Small-molecule inhibitor targeting USP1 and application of small-molecule inhibitor

Abstract

The invention relates to a USP1-targeting small-molecule inhibitor and application thereof. Specifically, the invention relates to a compound represented by a general formula (I), an enantiomer, a diastereomer, a tautomer, a salt, a crystal form, a solvate and / or an isotope substituted derivative thereof, and an application thereof in treatment of USP1 related diseases, and is expected to be used for treatment of cancers and other USP1 related diseases. # imgabs0 #

Description

A small molecule inhibitor targeting USP1 and its application

[0001] This application claims priority and benefits of the Chinese patent application No. 202211644240.6 filed with the State Intellectual Property Office of China on December 20, 2022, the entire contents of which are incorporated into this application by reference. Technical Field

[0002] The present application relates to the synthesis and pharmacological application of chemical drugs, and more particularly to inhibitors of ubiquitin-specific protease 1 (USP1) for treating diseases or disorders associated with the ubiquitin-specific protease 1 (USP1). In particular, the present application relates to compounds and compositions that inhibit USP1, methods for treating diseases or disorders associated with USP1, and chemical synthesis methods for the same. Background Art

[0003] Ubiquitin is a small protein composed of 76 amino acids. Ubiquitination refers to the process by which ubiquitin proteins are specifically modified on target proteins by a series of specialized enzymes. Ubiquitination and deubiquitination are dynamic and reversible processes. Deubiquitination involves the removal of ubiquitin chains, or ubiquitin, from substrate proteins. This process is specifically catalyzed by a large class of proteins known as deubiquitinating enzymes (DUBs). To date, approximately 100 human DUBs exist, and their genetic defects and dysfunctions affect or contribute to a variety of human diseases, such as neurological disorders and cancer.

[0004] USP1 is a deubiquitinase in the ubiquitin-specific protease (USP1) family. The full-length human USP1 protein consists of 785 amino acids, making it one of the largest proteins in the deubiquitinase family. Its deubiquitination activity is primarily derived from the catalytic triad consisting of Cys90, His593, and Asp751. USP1 itself is relatively catalytically inactive, and full deubiquitinase activity is achieved only when it associates with UAF1 (USP1-associated factor 1) to form a heterodimeric complex.

[0005] USP1 is involved in the deubiquitination of proliferating cell nuclear antigen (PCNA), Fanconi anemia complementation group protein D2 (FANCD2) and I (FANCI), and is therefore considered an important regulator in the DNA damage and repair process. It is essential for the repair of DNA damage caused by DNA cross-linking agents such as cisplatin, mitomycin C, diepoxybutane, ionizing radiation, and ultraviolet radiation. [1,2] .

[0006] In chicken DT40 cells, studies in which USP1 and UAF1 were knocked out separately or together showed that all three clones showed similar sensitivity to chemical cross-linkers, the topoisomerase poison camptothecin, and poly(ADP-ribose) polymerase (PARP) inhibitors, indicating that the USP1 / UAF1 complex is a regulator of the cellular response to DNA damage. [3] .

[0007] USP1 is overexpressed in osteosarcoma and is associated with maintaining the mesenchymal stem cell state. In human osteosarcoma U2OS cells, silencing USP1 attenuated the proliferation and invasion of U2OS, and the selective inhibitor of USP1, ML323, has the ability to sensitize resistant human osteosarcoma cells to cisplatin. The Hippo signaling pathway is an evolutionarily conserved pathway, and the key effector proteins YAP / TAZ in this pathway are dysregulated in cancer, leading to high cell proliferation, migration, metastasis, and cancer stem cells. USP1 directly interacts with the TAZ protein of the Hippo signaling pathway in human osteosarcoma cells. The pharmacological inhibition of USP1 by ML323 also affects this pathway, inhibiting the growth and metastasis of osteosarcoma both in vivo and in vitro. [4] .

[0008] In studies of breast cancer cells, USP1 has also been shown to be a regulatory protein of TAZ. USP1 interacts with TAZ and increases the stability of the TAZ protein. Loss of USP1 function reduces TAZ protein levels by increasing polyubiquitination, which can lead to reduced cell proliferation and migration of breast cancer cells. Therefore, targeting USP1 is a potential therapeutic intervention for breast cancer patients. [5] .

