XIAP e3 ligase as target for tumor treatment and use of targeted compound thereof

Abstract

The present application relates to an XIAP E3 ligase as a target for tumor treatment and the use of a targeted compound thereof; and further relates to a drug containing one or more compounds, or a stereoisomer or tautomer thereof, or an optical isomer or racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt, ester or amide thereof, or a prodrug thereof, or a metabolite thereof, or an analog thereof, or a derivative thereof, or a crystal form of the compound thereof, or a nitrogen oxide thereof, or a deuterated compound thereof, or a combination of these substances.

Description

XIAP E3 ligase as a tumor treatment target and application of its targeting compounds TECHNICAL FIELD

[0001] The present application relates to the application of XIAP E3 ligase as a tumor treatment target and its targeting compounds, in particular to a drug for treating tumors, belonging to the technical field of biochemistry and medicine. BACKGROUND

[0002] X-linked inhibitor of apoptosis protein (XIAP) is one of the main members of the apoptosis inhibitor (IAP) family, which contains three N-terminal BIR repeat domains and a C-terminal RING domain. XIAP has very wide significance, and it is involved in cell survival, immune regulation, apoptosis and other physiological and pathological processes. XIAP gene is overexpressed in various tumor cells, such as 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 leukemia and other malignant tumors, and the expression level of XIAP is closely related to the invasion and metastasis ability of tumors. Therefore, XIAP is widely used as a target for drug research and development of various tumors or immune diseases. Early research on XIAP inhibitors mainly focused on the BIR domain of XIAP, and some apoptosis-inducing tumor treatment drugs targeting the BIR domain of XIAP were developed. However, since the BIR domain plays an important role in maintaining normal cell function, and these tumor treatment drugs also target the BIR domains of other IAPs (such as cIAP1 and cIAP2) while targeting the BIR domain of XIAP, resulting in greater side effects, therefore, other research methods are urgently needed. SUMMARY

[0003] The inventors have found that the binding sites in the C-terminal of XIAP, especially the RING domain, which can change the E3 ligase activity, are important targets for screening anti-tumor or autoimmune disease treating drugs. Based on these targets, a number of compounds with anti-tumor effects have been screened and further functionally verified (see Table 1, Table 2, Table 8). In the present application, anti-tumor includes 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, renal cancer, etc., but also inhibiting the metastasis (e.g., bladder cancer cell lung metastasis) and invasion of these tumors. Therefore, the present application first provides the use of the C-terminal of XIAP, the RING domain of XIAP, the fragment of XIAP with E3 ubiquitin ligase activity, the amino acid site of XIAP corresponding to the 467H site of human XIAP, or any binding site of the RING domain of XIAP which, after being bound, changes the E3 ligase activity as a target in the preparation of a drug for treating tumors. Examples of the target are one or more of the 440th, 444th, 446th, 447th, 448th, 449th, 451st, 454th, 457th, 458th, 467th, 468th, 469th, 483rd, 494th, 495th, 496th, or 497th amino acid of human XIAP, and all the compounds of the present application are obtained according to these targets. Secondly, the present application also provides the use of an inhibitor (e.g., a compound or a preparation) that inhibits the E3 ubiquitin ligase activity of XIAP in the preparation of a drug for treating tumors. These inhibitors are, for example, all the compounds of the present application, including general compounds, specific compounds, or one or more combinations thereof. In some embodiments, the inhibitors are substances that bind to the C-terminal of XIAP, or bind to the RING domain of XIAP, or bind to the amino acid site of XIAP corresponding to the 467H site of human XIAP, or bind to the RING domain of human XIAP and then inhibit 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 administration methods mentioned in the present application. In some embodiments, the tumor can be any tumor defined in the art, such as the specific types of tumors or cancers mentioned in the present application. In some embodiments, the drug is in any form defined in the art, such as the pharmaceutical composition mentioned in the present application, or the composition formed by all the compounds of the present application, including general compounds, specific compounds, or one or more combinations thereof, together with a pharmaceutically acceptable adjuvant, diluent, carrier, and / or excipient. Specific descriptions are as follows.

[0004] The present application first provides a general compound shown in formula (I), or a stereoisomer thereof, a tautomer thereof, or an optical isomer thereof or a racemate thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, or an analogue thereof or a derivative thereof, or a crystal type compound thereof, or a nitrogen oxide thereof, or a deuterium 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 -(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 independently is 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, said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl or phenyl being 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, said C1-C6 alkyl, C4-C 10Cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl optionally substituted with one or more R7; R7is H, halogen, C1-C4alkyl, C1-C6alkoxy, C3-C6cycloalkyl, 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, said C1-C4alkyl, C1-C6alkoxy, C3-C6cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl optionally substituted with one or more R9; R8is H or C1-C4alkyl; R a and R b are each independently H, C1-C6alkyl, C3-C7cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylCo-C3alkyl, or phenyl, said C1-C6alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylCo-C3alkyl, C3-C7cycloalkyl, or phenyl optionally substituted with one or more R9; or, R a and R b together with the atoms to which they are attached form a 4-, 5-, 6-, or 7-membered cyclic amine moiety, said 4-, 5-, 6-, or 7-membered cyclic amine moiety optionally substituted with one or more R9, said 4-, 5-, 6-, or 7-membered cyclic amine moiety optionally containing zero or one additional heteroatom selected from O, N, and S; R9is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl, said C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl independently optionally substituted with 0, 1, or 2 C1-C6alkyl or C1-C6haloalkyl; X9is S or O; m, n, and t are each independently 0, 1, 2, or 3.

[0006] In some embodiments, R7is H, halogen, C1-C4alkyl, C1-C6alkoxy, C3-C6cycloalkyl, 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 bor CN, said Ci-C4alkyl, Ci-C6alkoxy, C3-C6cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl being optionally substituted with one or more R9.

[0007] In some embodiments, ring A is wherein: R1, R2, R3, and R4are 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 , Ci-C6alkyl, Ci-C6alkoxy, C3-C6cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl; wherein said Ci-C6alkyl, Ci-C6alkoxy, C3-C6cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl is independently optionally substituted with one or more R7; X1, X2, X3, X4, X5, X6, and X7are each independently N or CH. Preferably, R1, R2, R3, and R4are 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 , Ci-C4alkyl, Ci-C4alkoxy, C3-C6cycloalkyl, or phenyl; wherein said Ci-C4alkyl, Ci-C4alkoxy, C3-C6cycloalkyl, or phenyl is independently optionally substituted with one or more R7. Preferably, R5and R6are each independently C4-C6cycloalkyl, C4-C6cycloalkenyl, 4- to 6-membered azacycloalkyl, bicyclo[2.2.2]octane, or phenyl; wherein said C4-C6cycloalkyl, C4-C6cycloalkenyl, 4- to 6-membered azacycloalkyl, bicyclo[2.2.2]octane, or phenyl is optionally substituted with one or more R7. Preferably, R7is H, halogen, Ci-C4alkyl, Ci-C4alkoxy, C3-C6cycloalkyl, 4- to 6-membered azacycloalkyl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, or CN; wherein said Ci-C4alkyl, Ci-C4alkoxy, C3-C6cycloalkyl, 4- to 6-membered azacycloalkyl, or phenyl is independently optionally substituted with one or more R9.

[0008] In some embodiments, the compound of the present application is a compound of the general formula of formula (II-A), (II-B), or (II-C):

[0009] wherein R1, R2, R3, and R1a each independently H, halogen, -OH, -CN, -NO2, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, or C1-C4 hydroxyalkoxy; 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 C3-C7 cycloalkyl, C3-C7 cycloalkenyl, 4-7 membered heterocyclyl, 4-7 membered heteroaryl, 4-7 membered aryl, or 5-9 membered bicyclic; said ring G is optionally substituted with 0, 1, or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy; R a and R b each independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclyl Co-C3 alkyl, or phenyl, said C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclyl Co-C3 alkyl, C3-C7 cycloalkyl, or phenyl is optionally substituted with one or more R9; or, R a and R b together with the atom to which they are attached form a 4, 5, 6, or 7 membered cyclic amine group, said 4, 5, 6, or 7 membered cyclic amine group is optionally substituted with one or more R9, said 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, said 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, 5-9 membered bicyclic; preferably cyclohexyl, cyclohexenyl, phenyl, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane; said ring G is optionally substituted with 0, 1, or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy. Preferably, R a is H, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl; R bH, C1-C4alkyl, C3-C7cycloalkyl, phenyl, said C1-C4alkyl, C3-C7cycloalkyl or phenyl is optionally substituted with 0, 1, 2 or 3 halo, -OH or C1-C4alkoxy; or R a and R b together with the atoms to which they are attached form an azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole, triazole, said azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole or triazole is optionally substituted with 0, 1, 2 or 3 halo, -OH or C1-C4alkoxy.

[0010] In some embodiments, the compounds of the present application are of the structure of the general compounds described by Formula (III-A), (III-B), (III-C):

[0011] wherein R a and R b are each independently H, C1-C4alkyl, C1-C4haloalkyl or C1-C4hydroxyalkyl; or, R a and R b together with the atoms to which they are attached form a 4-, 5-, 6- or 7-membered cyclic amine group, said 4-, 5-, 6- or 7-membered cyclic amine group optionally substituted with one or more R 4b , said 4-, 5-, 6- or 7-membered cyclic amine group optionally containing zero or one additional heteroatom selected from O, N and S; said 4-, 5-, 6- or 7-membered cyclic amine group preferably being azetidine, tetrahydropyrrole, piperidine, piperazine or azepane; R4, R 4a , R 4b , R 4c are each independently H, halo, -OH, -CN, -NO2, -NH2, C1-C4alkyl, C1-C4haloalkyl, C1-C4hydroxyalkyl or C1-C4alkoxy; t and t1are independently optionally 0, 1, 2, 3, 4 or 5; m is 0, 1, 2 or 3; X7is N or CH; X9is S or O; R 4m and R 4n are each independently H, halo, C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, 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 aR b or CN, said Ci-C6alkyl, Ci-C6alkoxy, C3-C6cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl optionally substituted with one or more R9; R8is H or Ci-C4alkyl; R a and R b are each independently H, Ci-C6alkyl, C3-C7cycloalkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylCo-C3alkyl, or phenyl, said Ci-C6alkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylCo-C3alkyl, C3-C7cycloalkyl, or phenyl optionally substituted with one or more R9; or, R a and R b together with the atoms to which they are attached form a 4-, 5-, 6-, or 7-membered cyclic amine group, said 4-, 5-, 6-, or 7-membered cyclic amine group optionally substituted with one or more R9, said 4-, 5-, 6-, or 7-membered cyclic amine group optionally containing zero or one additional heteroatom selected from O, N, and S; R9is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, Ci-C6alkyl, Ci-C6haloalkyl, Ci-C6alkoxy, Ci-C6haloalkoxy, C3-C6cycloalkyl, 4- to 7-membered heterocyclyl, or phenyl, said C3-C6cycloalkyl, 4- to 7-membered heterocyclyl, or phenyl independently optionally substituted with 0, 1, or 2 Ci-C6alkyl or Ci-C6haloalkyl.

[0012] In some embodiments, the compounds of the present application are of the structure of the general compounds described by Formulae (IV-A), (IV-B), (IV-C), (IV-D):

[0013] wherein ring Ar is a 6- to 10-membered aryl ring or a 5- to 10-membered heteroaryl ring; ring P is a 4- to 10-membered cycloalkyl, 4- to 10-membered azacycloalkyl, 6- to 10-membered aryl ring, or 5- to 10-membered heteroaryl ring; R 5a , R 5b is H, halogen, Ci-C4alkyl, Ci-C4haloalkyl, Ci-C4hydroxyalkyl, or phenyl; R 5c is H, halogen, Ci-C4alkyl, Ci-C4haloalkyl, Ci-C4hydroxyalkyl, C3-C6cycloalkyl, -(C=O)-R8, tetrahydropyrrolyl, piperidinyl, piperazinyl; said C3-C6cycloalkyl, tetrahydropyrrolyl, piperidinyl, piperazinyl optionally substituted with 0, 1, or 2 halogen, -OH, -NH2, or Ci-C4alkyl; R 5d is H, Ci-C4alkyl, Ci-C4haloalkyl, Ci-C4hydroxyalkyl; R 5eIt is a 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 halogens, -OH, -NH2, or C1-C4 alkyl; X5a, X5b, X5c, X5d, or X5e are each independently N or CH.

[0014] This application also provides compounds of formula (Iⅰ), or their stereoisomers, tautomers, optical isomers or their racemates, or their solvates, or their prodrugs, or their metabolites, or their analogs or derivatives, or their crystalline compounds, or their nitrides, or their deuterated derivatives, or combinations thereof:

[0015] 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, -(CH2) n -R6 or -X9-(CH2) t -C(=O)-NR a R b ;R c It is a 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 heterocyclic, 4- to 10-membered heteroaryl or phenyl, wherein the C4-C 10 Cycloalkyl, 3- to 10-membered heterocyclic, 4- to 10-membered heteroaryl, or phenyl groups are optionally substituted with one or more R7 groups; R7 is H, halogen, C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclic, 4- to 10-membered heteroaryl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NR a R b Or CN, wherein the C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclic, 4- to 10-membered heteroaryl, or phenyl is optionally substituted by one or more R9; R8 is H or C1-C4 alkyl; R a and R beach independently H, C1-C6alkyl, C3-C7cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylCo-C3alkyl, or phenyl, said C1-C6alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclylCo-C3alkyl, C3-C7cycloalkyl or phenyl optionally substituted with one or more R9; or, R a and R b together with the atoms to which they are attached form a 4, 5, 6, or 7 membered ring amine group, said 4, 5, 6, or 7 membered ring amine group optionally substituted with one or more R9, said 4, 5, 6, or 7 membered ring amine group optionally containing zero or one additional heteroatom selected from O, N, and S; R9is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl, said C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl independently optionally substituted with 0, 1, or 2 C1-C6alkyl or C1-C6haloalkyl; X9is S or O; m, n, and t are each independently 0, 1, 2, or 3.

[0016] In some embodiments, ring A is R1, R2, R3, and R4are 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-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl, or phenyl; wherein said C1-C6alkyl, C1-C6alkoxy, C3-C6cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl, or phenyl is independently optionally substituted with one or more R7; X1, X2, X3, X4, X5, X6, and X7are each independently N or CH. Preferably, R1, R2, R3, and R4are 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-C4alkyl, C1-C4alkoxy, C3-C6cycloalkyl, or phenyl; wherein said C1-C4alkyl, C1-C4alkoxy, C3-C6cycloalkyl, or phenyl is independently optionally substituted with one or more R7. Preferably, R5and R6are each independently C4-C6cycloalkyl, C4-C6cycloalkenyl, 4- to 6-membered nitrogen heterocycloalkyl, bicyclo[2.2.2]octane, or phenyl; wherein said C4-C6cycloalkyl, C4-C6cycloalkenyl, 4- to 6-membered nitrogen heterocycloalkyl, bicyclo[2.2.2]octane, or phenyl is optionally substituted with one or more R7. R7is H, halogen, C1-C4alkyl, C1-C4alkoxy, C3-C6cycloalkyl, 4- to 6-membered nitrogen heterocycloalkyl, phenyl, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, or CN; wherein said C1-C4alkyl, C1-C4alkoxy, C3-C6cycloalkyl, 4- to 6-membered nitrogen heterocycloalkyl, or phenyl is independently optionally substituted with one or more R9.

