layer. Take care not to create bubbles when plating. Allow the solution to solidify at room temperature for approximately 2 hours. Once solidified, seal the plate with parafilm and incubate in a 37°C cell culture incubator. Observe cell colony formation regularly during this period. After approximately 2-4 weeks, photograph and analyze the colony formation rate using a microscope.

[0287] The above results show that under the action of compound 1 and its modified compounds, the malignant proliferation ability of human melanoma cells (A375), lung cancer cells (HCC827) and prostate cancer cells (PC-3) was inhibited (see Figure 9 , Figure 10 ).

[0288] Example 4 Effects of small molecule compounds on cell metastasis and invasion

[0289] Referring to the method of Example 2, the effects of compounds 1, X-6, X-10, X-20, and Y-9-3 on the migration and invasion abilities of T24T cells were determined. The results are shown in FIG. Figure 11 The effects of compounds B6, B8, B9, B10, C1-4, C5-2-2, C1-5, Y-9-2, 940532-51-4, and 714278-58-7 on the migration and invasion abilities of T24T cells were determined. The results are shown in FIG. Figure 25 shown.

[0290] Example 5 The XIAP RING domain plays a crucial role in the development and progression of BBN-induced basal muscle-invasive bladder cancer (BMIBC)

[0291] BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine) is an environmental chemical carcinogen that can induce invasive bladder cancer in mice and is widely used in bladder cancer research. Adding 0.05%-0.1% (v / v) BBN to the drinking water of mice causes abnormal proliferation of the urothelium to develop invasive bladder cancer with squamous differentiation after approximately 15 weeks. This model closely resembles the development and histological changes of human muscle-invasive bladder cancer. Given that BBN can induce primary basal-type muscle-invasive bladder cancer in the majority of exposed mice, we applied this mouse model to investigate mice lacking the RING domain of XIAP (△RING knock-in). Our results revealed that the RING domain, rather than the BIR domain, mediates the development and progression of BBN-induced primary BMIBC. Figure 12 (A) Comparison of basal-type muscle-invasive bladder cancer in BBN-induced XIAP wild-type and RING domain-deficient mice; (B) 23 weeks after BBN induction, HE staining and pathological analysis of mouse bladder tissue confirmed that bladder tumors in the urothelium led to muscle-invasive bladder cancer, and the basal-type bladder cancer marker KRT5 was highly expressed; XIAPRING domain deletion significantly inhibited the occurrence of basal-type muscle-invasive bladder cancer; (C and D) In ​​the mouse bladder epithelium, the number of KRT14-expressing cells increased and thickened with increasing BBN induction time; (E) Comparison of KRT14 and KRT20 expression in the bladder urothelium in XIAP wild-type and RING domain-deficient mice after BBN induction for 23 weeks.

[0292] like Figure 12As shown in Figure A, wild-type XIAP and RING domain-deficient mice were given water containing 0.05% BBN for 23 weeks. 75% (15 / 20) of the wild-type XIAP mice developed bladder cancer, while the RING domain-deficient mice had only a 4.5% (1 / 22) bladder cancer incidence rate. Therefore, RING domain-deficient mice blocked the development of bladder cancer by 94%. HE staining and immunohistochemical staining with antibodies against KRT5 and KRT14 showed that BBN induced BMIBC, and the expression of the basal marker KRT14 gradually increased with increasing BBN induction time ( Figure 12 C and 12D); the expression of the luminal-like molecular marker KRT20 gradually decreased, and the deletion of the RING domain significantly weakened the expression of KRT14 ( Figure 12 E). The results showed that the deletion of the XIAPRING domain in bladder epithelial cells significantly inhibited the development of BBN-induced BMIBC in mice, suggesting that the RING domain plays a crucial role in the development of BBN-induced primary BMIBC.