[0009] Studies have shown that the USP1 inhibitor ML323 can hinder the viability and colony formation of esophageal cancer cells. ML323 blocks esophageal cancer cells at the G0 / G1 phase, accompanied by decreased levels of c-Myc, cyclin D1, CDK4, and CDK6 proteins. ML323 treatment also induces DNA damage and active p53 in esophageal cancer cells. ML323 also stimulates protective autophagy, and co-treatment with two autophagy inhibitors, chloroquine or bafilomycin A1, enhances the cytotoxicity of ML323. [6] .

[0010] In summary, small molecule inhibitors targeting USP1 and their use in compositions have the potential to become treatments for a variety of cancers and other USP1-related diseases.

[0011] [1]Nijman,S.M.,Huang,T.T.,Dirac,A.M.,Brummelkamp,T.R.,Kerkhoven,R.M.,D'Andrea,A.D.,& Bernards,R.(2005).The deubiquitinating enzyme USP1 regulates the Fanconi anemiapathway.Mol Cell,17(3),331-339.

[0012] [2][1]T.,Nijman,S.M.,Mirchandani,K.D.,Galardy,P.J.,Cohn,M.A.,Haas,W.,D'Andrea,A.D.(2006).Regulation of monoubiquitinated PCNA by DUB autocleavage.Nat Cell Biol,8(4),339-347.

[0013] [3]Murai,J.,Yang,K.,Dejsuphong,D.,Hirota,K.,Takeda,S.,& D'Andrea,A.D.(2011).The USP1 / UAF1 complex promotes double-strand break repair through homologous recombination.Mol Cell Biol,31(12),2462-2469.

[0014] [4]Yuan,P.,Feng,Z.,Huang,H.,Wang,G.,Chen,Z.,Xu,G.,Sun,X.(2022).USP1 inhibition suppresses the progression of osteosarcoma via destabilizing TAZ.Int J Biol Sci,18(8),3122-3136.

[0015] [5]Mussell,A.,Shen,H.,Chen,Y.,Mastri,M.,Eng,KH,Bshara,W.,...Zhang,J.(2020).USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer.Cancers(Basel),12(11),3090.

[0016] [6] Sun, Y., Sha, B., Huang, W., Li, M., Zhao, S., Zhang, Y., Chen, P. (2022). ML323, a USP1 inhibitor triggers cell cycle arrest, apoptosis and autophagy in esophageal squamous cell carcinoma cells. Apoptosis, 27(7-8), 545-560.

[0017] Summary of the Invention

[0018] According to one aspect of the present invention, the present invention provides compounds of general formula (I), their enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotopically substituted derivatives as USP1 inhibitors:

[0019] in,

[0020] X is selected from N, CH;

[0021] Z is selected from O, -N(R 3 )-;

[0022] R 1 selected from hydrogen, hydroxy, amino, C1-C6 alkyl, C1-C6 alkoxy which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkylamino which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C3-C8 cycloalkyl, C3-C8 cycloalkylamino, C1-C6 alkylthio; in particular, selected from hydrogen, hydroxy, amino, C1-C3 alkyl, C1-C3 alkoxy which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C1-C3 alkylamino which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C3-C6 cycloalkylamino, C1-C3 alkylthio; more particularly, selected from hydrogen, hydroxy, amino, methyl, ethyl, methoxy, trideuteromethoxy, ethoxy, methylamino, trideuteromethylamino, ethylamino, cyclopropyl, cyclopropylamino, cyclobutylamino, methylthio;

[0023] Especially R 1 It may be selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C8 cycloalkylamino; in particular, selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylamino, C3-C6 cycloalkylamino; more particularly, selected from hydrogen, methyl, ethyl, methoxy, ethoxy, methylamino, ethylamino, cyclopropylamino, cyclobutylamino;

[0024] R 2 is selected from substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-10 membered heteroaryl; in particular, selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl (e.g. 5-6 membered heteroaryl), wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted quinolinyl; wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms Substituted C1-C6 alkoxy, C3-C8 cycloalkyl, amino, amino substituted by 1 or 2 C1-C6 alkyl groups; in particular, the substituents for substitution are each independently selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl, amino, amino substituted by 1 or 2 C1-C3 alkyl groups; for example, the substituents for substitution are each independently selected from 1, 2 or 3 of methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, methylamino, dimethylamino, cyclopropyl, trifluoromethyl, trifluoromethoxy, fluorine and chlorine;