[0017] In some embodiments, the compound is of the structure described by Formula (II-Ai): wherein R1, R 1a each independently is H, halogen, -OH, -CN, -NO2, -NH2, C1-C4alkyl, C1-C4haloalkyl, C1-C4hydroxyalkyl, C1-C4alkoxy, C1-C4haloalkoxy, or C1-C4hydroxyalkoxy; R5is H or C1-C4alkyl, C1-C4haloalkyl, or C1-C4hydroxyalkyl; ring Ar is a 6- to 10-membered aryl ring or a 5- to 10-membered heteroaryl ring; ring G is C3-C7cycloalkyl, C3-C7cycloalkenyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heteroaryl, 4- to 7-membered aryl, or 5- to 9-membered bicyclyl; said ring G is optionally substituted with 0, 1, or 2 halogen, -OH, C1-C4alkyl, C1-C4haloalkyl, C1-C4hydroxyalkyl, C1-C4alkoxy; R a and R b each independently is H, C1-C6alkyl, C3-C7cycloalkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylC0-C3alkyl, or phenyl, said C1-C6alkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylC0-C3alkyl, C3-C7cycloalkyl, or phenyl is optionally substituted with one or more R9; or, R a and R b together with the atom to which they are attached form a 4-, 5-, 6-, or 7-membered cyclic amine group, said 4-, 5-, 6-, or 7-membered cyclic amine group is optionally substituted with one or more R9, said 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 is of the structure described by Formula (II-Bi):

[0018] In some embodiments, the compound is a structure according to Formula (III-Ai): wherein 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 atoms to which they are attached form a 4-, 5-, 6-, or 7- membered cyclic amine group optionally substituted with one or more R 4b , said 4-, 5-, 6-, or 7- membered cyclic amine group optionally containing zero or one additional heteroatom selected from O, N, and S; said 4-, 5-, 6-, or 7- membered cyclic amine group preferably being azetidine, pyrrolidine, piperidine, piperazine, or azepane; R4, R 4a , R 4b are each 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 is a structure according to Formula (III-Bi):

[0019] In some embodiments, the compound is a structure according to 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, 4-10 membered azacycloalkyl, 6-10 membered aryl ring, or 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, pyrrolidinyl, piperidinyl, piperazinyl; said C3-C6 cycloalkyl, pyrrolidinyl, piperidinyl, piperazinyl 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; said C3-C6 cycloalkyl, phenyl, or C1-C4 alkylphenyl optionally substituted with 0, 1, or 2 halogen, -OH, -NH2, or C1-C4 alkyl. Preferably, the compound is a structure according to Formula (IV-Bi), (IV-Ci), or (IV-Di): wherein X 5a , X 5b , X 5c , X 5d or X 5e each independently is N or CH.

[0020] In some embodiments, wherein ring G is C4-C6cycloalkyl, C4-C6cycloalkenyl, phenyl, 5-9 membered bicyclic; preferably cyclohexyl, cyclohexenyl, phenyl, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane; said ring G is optionally substituted with 0, 1, or 2 halogen, -OH, C1-C4alkyl, C1-C4haloalkyl, C1-C4hydroxyalkyl, C1-C4alkoxy.

[0021] In some embodiments, wherein R a is H, C1-C4alkyl, C1-C4haloalkyl, C1-C4hydroxyalkyl; R b is H, C1-C4alkyl, C3-C7cycloalkyl, phenyl, said C1-C4alkyl, C3-C7cycloalkyl, or phenyl is optionally substituted with 0, 1, 2, or 3 halogen, -OH, or C1-C4alkoxy; or R a and R b together with the atoms to which they are attached form an azetidine, tetrahydrofuran, piperidine, azepane, piperazine, morpholine, pyridine, pyridazine, pyrazine, pyrimidine, pyrazole, triazole, said 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-C4alkoxy.

[0022] In some embodiments, the specific compounds of the present application have at least one of the following structures:

[0023] Table 1 Correspondence of general formula and compounds 1-65

[0024] In another aspect, the present application provides a composition, characterized in that, comprising 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 its derivative, or its crystal type compound, or its nitroxide, or its deuterium compound, or a combination of these substances; optionally, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, and / or excipient.

[0025] The aforementioned composition, further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent.

[0026] In another aspect, the present application provides use of the aforementioned compound, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, or the aforementioned composition in the preparation of a medicament for preventing and / or treating a XIAP-mediated disease and / or disorder.

[0027] In some embodiments, the disease and / or disorder is all types of diseases that cannot undergo apoptosis, such as tumors, cancers or immune diseases.

[0028] In some embodiments, the aforementioned compound, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, or the aforementioned composition is used as an active ingredient or a therapeutic agent, for example, as the only active ingredient or the main active ingredient, in achieving the aforementioned use. In some embodiments, “as an active ingredient or a therapeutic agent” means that the content thereof reaches a therapeutically effective amount; or “as the only active ingredient or the main active ingredient” means that the content thereof accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100% of all active ingredients, the percentages being mass ratios or molar ratios; or “as the only active ingredient or the main active ingredient” means that the contribution of the drug to the treatment of the disease is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100%.

[0029] In some embodiments, the aforementioned compound, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, or the aforementioned composition is formulated or designed into a form of continuous administration, or a form of simultaneous administration, or a form of sequential administration, or a form of alternating administration, or a form of interval administration, or a form of separate administration. In some embodiments, the administration is performed by systemic administration, or local administration, or parenteral administration (such as by mucosal administration, transdermal administration, microneedle administration), or non-invasive administration, or non-invasive administration.

[0030] In some embodiments, the concentration or proportion of the foregoing compound, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, in the foregoing composition is at least not less than 0.001%, preferably 0.01% to 25%, preferably 0.05% to 5%, more preferably 0.15%; or the concentration or proportion of the foregoing compound, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances is less than 0.001%, the percentage being expressed as a mass / volume concentration (ratio) or mass ratio or molar (number) ratio.

[0031] In some embodiments, the compound of the present application is prepared in the form of an injection solution, a tablet, a lyophilized powder, a capsule, an effervescent tablet, a chewable tablet, a buccal tablet, a granule, an ointment, a syrup, an aerosol, a nasal drop, a topical preparation, an ophthalmic preparation, an oral preparation, an oil-water mixture, a suspension, a liniment, a lotion, a cream, a drop, a bolus, a spray, a paste, a patch, a paste, a pill, a suppository, or an emulsion, a health product, a food, a dietary supplement, a nutritional product, or a beverage. The solid dosage form can generally contain 1% to 95% (w / w) of the active compound. In some embodiments, the active compound is in the range of 5% to 70% (w / w).

[0032] In some embodiments, the patient with a disease is a human, such as an infant, a child, an adolescent, a middle-aged person, or an elderly person.

[0033] In some embodiments, the XIAP-mediated disease and / or disorder is a tumor or an autoimmune disease, including but not limited to, carcinoma, sarcoma, Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma; brain cancer, skin cancer, adenocarcinoma, carcinoma, urological tumor, prostate cancer, urothelial cancer, locally advanced or metastatic urothelial cancer, bladder urothelial cancer, 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 tract cancer such as colorectal cancer, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal cell carcinoma, cervical cancer, cervical squamous and adenocarcinoma, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, breast infiltrating carcinoma, urethral cancer, melanoma, brain tumor, glioma, astrocytoma, meningioma, medulloblastoma, peripheral neuroectodermal tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, leukemia, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), adult T-cell leukemia lymphoma, B-cell lymphoma, polycythemia vera, hepatocellular carcinoma, gallbladder cancer, bronchopulmonary cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, head and neck tumor, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, thyroid tumor, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, plasmacytoma, systemic lupus erythematosus or rheumatoid arthritis.

[0034] 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 lung cancer, lung adenocarcinoma, lung squamous carcinoma, large cell lung cancer, bronchioloalveolar carcinoma), pleural mesothelioma, esophageal cancer (squamous cell carcinoma, adenocarcinoma, neuroendocrine carcinoma), gastric cancer (adenocarcinoma (intestinal / diffuse), signet ring cell carcinoma, gastric lymphoma (MALT lymphoma)), colorectal cancer (adenocarcinoma (mucinous adenocarcinoma / signet ring cell carcinoma), anal canal squamous carcinoma), hepatobiliary system (hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), hepatoblastoma, gallbladder cancer, ampullary cancer), pancreatic cancer (ductal adenocarcinoma, acinar cell carcinoma, pancreatic blastoma), renal cancer (clear cell carcinoma, papillary renal cell carcinoma, chromophobe 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-stage bladder cancer, intermediate-stage bladder cancer, metastatic bladder cancer, or advanced bladder cancer), prostate cancer (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 neoplasm (polycythemia vera, primary myelofibrosis), lymphoma (Hodgkin lymphoma (nodular sclerosis / hybrid 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 (schwannoma), malignant peripheral nerve sheath tumor (MPNST), teratoma, yolk sac tumor, embryonal carcinoma, choriocarcinoma, thyroid cancer (papillary carcinoma, follicular carcinoma, medullary carcinoma, undifferentiated carcinoma), 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, gastrointestinal pancreatic neuroendocrine tumor (GEP-NETs), small cell neuroendocrine carcinoma, metastatic carcinoma, brain metastatic carcinoma, bone metastatic carcinoma, liver metastatic carcinoma, primary tumor unknown, metastatic poorly differentiated carcinoma, metastatic adenocarcinoma, retinoblastoma, nephroblastoma (Wilms tumor), hepatoblastoma, primitive neuroectodermal tumor (PNET).

[0035] The present application also provides the use of the aforementioned compound, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, or the aforementioned composition in the preparation of a medicament or a preparation for binding to XIAP. In some embodiments, binding to XIAP is binding to the C-terminal of XIAP, or binding to the RING domain of XIAP, or binding to the amino acid site of XIAP corresponding to the 467H site of human XIAP (XIAP protein sequence as Uniprot ID: P98170), or binding to the RING domain of human XIAP and then changing (such as inhibiting) the E3 ligase activity.

[0036] The present application further provides the use of the C-terminal of XIAP, or the RING domain of XIAP, or the fragment of XIAP with E3 ubiquitin ligase activity, or the amino acid site of XIAP corresponding to the 467H site of human XIAP, or the binding site in the RING domain of XIAP which is bound and then changes the E3 ligase activity as a tumor treatment target.

[0037] The present application further provides the use of the C-terminal of XIAP, or the RING domain of XIAP, or the fragment of XIAP with E3 ubiquitin ligase activity, or the amino acid site of XIAP corresponding to the 467H site of human XIAP, or the binding site in the RING domain of XIAP which is bound and then changes the E3 ligase activity as a target in the preparation of a medicament for treating tumors.

[0038] The present application further provides the use of a compound or a preparation for inhibiting the E3 ubiquitin ligase activity of XIAP in the preparation of a medicament for treating tumors. In some embodiments, the compound or the preparation for inhibiting the E3 ubiquitin ligase activity of XIAP is a substance that binds to the C-terminal of XIAP, or binds to the RING domain of XIAP, or binds to the amino acid site of XIAP corresponding to the 467H site of human XIAP, or binds to the RING domain of human XIAP and then changes (such as inhibits) the E3 ligase activity. In some preferred embodiments, the compound or the preparation for inhibiting the E3 ubiquitin ligase activity of XIAP is a compound of the present application, or a stereoisomer thereof, a tautomer thereof, or 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 crystal form compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, or a composition of the present application.

[0039] In some embodiments, the tumor or tumor treatment drug is capable of inhibiting the occurrence or development of a tumor by inhibiting the E3 ubiquitin ligase activity of XIAP. In some embodiments, the inhibition of the E3 ubiquitin ligase activity of XIAP can be achieved by any known technique in the art, such as mutating the active site of the key enzyme, knocking out the key fragment, binding small molecule compounds or binding macromolecular substances, etc. In one embodiment, the above embodiments can be implemented alone or in combination with other embodiments or embodiments disclosed herein. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Western blot experiment of candidate compounds on human bladder cancer T24T cells.

[0041] Figure 2 Results of detecting the migration and invasion ability of T24T cells under the action of candidate compounds.

[0042] Figure 3 Results of compound 1 on primary basal muscle-infiltrating bladder cancer in mice.

[0043] Figure 4 Results of compound 1 administration on BBN-induced primary basal muscle-infiltrating bladder cancer in mice. Wherein: (A) After the treatment, the mice were sacrificed and the bladder was removed; (B) Weight statistics of the bladders of mice in each group; (C) Bladder / mouse weight ratio of mice in each group; (D) Body weight change of mice during the treatment period.

[0044] Figure 5 Inhibitory effect of compound 1 on subcutaneous tumor formation of human BMIBC cells in nude mice. Wherein: (A) Tumor bodies removed after the nude mice were sacrificed after 33 days. (B) Weight of subcutaneous tumor formed in nude mice and statistical analysis. (C) Long diameter and short diameter of the tumor were measured every 4 days, and the tumor volume was calculated by the formula V = π / 6 × (long diameter) × (short diameter)2for statistical analysis.

[0045] Figure 6 Results of compound 2 on primary basal muscle-infiltrating bladder cancer in mice.

[0046] Figure 7 Diagram of gene expression difference analysis of XIAP in various tumor tissues.

[0047] Figure 8 Western blot experiment of compound 1 on different tumor cells.

[0048] Figure 9 Compound 1 and its modified compounds inhibit the malignant proliferation of human melanoma cells. Wherein: (A) Soft agar colony formation experiment of A375 cells treated with different compounds to detect the malignant proliferation of cells. (B) Corresponding colony formation number and data analysis results.

[0049] Figure 10. Compound 1 inhibits the malignant proliferation of human lung cancer cells and prostate cancer cells. Among them: (A-B) soft agar colony formation experiment of human prostate cancer PC-3 cells treated with compound 1 and corresponding colony formation number and data analysis results. (C-D) soft agar colony formation experiment of human lung adenocarcinoma HCC827 cells treated with compound 1 and corresponding colony formation number and data analysis results.

[0050] Figure 11. The invasion and metastasis ability of human bladder cancer T24T cells is inhibited under the action of compound 1 and its modified and analog compounds. Among them: (A)-(D) are the structural formulas of the compounds, (E)-(H) are the Transwell experiments of T24T cells treated with different compounds, detecting the changes of cell migration and invasion ability, and comparing with the effect of No. 1 compound treatment. (I)-(L) are the corresponding statistical results of the Transwell experiment.

[0051] Figure 12. XIAP RING domain plays an important role in BBN-induced basal muscle-infiltrating bladder cancer.

[0052] Figure 13. XIAP RING domain takes DNMT3B protein as substrate and plays an important role in basal muscle-infiltrating bladder cancer.

[0053] Figure 14. Results of the inhibitory effect of compound 2 (Com2) on T24T bladder cancer subcutaneous transplanted tumor. Among them, (A) time axis of nude mouse subcutaneous tumor experiment; (B) comparison of tumor size and shape between control group and drug treatment group; (C) dynamic change curve of tumor volume of control group and drug treatment group mice; (D) the subcutaneous tumor of nude mice was weighed and statistically analyzed.