[0293] Example 6: RING domain E3 ligase is a key site for XIAP to promote the development and invasion of BMIBCs in mice

[0294] Further studies found that the XIAP RING domain can specifically downregulate the expression of DNMT3B protein in BMIBC cells. Therefore, we explored the role of the XIAP RING domain in the degradation of DNMT3B. Figure 13, where (A) Western blot assay was used to detect the role of the XIAP RING domain in DNMT3B protein degradation; (B) Real-time fluorescence quantitative PCR assay was used to detect the expression level of DNMT3B in the indicated cells; (C and D) U5637 cells after XIAP knockout successfully reversed the RING domain (Myc-RING) and the RING domain containing a point mutation in the E3 ligase active site (H467A), respectively. The effect of DNMT3B protein degradation was detected by blot analysis; (E) The interaction between DNMT3B and the RING domain was detected by immunoprecipitation; (F) The ubiquitination level of DNMT3B was detected by immunoprecipitation; (G and H) The effects of XIAP knockout, including the reversed RING domain and the RING domain containing a point mutation in the E3 ligase active site, on the migration and invasion of U5637 cells were detected by Transwell assay; (I) The expression level of SNHG1 in the indicated cells was detected by real-time quantitative PCR; (J) Wild-type XIAP and H466A mutant XIAP mice (equivalent to the human H467A site) were treated with control or BBN for 16 weeks (n=10). The mouse bladders were harvested and the bladder tissues were pathologically analyzed by HE staining to calculate the incidence of basal-type muscle-invasive bladder cancer in mice; (K) The expression level of SNHG1 was detected by real-time quantitative PCR.

[0295] The results showed that compared with BMIBC U5637 (Vector), knockout of XIAP significantly inhibited the degradation rate of DNMT3B, while rotation of the XIAP RING domain completely restored the degradation rate of DNMT3B protein ( Figure 13 A). Compared with protein degradation, mRNA levels did not change significantly in U5637(Vector), U5637(KOXIAP / Vector) and U5637(KOXIAP / RING) cells ( Figure 13 B). This result indicates that the XIAP RING domain regulates DNMTB expression by mediating the degradation of DNMT3B protein. The RING domain has E3 ligase activity, so is DNMT3B protein degradation related to E3 ligase? To this end, a point mutation (H467A) was made in the E3 ligase active site to inactivate its E3 ligase activity. The RING domain and the wild-type RING domain were then re-transfected into XIAP knockout cells. The results showed that compared with the wild-type RING domain, the RING domain with the H467A point mutation lost the ability to mediate the degradation of DNMT3B protein ( Figure 13 CD), indicating that the E3 ligase activity of the XIAP RING domain plays an important role in the degradation of DNMT3B protein.

[0296] To determine whether the RING domain E3 ligase uses DNMT3B protein as a substrate and mediates the ubiquitination degradation of DNMT3B protein, we transfected U5637 (KOXIAP / myc-DNMT3B) cells with wild-type RING and its H467A mutant plasmids, lysed the transfected cells, and used co-immunoprecipitation to detect the interaction between DNMT3B and the RING domain. Co-immunoprecipitation experiments revealed that DNMT3B can bind to the RING domain, but the H467A mutant RING domain loses its ability to bind to DNMT3B ( Figure 13 E). Meanwhile, the ubiquitination level of DNMT3B was significantly increased in cells transfected with wild-type RING plasmid, while the ubiquitination level of DBMT3B was significantly decreased in cells transfected with H467A mutant RING ( Figure 13 F). This suggests that the RING domain E3 ligase of XIAP not only binds to DNMT3B protein, but also mediates the ubiquitination and degradation of DNMT3B protein. In terms of cellular function, cells transfected with the H467A mutant RING plasmid were unable to restore their invasive ability as with the wild-type RING plasmid ( Figure 13 GH); In U5637 (KOXIAP), reversion of the XIAP RING plasmid completely restored the expression of SNHG1, whereas reversion of the H467A mutant RING plasmid failed to restore the expression of SNHG1 ( Figure 13 I). In addition, in mice, the incidence of BMIBC induced by BBN was significantly reduced in bladder epithelial conditionally inducible transgenic mice with the H466A mutant (equivalent to the human H467A site) compared with wild-type XIAP mice (10% vs. 50%, as shown in Figure 1). Figure 13 J); BBN-induced SNHG1 expression in urothelial cells of XIAP H466A mutant mice was also significantly inhibited ( Figure 13 K).