[0025] Especially R 2is selected from substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-10 membered heteroaryl; in particular, selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted quinolinyl, wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, 1-C6 alkoxy, C3-C8 cycloalkyl, amino, amino substituted by 1 or 2 C1-C6 alkyl groups; in particular, the substituents for substitution are each independently selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl, amino, amino substituted by 1 or 2 C1-C3 alkyl groups; for example, the substituents for substitution are each independently selected from 1, 2 or 3 of methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, methylamino, dimethylamino, trifluoromethyl, trifluoromethoxy, fluorine and chlorine; R 3 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C1-C6 alkoxy C1-C6 alkyl; in particular, selected from hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C1-C3 alkoxy C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, trideuteriomethyl, cyclopropyl, methoxymethyl, methoxyethyl;

[0026] Especially R 3 Can be selected from hydrogen, C1-C6 alkyl; in particular, selected from hydrogen, C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl;

[0027] R 4 、R 5 The same or different and independently selected from hydrogen, C1-C6 alkyl; in particular, selected from hydrogen, C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl;

[0028] R 6 Selected from R 7 Substituted or unsubstituted C6-C12 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl, R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7Substituted or unsubstituted cubanes; in particular, selected from R 7 Substituted or unsubstituted C6-C10 aryl, replaced by R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubane, R 7 Substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from R 7 Substituted or unsubstituted phenyl, replaced by R 7 Substituted or unsubstituted pyridyl, R 7 Substituted or unsubstituted quinolinyl, R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubane, wherein unsubstituted means R 7 does not exist,

[0029] In particular, R 6 Can be selected from R 7 Substituted or unsubstituted C6-C12 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl; in particular, selected from R 7 Substituted or unsubstituted C6-C10 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from R 7 Substituted or unsubstituted phenyl, replaced by R 7 Substituted or unsubstituted pyridyl, R 7 Substituted or unsubstituted quinolinyl, wherein unsubstituted represents R 7 does not exist,

[0030] R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-10 membered heteroaryl, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6-alkyl, C1-C6-alkoxy, halogen, C1-C6-alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6-alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C6-alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8-cycloalkyl; in particular, optionally substituted by 1, 2 or 3 of the following groups: C1-C6-alkyl, C1-C6-alkoxy, halogen, C1-C6-alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6-alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8-cycloalkyl; in particular, R 7 Represents R 61, 2 or 3 substituents are independently selected from 5-7 membered heteroaryl groups, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; in particular optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen (e.g. fluorine, chlorine), CF3, CHF2, trideuteromethyl, pentadeuteroethyl, cyclopropyl, cyclobutyl, cyclopentyl; in particular optionally substituted by 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, cyclopropyl, cyclobutyl, cyclopentyl.

[0031] For example, in one embodiment,

[0032] R 1 is selected from hydrogen, hydroxy, amino, C1-C6 alkyl, C1-C6 alkoxy which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkylamino which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C3-C8 cycloalkyl, C3-C8 cycloalkylamino, C1-C6 alkylthio;

[0033] Other substituents are as described above.

[0034] For example, in one embodiment,

[0035] R 2 is selected from substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-10 membered heteroaryl; in particular, selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl (e.g. 5-6 membered heteroaryl), wherein the heteroatom is 1, 2 or 3 nitrogen atoms;

[0036] Other substituents are as described above.

[0037] For example, in one embodiment,

[0038] R 2is selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, and substituted or unsubstituted quinolyl; the substituents for substitution are each independently selected from 1, 2 or 3 of methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, methylamino, dimethylamino, cyclopropyl, trifluoromethyl, trifluoromethoxy, fluorine and chlorine;

[0039] Other substituents are as described above.

[0040] For example, in one embodiment,

[0041] R 3 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C1-C6 alkoxy C1-C6 alkyl;

[0042] Other substituents are as described above.

[0043] For example, in one embodiment,

[0044] R 3 Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, trideuteromethyl, cyclopropyl, methoxymethyl, methoxyethyl;

[0045] Other substituents are as described above.

[0046] For example, in one embodiment,

[0047] R 6 Selected from R 7 Substituted or unsubstituted C6-C12 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl, R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubanes;

[0048] R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-10 membered heteroaryl, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl;

[0049] Other substituents are as described above.