[0054] Figure 15. Results of the inhibitory effect of compound 1 (Com1) on lung metastasis of human bladder cancer T24T cells. Among them, (A) the lung tissue was fixed after being saturated with picric acid solution, and the lung metastasis was photographed; (B) the tissue was paraffin-embedded and sectioned for HE staining; (C) the number of metastatic foci on the lung tissue of nude mice was counted.

[0055] Figure 16. The results of the effect of compound 1 (Coml) on human bladder cancer T24T cell target dependence. Among them, (A) T24T (KOXIAP / XIAP) Vehicle control group, T24T (KOXIAP / XIAP) drug group (10 mg / kg / day), T24T (KOXIAP / H467A) Vehicle control group, T24T (KOXIAP / H467A) drug group (10 mg / kg / day), the subcutaneous tumors of the four groups of nude mice were taken after the nude mice were sacrificed, and the tumor size and morphology were compared; (B) The subcutaneous tumors of the nude mice were weighed and statistically analyzed. (C) The tumor volume size change curve of the mice; (D) According to the tumor volume size, the tumor inhibition percentage of the drug group was calculated.

[0056] Figure 17. The results of the inhibitory effect of different concentrations of compound 1 (Coml) on human gastric cancer AGS cell subcutaneous tumors. Among them, (A) The photos of the control group and the drug group (5 mg / kg / day, 20 mg / kg / day, 100 mg / kg / day) mice on the 24th day; (B, C) Tumor volume and tumor growth curve of the control group and the drug group; (D) Tumor weight of the control group and the drug group; (E) According to the tumor volume size, the tumor inhibition percentage of the drug group was calculated; (F) The body weight change curve of the control group and the drug group mice; (G) The tumor photos of the control group and the drug group mice.

[0057] Figure 18. The results of the inhibitory effect of different concentrations of compound 1 (Coml) on mouse gastric cancer MFC cell subcutaneous tumors. Among them, (A) The photos of the control group and the drug group (5 mg / kg / day, 10 mg / kg / day, 40 mg / kg / day) mice on the 24th day; (B, C) Tumor volume and tumor growth curve of the control group and the drug group; (D) Tumor weight of the control group and the drug group; (E) According to the tumor volume size, the tumor inhibition percentage of the drug group was calculated; (F) The body weight change curve of the control group and the drug group mice; (G) The tumor photos of the control group and the drug group mice.

[0058] Figure 19. The results of the inhibitory effect of different concentrations of compound 1 (Coml) on mouse pancreatic cancer KPC cell subcutaneous tumors. Among them, (A) The tumor size and morphology comparison after the action of the control group and the drug group (10 mg / kg / day, 40 mg / kg / day, 100 mg / kg / day, intraperitoneal injection); (B, C) The tumor size and volume quantitative comparison of the control group and each drug group; (D) According to the tumor volume size, the tumor inhibition percentage of the drug group was calculated; (E) The tumor volume size change curve of the control group and the drug group mice; (F) The body weight change curve of the control group and the drug group mice.

[0059] Figure 20. The results of the inhibitory effect of compound 1 (Coml) on human melanoma A375 cell subcutaneous tumor. Among them, (A) the photos of the control group and the drug administration group (Coml, 50 mg / kg / day) mice on the 25th day; (B) the tumor size and morphology comparison of the control group and the drug administration group; (C, D) the tumor size and volume quantitative statistics of the control group and the drug administration group after treatment; (E) according to the tumor size, the tumor inhibition percentage of the drug administration group was calculated; (F) the tumor size change curve of the control group and the drug administration group mice.

[0060] Figure 21. The results of the inhibitory effect of compound 9 (X-10) of different concentrations on mouse renal cell carcinoma RENCA cell subcutaneous tumor. Among them, (A) the tumor size and morphology comparison of the control group, different concentration intraperitoneal injection groups (5 mg / kg / day, 20 mg / kg / day, 40 mg / kg / day) and drinking water administration group (40 mg / kg / day) after treatment; (B, C) the tumor weight and volume comparison of each group after treatment; (D) the results of calculating the tumor inhibition percentage of each group after treatment according to the tumor weight; (E) the tumor size change curve of each group; (F) the body weight change curve of each group of mice.

[0061] Figure 22. The results of the inhibitory effect of compound 9 (X-10) on human lung adenocarcinoma H1299 cell subcutaneous tumor. Among them, (A) the photos of the control group and the drug administration group (intraperitoneal injection, 40 mg / kg / day) mice on the 32nd day; (C) the tumor size and morphology comparison of the control group and the drug administration group; (C, D) the tumor size and volume quantitative statistics of the control group and the drug administration group after treatment; (E) the tumor size change curve of the control group and the drug administration group mice; (F) according to the tumor size, the tumor inhibition percentage of the drug administration group was calculated.

[0062] Figure 23. The results of the inhibitory effect of compound 9 (X-10) on mouse colorectal cancer MC38 cell subcutaneous tumor. Among them, (A) the control group and the drug administration group (X-10, 40 mg / kg / day); (B) the tumor size and morphology comparison of the control group and the drug administration group after treatment; (C, E) the tumor size and volume quantitative statistics of the control group and the drug administration group after treatment; (E) the tumor size change curve of the control group and the drug administration group mice; (F) according to the tumor size, the tumor inhibition percentage of the drug administration group was calculated.

[0063] Figure 24. The nuclear magnetic resonance hydrogen spectrum of compound 3 (T4489).

[0064] Figure 25. The invasion and metastasis abilities of human bladder cancer cells T24T are inhibited under the action of compound 1 and its modified analogs. (A)-(D) are the structural formulas of the compounds, (E)-(H) are the Transwell experiments of T24T cells treated with different compounds to detect the changes of cell migration and invasion abilities, and the comparison with the effect of compound 1 treatment. (I)-(L) are the statistical results of the corresponding Transwell experiments.

[0065] Figure 26. Amino acid sites involved in binding to small molecule compounds of TargetMol database and the frequency of binding to small molecule compounds.

[0066] Figure 27. Amino acid sites involved in binding to small molecule compounds of Chemdiv database and the frequency of binding to small molecule compounds. DETAILED DESCRIPTION

[0067] Embodiments of the present application are described below with reference to the accompanying drawings. The elements and features described in one drawing or embodiment of the present application can be combined with the elements and features shown in one or more other drawings or embodiments. It should be noted that, for the purpose of clarity, the representations and descriptions irrelevant to the present application, which are known to those skilled in the art, are omitted from the drawings and the description. The present application is further described below in conjunction with the drawings.

[0068] Table 2 represents the functional verification of the compounds

[0069] Terminology. The abbreviations used herein have their conventional meanings in the chemical and biological arts. The chemical structures and formulas set forth herein are constructed in accordance with standard rules of chemical valence known in the art of chemistry, and for terms not specifically defined herein, the meaning to be given such terms is to be found in the disclosure and context in which they are used.

[0070] A "XIAP inhibitor" or "XIAP antagonist" refers to an agent that targets the inhibition of the RING structure or E3 ligase biological function of XIAP protein, thereby exerting an anti-tumor effect. In some embodiments, the inhibitors are one or more selected from the group consisting of a nucleic acid molecule, a small molecule, an antibody drug, a polypeptide, a protein, a nucleic acid construct, an interfering lentivirus, an interfering adeno-associated virus, and a gene editing system. In some embodiments, the inhibitors are a drug, a compound, a composition, or a formulation, such as a compound or a composition (e.g., a pharmaceutical composition) of the present application. Thus, 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. Still further, the compounds and related compositions of the present application can bind to the 467H site of human XIAP, or an amino acid site of XIAP of another species that corresponds to the 467H site of human XIAP, such as the 466H site of mouse XIAP. Further, the compounds and related compositions of the present application bind to the RING domain of human XIAP, thereby altering E3 ligase activity.

[0071] The term "treatment" refers to alleviating, preventing, ameliorating, or delaying the onset of a symptom of one or more unwanted conditions or diseases in a patient; or refers to controlling the progression of a disease or maintaining a disease in a stable state.

[0072] As used herein, "subject" or "patient" refers to a human or non-human animal (e.g., a mammal), including, but not limited to, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, a monkey, a rabbit, a rat, and a mouse.

[0073] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of a compound, or a pharmaceutically acceptable salt thereof (typically as part of a pharmaceutical composition), that is sufficient to inhibit, stop, ameliorate, alleviate, delay the onset of, or produce an improvement in a symptom of one or more diseases to be treated, when administered alone or in conjunction with another drug used to treat a particular subject or population of subjects.

[0074] A "pharmaceutical composition" refers to a compound suitable for administration in a medical or veterinary use. A pharmaceutical composition includes 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 appropriate dosages and regimes, as well as formulating a pharmaceutical composition, are well within the level of ordinary skill in the pharmaceutical and medical arts.

[0075] 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 not responded favorably to conventional therapy, such as radiotherapy or chemotherapy, or who have developed resistance to such therapy. The other pharmaceutically active substances can be used to treat the same or different disease or disorder as the disease or disorder of the present application. If a patient is receiving or is to receive multiple pharmaceutically active substances, the substances can be administered simultaneously or sequentially. Common methods of treating tumors include surgery, chemotherapy, radiotherapy, immunotherapy, photodynamic therapy, and targeted therapy, among others, and drugs for treating tumors include chemotherapeutic drugs, immunotherapeutic drugs, and targeted therapy drugs, among others. The compounds and compositions of the present application can be used in conjunction with these drugs or methods. Administration of the compounds and compositions of the present application can precede, be concurrent with, or follow the administration of the combination therapy. A variety of routes of administration can be used. Non-limiting methods of administering the compounds and related compositions to a patient include oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) infusion, intracisternal, intradermal, inhalation, extra-digestive tract routes, intravaginal, intraperitoneal, intravesical, topical (powders, ointments, or drops) or as buccal or nasal sprays. Additionally, the substances or compositions comprising the active substances can be administered once, for example, by bolus injection, can be administered multiple times, for example, by a series of tablets or delivered substantially uniformly over a period of time, for example, using transdermal delivery. It is noted that the dosage of the substances can vary 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 (such as the use of nanocarriers).

[0076] The present application also includes isotopically enriched compounds, which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. The compounds of the present application can exist in non-solvated or solvated forms (e.g., hydrates). The compounds of the present application can be administered as pharmaceutically acceptable salts, esters, amides, or prodrugs. Among these, the 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 can also exist in the form of tautomers, such as the enol or imine form, and their corresponding keto and enamine forms, and their geometric isomers and compounds. The tautomers exist as mixtures of tautomers in solution. The compounds of the present application can contain asymmetric or chiral centers, and therefore, exist in different stereoisomeric forms. All stereoisomeric forms of the compounds, and mixtures thereof, including racemates, form part of the present application. In addition, the present application includes all stereoisomers and positional isomers. For example, if a compound contains a double bond, the cis- and trans-forms (designated Z and E, respectively), as well as mixtures, are included.

[0077] The compounds of the application can be administered to a patient by themselves or as part of a pharmaceutical composition in a therapeutically effective amount. Generally, the dosage of active compound is from about 0.01 mg / kg to 1000 mg / kg per day. It is contemplated that a dosage range of 50-500 mg / kg is suitable, preferably intravenous, intramuscular or intradermal administration, and given once or several times per day. In some embodiments, the dosing regimen of the compounds or compositions of the application can be 1 mg to 2000 mg per day, preferably 1 to 1000 mg per day, more preferably 50 to 600 mg per day, in two to four (preferably two) divided doses; periodic treatment (e.g., one week in three weeks, or three weeks in four weeks) can also be used.

[0078] Preparation of the compound of Example 1 Y205-0880

[0079] Reaction scheme

[0080] Dissolve bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic anhydride (1.0 g, 5.6 mmol) in ethanol, add 10 mL of 20% NaOH solution, stir the reaction at 70 °C for 5 h, remove ethanol under reduced pressure, adjust the pH to 2-3 with 6N-HCl, wash out the crystals in an ice water bath, filter under reduced pressure, and dry to obtain bicyclo[2.2.2]oct-5-ene-2,3-dicarboxylic acid (Intermediate 2) in 50% yield.

[0081] Dissolve 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) in N,N-dimethylformamide, stir at room temperature for 10 min, add tetrahydropyrrole (307 mg, 4.38 mmol), and stir the reaction overnight. After the reaction is complete, remove the solvent under reduced pressure, extract with ethyl acetate / water, collect the organic layer, dry over anhydrous magnesium sulfate, filter, and dry in vacuo. Purify the crude product on a silica gel column to obtain the off-white intermediate 4-pyrrolidinoylaniline (Intermediate 3) in 81% yield.

[0082] Intermediate 2 (500 mg, 2.80 mmol), benzotriazol-1 -yl-oxytris-pyrrolidino-phosphonium 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 before 4-pyrrolidinylphenylamine (0.57 g, 2.8 mmol) was added. The reaction was transferred to 70 °C and left to react for 5 h. After the reaction was complete, the solvent was removed by distillation under reduced pressure, extracted with ethyl acetate / water system, the organic layer was collected, dried over anhydrous magnesium sulfate, suction filtered and rotary evaporated. The crude product was purified by silica gel column to obtain intermediate 4 with a yield of 71 %.

[0083] Intermediate 4 (500 mg, 2.80 mmol) was dissolved in methanol, a catalytic amount of Pd / C and triethylamine were added, and hydrogen was bubbled for 30 minutes. After the reaction was complete, the reaction was terminated by adding water. The methanol was removed by distillation under reduced pressure, extracted with ethyl acetate / water system, the organic layer was collected, dried over anhydrous magnesium sulfate, suction filtered and rotary evaporated. 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 NMR data of product Y205-0880 are shown in Table 8, and the retention time in LC-MS was 0.861 min, m / z (ESI) = 371.1 (M+H) + .

[0084] For those compounds with similar structures and synthetic routes, they can be prepared according to the preparation methods of specific compounds in this application or according to the conventional methods in the art. The NMR data of compound E587-0499 are shown in Table 8, and the retention time in LC-MS was 0.595 min, m / z (ESI) = 450.3 (M+H) + .

[0085] The NMR spectrum of compound T4489 is shown in Figure 24.

[0086] Preparation of compounds X-1, X-4, X-3 and X-12 of Preparation Example 2

[0087] Reagents and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH H2O, THF / H2O 1 :1, rt;

[0088] Aminobenzoic acids with different substitutions (X-1 : R1is H, p-; X-4: R1is H, m-; X-3: R1is CH3, m-; X-12: R1is H, o-, X-2: R1is CH3, R1and To a solution of compound a (2.8 mmol), monomethyl phthalate (3.4 mmol), 1H- benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.8 mmol) and N,N- diisopropylethylamine (4.2 mmol) in 6 mL of DMF, the reaction was stirred at 90 °C, TLC was used to monitor the reaction, and the reaction was completed after about 4 h. The reaction was distilled under reduced pressure, extracted with ethyl acetate / saturated brine, the organic layers were combined, dried, and concentrated under reduced pressure. Compound b was obtained by column chromatography.