[0297] These results suggest that the RING domain and its E3 ligase can use DNMT3B as a direct substrate, mediating its ubiquitination and degradation, thereby promoting SNHG1 expression, the development and progression of primary BMIBC, and cancer cell invasion. These findings fully demonstrate that the XIAP RING domain E3 ligase plays a crucial role in the development and progression of BMIBC and cancer cell invasion. It is known that XIAP is ubiquitously expressed in most adult and fetal tissues. XIAP expression is elevated in various tumors and positively correlates with tumor malignancy: expression is significantly higher in poorly differentiated tumors than in well-differentiated tumors, and significantly higher in metastatic tumors than in primary tumors. Furthermore, high XIAP expression is closely associated with poor clinical prognosis, including a strong correlation between XIAP protein levels and survival in patients with acute myeloid leukemia. Because the compounds and compositions of the present application inhibit X-linked inhibitor of apoptosis protein (XIAP), specifically by targeting the XIAP RING domain and altering E3 ligase activity, they exert key biological activities such as inhibiting cell invasion and metastasis. Therefore, XIAP antagonists can be used to treat all types of diseases mediated by XIAP, including but not limited to cancer, autoimmune diseases (such as systemic lupus erythematosus or rheumatoid arthritis) and other diseases involving XIAP mediation.

[0298] Example 7: Studying the Antitumor Activity of Small Molecule Compounds Using a Bladder Cancer Model

[0299] 1. Detection of the tumor inhibitory effect of the small molecule compound Com2 in mice using a subcutaneous tumor model

[0300] The antitumor activity of the small molecule compound Com2 was evaluated using a subcutaneous tumor model of human bladder cancer cells T24T in nude mice. 6 The mice were randomly divided into a control group (Vehicle) and a drug-treated group (Com2, 100 mg / kg / day), with 9 nude mice in each group. The mice were given the drug once a day via drinking water starting from the day after inoculation for 33 days, and the tumor volume was measured every 4 days starting from the 9th day. The results showed (see Figure 14 ), compared with the control group, Com2 showed a significant tumor growth inhibition effect, with a tumor inhibition rate of 94.9%.

[0301] 2. Lung metastasis model to detect the inhibitory activity of small molecule compound Com1 on tumor lung metastasis in mice

[0302] BALB / C-nu nude mice aged 3 to 4 weeks were selected. T24T bladder cancer cells were amplified, digested with trypsin, counted, and resuspended in PBS to adjust the cell density to 3×10 7 / ml. Use an insulin injection needle to draw 100μl (3×10 6 ) cell suspension was injected into nude mice through the tail vein. After 5 weeks of normal feeding, the mice were randomly divided into 2 groups, including a vehicle control group and a small molecule compound treatment group to be tested (Com1, 100 mg / kg / day), with 5 mice in each group. The drug was administered by drinking water for 8 weeks. After administration, physiological indicators (such as body weight, heart rate, respiratory rate, etc.) and biochemical indicators (such as blood sugar, blood lipids, liver function, etc.) were monitored every 3 days, and the status of the mice was closely observed. After the drug treatment, the mice were killed, and the lung tissues were fixed with picric acid for 24 hours, and the number of lung metastases was counted and photographed. The small molecule compounds were embedded in paraffin and HE stained. The activity of inhibiting tumor lung metastasis in mice was evaluated. The results are shown (see Figure 15 ), the Com1-treated group showed a significant decrease in lung metastases and a reduction in size. Preliminary observation of tissue morphology using HE staining revealed that the normal hierarchical structure of tissue cells was restored after drug treatment, indicating that Com1 significantly inhibits the formation of tumor lung metastases in mice.