[0050] For example, in one embodiment,

[0051] R 6 Selected from Selected from R 7 Substituted or unsubstituted phenyl, replaced by R 7 Substituted or unsubstituted pyridyl, R 7 Substituted or unsubstituted quinolinyl, R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubanes,

[0052] R 7 Represents R 6 1, 2 or 3 substituents are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, trideuteromethyl, pentadeuteroethyl, cyclopropyl, cyclobutyl, cyclopentyl;

[0053] Other substituents are as described above.

[0054] For example, in one embodiment,

[0055] X is selected from N, CH;

[0056] Z is selected from -N(R 3 )-;

[0057] R 1 Selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylamino, C3-C6 cycloalkylamino;

[0058] R 2 a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted 5-10 membered heteroaryl group, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; wherein the substituents for substitution are each independently selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl, amino, and amino substituted by 1 or 2 C1-C3 alkyl groups;

[0059] R 3 Selected from hydrogen, C1-C3 alkyl;

[0060] R 4 、R 5 are the same or different and are independently selected from hydrogen, C1-C3 alkyl;

[0061] R 6 Selected from R 7 Substituted or unsubstituted C6-C10 aryl, replaced by R 7Substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; wherein unsubstituted means R 7 does not exist,

[0062] R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-7 membered heteroaryl groups, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl.

[0063] For example, in another embodiment,

[0064] X is selected from N, CH;

[0065] Z is selected from -N(R 3 )-;

[0066] R 1 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C8 cycloalkylamino;

[0067] R 2 Selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted quinolinyl, wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl, amino, amino substituted by 1 or 2 C1-C6 alkyl groups;

[0068] R 3 Selected from hydrogen, C1-C6 alkyl;

[0069] R 4 、R 5 are the same or different and are independently selected from hydrogen, C1-C6 alkyl;

[0070] R 6 Selected from R 7 Substituted or unsubstituted C6-C10 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; wherein unsubstituted means R 7 does not exist,

[0071] R 7 Represents R6 1, 2 or 3 substituents are independently selected from 5-7 membered heteroaryl groups, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl.

[0072] For example, in another embodiment,

[0073] X is selected from N, CH;

[0074] Z is selected from -N(R 3 )-;

[0075] R 1 Selected from hydrogen, methyl, ethyl, methoxy, ethoxy, methylamino, ethylamino, cyclopropylamino, cyclobutylamino;

[0076] R 2 Selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted quinolinyl, wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl, amino, amino substituted by 1 or 2 C1-C6 alkyl groups;

[0077] R 3 Selected from hydrogen, methyl, ethyl, n-propyl, isopropyl;

[0078] R 4 、R 5 are the same or different and are independently selected from hydrogen, C1-C6 alkyl;

[0079] R 6 Selected from R 7 Substituted or unsubstituted phenyl, replaced by R 7 Substituted or unsubstituted pyridyl, R 7 Substituted or unsubstituted quinolinyl, wherein unsubstituted represents R 7 does not exist,

[0080] R 7 Represents R 6 1, 2 or 3 substituents are independently selected from pyrazole, imidazole, triazole, wherein R 7Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, cyclopropyl, cyclobutyl, cyclopentyl.

[0081] Preferably, R 2 Selected from:

[0082] Other substituents are as described above.

[0083] Preferably, R 3 Selected from: hydrogen, methyl, ethyl;

[0084] R 4 、R 5 are the same or different and independently selected from hydrogen, methyl; other substituents are as described above.

[0085] Preferably, Selected from:

[0086] Other substituents are as described above.

[0087] According to the present invention, preferably, the general formula (I) is represented by the following general formula (IA),

[0088] wherein each substituent is as described above.

[0089] According to the present invention, preferably, the general formula (I) is represented by the following general formula (IB),

[0090] wherein each substituent is as described above,

[0091] R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from 5-10 membered heteroaryl groups, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl; in particular, R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from a 5-7 membered heteroaryl group, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 represents 1, 2 or 3 substituents on the benzene ring and are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, trideuteromethyl, pentadeuteroethyl, cyclopropyl, cyclobutyl, cyclopentyl; or

[0092] R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from 5-10 membered heteroaryl groups, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl; in particular, R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from a 5-7 membered heteroaryl group, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 represents 1, 2 or 3 substituents on the benzene ring and are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, cyclopropyl, cyclobutyl, cyclopentyl;

[0093] Or two adjacent R 7 They connect to each other and the benzene ring to form benzopyridine.