[0089] To a solution of compound a (2.8 mmol), monomethyl phthalate (3.4 mmol), 1H- benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.8 mmol) and N,N- diisopropylethylamine (4.2 mmol) in 6 mL of DMF, the reaction was stirred at 90 °C, TLC was used to monitor the reaction, and the reaction was completed after about 4 h. The reaction was distilled under reduced pressure, extracted with ethyl acetate / saturated brine, the organic layers were combined, dried, and concentrated under reduced pressure. Compound b was obtained by column chromatography.

[0090] To a solution of compound b (1.4 mmol), lithium hydroxide hydrate (2.8 mmol) in 6 mL of a mixture of tetrahydrofuran / water (1:1), the reaction was 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, the reaction was acidified with 1 mol / L dilute hydrochloric acid, and 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 the product, and the product was allowed to precipitate. The product was filtered, and 2-((4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-1, white powder, yield 45%), 2-((3-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-4, pink powder, yield 40%), 2-((2-methyl-5-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-3, white powder, yield 41%), and 2-((2-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-12, white powder, yield 43%) were obtained. The structural formulas and NMR characterization results of compounds X-1, X-4, X-3, and X-12 are shown in Table 8.

[0091] Preparation of compounds X-5, X-6, X-7, X-8, and X-10 of Preparation Example 3

[0092] Reagents and conditions: (IV) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0093] Dissolve different substituted p-aminobenzoic acid (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-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (3.6 mmol) and N,N-diisopropylethylamine (5.5 mmol) in 6 mL DMF, stir at room temperature, and react overnight. Monitor the reaction by TLC, after the reaction is completed, distill under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry, and concentrate under reduced pressure. Purify the compound c by column chromatography.

[0094] Synthetic route II is referenced to the synthetic route II in Preparation Example 2, and compound d is synthesized.

[0095] Synthetic route III is referenced to the synthetic route III in Preparation Example 2, and products 2-((3-methyl-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-5, white powder, yield 35%), 2-((3-chloro-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-6, white powder, yield 19%), 2-((3-hydroxy-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-7, brown powder, yield 10%), 2-((2-methoxy-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-8, white powder, yield 40%), 2-((2-hydroxy-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)benzoic acid (X-10, white powder, yield 15%) are synthesized. 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.

[0096] Preparation of compounds LYS10-LYS25 of Preparation Example 4

[0097] Reagents and conditions: (V) PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH·H2O, THF / H2O 1:1, rt;

[0098] Table 3 Amines in synthetic route V

[0099] 4-amino-2-chlorobenzoic acid (3.6 mmol), various amines (see Table 3) (4.4 mmol), lH-benzotriazole-l-yloxytris-pyrrolidino-phosphonium 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 and after completion, the reaction mixture was distilled under reduced pressure, extracted with ethyl acetate / saturated brine, the organic layers were combined, dried, and concentrated under reduced pressure. The compound e was isolated and purified by column chromatography.

[0100] Synthetic Scheme II was followed to synthesize compound f.

[0101] Synthetic Scheme III was followed to synthesize:

[0102] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0103] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0104] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0105] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0106] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0107] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0108] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0109] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0110] 2-((3-chloro-4-(cyclopentylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-11, white powder, 35%),

[0111] 2-((3-chloro-4-(ethylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-19, white powder, 40%),

[0112] 2-((3-chloro-4-(4-(4-methoxybenzoyl)piperidine-l-carbonyl)phenyl)carbamoyl)benzoic acid (LYS-20, white powder, 20%),

[0113] 2-((3-chloro-4-(diethylcarbamoyl)phenyl)carbamoyl)benzoic acid (LYS-21, white powder, 28%),

[0114] 2-((3-chloro-4-((3,5-dimethoxyphenyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-22, white powder, 45%),

[0115] 2-((3-chloro-4-((3-chlorophenyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-23, white powder, 35%),

[0116] 2-((3-chloro-4-((4-morpholinophenyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-24, white powder, 24%),

[0117] 2-((3-chloro-4-((thiophen-2-ylmethyl)carbamoyl)phenyl)carbamoyl)benzoic acid (LYS-24, white powder, 15%). The structural formulas and NMR characterization results of compounds LYS10-25 are shown in Table 8.

[0118] Preparation of compounds Y-9-2 and Y-9-3 of Preparation Example 5

[0119] Reagents and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (VI) PyBOP, DIPEA, DMF, 70 °C; (III) LiOH H2O, THF / H2O 1:1, rt;

[0120] Synthetic Route I was synthesized according to Synthetic Route I in Preparation Example 2 to obtain compound g.

[0121] Compound g (2.6 mmol), (1R,2R)-2-(methoxycarbonyl)cyclohexanecarboxylic acid (2.94 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium 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. Then (4-amino-3-methylphenyl)(pyrrolidin-1-yl)methanone (2.45 mmol) was added. The reaction was transferred to 70 °C and the progress of the reaction was monitored by TLC. The reaction was completed in about 4 hours. The reaction mixture was distilled under reduced pressure, extracted with ethyl acetate / saturated brine, the organic layers were combined, dried and concentrated under reduced pressure. The product, methyl (1R,2R)-2-((3-methyl-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)cyclohexane-1-carboxylate (Y-9-2, white powder, yield 60%) was isolated and purified by column chromatography.

[0122] Synthetic route III refers to the synthetic route III in the preparation example 2. Finally, (1R,2R)-2-((3-methyl-4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)cyclohexane-1-carboxylic acid (Y-9-3, pink solid powder, yield 30%) was synthesized. The structural formula and NMR characterization results of compounds Y-9-2 and Y-9-3 are shown in Table 8.

[0123] Preparation of compounds X-15, X-16, X-17, X-18 and X-19 of preparation example 6

[0124] Reagents and conditions: (I) PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH H2O, THF / H2O 1:1, rt;

[0125] Table 4 Amine in synthetic route I and 2-(methoxycarbonyl)cycloalkane carboxylic acid in synthetic route II

[0126] P-Aminobenzoic acid (3.6 mmol), different amine (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 progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was distilled under reduced pressure, 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.

[0127] Compound a (2.8 mmol), different 2-(methoxycarbonyl)cycloalkanecarboxylic acids (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium 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 detected by TLC, and after about 4 h of reaction, the reaction was completed. Distillation under reduced pressure, extraction with ethyl acetate / saturated brine, drying, and concentration under reduced pressure. Compound b was separated and purified by column chromatography.

[0128] Compound b (1.4 mmol), lithium hydroxide hydrate (2.8 mmol) was dissolved in 6 mL of a mixture of tetrahydrofuran / water (1:1), 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, the reaction solution was acidified with 1 mol / L dilute hydrochloric acid, and 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 it, and the product was precipitated by standing. After suction filtration, the products 2-((4-(diethylcarbamoyl)phenyl)carbamoyl)cyclohexane-1-carboxylic acid (X-15, white powder, yield 21%), 2-((4-(4-methylpiperidine-1-carbonyl)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-(piperidine-1-carbonyl)phenyl)carbamoyl)bicyclo[2.2.1]heptane-2-carboxylic acid (X-18, white powder, yield 15%) and (1R,6S)-6-((4-(pyrrolidine-1-carbonyl)phenyl)carbamoyl)cyclohex-3-ene-1-carboxylic acid (X-19, white powder, yield 10%) were obtained. The structural formulas and NMR characterization results of compounds X-15, X-16, X-17, X-18 and X-19 are shown in Table 8.

[0129] Preparation of compound X-20 of preparation example 7

[0130] Reagents and conditions: (IV) 4-Methylpiperazine, STAB, CH2Cl2, CH3COOH, rt; (V) Pd(DPPF)Cl2.CH2Cl2, K2CO3, dioxane / H2O 5:1, N2, 100°C;

[0131] To a solution of 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]dichloropalladium (0.04 mmol) and potassium carbonate (2.49 mmol) in 6 mL of dioxane: water (5:1) mixture under nitrogen protection, the reaction was carried out at 100 °C for 2 h. After the reaction was completed, the solvent was distilled off under reduced pressure, extracted with ethyl acetate / water system, combined organic layers, dried, concentrated under reduced pressure. Add appropriate amount of dichloromethane, precipitate was separated out, stand, suction filtration, to obtain the final product 4-(5-((4-methylpiperazin-1-yl)methyl)pyridin-2- yl)benzamide (X-20, white solid powder, yield 60%). The structural formula and NMR characterization results of compound X-20 are shown in Table 8.

[0132] To a solution of 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]dichloropalladium (0.04 mmol) and potassium carbonate (2.49 mmol) in 6 mL of dioxane: water (5:1) mixture under nitrogen protection, the reaction was carried out at 100 °C for 2 h. After the reaction was completed, the solvent was distilled off under reduced pressure, extracted with ethyl acetate / water system, combined organic layers, dried, concentrated under reduced pressure. Add appropriate amount of dichloromethane, precipitate was separated out, stand, suction filtration, to obtain the final product 4-(5-((4-methylpiperazin-1-yl)methyl)pyridin-2- yl)benzamide (X-20, white solid powder, yield 60%). The structural formula and NMR characterization results of compound X-20 are shown in Table 8.

[0133] Preparation of compound X-21 of Preparation Example 8

[0134] Reagents and conditions: (I) 4-Methylbenzylamine, PyBOP, DIPEA, DMF, rt; (VI) Pd(PPh3)4, Cs2CO3, dioxane / H2O 5:1, N2, 100 °C;

[0135] To a solution of 6-bromonicotinaldehyde (2.2 mmol), 4-methylpiperazine (2.2 mmol) and sodium triacetoxyborohydride (4.3 mmol) in 6 mL of 1,2-dichloroethane, two drops of acetic acid were added dropwise with stirring, and the reaction was carried out at room temperature for 4 h. TLC was used to monitor the reaction, and after the reaction was completed, the solvent was distilled off under reduced pressure, extracted with water / dichloromethane:methanol system, combined organic layers, dried, and concentrated under reduced pressure. Column purification gave colorless transparent oil c.

[0136] Compound d (0.66 mmol), 2-(4-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (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) system, under nitrogen protection, 100 °C for 2 h. After the reaction was completed, the solvent was distilled off under reduced pressure, extracted with ethyl acetate / water system, the organic layers were combined, dried, concentrated under reduced pressure. The crude product was purified by silica gel column to obtain the final product 6-(4-fluorophenyl)-N-(4-methylbenzyl)nicotinamide (X-21, white solid powder, 62%). The structural formula and NMR characterization results of compound X-21 are shown in Table 8.

[0137] Preparation of compound C1-2-C3-5 of Preparation Example 9

[0138] Reagents and conditions: (VI) Pyrrolidine (Cyclopropylamine), PyBOP, DIPEA, DMF, 70 °C; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH H2O, THF / H2O 1:1, rt; (V) SOCl2, DMF, DCM, rt; (VI) Amines, DCM, rt.

[0139] Dissolve 4-amino-2-chlorobenzoic acid A (2.9 mmol), 1H-benzotriazol-1- yloxytripyrrolidinophosphonium hexafluorophosphate (2.9 mmol) and N,N- diisopropylethylamine (4.4 mmol) in 6 mL of anhydrous DMF, stir at room temperature for 5 min, then add cyclopropylamine (3.5 mmol), heat the reaction to 70 degrees Celsius in an oil bath, and react for 4 h. Monitor the reaction by TLC, and after the reaction is complete, remove most of the anhydrous DMF under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry with anhydrous magnesium sulfate, filter, and remove the solvent under reduced pressure. The crude product is separated and purified by silica gel chromatography (dichloromethane:methanol = 90:1) to obtain compound B.

[0140] Dissolve monomethyl phthalate (3.4 mmol), lH-benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate (2.8 mmol) and N,N-diisopropylethylamine (4.2 mmol) in 10 mL of anhydrous DMF, after stirring for 5 min at room temperature, add compound B (2.8 mmol), heat the reaction to 90 °C in an oil bath, stir for 4 h. Monitor the reaction progress by TLC, after the reaction is complete, remove the anhydrous DMF by distillation under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry with anhydrous MgS04, filter, remove the solvent from the crude product by distillation under reduced pressure. Purify the crude product by silica gel column chromatography (dichloromethane:methanol = 75:1) to obtain compound C1-2. Replace monomethyl phthalate with monomethyl isophthalate and monomethyl terephthalate, respectively, and keep other conditions unchanged to obtain C2-2 and C3-2, respectively.

[0141] Dissolve C2-2 (1.4 mmol) in 6 mL of a tetrahydrofuran:water (1:1) solution, add lithium hydroxide hydrate (2.8 mmol), stir for 2 h at room temperature, monitor by TLC, after the reaction is complete, remove the solvent by distillation under reduced pressure, adjust the pH to weakly acidic with 1 M dilute hydrochloric acid solution, extract with ethyl acetate / saturated brine, remove the solvent by distillation under reduced pressure. Add an appropriate amount of ethyl acetate, stand until the crystals are stable and precipitate. Filter to obtain compound C2-3, and place it in an oven for low-temperature drying. Replace C2-2 with C3-2, and keep other conditions unchanged to obtain C3-3.

[0142] Dissolve different position substituted (1.4 mmol) in 6 mL of anhydrous dichloromethane, add dichlorosulfoxide (2.8 mmol) dropwise, seal the bottle with a rubber plug, and add a catalytic amount of DMF dropwise with a syringe. Stir at room temperature, react for 4 h, monitor the reaction by TLC, after the reaction is complete, remove the solvent by distillation under reduced pressure, seal the cap, and wait for the next reaction.

[0143] The product of the previous step was dissolved in 6 mL of anhydrous dichloromethane and placed in a low temperature reaction box and stirred at -20 °C. Different amine compounds (1.7 mmol) were quickly transferred into the reaction bottle using a pipette, and after 5 min, the reaction was stirred at room temperature for 5 h. The reaction progress was monitored by TLC, and after the reaction was completed, the solvent was removed by distillation under reduced pressure, extracted with ethyl acetate / saturated brine, combined organic layers, 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 = 25:1) to obtain compounds C1-4, C1-5, C2-4, C2-5, C3-4, C3-5. The substitution of the product and the corresponding relationship of the different amine compounds in the sixth step are as follows: (C1-4: o-, diethylamine; C1-5: o-, azetidine; C2-4: m-, diethylamine; C2-5: m-, azetidine; C3-4: p-, diethylamine; C3-5: p-, azetidine)

[0144] Preparation of compounds C5-2-1, C5-2-2, C6-2-1 of Preparation Example 10

[0145] Reagents and conditions: (V) SOCl2, DMF, DCM, rt; (VI) B, DCM, rt.

[0146] The product of the previous step was dissolved in 6 mL of anhydrous dichloromethane and placed in a low temperature reaction box and stirred at -20 °C. Different amine compounds (1.7 mmol) were quickly transferred into the reaction bottle using a pipette, and after 5 min, the reaction was stirred at room temperature for 5 h. The reaction progress was monitored by TLC, and after the reaction was completed, the solvent was removed by distillation under reduced pressure, extracted with ethyl acetate / saturated brine, combined organic layers, 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 = 25:1) to obtain compounds C1-4, C1-5, C2-4, C2-5, C3-4, C3-5. The substitution of the product and the corresponding relationship of the different amine compounds in the sixth step are as follows: (C1-4: o-, diethylamine; C1-5: o-, azetidine; C2-4: m-, diethylamine; C2-5: m-, azetidine; C3-4: p-, diethylamine; C3-5: p-, azetidine) (1.5 mmol) was dissolved in 5 mL of anhydrous dichloromethane, and thionyl chloride (3.0 mmol) was added dropwise. The bottle was capped with a rubber plug, and a catalytic amount of DMF was added dropwise using a syringe. The reaction was stirred at room temperature for 5 h, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the bottle was sealed with a cap. The product was used in the next step.