[0303] 3. Detection of the target dependence of the small molecule compound Com1 in inhibiting bladder cancer growth in mice

[0304] To verify whether the antitumor activity of the small molecule compound Com1 in bladder cancer is mediated by its effect on XIAP E3 ligase activity, subcutaneous tumor formation experiments were performed in 3-4 week-old BALB / C-nu nude mice using human bladder cancer cell line T24T. T24T cells were expanded, trypsinized, counted, and resuspended in PBS to adjust the cell density to 2×10 7 / ml. Use a 1ml sterile injection needle to draw up 100μl of cell suspension and inoculate it subcutaneously into nude mice. Generally, it should be completed within half an hour. During the process, the cell suspension should be placed on ice to reduce cell metabolism and maintain cell activity. The mice were randomly divided into 4 groups (5 mice in each group), including ①T24T (KOXIAP / XIAP) Vehicle control group; ②T24T (KOXIAP / XIAP) drug-added group (10mg / kg / day); ③T24T (KOXIAP / H467A) Vehicle control group; ④T24T (KOXIAP / H467A) drug-added group (10mg / kg / day). The drugs were administered by intraperitoneal injection starting from the second day after tumor cell inoculation, once a day, for 30 days. The results showed (see Figure 16), the T24T (KOXIAP / XIAP)-treated group (10 mg / kg / day) significantly inhibited subcutaneous tumor growth compared to the control group, with a tumor inhibition rate of approximately 70%. T24T (KOXIAP / H467A) cells showed reduced subcutaneous tumorigenicity compared to T24T (KOXIAP / XIAP) cells, but no significant difference in tumor growth was observed in the T24T (KOXIAP / H467A)-treated group (10 mg / kg / day) compared to the control group. This suggests that Com1 inhibits bladder cancer growth in mice by specifically targeting and inhibiting the XIAP E3 ubiquitin ligase site.

[0305] Example 8: Studying the Antitumor Activity of Small Molecule Compounds Using a Gastric Cancer Model

[0306] The anti-tumor activity of the small molecule compound Com1 against gastric cancer was tested using a subcutaneous tumor model of human gastric adenocarcinoma AGS cells in mice. 16 BALB / C-nu nude mice were inoculated with 5×10 6 AGS cells were randomly divided into four groups: control group (Vehicle) and different concentrations of drug-treated groups (5 mg / kg / day, 20 mg / kg / day, 100 mg / kg / day), with 4 mice in each group. Drug administration began on the day after inoculation, once a day, intraperitoneally, for 24 days. The results showed (see Figure 17 ), compared with the control group, the drug-added groups (5 mg / kg / day, 20 mg / kg / day, 100 mg / kg / day) all had inhibitory effects, among which compound 1 at a dose of 20 mg / kg / day had an inhibitory effect of 90%, and at a dose of 100 mg / kg / day, human gastric adenocarcinoma AGS cells hardly grew in nude mice.

[0307] In addition, the subcutaneous tumor model of mouse gastric cancer MFC cells was used to further test the anti-tumor activity of the small molecule compound Com1 against gastric cancer in vivo. 20 C57BL6J mice were subcutaneously inoculated with 5×10 6 MFC cells were randomly divided into four groups: control group (Vehicle) and different concentrations of drug-treated groups (5 mg / kg / day, 10 mg / kg / day, 40 mg / kg / day), with 5 mice in each group. Drug administration began on the day after inoculation, once a day, intraperitoneally, for 22 days. The results showed (see Figure 18 ), compared with the control group, the drug-added groups (5 mg / kg / day, 10 mg / kg / day, 40 mg / kg / day) all had inhibitory effects, among which compound 1 at a dose of 10 mg / kg / day caused almost no growth of mouse gastric cancer MFC cells in the mice, showing a very significant tumor inhibitory effect.

[0308] Example 9: Studying the Antitumor Activity of Small Molecule Compounds Using a Pancreatic Cancer Model

[0309] The anti-tumor activity of the small molecule compound Com1 against pancreatic cancer was further tested by using a subcutaneous tumor model of pancreatic cancer mouse KPC cells. 24 C57BL / 6J mice were subcutaneously inoculated with 1×10 6 KPC cells were randomly divided into four groups: control group (Vehicle) and different concentrations of drug-treated groups (10 mg / kg / day, 40 mg / kg / day, 100 mg / kg / day), with 6 mice in each group. The drug was administered once a day by intraperitoneal injection on the day after inoculation for 24 days. The results showed (see Figure 19 ), compared with the control group, the drug-added group showed a certain tumor inhibition effect at high doses (40 mg / kg / day and 100 mg / kg / day), and the inhibition rate of the drug-added group (100 mg / kg / day) reached 90%, which indicates that compound 1 has the effect of inhibiting the growth of pancreatic cancer cells in mice.