[0094] In a preferred embodiment, the present invention relates to compounds of formula (I) of USP1 inhibitors, their enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotopically substituted derivatives, wherein: the compound is selected from:

[0095] According to another aspect of the present invention, there is provided a method for preparing a compound of formula (IA), which is carried out by the following route:

[0096] Starting from compound IA, when R 1 When is methylthio and X is N, compound I-A' can be synthesized from compound IA by the following steps:

[0097] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 , X are as described above, and Y is halogen.

[0098] According to another aspect of the present invention, the present invention also relates to a pharmaceutical composition comprising one or more compounds of the above-mentioned general formula (I) according to the present invention, or their enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, and optionally a pharmaceutically acceptable carrier, excipient or diluent.

[0099] According to another aspect of the present invention, the present invention also relates to a pharmaceutical composition comprising one or more compounds of the above-mentioned general formula (I) according to the present invention, or their enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, and optionally a pharmaceutically acceptable carrier, excipient or diluent, for treating and / or preventing diseases or disorders caused by or associated with the activity of ubiquitin-specific protease 1 (USP1).

[0100] According to another aspect of the present invention, the present invention also relates to a method for treating and / or preventing diseases or disorders caused by or related to the activity of ubiquitin-specific protease 1 (USP1), the method comprising administering to a subject an effective amount of a compound of the above-mentioned general formula (I), or its enantiomer, diastereomer, tautomer, salt, crystal form, solvate and / or isotope-substituted derivative, or the pharmaceutical composition.

[0101] According to another aspect of the present invention, the present invention also relates to the use of one or more compounds of the above-mentioned general formula (I) according to the present invention, or their enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, or the pharmaceutical compositions for the preparation of drugs for treating and / or preventing diseases or disorders caused by the activity of ubiquitin-specific protease 1 (USP1).

[0102] In a preferred embodiment, the disease or disorder is selected from the group consisting of cancer; preferably, the cancer is breast cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, liver cancer, esophageal cancer, bladder cancer, kidney cancer, prostate cancer, renal pelvis and ureter cancer, testicular cancer, urethral cancer, Wilms tumor, thyroid cancer, head and neck cancer, nasopharyngeal cancer, acute lymphoblastic leukemia, Acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, melanoma, skin cancer, cutaneous T-cell lymphoma, Hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, chondrosarcoma, Ewing's sarcoma, osteosarcoma and malignant fibrous histiocytoma of bone, childhood rhabdomyosarcoma, soft tissue sarcoma, brain tumor, astrocytoma, brain stem glioma, neuroblastoma, pituitary tumor. Further preferably, the cancer is breast cancer or ovarian cancer.

[0103] As used herein, the term "alkyl" when used alone or as part of another group refers to a straight-chain or branched aliphatic saturated hydrocarbon group. In some embodiments, for example, "C1-C6 alkyl" refers to a straight-chain or branched alkyl group containing 1-6 (1, 2, 3, 4, 5, 6) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0104] As used herein, the term "alkoxy", when used alone or as part of another group, refers to a group of formula -ORa1, wherein Ra1 is an alkyl group. The "C1-C6 alkoxy" herein refers to a "C1-C6 alkyl-O-" group, such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, 1,1-dimethylethoxy, pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, hexyloxy, 1-methylpentoxy, oxy, 2-methylpentyloxy, 3-methylpentyloxy, 4-methylpentyloxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, 1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy and 1-ethyl-2-methylpropoxy.

[0105] As used herein, the term "alkylthio," when used alone or as part of another group, refers to a group of the formula -SR a1, wherein R a1 is alkyl. The term "C1-C6 alkylthio" as used herein refers to a "C1-C6 alkyl-S-" group, such as methylthio, ethylthio, propylthio, butylthio, and the like.

[0106] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, iodine, and the like.

[0107] As used herein, the term "halogenated" means that any one or more substitutable atoms in the group are substituted by halogen, and may be perhalogenated, that is, halogen atoms replace all substitutable positions in the group.