[0147] 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 and stirred at room temperature overnight. The reaction progress was monitored by TLC, and after the reaction was completed, the solvent was removed by distillation under reduced pressure, extracted with ethyl acetate / saturated brine, combined organic layers, 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 compound C5-2-1. When were replaced with C5-2-2 and C6-2-1 were prepared under the same conditions, respectively.

[0148] Preparation of compounds C7-3, C8-3, C9-3 of Preparation Example 11

[0149] Reagents and conditions: (VI) Pyrrolidine, (Cyclopropylamine) PyBOP, DIPEA, DMF, 70℃; (VII) PyBOP, DIPEA, DMF, 100℃; (III) LiOH·H2O, THF / H2O 1:1, rt.

[0150] Will Compound B (2.7 mmol), 1H-benzotriazol-1-yloxytripyrrolylphosphonium 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 min, compound B (2.2 mmol) was added, and the mixture was heated in an oil bath to 100 °C and stirred for 4 h. The reaction progress was monitored by TLC. After the reaction was complete, the anhydrous DMF was removed by vacuum distillation, and the mixture was extracted with ethyl acetate / saturated brine. The organic layers were combined, dried over anhydrous MgSO4, filtered, and the crude product was purified by vacuum distillation to remove the solvent. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 75:1) to obtain compound B.

[0151] The product from the previous step (1.4 mmol) was dissolved in 6 mL of tetrahydrofuran:water (1:1) solution, and lithium hydroxide hydrate (2.8 mmol) was added. The mixture was stirred at room temperature for 2 h, monitored by TLC. After the reaction was complete, the solvent was removed by vacuum distillation. The pH was adjusted to weakly acidic with 1 M dilute hydrochloric acid solution, and the mixture was extracted with ethyl acetate / saturated brine. The solvent was removed by vacuum distillation. An appropriate amount of ethyl acetate was added, and the mixture was allowed to stand until crystals stabilized and precipitated. The product was filtered to obtain compound C7-3, which was then dried in an oven at low temperature. Replace with With other conditions remaining unchanged, C8-3 and C9-3 can be prepared respectively.

[0152] General synthetic route for compound C1-2-C3-5:

[0153] 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.

[0154] Dissolve 4-amino-2-chlorobenzoic acid A (2.9 mmol), 1H-benzotriazole-1- yloxytripyrrolidinophosphonium hexafluorophosphate (2.9 mmol) and N,N- diisopropylethylamine (4.4 mmol) in 6 mL of anhydrous DMF, stir for 5 min at room temperature, then add cyclopropylamine (3.5 mmol), heat the reaction to 70 °C in an oil bath, and stir for 4 h. Monitor the reaction by TLC, and when the reaction is complete, remove most of the anhydrous DMF by distillation under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry over anhydrous MgSO4, filter, and concentrate under reduced pressure to remove the solvent. Purify the crude product by silica gel chromatography (dichloromethane:methanol = 90:1) to obtain compound B.

[0155] Dissolve monomethyl phthalate (3.4 mmol), 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.8 mmol) and N,N- diisopropylethylamine (4.2 mmol) in 10 mL of anhydrous DMF, stir for 5 min at room temperature, then add compound B (2.8 mmol), heat the reaction to 90 °C in an oil bath, and stir for 4 h. Monitor the reaction by TLC, and when the reaction is complete, remove the anhydrous DMF by distillation under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry over anhydrous MgSO4, filter, and concentrate under reduced pressure to remove the solvent. Purify the crude product by silica gel chromatography (dichloromethane:methanol = 75:1) to obtain compounds C1-2, C2-2, C3-2.

[0156] Dissolve the compound ((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)methyl benzoate (1.4 mmol) substituted at different positions in 6 mL of a tetrahydrofuran:water (1:1) solution, add lithium hydroxide hydrate (2.8 mmol), stir at room temperature for 2 h, monitor the reaction by TLC, and when the reaction is complete, remove the solvent by distillation under reduced pressure, adjust the pH to weakly acidic with 1M 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 the crystals to precipitate and stabilize. Filter to obtain compounds C2-3, C3-3, and place them in an oven to dry at low temperature.

[0157] Dissolve the compound ((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)methyl benzoate (1.4 mmol) substituted at different positions in 6 mL of anhydrous dichloromethane, add dichlorosulfoxide (2.8 mmol) dropwise, seal the bottle with a rubber stopper, and add a catalytic amount of DMF dropwise using a syringe. Stir at room temperature for 4 h, monitor the reaction by TLC, and when the reaction is complete, remove the solvent by distillation under reduced pressure, seal the bottle, and allow it to stand until the next reaction.

[0158] The product of the previous reaction was dissolved in 6 mL of dry dichloromethane and placed in a low temperature reaction block and stirred at -20 °C. Different amine compounds (1.7 mmol) were quickly transferred into the reaction vial using a syringe and stirred at room temperature after 5 min. The reaction was monitored by TLC and upon completion, the solvent was removed under reduced pressure, extracted with ethyl acetate / saturated brine, combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (dichloromethane:methanol = 25:1) to obtain compounds C1-4, C1-5, C2-4, C2-5, C3-4, C3-5.

[0159] General synthetic route for compounds C5-2-1-C6-2-1:

[0160] Reagents and conditions: (V) SOCl2, DMF, DCM, rt; (VI) B, DCM, rt.

[0161] The sulfonamide substituted benzoic acid (1.5 mmol) was dissolved in 5 mL of dry dichloromethane and dropwise added with dichlorosulfoxide (3.0 mmol). The vial was capped with a rubber septum and a catalytic amount of DMF was added dropwise using a syringe. The reaction was stirred at room temperature for 5 h and monitored by TLC. Upon completion, the solvent was removed under reduced pressure and the vial was capped and ready for the next step.

[0162] The product of the previous reaction and 4-amino-2-chloro-N- cyclopropylbenzamide B (1.8 mmol) were dissolved in 5 mL of dry dichloromethane and stirred at room temperature overnight. The reaction was monitored by TLC and upon completion, the solvent was removed under reduced pressure, extracted with ethyl acetate / saturated brine, combined organic layers were dried over anhydrous magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (dichloromethane:methanol = 30:1) to obtain compounds C5-2-1, C5-2-2, C6-2-1.

[0163] General synthetic route for compounds C7-3-C9-3:

[0164] Reagents and conditions: (VI) Pyrrolidine, PyBOP, DIPEA, DMF, 70 °C; (VII) PyBOP, DIPEA, DMF, 100 °C; (III) LiOH H2O, THF / H2O 1:1, rt.

[0165] Dissolve 4-amino-2-chlorobenzoic acid A (2.9 mmol), 1H-benzotriazole-1- yloxytripyrrolidinophosphonium hexafluorophosphate (2.9 mmol) and N,N- diisopropylethylamine (4.4 mmol) in 6 mL of anhydrous DMF, after stirring for 5 min at room temperature, add cyclopropylamine (3.5 mmol), heat the reaction to 70 °C in an oil bath, stir for 4 h. Monitor the reaction by TLC, after the reaction is complete, remove most of the anhydrous DMF by distillation under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry over anhydrous MgSO4, filter, concentrate under reduced pressure to remove the solvent, purify the crude product by silica gel column chromatography (dichloromethane:methanol = 90:1) to obtain compound B.

[0166] Dissolve different substituted carboxylic acids (2.7 mmol), 1H-benzotriazole-1- yloxytripyrrolidinophosphonium hexafluorophosphate (2.2 mmol) and N,N- diisopropylethylamine (3.4 mmol) in 10 mL of anhydrous DMF, after stirring for 5 min at room temperature, add compound B (2.2 mmol), heat the reaction to 100 °C in an oil bath, stir for 4 h. Monitor the reaction by TLC, after the reaction is complete, remove the anhydrous DMF by distillation under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry over anhydrous MgSO4, filter, concentrate under reduced pressure to remove the solvent. Purify the crude product by silica gel column chromatography (dichloromethane:methanol = 75:1) to obtain compounds E1, E2, E3.

[0167] 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), stir for 2 h at room temperature, monitor by TLC, after the reaction is complete, remove the solvent by distillation under reduced pressure, adjust the pH to weakly acidic with 1M dilute hydrochloric acid solution, extract with ethyl acetate / saturated brine, remove the solvent by distillation under reduced pressure. Add an appropriate amount of ethyl acetate, stand until the crystals are stable and precipitate. Filter to obtain compounds C7-3, C8-3, C9-3, and place them in an oven for low-temperature drying.

[0168] X-2 Solvent: deuterated DMSO

[0169] Methyl 2-((3-chloro-4-(cyclopropylcarbamoyl)phenyl)carbamoyl)benzoate C1-2

[0170] Prepare compound LYS26 according to the preparation method of compound C1, using B18 instead of B1.

[0171] General synthetic route of compounds in series A

[0172] Reagent and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH-H2O, THF / H2O 1:1, rt;

[0173] Dissolve different substituted aminobenzoic acid (3.6 mmol), pyrrolidine (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (3.6 mmol) and N,N-diisopropylethylamine (5.5 mmol) in 6 mL DMF, stir at room temperature, and react overnight. Monitor the reaction by TLC, and after the reaction is completed, distill under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry, and concentrate under reduced pressure. Purify the compound a by column chromatography.

[0174] Dissolve compound a (2.8 mmol), monomethyl phthalate (3.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (2.8 mmol) and N,N-diisopropylethylamine (4.2 mmol) in 6 mL DMF, stir the reaction system at 90 °C, monitor the reaction progress by TLC, and after about 4 h, the reaction is completed. Distill under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry, and concentrate under reduced pressure. Purify the compound b by column chromatography.

[0175] Dissolve compound b (1.4 mmol), lithium hydroxide hydrate (2.8 mmol) in 6 mL tetrahydrofuran / water (1:1) mixed solution, stir at room temperature, monitor the reaction by TLC, and after about 2 h, the reaction is completed. Distill the solvent under reduced pressure, acidify the reaction solution with 1 mol / L dilute hydrochloric acid, extract with ethyl acetate / water, combine the organic layers, dry, and concentrate under reduced pressure. Dissolve a small amount of ethyl acetate, stand to precipitate the product, and finally obtain the compound LYS1-4 in series A by suction filtration.

[0176] Table 5 Compounds in series A

[0177] General synthetic route of compounds in series B

[0178] Reagent and conditions: (IV) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH-H2O, THF / H2O 1:1, rt;

[0179] Dissolve the various substituted p-aminobenzoic acid (3.6 mmol), pyrrolidine (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (3.6 mmol) and N,N-diisopropylethylamine (5.5 mmol) in 6 mL of DMF, stir at room temperature overnight. Monitor the reaction by TLC, after the reaction is complete, distill under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry, and concentrate under reduced pressure. Purify the compound c by column chromatography.

[0180] Synthetic route II is the same as the previous, to synthesize compound d.

[0181] Synthetic route III is the same as the previous, to synthesize compound LYS 5-9.

[0182] Table 6

[0183] General synthetic route of C series compounds

[0184] Reagent and conditions: (V) PyBOP, DIPEA, DMF, rt; (II) PyBOP, DIPEA, DMF, 90 °C; (III) LiOH H2O, THF / H2O 1:1, rt;

[0185] Dissolve 4-amino-2-chlorobenzoic acid (3.6 mmol), various amines (4.4 mmol), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (3.6 mmol) and N,N-diisopropylethylamine (5.5 mmol) in 6 mL of DMF, stir at room temperature overnight. Monitor the reaction by TLC, after the reaction is complete, distill under reduced pressure, extract with ethyl acetate / saturated brine, combine the organic layers, dry, and concentrate under reduced pressure. Purify the compound e by column chromatography.

[0186] Synthetic route II is the same as the previous, to synthesize compound f.

[0187] Synthetic route III is the same as the previous, to synthesize compound LYS 10-25.

[0188] Table 7

[0189] General synthetic route of D series compounds

[0190] Reagent and conditions: (I) Pyrrolidine, PyBOP, DIPEA, DMF, rt; (VI) PyBOP, DIPEA, DMF, 70 °C; (III) LiOH-H2O, THF / H2O 1 :1, rt;

[0191] Synthetic route I is the same as the previous, the synthesis of compound g.

[0192] Compound g (2.6 mmol), (1R,2R)-2-(methoxycarbonyl)cyclohexanecarboxylic acid (2.94 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (2.45 mmol) and N,N-diisopropylethylamine (3.67 mmol) were dissolved in 8 mL of N,N-dimethylformamide, stirred at room temperature for 10 minutes, then (4-amino-3-methylphenyl)(pyrrolidin-1-yl)methanone (2.45 mmol) was added. The reaction was transferred to 70 °C for reaction, TLC monitoring reaction progress, about 4 h reaction was completed, reduced pressure distillation, extraction with ethyl acetate / saturated brine, combined organic layers, dried, concentrated under reduced pressure. Separated and purified by column chromatography to obtain compound LYS26 (Y-9-2, i.e. 11), white powder, yield 60%.

[0193] Synthetic route III is the same as the previous, finally synthesized compound LYS27 (Y-9-3), pink solid powder, yield 30%.

[0194] Example 1 Screening of small molecule drugs targeting XIAP RING domain

[0195] The Site Finder module of MOE software (version 2022) was used to analyze the possible binding sites on the surface of human XIAP protein (PDB ID: 4IC2, 4IC3 and 5O6T). It was found that the co-crystal structure of 4IC3 near the 467th amino acid and the 495th amino acid predicted the binding pockets Site 1 (LEU444 GLU446 GLU447 LYS448 LEU449 LYS451 MET454 ASN457 ILE458 HIS467 LEU468 VAL469 MET483 ILE494 MET496) and Site 2: (LEU444 GLU447 LYS448 LEU468 LEU495 MET496 SER497). Among them, the PLB of Site1 is greater than 1, therefore, the Site1 site of the co-crystal structure 4IC3 was selected to carry out virtual screening, and the XIAP protein sequence is referred to Uniprot ID: P98170. The present application selects a TargetMol compound library containing 36861 bioactive compounds and a Chemdiv compound library containing 1,583,358 structurally diverse compounds as screening databases, a total of 1.62 million molecules, and selects compounds that can form direct interaction with XIAP RING domain (E3 ligase) as screening indicators in terms of affinity, drugability attributes, structural diversity and binding specificity. The high-frequency interaction amino acid sites near the 467th amino acid and the 495th amino acid (key sites of E3 ligase activity) are Asn457, Ile458, Lys448, Met496 and Ser497 (see FIG. 26-FIG. 27), for example, the amino acid Ile458 is involved in the interaction in the interaction mode of 68 compounds. Finally, 65 compounds with relatively strong affinity score, good drugability attributes, and strong binding specificity (such as the affinity score of compound 1 with XIAP E3 ligase is -8.40 kcal / mol) were screened, and subsequent biological activity tests were carried out. When the person skilled in the art needs to screen XIAP inhibitors from human XIAP with other amino acid sequences or from XIAP of other organisms other than humans using a method similar to the present application, the amino acid sites corresponding to the above-mentioned human XIAP protein can be screened without creative labor, for example, H467 of human XIAP corresponds to H466 of mouse XIAP.