[0310] Example 10: Studying the Anti-tumor Activity of Small Molecule Compounds Using a Melanoma Model

[0311] The anti-tumor activity of the small molecule compound Com1 against melanoma was tested using a subcutaneous tumor model of human melanoma A375 cells in nude mice. 6 A375 cells were then divided into a control group (Vehicle) and a drug-treated group (Com1, 50 mg / kg / day), with 6 mice in each group. The drug was administered via drinking water starting from the day after inoculation, once a day for 25 days. The results showed (see Figure 20 ), compared with the control group, the small molecule compound Com1 has a certain inhibitory effect on the growth of human melanoma A375 cells in nude mice, and the tumor inhibition rate reaches 40%.

[0312] Example 11: Studying the Antitumor Activity of Small Molecule Compounds Using a Renal Cancer Model

[0313] The anti-tumor activity of the small molecule compound X-10 against renal cancer was tested using a subcutaneous tumor model of mouse renal cancer RENCA cells. 25 male BALB / c mice aged 4-6 weeks were inoculated with 6×10 5 RENCA cells were randomly divided into a control group, a group injected with different concentrations of intraperitoneal injection (5 mg / kg / day, 20 mg / kg / day, 40 mg / kg / day), and a drinking water administration group (40 mg / kg / day), with 5 mice in each group. The drug was administered once a day starting from the day after inoculation for 18 consecutive days. The results showed (see Figure 21), compared with the control group, the drug-treated groups showed significant inhibitory effects on tumor growth. The tumor inhibition rate of the intraperitoneal injection group (5 mg / kg / day) reached more than 90%, while tumor cells did not grow in the intraperitoneal injection groups (20 mg / kg / day, 40 mg / kg / day) and the drinking water administration group (40 mg / kg / day).

[0314] Example 12: Studying the Antitumor Activity of Small Molecule Compounds Using a Lung Cancer Model

[0315] The antitumor activity of the small molecule compound X-10 against lung adenocarcinoma was tested by subcutaneous tumor formation experiment of human lung adenocarcinoma H1299 cells in nude mice. 6 H1299 cells were randomly divided into a control group and a drug-treated group (40 mg / kg / day), with 5 mice in each group. The drug was administered on the day after inoculation, once a day, by intraperitoneal injection, for 32 days. The results showed (see Figure 22 ), compared with the control group, the tumor inhibition rate of compound X-10 at a dose of 40 mg / kg / day reached 70%, indicating that X-10 also has significant anti-tumor activity against lung adenocarcinoma in mice.

[0316] Example 13: Studying the Antitumor Activity of Small Molecule Compounds Using a Colorectal Cancer Model

[0317] The anti-tumor activity of the small molecule compound X-10 against colorectal cancer was tested using a subcutaneous tumor model of MC38 cells in mice. Ten C57BL / 6J mice were subcutaneously inoculated with 1×10 6 MC38 cells were randomly divided into a control group (Vehicle) and a drug group (X-10, 40 mg / kg / day), with 5 mice in each group. The drug was administered via drinking water starting from the day after inoculation, once a day for 19 days. The results showed (see Figure 23 ), compared with the control group, the drug-treated group (40 mg / kg / day) showed a certain tumor inhibition effect, and its tumor inhibition rate reached 80%, which indicates that compound X-10 has the effect of inhibiting the growth of colorectal cancer cells in mice.

Claims

1. A method for screening XIAP inhibitors, comprising screening for inhibitors that bind to one or more amino acids corresponding to positions 440, 444, 446, 447, 448, 449, 451, 454, 457, 458, 467, 468, 469, 483, 494, 495, 496, or 497 of human XIAP, wherein the amino acid sequence of XIAP is referenced to Uniprot ID: P98170; preferably, the method comprises screening for inhibitors that bind to one or more amino acids corresponding to positions 458, 448, 457, 467, 495, 496, or 497 of human XIAP.