[0108] As used herein, the term "haloalkyl", when used alone or as part of another group, refers to an alkyl group substituted with one or more fluorine, chlorine, bromine and / or iodine atoms. In a preferred embodiment, the haloalkyl group is an alkyl group substituted with 1, 2 or 3 halogen atoms, including, but not limited to, trifluoromethyl, difluoromethyl, 1,1,1-trifluoropropyl, and the like.

[0109] As used herein, the term "cycloalkyl," when used alone or as part of another group, refers to a monocyclic saturated carbocyclic group having, for example, 3 to 8 ring carbon atoms (e.g., "C3-C8 cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 6 ring carbon atoms ("C5-C6 cycloalkyl"). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0110] As used herein, the term "aryl", when used alone or as part of another group, refers to a group having a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in the ring array) with 6-14 carbon atoms and 0 heteroatoms provided in the aromatic ring system ("C6-14 aryl"). In some embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl"; for example, phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C10 aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C14 aryl"; for example, anthracenyl).

[0111] As used herein, the term "heteroaryl", when used alone or as part of another group, refers to a group ("5-10 membered heteroaryl") of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in the ring array) having ring carbon atoms and 1-4 ring heteroatoms (wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur) provided in the aromatic ring system. For example, 5-6 membered heteroaryl groups are included. Where valence permits, in heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom. Heteroaryl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in this case, the number of ring members continues to represent the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on either the aryl or heteroaryl ring, and in such cases, the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. For bicyclic heteroaryls in which one ring does not contain heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., the ring with the heteroatom (e.g., 2-indolyl) or the ring without the heteroatom (e.g., 5-indolyl).

[0112] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, but are not limited to tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, aza Azepinyl, oxa Oxepinyl and thia Exemplary 9-membered heteroaryl groups (5,6-bicyclic heteroaryl groups) include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 10-membered heteroaryl groups (6,6-bicyclic heteroaryl groups) include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0113] As used herein, "substituted" refers to the replacement of one or more hydrogen atoms in another group by one group;

[0114] As used herein, group AB means that group A replaces the hydrogen on group B and connects to the parent core or other groups through the carbon of group B. For example, C1-C6 alkylamino means that the hydrogen on the amino group is replaced by C1-C6 alkyl and connected to the parent core or other groups through N.

[0115] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response) at a reasonable benefit / risk ratio.

[0116] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable solvent, suspending agent or excipient for delivering the active substance of the present invention or a physiologically acceptable salt thereof to animals or humans. The carrier can be liquid or solid.

[0117] Beneficial effects: The compounds of the present invention show excellent inhibitory activity against USP1 / UAF1 enzymes and are expected to be used in the treatment of cancer and other USP1-related diseases. The compounds of the present invention can produce a relatively obvious growth inhibitory effect on related cancer cells. Preferably, the cancer is breast cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, liver cancer, esophageal cancer, bladder cancer, kidney cancer, prostate cancer, renal pelvis and ureter cancer, testicular cancer, urethral cancer, Wilms' tumor, thyroid cancer, head and neck cancer, nasopharyngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, Preferably, the cancer is a leukemia, a chronic myeloid leukemia, a hairy cell leukemia, a melanoma, a skin cancer, a cutaneous T-cell lymphoma, a Hodgkin's lymphoma, a mycosis fungoides, a non-Hodgkin's lymphoma, a primary central nervous system lymphoma, a chondrosarcoma, an Ewing's sarcoma, an osteosarcoma and a malignant fibrous histiocytoma of bone, a childhood rhabdomyosarcoma, a soft tissue sarcoma, a brain tumor, an astrocytoma, a brain stem glioma, a neuroblastoma, a pituitary tumor. Further preferably, the cancer is a breast cancer or an ovarian cancer. DETAILED DESCRIPTION

[0118] Synthesis of the compounds of Example 1 and Example 2:

[0119] Step 1: Compound 1 (1.2 equivalents), compound 2 (1.0 equivalents), and NaHCO₃ (3.0 equivalents) were dissolved in THF and reacted at 0°C for 12 hours. The mixture was analyzed by thin-layer chromatography. After completion of the reaction, the mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 3 in a 47% yield. 1 H NMR(600MHz,Chloroform-d)δ7.44(d,J=7.8Hz,2H),7.34(d,J=9.6Hz,3H),6.98–6.46(m,1H),4.66 (d,J=6.1Hz,2H),4.47(h,J=6.6Hz,1H),3.89(d,J=18.6Hz,3H),1.38(d,J=6.7Hz,6H).MS(APCI)m / z 427.1[M+1] +