[0196] Example 2 Functional verification of small molecule drugs targeting XIAP RING domain

[0197] 1.1 In vitro experiment to detect the anti-tumor activity of small molecule compounds

[0198] 1.1.1 Screening and determination of small molecule compounds

[0199] The small molecule compounds obtained from several screenings were respectively applied to bladder cancer cells, and the anti-tumor activity was detected by CCK8 experiment. In the preliminary screening experiment, the compounds were applied at a concentration of 1 μM for 72 h, and then IC50determination and further experiments were performed on them, and the results are as shown in Table 8.

[0200] Table 8

[0201] 1.1.2 Detection of the targeting specificity and the inhibition of the invasion ability of cancer cells of the small molecule compounds

[0202] The reported direct substrate proteins of XIAP E3 ligase include p62, Bcl-2, PTEN and DNMT3B found in the previous research of the team. The compounds obtained from the above preliminary screening were applied to T24T cancer cells at a safe and non-cytotoxic concentration by CCK8 experiment, and the action time was 24 h, and the protein was collected. The changes of DNMT3B and other reported direct substrate proteins of XIAP E3 ligase after the action of the compounds were detected by Western blotting, and the changes of the invasion-related marker proteins (such as MMP2, MMP9 and N-cadherin) were also detected, and the compounds with consistent up-regulation of substrate proteins and consistent decrease of invasion-related protein indicators were screened. We finally screened the 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 FIG. 1). In the Western blot experiment of human bladder cancer T24T cells after the action of the candidate compounds, the changes of DNMT3B and other reported direct substrate proteins of XIAP E3 ligase were detected, and the changes of the invasion-related marker proteins were also detected. It can be seen that the expression of the substrate proteins is consistently up-regulated after the action of compounds 1 and 2, and the expression of the invasion-related proteins is consistently decreased.

[0203] Based on the fact that cell invasion is an important feature of cancer progression, and cancer cell metastasis is the main cause of death in bladder tumors, finding small molecule compounds that have a significant inhibitory effect on tumor cell infiltration and metastasis is a focus of research efforts. Because the team had previously successfully constructed the XIAP stable knockout cell line T24T(KOXIAP) and the control cell line T24T(vector), and had constructed the corresponding cell models by returning the HA-labeled XIAP plasmid and the HA-labeled XIAP RING H467 mutant plasmid in the XIAP-knockout cells, namely T24T(KOXIAP / XIAP) and T24T(KOXIAP / H467A), respectively. Further, the effects of these compounds on the migration and invasion abilities of T24T(vector), T24T(KOXIAP), T24T(KOXIAP / XIAP), and T24T(KOXIAP / H467A) cells were detected by Transwell experiments. The 5% FBS medium containing the compound to be detected was added to the lower chamber, and the cells were diluted to a uniform single-cell suspension (20,000 cells / well) in 0.1% FBS medium containing the same concentration of compound and added to the upper chamber, and acted for 24 h, with the medium containing the corresponding DMSO working concentration as the control. Figure 2 shows the results of detecting the migration and invasion abilities of T24T cells under the action of small molecule inhibitors. Among them, (A & F) are photographs 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 2 kinds of small molecule inhibitors (1, 2) detected by Transwell experiments; (B & G) are the column chart results of the migration abilities corresponding to A and F; (C & H) are the column chart results of the invasion abilities corresponding to A and F; the column chart represents the average value ± standard deviation of 5 independent fields; (D) is a photograph of the migration and invasion abilities of T24T cells under the action of DMSO and small molecule inhibitor A at different concentrations detected by Transwell experiments; (E) is the line graph results of the migration and invasion abilities corresponding to D. The asterisk (*) indicates that the results have statistical significance compared with the control group (P<0.05).

[0204] It can be seen that when compound 1 and compound 2 are used at concentrations of 0.5 μM and 2 μM respectively, the invasion abilities of T24T(Vector) cells and T24T(KOXIAP / XIAP) cells are significantly reduced compared with cells treated with the same dose of DMSO, indicating that the compounds can effectively inhibit cell invasion. At the same time, T24T(KOXIAP) cells and T24T(KOXIAP / H467A) cells do not show obvious inhibition of cell invasion under the same grouping treatment. Compound 1 is used at concentration gradients (0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM) to act on T24T cells, and the cell invasion ability shows concentration-dependent inhibition. The results suggest that the compound inhibits the invasion ability of bladder cancer cells by specifically targeting the E3 ubiquitin ligase site of XIAP.

[0205] 1.2 In vivo experiment for detecting the anti-tumor effect and pharmacokinetics of the small molecule compound

[0206] 1.2.1 In vivo detection of the anti-tumor activity of the small molecule compound

[0207] 1.2.1.1 Detection of the anti-tumor activity of the small molecule compound on primary basal muscle-invasive bladder cancer in mice

[0208] 5-6-week-old wild-type mice were randomly divided into 3 groups, 10 mice in each group, including a vehicle control group, a 0.05% BBN-containing drinking water group, and a 0.05% BBN-containing drinking water + small molecule compound to be tested treatment group (100 mg / kg / day). After administration, physiological indicators (such as body weight and respiratory rate, etc.) were monitored every 3 days, and the state of the mice was closely observed. After drug treatment, the mice were sacrificed, the bladder was removed, the changes in the bladder were observed, the weight of the bladder was measured, and the bladder / mouse weight ratio was calculated. They were 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.

[0209] The therapeutic effect of the targeted small molecule compound on primary basal muscle-invasive bladder cancer was evaluated.

[0210] Specifically, 5-6 week old wild type C57 / B6 male mice were randomly divided into 3 groups (5 mice in each group), including: ① vehicle drinking water control group, ② 0.05% BBN drinking water group, ③ 0.05% BBN drinking water group plus small molecule compound group. The ① group was given vehicle drinking water until 25 weeks, the ② group was given 0.05% BBN drinking water until 25 weeks, and the ③ group was given BBN plus the small molecule compound (100 mg / kg / day) until 25 weeks after BBN exposure for 16 weeks. The BBN drinking water and the small molecule compound were replaced every other day. After the treatment, the mice were sacrificed, the bladder was removed, and the changes in bladder morphology and volume were observed, and the weight of the bladder was measured and the bladder / mouse weight ratio was calculated. Figure 3 shows the results of the blocking effect of candidate small molecule compound 1 on primary basal muscle layer invasive bladder cancer in mice. (A-D) Comparison of bladder tissue morphology size (A), tumor weight (B), tumor size to mouse weight ratio (C), and mouse weight (D) in the vehicle control group, 0.05% BBN drinking water group, and 0.05% BBN drinking water group plus small molecule compound 1 group; (E) HE staining to observe the inhibitory effect of small molecule compound 1 on the pathological changes of BMIBC caused by BBN exposure.

[0211] The results showed that: 80% (4 / 5) of the mice in the ② group developed basal muscle layer invasive bladder cancer. In the ③ group, compound 1 at a dose of 100 mg / kg / day had no significant difference in bladder morphology, volume, and bladder / mouse weight ratio compared with the ① group, and the BBN-induced decrease in mouse weight was also improved. HE staining showed that BBN induced pathological changes in BMIBC, and compound 1 significantly reduced the pathological damage of BMIBC after intervention. The above results showed that compound 1 had a significant therapeutic effect on BBN-induced primary basal muscle layer invasive bladder cancer in mice.

[0212] Specifically, 5-6 week old wild type C57 / B6 male mice were randomly divided into 3 groups (5 mice in each group), including: ① vehicle drinking water control group, ② 0.05% BBN drinking water group, ③ 0.05% BBN drinking water group plus small molecule compound group. The ① group was given vehicle drinking water until 25 weeks, the ② group was given 0.05% BBN drinking water until 25 weeks, and the ③ group was given BBN plus the small molecule compound (100 mg / kg / day) until 25 weeks after BBN exposure for 16 weeks. The BBN drinking water and the small molecule compound were replaced every other day. After the treatment, the mice were sacrificed, the bladder was removed, and the changes in bladder morphology and volume were observed, and the weight of the bladder was measured and the bladder / mouse weight ratio was calculated. Figure 3 shows the results of the blocking effect of candidate small molecule compound 1 on primary basal muscle layer invasive bladder cancer in mice. (A-D) Comparison of bladder tissue morphology size (A), tumor weight (B), tumor size to mouse weight ratio (C), and mouse weight (D) in the vehicle control group, 0.05% BBN drinking water group, and 0.05% BBN drinking water group plus small molecule compound 1 group; (E) HE staining to observe the inhibitory effect of small molecule compound 1 on the pathological changes of BMIBC caused by BBN exposure.

[0213] Results show that: in group ③, compound 1 at a dose of 100 mg / kg / day, the bladder shape, volume and bladder / mouse weight ratio of mice have a significant inhibitory effect compared with group ①, suggesting that compound 1 significantly inhibits the occurrence of BMIBC. The above results all show that compound 1 can effectively inhibit the occurrence and development of primary basal muscle layer invasive bladder cancer in mice.

[0214] Experiment 3 is specifically as follows: 5-6 week-old wild-type C57 / B6 male mice are randomly divided into 3 groups, 5 in each group, including: ① vehicle containing drinking water control group, ② 0.05% BBN containing drinking water group, ③ 0.05% BBN containing drinking water group plus drug group. Group ① is given vehicle containing drinking water until 25 weeks, group ② is given 0.05% BBN containing drinking water until 25 weeks, and group ③ is given BBN plus compound 2 (Com2 100 mg / kg / day) after BBN exposure for 17 weeks until 25 weeks. BBN drinking water and Com2 are replaced every other day.

[0215] Results show (see Figure 6) the administration results of compound 2 in a mouse model of BBN-induced primary basal muscle layer invasive bladder cancer. Among them, in group ③, compound 2 at a dose of 100 mg / kg / day, the bladder shape, volume and bladder / mouse weight ratio of mice have decreased compared with group ②, and the occurrence of BBN-induced BMIBC in mice has been improved. The above results show that compound 2 has a therapeutic effect on BBN-induced primary basal muscle layer invasive bladder cancer in mice.

[0216] 1.2.1.2 Detection of the inhibitory effect of small molecule compounds on the subcutaneous tumor formation of BMIBC cells in nude mice

[0217] BALB / C-nu nude mice aged 3-4 weeks are selected. T24T bladder cancer cells are expanded, counted after trypsin digestion, resuspended with PBS, and the cell density is adjusted to 2×10 7 / ml. 100 μl of cell suspension is taken with a 1 ml sterile injection needle and inoculated subcutaneously in nude mice, generally within half an hour, and the cell suspension is placed on ice during the process to reduce cell metabolism and maintain cell activity. The mice are randomly divided into 4 groups (9 in each group), including ① vehicle control group; ② 10 mg / kg compound 1 intraperitoneal injection administration group; ③ 40 mg / kg compound 1 administration group via drinking water; ④ 10 mg / kg compound 1 intraperitoneal injection administration treatment group after subcutaneous injection of tumor cells for 5 days and obvious tumor growth. After 4 weeks of drug treatment, the mice are sacrificed, the changes in subcutaneous tumors are observed, and the inhibitory effect of small molecule compounds on the subcutaneous tumor formation of BMIBC cells in nude mice is evaluated.

[0218] The results show (see Figure 5) that the weight and size of the subcutaneous tumors of the nude mice treated with compound 1 are significantly reduced compared with the control group, indicating that compound 1 has a good inhibitory effect and therapeutic effect on the subcutaneous tumor formation of BMIBC cells in vivo.

[0219] 1.2.1.3 Human xenograft model (PDX) for detecting the anti-tumor activity of small molecule compounds in mice

[0220] Fresh tumor tissue samples of bladder cancer patients are taken, the capsule and necrotic tissue are removed, washed with sterile PBS for 3 times, and cut into fragments with a diameter of about 2-3 mm on a sterile plate. A small incision is made on the kidney of a 4-8 week old nude mouse to find the kidney position, and the tumor tissue sample is filled into the tip of a customized trocar needle with forceps and transplanted under the kidney capsule of the mouse. 3-4 nude mice are inoculated for each tumor, and the inoculation process must be completed within 2 hours after the tumor sample is removed from the body. This model retains the microenvironment and basic characteristics of the primary tumor cells, forming the first generation of transplanted tumors (F1). When the transplanted tumor tissue grows to a certain size, it is sterilely operated and transplanted into a new batch of mice to form the second generation of transplanted tumors (F2). Repeat the operation to form the third generation (F3), fourth generation (F4), etc. of transplanted tumors, and the F3 and above mice can be used for drug treatment research.

[0221] The F4 generation of transplanted tumors is transplanted into 40 BALB / C-nu nude mice to construct a bladder cancer PDX model for efficacy evaluation. After 18 days of normal feeding, CT evaluation is performed to assess tumor formation. The tumor-bearing mice are randomly divided into 4 groups, including a vehicle control group, a small molecule compound to be tested treatment group, and 3 dose groups of 50 mg / kg / day, 100 mg / kg / day, and 200 mg / kg / day. The gastric gavage method is used for drug administration for 30 days. The physiological indicators (such as body weight, heart rate, and respiratory rate) and biochemical indicators (such as blood glucose, blood lipids, and liver function) are monitored every 3 days after drug administration, and the state of the mice is closely observed. After drug treatment, the mice are sacrificed, the changes in the bladder are observed, the weight of the bladder is measured, and the bladder / mouse weight ratio is calculated. One part is used for pathological staining analysis such as H&E and immunohistochemistry (IHC), and the other part is frozen for future RNA and protein analysis. The therapeutic effect of the small molecule compound on bladder cancer PDX is evaluated.

[0222] Example 3 Screening and identification of the anti-tumor spectrum of small molecule compounds

[0223] Based on the overexpression of XIAP in various epithelial tumors and the possible important biological function, the screened small molecule inhibitors targeting human XIAP E3 ligase with anti-bladder cancer activity were applied to different tumor cells to detect whether they have the same anti-tumor activity on other XIAP high-expression tumors. According to the analysis of TCGA database (as shown in Figure 7), the expression of XIAP in 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 screened are applied to each tumor cell to detect their anti-tumor activity.

[0224] Experiment 1: The compound 1 obtained by preliminary screening was applied to AGS (human gastric adenocarcinoma cells) and HGC-27 gastric cancer cells, U2-OS human osteosarcoma cells and MB-231 breast cancer cells at different concentrations, and the action time was 24 h. The protein was collected. Figure 8 shows the Western blot experiment after the candidate small molecule inhibitor 1 acts on different tumor cells. The change of the protein of the reported XIAP E3 ligase direct substrate and the change of the marker protein related to cell invasion were detected. (A and B) The results of the up-regulation of the substrate protein and the decrease of the invasion index after the compound 1 acts on the gastric cancer cells AGS and HGC-27. (C) The results of the up-regulation of the substrate protein and the decrease of the invasion index after the compound 1 acts on the human osteosarcoma cells U2-OS. (D) The results of the up-regulation of the substrate protein and the decrease of the invasion index after the compound 1 acts on the breast cancer cells MB-231.