2. Use of a XIAP inhibitor in the preparation of a drug for treating tumors or autoimmune diseases, characterized in that: The inhibitor binds to one or more amino acids corresponding to positions 440, 444, 446, 447, 448, 449, 451, 454, 457, 458, 467, 468, 469, 483, 494, 495, 496 or 497 of human XIAP, the amino acid sequence of which is referenced to Uniprot ID: P98170; preferably, the XIAP inhibitor binds to one or more amino acids corresponding to positions 458, 448, 457, 467, 495, 496 or 497 of human XIAP.

3. The use according to claim 2, characterized in that The tumor or autoimmune disease is selected from the group consisting of carcinoma, sarcoma, Kaposi's sarcoma, erythroblastoma, malignant glioma, meningioma, astrocytoma, melanoma and myoblastoma; Brain cancer, skin cancer, adenocarcinoma, malignant epithelial tumors, urinary tract cancer, prostatic hyperplasia, prostate cancer, urothelial carcinoma, locally advanced or metastatic urothelial carcinoma, bladder urothelial carcinoma, bladder cancer (such as muscle-invasive bladder cancer, non-muscle-invasive bladder cancer, metastatic bladder cancer or advanced bladder cancer), ovarian cancer, breast hyperplasia, breast cancer, uterine cancer, pancreatic cancer, liver cancer, colon cancer, blood cancer, lung adenocarcinoma, lung cancer, small cell lung cancer, bone cancer, neuroblastoma, intestinal cancer such as colorectal cancer, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal thin-walled cell carcinoma, cervical cancer, cervical squamous cell carcinoma and adenocarcinoma, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, invasive breast cancer , urethral cancer, melanoma, brain tumor, glioma, astrocytoma, meningioma, medulloblastoma, peripheral neuroectodermal tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia), adult T-cell leukemia lymphoma, B-cell lymphoma, polycythemia vera, hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, head and neck cancer, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharynx tumor, thyroid tumor, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, plasmacytoma, systemic lupus erythematosus, or rheumatoid arthritis.

4. The use according to any one of claims 2 to 3, wherein the inhibitor is one or more compounds, or stereoisomers or tautomers thereof, or optical isomers or racemates thereof, or solvates thereof, or pharmaceutically acceptable salts, esters, amides, or prodrugs thereof, or metabolites thereof, or analogs or derivatives thereof, or crystalline compounds thereof, or nitrogen oxides thereof, or deuterated compounds thereof, or combinations thereof; the compounds are represented by Formula I: in: Ring A is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Y1 is -(CH2) m -C(=O)-NR a R b 、-(CH2) n -R6 or -(CH2) m -C(=O)-NH-C(=O)-R c ; Y2 is -NH-C(=O)-R5, -(CH2) n -R6, -X9-(CH2) t -R 4n or -X9-(CH2) t -C(=O)-NR a R b ; R c is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 aminoalkyl, C1-C6 cycloalkyl, C1-C6 halocycloalkyl, C1-C6 hydroxycycloalkyl, C1-C6 aminocycloalkyl or phenyl; R 4n Each is independently H, halogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b 、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R b or CN, the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl or phenyl is optionally substituted with one or more R9; R5 and R6 are each independently H, halogen, -OH, C1-C6 alkyl, C4-C 10 Cycloalkyl, C4-C6 cycloalkenyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl, the C1-C6 alkyl, C4-C 10 Cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl or phenyl is optionally substituted with one or more R7; R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b or CN, the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is optionally substituted with one or more R9; R8 is H or C1-C4 alkyl; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connected atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; R9 is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C (=O)NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl, wherein the C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl is independently optionally substituted by 0, 1 or 2 C1-C6 alkyl or C1-C6 haloalkyl; X9 is S or O; m, n and t are each independently 0, 1, 2 or 3.