[0120] Step 2: Compound 3 (1.0 equivalent), boronic acid compound 4 (1.2 equivalents), Pd(dppf)Cl2 (10% equivalents), and potassium carbonate (3.0 equivalents) were dissolved in 1,4-dioxane and water (3:1 v / v). The atmosphere was replaced with argon three times and the temperature was raised to 90°C under argon protection for 3 hours. After completion of the reaction, the insoluble matter was filtered off and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed once with brine and once with water, and dried over anhydrous sodium sulfate, filtered, concentrated, and then separated by silica gel column chromatography to obtain Example 1 in a yield of 73%.

[0121] Step 3: Dissolve the compound of Example 1 (1.0 equivalent) in DMF, cool to 0°C, add sodium hydride (3.0 equivalents), stir for 30 minutes, add iodomethane (3.0 equivalents), and react at room temperature for 1 hour. After completion, quench with saturated aqueous ammonium chloride, extract with ethyl acetate, and wash the organic phase three times with saturated brine. The combined organic phases are dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to obtain the compound of Example 2 in a 49% yield.

[0122] Synthesis of Example 59 Compound:

[0123] Step 1: To a solution of Example 78 (1.0 equiv) in dichloromethane was added 3-chlorophenylcarboperoxyacid (2.5 equiv), followed by stirring at room temperature for 3 hours. The mixture was quenched with water, diluted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product, Intermediate 2, was used directly in the next reaction without further purification.

[0124] Step 2: To a THF solution of intermediate 2 (1.0 equiv.) and methylamine hydrochloride (3.0 equiv.) was added N,N-diisopropylethylamine (3.0 equiv.). The mixture was stirred at 40°C for 16 hours, cooled to room temperature, poured into water, and extracted with ethyl acetate. The combined organic phases were washed three times with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by flash column chromatography gave Example 59 in a two-step total yield of 38%.

[0125] According to the synthesis steps of Example 1 and / or Example 59, using appropriate dichlorotriazine / pyrimidine, (deuterated) amino / hydroxyl compounds, boronic acid / boronic esters and (deuterated) alkyl halides, the example compounds shown in Table 1 below were synthesized:

[0126] Table 1

[0127] Test Example 1: Half maximal inhibitory concentration (IC) of the compound on USP1 / UAF1 enzyme 50 )

[0128] The half-maximal inhibitory concentration (IC50) of the compound on USP1 / UAF1 50 The method for evaluating the fluorescence intensity (V) is to measure the deubiquitinase activity of USP1 / UAF1 using Ub-AMC as a substrate. The assay principle is that USP1 / UAF1 cleaves the amide bond between AMC and the C-terminal glycine of ubiquitin, which increases the fluorescence signal.

[0129] The assay conditions are as follows: to 35 μl of 2.5 nM USP1 / UAF1 enzyme solution, add 70 μl of buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.05% Tween 20) and 35 μl of a series of small molecule compound solutions (containing 5% DMSO) at different concentrations (500 μM, 150 μM, 50 μM, 15 μM, 5 μM, 1.5 μM, 0.5 μM, 0.15 μM, 0.05 μM, 0.015 μM, 0.005 μM), gently mix 140 μl of the mixture, and incubate at the assay temperature for 20 min. Aspirate 40 μl of the mixture and add 10 μl of 5 μM substrate solution to initiate the reaction. Immediately place the plate in a microplate reader and read the reaction in kinetic mode at 25°C using the Ex360 / Em460 setup, reading every 30 seconds for 10 minutes. A control is replaced with 35 μl of 5% DMSO-containing buffer without compound. Calculate the relative percentage activity of USP1 / UAF1 using the data. One blank well and three sample wells are included in the assay.

[0130] The relative activity of USP1 / UAF1 at different concentrations of compounds was calculated as follows:

[0131] Activity data were analyzed and plotted using GraphPad Prism statistical software. A nonlinear regression model ([Inhibitor] vs. response--Variable slope four parameters) was used to draw S-shaped dose-activity curves and calculate IC 50 value.