[0225] The results of Western blotting showed that the expression of DNMT3B and other reported XIAP E3 ligase direct substrate proteins increased after the compound acted, and the expression of the marker proteins related to cell invasion (such as MMP2, MMP9) decreased significantly.

[0226] Experiment 2: 1.25% agarose solution and BME cell culture medium (2X) were prepared, and the 42℃ preheated medium was mixed with 1.25% agarose solution at a ratio of 3:2, and then added into the six-well plate (3mL / well) as soon as possible. Attention should be paid to not generate bubbles during plating. After plating, it was solidified at room temperature for about 2h; the prepared cells were digested and counted, and 8000 cells per well were added to the prepared medium. The 1.25% agarose solution was added to the mixed cell suspension in the medium in proportion, and then immediately added to the corresponding lower gel. Attention should be paid to not generate bubbles during plating. After plating, it was solidified at room temperature for about 2h. After solidification, it was sealed with sealing film and placed in a 37℃ cell incubator for culture. During the period, the cell colony formation was observed regularly, and after about 2-4 weeks, the microscope was used to take pictures and statistically analyze the cell colony formation rate.

[0227] The above results show that the malignant proliferation of human melanoma cells (A375), lung cancer cells (HCC827) and prostate cancer cells (PC-3) is inhibited under the action of compound 1 and its modified compounds (see Figures 9 and 10).

[0228] Effect of small molecule compounds on cell metastasis and invasion

[0229] The effects of compounds 1, X-6, X-10, X-20, Y-9-3 on the metastasis and invasion ability of T24T cells were determined by the method of Reference Example 2, and the results are shown in Figure 11. The effects of compounds B6, B8, B9, B10, C1-4, C5-2-2, C1-5, Y-9-2, 940532-51-4, 714278-58-7 on the metastasis and invasion ability of T24T cells were determined, and the results are shown in Figure 25.

[0230] XIAP RING domain plays a crucial role in the development of BBN-induced basal muscle-invasive bladder cancer (BMIBC)

[0231] BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine) is an environmental chemical carcinogen that can induce mouse muscle-invasive bladder cancer and is widely used in the study of bladder cancer. When 0.05%-0.1% (v / v) of BBN is added to the drinking water of mice, about 15 weeks later, the abnormal hyperplasia of the mouse urothelium develops into squamous differentiation and invasive bladder cancer. This model is highly consistent with the development process and histological changes of human muscle-invasive bladder cancer. Since BBN can induce primary basal muscle-invasive bladder cancer in most mice exposed to it, we used this mouse model to study mice with deletion of the RING domain of XIAP (△RING knock-in), and found that the RING domain, rather than the BIR domain, mediates the development of BBN-induced primary BMIBC. In Figure 12, (A) Comparison of the development of basal muscle-invasive bladder cancer in XIAP wild-type and RING domain deletion mice induced by BBN; (B) After 23 weeks of BBN induction, HE staining and pathological analysis of mouse bladder tissue confirmed that the urothelium developed into muscle-invasive bladder cancer, and the basal bladder cancer marker KRT5 was highly expressed; deletion of the XIAP RING domain can significantly inhibit the development of basal muscle-invasive bladder cancer; (C and D) In the mouse urothelium, the expression of KRT14 cells increases and thickens with the increase of BBN induction time; (E) Comparison of the expression of KRT14 and KRT20 in the urothelium of the bladder of XIAP wild-type and RING domain deletion mice after 23 weeks of BBN induction.

[0232] As shown in Figure 12A, wild-type XIAP and RING domain-deleted mice were given 0.05% BBN in drinking water for 23 weeks. 75% (15 / 20) of XIAP wild-type mice developed bladder cancer, while only 4.5% (1 / 22) of RING domain-deleted mice developed bladder cancer. Thus, RING domain deletion blocked 94% of bladder cancer development. HE staining and immunohistochemical staining with anti-KRT5 and KRT14 antibodies showed that BBN induced BMIBC, and the expression of basal-type marker KRT14 gradually increased as the BBN induction time increased (Figures 12C and 12D); while the expression of luminal-type molecular marker KRT20 gradually decreased, and RING domain deletion significantly reduced the expression of KRT14 (Figure 12E). The results show that deletion of the XIAP RING domain in bladder epithelial cells can significantly inhibit the development of BBN-induced mouse BMIBC, suggesting that the RING domain plays a crucial role in the development of BBN-induced primary BMIBC.

[0233] Example 6 RING domain E3 ligase is a key site for XIAP to promote the development of mouse BMIBCs and cancer cell invasion

[0234] Further studies found that XIAP RING domain can specifically down-regulate the expression of DNMT3B protein in BMIBC cells, therefore, we explored the role of XIAP RING domain in the degradation of DNMT3B. The results are shown in Figure 13, wherein, (A) the role of XIAP RING domain in the degradation of DNMT3B protein was detected by Western blot experiment; (B) the expression level of DNMT3B in the indicated cells was detected by real-time fluorescent quantitative PCR experiment; (C and D) the U5637 cells after knocking out XIAP were successfully returned to RING domain (Myc-RING) and RING domain containing E3 ligase active site point mutation (H467A), respectively, and the influence of Western blot experiment on the degradation of DNMT3B protein was detected; (E) the interaction between DNMT3B and RING domain was detected by immunoprecipitation; (F) the ubiquitination level of DNMT3B was detected by immunoprecipitation; (G and H) the influence of the returned RING domain and the RING domain containing E3 ligase active site point mutation on the migration and infiltration of U5637 cells after knocking out XIAP was detected by Transwell experiment; (I) the expression level of SNHG1 in the indicated cells was detected by real-time fluorescent quantitative PCR experiment; (J) wild type XIAP and H466A mutant XIAP mice (equivalent to human H467A site) were treated with control or BBN for 16 weeks (n=10), the bladder of the mice was taken, and the pathological analysis of the bladder tissue was carried out by HE staining, and the incidence of basal muscle invasive bladder cancer formation in the mice was calculated; (K) the expression level of SNHG1 was detected by real-time fluorescent quantitative PCR.

[0235] Results showed that: compared with BMIBC U5637(Vector), knock-out XIAP could significantly inhibit the degradation rate of DNMT3B, while the return of XIAP RING domain could completely restore the degradation rate of DNMT3B protein (Figure 13A). Compared with protein degradation, mRNA levels did not change significantly in U5637(Vector), U5637(KOXIAP / Vector) and U5637(KOXIAP / RING) three cells (Figure 13B). This result showed that XIAP RING domain regulated the expression of DNMTB by mediating the degradation of DNMT3B protein. RING domain has E3 ligase activity, so is the degradation of DNMT3B protein related to E3 ligase? For this, the E3 ligase active site was point-mutated (H467A) to lose E3 ligase activity, and then the wild-type RING domain was returned to the XIAP knockout cell respectively. The results showed that: compared with the wild-type RING domain, the H467A point-mutated RING domain lost the ability to mediate the degradation of DNMT3B protein (Figure 13C-D), indicating that the E3 ligase activity of XIAP RING domain played an important role in the degradation of DNMT3B protein.

[0236] To determine whether the RING domain E3 ligase uses DNMT3B protein as a substrate and mediates the ubiquitination and degradation of DNMT3B protein, we transfected U5637 (KO XIAP / myc-DNMT3B) cells with wild-type RING and its H467A mutant plasmids, respectively, and lysed the transfected cells. We detected the interaction between DNMT3B and the RING domain by immunoprecipitation. We found that DNMT3B could bind to the RING domain, but the H467A mutant RING domain lost the ability to bind to DNMT3B (Figure 13E). At the same time, the ubiquitination level of DNMT3B in cells transfected with wild-type RING plasmid was significantly increased, while the ubiquitination level of DBMT3B in cells transfected with H467A mutant RING was significantly reduced (Figure 13F). This suggests that the RING domain E3 ligase of XIAP not only binds to the DNMT3B protein, but also mediates the ubiquitination and degradation of the DNMT3B protein. In terms of cell function, cells transfected with the H467A mutant RING plasmid could not restore the invasiveness of cells as well as the wild-type RING plasmid (Figures 13G-H); the restoration of SNHG1 expression in U5637 (KO XIAP) by the XIAP RING plasmid could not be restored by the H467A mutant RING plasmid (Figure 13I). In addition, compared with XIAP wild-type mice, the incidence of BBN-induced BMIBC in bladder epithelial conditionally induced transgenic mice with H466A mutation (equivalent to human H467A site) was significantly reduced (10% vs. 50%, as shown in Figure 13J); the expression of SNHG1 in the urothelial cells of BBN-induced XIAP H466A mutant mice was also significantly inhibited (Figure 13K).

[0237] The above results suggest that the RING domain and its E3 ligase can mediate ubiquitination and degradation of DNMT3B protein as a direct substrate, thereby promoting the expression of SNHG1 and the occurrence and development of primary BMIBC and cancer cell invasion. These findings allow us to fully understand the crucial role of XIAP RING domain E3 ligase in the occurrence and development of BMIBC and cancer cell invasion. It is known in the art that XIAP expression is ubiquitous in most adult and fetal tissues. XIAP expression is increased in various tumors and positively correlated with tumor malignancy: expression in poorly differentiated tumors is significantly higher than in well-differentiated tumors; expression in metastatic tumors is significantly higher than in primary tumors. Moreover, high expression of XIAP is closely related to poor clinical prognosis of tumors, including a strong correlation between XIAP protein levels and survival in patients with acute myeloid leukemia. Since the compounds and compositions of the present application inhibit X-linked inhibitor of apoptosis protein (XIAP), specifically by targeting the XIAP RING domain to alter E3 ligase activity, thereby exerting key biological activities such as inhibition of 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.

[0238] Example 7 Application of bladder cancer model to study the anti-tumor activity of small molecule compounds

[0239] 1. Subcutaneous tumor model to detect the tumor inhibition effect of small molecule compound Com2 in mice in vivo

[0240] The anti-tumor activity of small molecule compound Com2 was evaluated using a subcutaneous tumor model of human bladder cancer cells T24T in nude mice. After 18 nude mice were subcutaneously inoculated with 2x106 T24T cells, they were randomly divided into a control group (Vehicle) and a drug administration group (Com2, 100 mg / kg / day), with 9 nude mice in each group. Drug administration was started the next day after inoculation, once a day for 33 days, and tumor volume was measured every 4 days starting on the 9th day. The results showed (see Figure 14) that after the action of Com2, there was a significant tumor growth inhibition effect compared with the control group, with a tumor inhibition rate of 94.9%.

[0241] 2. Lung metastasis model to detect the tumor lung metastasis inhibition activity of small molecule compound Com1 in mice in vivo

[0242] BALB / C-nu nude mice 3-4 weeks old were selected. T24T bladder cancer cells were expanded, counted after trypsin digestion, resuspended with PBS, and adjusted to a cell density of 3x10 7 / ml. 100 μl (3x106 The cell suspension was injected into the tail vein of the nude mice. After 5 weeks of normal feeding, the mice were randomly divided into two groups, including a vehicle control group and a small molecule compound treatment group (Coml, 100 mg / kg / day), each group of 5. The drug was administered by drinking water for 8 weeks. The physiological indicators (such as body weight, heart rate, respiratory rate, etc.), biochemical indicators (such as blood sugar, blood lipids, liver function, etc.) were monitored every 3 days after administration, and the state of the mice was closely observed. After the drug treatment was completed, the mice were sacrificed, the lung tissue was fixed with picric acid for 24 h, and the number of lung metastases was calculated by taking pictures, paraffin-embedded and HE staining. The inhibitory activity of small molecule compounds on tumor lung metastasis in vivo was evaluated. The results are shown (see Figure 15) that the lung metastases in the Coml treatment group were significantly reduced, and the volume was smaller. The morphological characteristics of the tissue were observed by HE staining, and it was found that the normal tissue cell structure was restored after drug treatment, indicating that Coml can significantly inhibit the formation of tumor lung metastases in mice.

[0243] 3. Target point dependence of the inhibitory effect of small molecule compound Coml on bladder cancer growth in mice

[0244] To verify whether the anti-tumor activity of small molecule compound Coml in bladder cancer is mediated by acting on XIAP E3 ligase activity, human bladder cancer cells T24T were selected for subcutaneous tumor formation experiment in 3-4 week old BALB / C-nu nude mice. The T24T cells were expanded, counted after trypsin digestion, resuspended with PBS, and the cell density was adjusted to 2x10 7ml sterile syringe, 100 μl of the cell suspension was taken up with a 1 ml sterile syringe needle and inoculated subcutaneously into the nude mice, generally within half an hour, with the cell suspension being kept on ice in between to reduce the metabolism of the cells and to maintain the activity of the cells. The mice were randomly divided into four groups (5 mice in each group), including 1) T24T (KOXIAP / XIAP) Vehicle control group; 2) T24T (KOXIAP / XIAP) drug group (10 mg / kg / day); 3) T24T (KOXIAP / H467A) Vehicle control group; and 4) T24T (KOXIAP / H467A) drug group (10 mg / kg / day). The drug was injected intraperitoneally starting from the day after the inoculation of the tumor cells, once a day, for 30 days. The results (see Figure 16) show that the T24T (KOXIAP / XIAP) drug group (10 mg / kg / day) significantly inhibited the growth of the subcutaneous tumors as compared with the control group, with the tumor inhibition rate reaching about 70%, and the T24T (KOXIAP / H467A) cells had a reduced ability to form subcutaneous tumors as compared with the T24T (KOXIAP / XIAP) cells, but no significant change in the growth of the tumors was observed in the T24T (KOXIAP / H467A) drug group (10 mg / kg / day) as compared with the control group. This indicates that Coml inhibits the growth of bladder cancer in mice by specifically targeting the E3 ubiquitin ligase site of XIAP.

[0245] Example 8 Anti-tumor activity of the small molecule compound in a gastric cancer model

[0246] The anti-tumor activity of the small molecule compound Coml against gastric cancer was tested using a subcutaneous tumor model of human gastric adenocarcinoma AGS cells in mice. Sixteen BALB / C-nu nude mice were inoculated subcutaneously with 5 x 10 6 The mice were randomly divided into four groups, a control group (Vehicle) and different drug groups (5 mg / kg / day, 20 mg / kg / day, 100 mg / kg / day), with 4 mice in each group. The drug was administered once a day, intraperitoneally, starting from the day after the inoculation, for 24 days. The results (see Figure 17) show that the drug groups (5 mg / kg / day, 20 mg / kg / day, 100 mg / kg / day) all had an inhibitory effect as compared with the control group, with the inhibition rate reaching 90% at a dose of 20 mg / kg / day of the compound 1 and the human gastric adenocarcinoma AGS cells hardly growing in the nude mice at a dose of 100 mg / kg / day.

[0247] In addition, the anti-tumor activity of the small molecule compound Coml against gastric cancer was further tested using a subcutaneous tumor model of mouse gastric cancer MFC cells in mice. Twenty C57BL6J mice were inoculated subcutaneously with 5 x 10 6MFC cells were randomly divided into four groups: a control group (Vehicle) and groups receiving different concentrations of the drug (5 mg / kg / day, 10 mg / kg / day, and 40 mg / kg / day), with five mice in each group. Drug administration began the day after inoculation and was administered intraperitoneally once daily for 22 days. Results (see Figure 18) showed that, compared to the control group, the drug-treated groups (5 mg / kg / day, 10 mg / kg / day, and 40 mg / kg / day) all exhibited inhibitory effects. Specifically, compound 1 at a dose of 10 mg / kg / day showed almost no growth of gastric cancer MFC cells in mice, demonstrating a highly significant tumor-inhibiting effect.