5. The use according to claim 4, characterized in that The compound is one or more of the following formulas (II-A), (II-B), (II-C), (III-A), (III-B), (III-C), (IV-A), (IV-B), (IV-C) or (IV-D): In (Ⅱ-A), (Ⅱ-B), and (Ⅱ-C), R1, R 1a R5 is H or C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 hydroxyalkyl; Ring Ar is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Ring G is a C3-C7 cycloalkyl, C3-C7 cycloalkenyl, a 4-7 membered heterocyclyl, a 4-7 membered heteroaryl, a 4-7 membered aryl or a 5-9 membered bicyclyl; Ring G is optionally substituted with 0, 1 or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or C1-C4 alkoxy; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the atoms connected thereto, they form a 4-, 5-, 6- or 7-membered cyclic amine group, which is optionally substituted by one or more R9, and which optionally contains zero or one additional heteroatom selected from O, N and S; R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b 、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R b or CN, the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl is optionally substituted with one or more R9; In (III-A), (III-B), and (III-C), R a and R b are each independently H, C1-C4 alkyl, C1-C4 haloalkyl or C1-C4 hydroxyalkyl; or, R a and R b Together with the connecting atoms, a 4-, 5-, 6- or 7-membered cyclic amine group is formed, wherein the 4-, 5-, 6- or 7-membered cyclic amine group is optionally substituted by one or more R 4b substituted, the 4, 5, 6 or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; the 4, 5, 6 or 7-membered cyclic amine group is preferably azetidine, tetrahydropyrrole, piperidine, piperazine or azepane; R4, R 4a 、R 4b 、R 4c Each is independently H, halogen, -OH, -CN, -NO2, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or C1-C4 alkoxy; t and t1 are independently optionally 0, 1, 2, 3, 4 or 5; m is 0, 1, 2 or 3; X7 is N or CH; X9 is S or O; R 4m and R 4n Each is independently H, halogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b 、-S(=O)2R8、-S(=O)2NR a R b 、-S(=O)2NHC(=O)NR a R b or CN, the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10-membered heterocyclyl, 4 to 10-membered heteroaryl or phenyl is optionally substituted with one or more R9; R8 is H or C1-C4 alkyl; R a and R b Each is independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, or phenyl, and the C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylC0-C3 alkyl, C3-C7 cycloalkyl or phenyl is optionally substituted with one or more R9; or, R a and R b Together with the connecting atoms, they form a 4-, 5-, 6- or 7-membered cyclic amine group, which is optionally substituted by one or more R9, and the 4-, 5-, 6- or 7-membered cyclic amine group optionally contains zero or one additional heteroatom selected from O, N and S; R9 is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C6 cycloalkyl, 4-7 membered heterocyclyl or phenyl, which is independently optionally substituted by 0, 1 or 2 C1-C6 alkyl or C1-C6 haloalkyl; In (IV-A), (IV-B), (IV-C), and (IV-D), ring Ar is a 6-10-membered aryl ring or a 5-10-membered heteroaryl ring; ring P is a 4-10-membered cycloalkyl, a 4-10-membered azacycloalkyl, a 6-10-membered aryl ring, or a 5-10-membered heteroaryl ring; R 5a 、R 5b is H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl or phenyl; R 5c is H, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C3-C6 cycloalkyl, -(C=O)-R8, tetrahydropyrrolyl, piperidinyl, piperazinyl; the C3-C6 cycloalkyl, tetrahydropyrrolyl, piperidinyl, piperazinyl are optionally substituted with 0, 1 or 2 halogen, -OH, -NH2 or C1-C4 alkyl; R 5d is H, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl; R 5e is C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C3-C6 cycloalkyl, phenyl or C1-C4 alkylphenyl; the C3-C6 cycloalkyl, phenyl or C1-C4 alkylphenyl is optionally substituted with 0, 1 or 2 halogen, -OH, -NH2 or C1-C4 alkyl; X5a, X5b, X5c, X5d or X5e are each independently N or CH.

6. The use according to claim 5, characterized in that The compound is one or more of the following numbered 1-65:

7. The use according to any one of claims 2 to 6, characterized in that Additional therapeutic agents are further included.

8. The use according to any one of claims 2 to 7, characterized in that The compound, or its stereoisomer or tautomer, or its optical isomer or racemate, or its solvate, or its pharmaceutically acceptable salt, ester, amide, or prodrug, or its metabolite, or its analog, or its derivative, or its crystalline compound, or its nitrogen oxide, or its deuterated substance, or a combination of these substances, is used as a direct active ingredient or therapeutic agent in achieving the use; for example, as the sole active ingredient or the main active ingredient.

9. The use according to any one of claims 2 to 8, characterized in that The content of the XIAP inhibitor is a therapeutically effective amount.

10. The use according to any one of claims 2 to 9, characterized in that The drug may be administered systemically, topically, parenterally, invasively, non-invasively, or non-invasively.

11. Use of the C-terminus of XIAP, or the RING domain of XIAP, or a fragment of XIAP having E3 ubiquitin ligase activity, or an amino acid site of XIAP corresponding to the 467H site of human XIAP, or a binding site of the RING domain of XIAP that, upon binding, alters E3 ligase activity as a target in the preparation of a drug for treating tumors; the amino acid sequence of human XIAP is referenced in Uniprot ID: P98170;The tumors include lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, bronchioalveolar carcinoma), pleural mesothelioma, esophageal cancer (squamous cell carcinoma, adenocarcinoma, neuroendocrine carcinoma), gastric cancer (adenocarcinoma (intestinal type / diffuse type), signet ring cell carcinoma, gastric lymphoma (MALT lymphoma)), colorectal cancer (adenocarcinoma (mucinous adenocarcinoma / signet ring cell carcinoma), anal canal squamous cell carcinoma), hepatobiliary system (hepatocellular carcinoma (HCC) ), cholangiocarcinoma (CCC), hepatoblastoma, gallbladder cancer, ampullary cancer), pancreatic cancer (ductal adenocarcinoma, acinar cell carcinoma, pancreatoblastoma), renal cancer (clear cell carcinoma, papillary renal cell carcinoma, chromophobe cell carcinoma), gastric adenocarcinoma, bladder cancer (urothelial carcinoma (transitional cell carcinoma), squamous cell carcinoma, adenocarcinoma, muscle invasive bladder cancer, non-muscle invasive bladder cancer, primary bladder cancer, invasive bladder cancer, early bladder cancer, intermediate bladder cancer, metastatic bladder cancer or advanced bladder cancer), prostate cancer Adenocarcinoma (adenocarcinoma (ductal / acinar), neuroendocrine carcinoma), testicular cancer (seminoma, embryonal carcinoma, teratoma, choriocarcinoma), breast cancer, ovarian cancer, cervical cancer (squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma), endometrial cancer (endometrioid adenocarcinoma, serous carcinoma, clear cell carcinoma), leukemia (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)), myeloproliferative Tumors (polycythemia vera, primary myelofibrosis), lymphoma (Hodgkin lymphoma (nodular sclerosis type / mixed cell type, etc.), non-Hodgkin lymphoma (diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, etc.)), multiple myeloma (plasmacytoma), liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), glioma (glioblastoma (GBM), astrocytoma (WHO I-IV), oligodendroglioma, medulloblastoma, ependymoma, meningioma, neuroblastoma, schwannoma, malignant peripheral nerve sheath tumor (MPNST), teratoma, yolk sac tumor, embryonal carcinoma, choriocarcinoma, thyroid cancer (papillary, follicular, medullary, undifferentiated), adrenal cortical carcinoma, pheochromocytoma / paraganglioma, pituitary adenoma, melanoma, basal cell carcinoma, squamous cell carcinoma, Skin T-cell lymphoma (mycosis fungoides), osteosarcoma, chondrosarcoma, Ewing sarcoma, chordoma, pulmonary carcinoid, gastroenteropancreatic neuroendocrine tumors (GEP-NETs), small cell neuroendocrine carcinoma, metastatic carcinoma, brain metastasis, bone metastasis, liver metastasis, tumor of unknown primary site, metastatic poorly differentiated carcinoma, metastatic adenocarcinoma, retinoblastoma, Wilms tumor, hepatoblastoma, primitive neuroectodermal tumor (PNET).

12. The use according to claim 11, wherein the target site corresponds to one or more amino acids at positions 440, 444, 446, 447, 448, 449, 451, 454, 457, 458, 467, 468, 469, 483, 494, 495, 496 or 497 of human XIAP, the amino acid sequence of which is referenced to Uniprot ID: P98170; preferably, the target site corresponds to one or more amino acids at positions 467, 457, 458, 448, 495, 496 or 497 of human XIAP.

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