[0248] Example 9: Studying the antitumor activity of small molecule compounds using a pancreatic cancer model.

[0249] Using a subcutaneous tumorigenesis model of pancreatic cancer in mice with KPC cells, the antitumor activity of the small molecule compound Com1 against pancreatic cancer was further investigated. Twenty-four C57BL / 6J mice were subcutaneously inoculated with 1×10- KPC cells. 6 After inoculating KPC cells, mice were randomly divided into four groups: a control group (Vehicle) and groups receiving different concentrations of the drug (10 mg / kg / day, 40 mg / kg / day, and 100 mg / kg / day), with six mice in each group. Drug administration began the day after inoculation and was administered intraperitoneally once daily for 24 days. Results (see Figure 19) showed that, compared to the control group, the drug-treated groups exhibited tumor-inhibiting effects at high doses (40 mg / kg / day and 100 mg / kg / day), with the drug-treated group (100 mg / kg / day) achieving an inhibition rate of 90%. This indicates that compound 1 has an inhibitory effect on the growth of pancreatic cancer cells in mice.

[0250] Example 10: Studying the antitumor activity of small molecule compounds using a melanoma model.

[0251] Using a subcutaneous tumorigenesis model of human melanoma A375 cells in nude mice, the antitumor activity of the small molecule compound Com1 against melanoma was detected. Twelve nude mice were subcutaneously inoculated with 2 × 10⁶ cells. 6 After inoculating A375 cells, mice were divided into a control group (Vehicle) and a drug-treated group (Com1, 50 mg / kg / day), with 6 mice in each group. Medication was administered via drinking water starting the day after inoculation, once daily for 25 days. Results (see Figure 20) showed that, compared to the control group, the small molecule compound Com1 had a certain inhibitory effect on the growth of human melanoma A375 cells in nude mice, with a tumor inhibition rate of 40%.

[0252] Example 11: Studying the antitumor activity of small molecule compounds using a renal cell carcinoma model.

[0253] The antitumor activity of the small molecule compound X-10 against renal cell carcinoma was detected using a mouse subcutaneous tumorigenesis model of RENCA cells in vivo. 6 × 10⁶ cells were injected into the right ventral region of 25 male 4-6 week old BALB / c mice. 5 Five RENCA cells were randomly divided into a control group, intraperitoneal injection groups with different concentrations (5 mg / kg / day, 20 mg / kg / day, 40 mg / kg / day), and a drinking water administration group (40 mg / kg / day), with five mice in each group. Drug administration began the day after inoculation and was administered once daily for 18 consecutive days. The results (see Figure 21) showed that, compared to the control group, all drug-administered groups exhibited significant inhibitory effects on tumor growth. The tumor inhibition rate in the intraperitoneal injection group (5 mg / kg / day) exceeded 90%, while no tumor cells grew in the intraperitoneal injection groups (20 mg / kg / day, 40 mg / kg / day) and the drinking water administration group (40 mg / kg / day).

[0254] Example 12: Studying the antitumor activity of small molecule compounds using a lung cancer model.

[0255] The antitumor activity of the small molecule compound X-10 against lung adenocarcinoma was detected by subcutaneous tumorigenesis of human lung adenocarcinoma H1299 cells in nude mice. Ten nude mice were subcutaneously inoculated with 6 × 10⁶ cells. 6 After inoculating H1299 cells, mice were randomly divided into a control group and a drug-treated group (40 mg / kg / day), with 5 mice in each group. Drug administration began the day after inoculation and was administered intraperitoneally once daily for 32 days. The results (see Figure 22) showed that, compared to the control group, compound X-10 at a dose of 40 mg / kg / day achieved a tumor inhibition rate of 70%, indicating that X-10 also has significant antitumor activity against lung adenocarcinoma in mice.

[0256] Example 13: Studying the antitumor activity of small molecule compounds using a colorectal cancer model.

[0257] Using a subcutaneous tumorigenesis model of colorectal cancer in mice with MC38 cells, the antitumor activity of the small molecule compound X-10 against colorectal cancer was detected. Ten C57BL / 6J mice were subcutaneously inoculated with 1×10⁻⁶ cells. 6 After inoculating MC38 cells, mice were randomly divided into a control group (Vehicle) and a treatment group (X-10, 40 mg / kg / day), with 5 mice in each group. Treatment began the day after inoculation via drinking water, once daily for 19 days. Results (see Figure 23) showed that, compared to the control group, the treatment group (40 mg / kg / day) exhibited a certain inhibitory effect on tumors, with a tumor inhibition rate reaching 80%. This indicates that compound X-10 has an inhibitory effect on the growth of colorectal cancer cells in mice.

Claims

A method for screening an inhibitor of XIAP, comprising screening for an inhibitor that 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 by Uniprot ID: P98170; preferably, screening for an inhibitor that binds to one or more amino acids corresponding to positions 467, 457, 458, 448, 495, 496 or 497 of human XIAP. Use of an inhibitor of the E3 ubiquitin ligase activity of XIAP for the manufacture of a medicament for the treatment of a tumor or an autoimmune disease. Use according to claim 2, characterized in that The inhibitor of the E3 ubiquitin ligase activity of XIAP is a substance that binds to the C-terminal of XIAP, or binds to the RING domain of XIAP, or binds to the amino acid position of XIAP corresponding to the 467H position of human XIAP, or binds to the RING domain of human XIAP and then inhibits the E3 ligase activity. Use according to claim 3, 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 by Uniprot ID: P98170; preferably, the XIAP inhibitor binds to one or more amino acids corresponding to positions 467, 457, 458, 448, 495, 496 or 497 of human XIAP. Use according to one of claims 2 to 4, 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, myoblastoma; brain cancer, skin cancer, adenocarcinoma, carcinoma, urological tumor, prostate hyperplasia, prostate cancer, urothelial cancer, locally advanced or metastatic urothelial cancer, bladder urothelial cancer, bladder cancer (such as muscle-invasive bladder cancer, non-muscle invasive bladder cancer, primary bladder cancer, invasive bladder cancer, early stage bladder cancer, intermediate stage bladder cancer, metastatic bladder cancer, or advanced bladder cancer), ovarian cancer, gastric adenocarcinoma, breast hyperplasia, 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 nonpolyposis colorectal cancer), esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal cell carcinoma, cervical cancer, cervical squamous carcinoma and adenocarcinoma, uterine body cancer, endometrial cancer, choriocarcinoma, testicular cancer, breast invasive carcinoma, urethral cancer, melanoma, brain tumor, glioma, astrocytoma, meningioma, medulloblastoma, peripheral neuroectodermal tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, leukemia (such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia), adult T-cell leukemia lymphoma, B-cell lymphoma, polycythemia vera, hepatocellular carcinoma, gallbladder cancer, bronchial lung cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, head and neck tumor, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, thyroid tumor, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, plasmacytoma, systemic lupus erythematosus, or rheumatoid arthritis. The use of any one of claims 2-5, wherein the inhibitor is one or more compounds, or a stereoisomer or a tautomer thereof, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof, or a metabolite thereof, or an analog thereof, or a derivative thereof, or a crystal polymorph thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these; the compound is represented by Formula I: wherein: Ring A is a 6-10 membered aryl ring or a 5-10 membered heteroaryl ring; Y1is -(CH2) m -C(=O)-NR a R b -(CH2) n -R6or -(CH2) m -C(=O)-NH-C(=O)-R c ; Y2is -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 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, said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl being optionally substituted by 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, said C1-C6 alkyl, C4-C 10 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl being optionally substituted by 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, said C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl being optionally substituted by one or more R9; R8 is H or C1-C4 alkyl; R a and R b are each independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylCo-C3 alkyl, or phenyl, said C1-C6 alkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylCo-C3 alkyl, C3-C7 cycloalkyl, or phenyl being optionally substituted by one or more R9; or, R a and R b with the attached atoms form a 4-, 5-, 6-, or 7-membered ring amine group, said 4-, 5-, 6-, or 7-membered ring amine group optionally containing zero or one additional heteroatom selected from O, N, and S; R9is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl, said C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl being independently optionally substituted with 0, 1, or 2 C1-C6alkyl or C1-C6haloalkyl; X9is S or O; m, n, and t are each independently 0, 1, 2, or 3. Use according to claim 6, characterized in that The compounds are one or more of the following formulae (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), (II-C), R1, R 1a each independently H, halogen, -OH, -CN, -NO2, -NH2, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, or C1-C4 hydroxyalkoxy; R5is 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 C3-C7 cycloalkyl, C3-C7 cycloalkenyl, 4-7 membered heterocyclyl, 4-7 membered heteroaryl, 4-7 membered aryl, or 5-9 membered bicyclyl; said ring G is optionally substituted with 0, 1, or 2 halogen, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy; R a and R b each independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclyl Co-C3 alkyl, or phenyl, said C1-C6 alkyl, 4-7 membered heterocyclyl, 4-7 membered heterocyclyl Co-C3 alkyl, C3-C7 cycloalkyl, or phenyl optionally substituted with one or more R9; or, R a and R b together with the atom to which they are attached form a 4, 5, 6, or 7 membered cyclic amine moiety, said 4, 5, 6, or 7 membered cyclic amine moiety optionally substituted with one or more R9, said 4, 5, 6, or 7 membered cyclic amine moiety optionally containing zero or one additional heteroatom selected from O, N, and S; R7is 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, said C1-C4 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3 to 10 membered heterocyclyl, 4 to 10 membered heteroaryl, or phenyl optionally substituted with one or more R9; In (III-A), (III-B), (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 atoms to which they are attached form a 4-, 5-, 6-, or 7-membered cyclic amine group, optionally substituted with one or more R 4b , said 4-, 5-, 6-, or 7-membered cyclic amine group optionally containing zero or one additional heteroatom selected from O, N, and S; said 4-, 5-, 6-, or 7-membered cyclic amine group preferably being azetidine, pyrrolidine, piperidine, piperazine, or azepane; R4, R 4a , R 4b , R 4c are each 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 are each 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, said C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3- to 10-membered heterocyclyl, 4- to 10-membered heteroaryl, or phenyl being optionally substituted with one or more R9; R8 is H or C1-C4 alkyl; R a and R b are each independently H, C1-C6 alkyl, C3-C7 cycloalkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylCo-C3 alkyl, or phenyl, said C1-C6 alkyl, 4- to 7-membered heterocyclyl, 4- to 7-membered heterocyclylCo-C3 alkyl, C3-C7 cycloalkyl, or phenyl being optionally substituted with one or more R9; or, R a and R b with the attached atom(s) form a 4-, 5-, 6-, or 7-membered ring amine group, said 4-, 5-, 6-, or 7-membered ring amine group optionally containing zero or one additional heteroatom selected from O, N, and S; R9is H, halogen, OH, -NO2, -NH2, -CN, -C(=O)R8, -OC(=O)R8, -C(=O)OR8, -C(=O)NH2, C1-C6alkyl, C1-C6haloalkyl, C1-C6alkoxy, C1-C6haloalkoxy, C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl, said C3-C6cycloalkyl, 4-7 membered heterocyclyl, or phenyl being independently optionally substituted with 0, 1, or 2 C1-C6alkyl or C1-C6haloalkyl; In (IV-A), (IV-B), (IV-C), (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 nitrogen heterocycloalkyl, 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; said 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; said C3-C6 cycloalkyl, phenyl, or C1-C4 alkylphenyl are 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. Use according to claim 7, characterized in that The compound is one or more of the following numbered 1-65: Use according to one of claims 2 to 8, characterized in that Further comprising an additional therapeutic agent. Use according to one of claims 2 to 9, characterized in that The compound, or a stereoisomer or a tautomer thereof, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt, ester, amide thereof, or a prodrug thereof, or a metabolite thereof, or an analog thereof, or a derivative thereof, or a crystal compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances, is used as a direct active ingredient or a therapeutic agent in achieving the use; for example, as a sole active ingredient or a main active ingredient. Use according to one of claims 2 to 10, characterized in that The XIAP inhibitor is in a therapeutically effective amount. Use according to one of claims 2 to 11, characterized in that The administration is by systemic administration, local administration, parenteral administration, invasive administration, non-invasive administration, or non-invasive administration. A medicament, characterized by comprising The medicament comprises the compound of any one of claims 6-8, or a stereoisomer or a tautomer thereof, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt, ester, amide thereof, or a prodrug thereof, or a metabolite thereof, or an analog thereof, or a derivative thereof, or a crystal compound thereof, or a nitroxide thereof, or a deuterated compound thereof, or a combination of these substances. XIAP C-terminal, or XIAP RING domain, or XIAP fragment with E3 ubiquitin ligase activity, or XIAP amino acid site corresponding to human XIAP 467H site, or XIAP RING domain binding site which changes E3 ligase activity after binding as a target in the preparation of a drug for treating tumors; the amino acid sequence of human XIAP is referred to Uniprot ID: P98170.The tumor includes lung cancer (small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic lung cancer, lung adenocarcinoma, lung squamous carcinoma, large cell lung cancer, bronchiolar alveolar 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 carcinoma), hepatobiliary system (hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), hepatoblastoma, gallbladder cancer, ampullary cancer), pancreatic cancer (ductal adenocarcinoma, acinar cell carcinoma, pancreatic blastoma), renal cancer (clear cell carcinoma, papillary renal cell carcinoma, chromophobe 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 (ductal type / acinar type), 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 neoplasm (polycythemia vera, primary myelofibrosis), lymphoma (Hodgkin's lymphoma (nodular sclerosis type / mixed cell type, etc.), non-Hodgkin's 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 (schwannoma), malignant peripheral nerve sheath tumor (MPNST), teratoma, yolk sac tumor, embryonal carcinoma, choriocarcinoma, thyroid cancer (papillary carcinoma, follicular carcinoma, medullary carcinoma, undifferentiated carcinoma), adrenal cortex cancer, pheochromocytoma / paraganglioma, pituitary adenoma, melanoma, basal cell carcinoma, squamous cell carcinoma, cutaneous T-cell lymphoma (mycosis fungoides), osteosarcoma, chondrosarcoma, Ewing's sarcoma, chordoma, lung carcinoid, gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), small cell neuroendocrine carcinoma, metastatic carcinoma, brain metastasis, bone metastasis, liver metastasis, primary tumor, metastatic poorly differentiated carcinoma, metastatic adenocarcinoma, retinoblastoma, nephroblastoma (Wilms tumor), hepatoblastoma, primitive neuroectodermal tumor (PNET). Use according to claim 14, said target site(s) corresponding to one or more amino acids at position 440, 444, 446, 447, 448, 449, 451, 454, 457, 458, 467, 468, 469, 483, 494, 495, 496 or 497 of the human XIAP, the amino acid sequence of which is referred to Uniprot ID: P98170; preferably, to one or more amino acids at position 467, 457, 458, 448, 495, 496 or 497 of the human XIAP.

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