[0132] Note: cpd_Time 1 is the average of the initial readings of the experimental group, cpd_Time 2 is the average of the readings of the experimental group after 10 minutes of reaction, ctrl_Time 1 is the average of the initial readings of the 1% DMSO control group, and ctrl_Time 2 is the average of the readings of the 1% DMSO control group after 10 minutes of reaction.

[0133] Reagents: Ub-AMC, Hefei KS-V Peptide Biotechnology Co., Ltd; 5M Nacl, Sango Biotech, B548121; 2M Tris-Hcl 7.5, Sango Biotech, Cat#B548139; 7.3μM USP1 / UAF1, R&D Systems.

[0134] The half-maximal inhibitory concentration (IC50) of some compounds of the present invention on USP1 / UAF1 enzyme 50 The half-maximal inhibitory concentration (IC) of the compound on USP1 / UAF1 enzyme 50 , μM): “+” indicates IC 50 >5μM; “++” means 1μM <IC 50 ≤5μM; “+++” means 100nM <IC 50 ≤1μM; “++++” indicates IC 50 ≤100nM.

[0135] Experimental Example 2: The half-maximal effective concentration (EC50) of the compound on the colony formation of breast cancer cells MDA-MB-436 50 )

[0136] 1. Cell plating: Digest MDA-MB-436 cells in the logarithmic growth phase, count, and plate 6×10 cells in a 6-well plate. 3 For cells, add 1.5-2 mL of culture medium;

[0137] 2. Compound treatment: On the second day after plating cells, treat the cells with a gradient of compound concentrations (usually 0, 0.1, 0.3, 1, 3, 10 μM, and 0.001, 0.01, and 0.03 μM concentrations can be added). Treat for 14 days, changing the culture medium every 3 days.

[0138] 3. Cell fixation: Remove the culture medium, add 1 mL of 10% neutral formalin solution (Sanggong E672001-0500), and fix at room temperature for 10 minutes;

[0139] 4. Color development: Remove the fixative and add 1 mL of 0.05% crystal violet (Sanggong A100528-0025, prepared with 20% methanol) and stain at room temperature for 30 min.

[0140] 5. Washing: Remove the staining solution and wash the plate with tap water until the blank area at the bottom of the well is free of color. Turn the plate upside down until it is dry.

[0141] 6. Scan: Place the 6-well plate in the middle of the scanner, scan, and save the image;

[0142] 7. Quantitative calculation of EC50: Add 1 mL of 10% acetic acid solution to each well, shake until the crystal violet is completely dissolved, take 100 μL to a 96-well plate, measure the absorbance at 590 nm, and calculate the EC50 by fitting. 50 .

[0143] The half-maximal effective concentration (EC50) of some compounds of the present invention on breast cancer cells MDA-MB-436 50 ). “+” indicates EC 50 >100nM; “++” indicates EC 50 ≤100nM.

[0144] Experimental Example 3: The half-maximal effective concentration (EC50) of the compound on the colony formation of ovarian cancer cells Caov-3 50 )

[0145] 1. Cell plating: Digest Caov-3 cells in the logarithmic growth phase, count, and plate 6×10 cells in a 6-well plate. 4 For cells, add 1.5-2 mL of culture medium;

[0146] 2. Compound treatment: On the second day after plating cells, treat the cells with a gradient of compound concentrations (usually 0, 0.1, 0.3, 1, 3, 10 μM, and 0.001, 0.01, and 0.03 μM concentrations can be added). Treat for 14 days, changing the culture medium every 3 days.

[0147] 3. Cell fixation: Remove the culture medium, add 1 mL of 10% neutral formalin solution (Sanggong E672001-0500), and fix at room temperature for 10 minutes;

[0148] 4. Color development: Remove the fixative and add 1 mL of 0.05% crystal violet (Sanggong A100528-0025, prepared with 20% methanol) and stain at room temperature for 30 min.

[0149] 5. Washing: Remove the staining solution and wash the plate with tap water until the blank area at the bottom of the well is free of color. Turn the plate upside down until it is dry.

[0150] 6. Scan: Place the 6-well plate in the middle of the scanner, scan, and save the image;

[0151] 7. Quantitative calculation of EC50: Add 1 mL of 10% acetic acid solution to each well, shake until the crystal violet is completely dissolved, take 100 μL to a 96-well plate, measure the absorbance at 590 nm, and calculate the EC50 by fitting. 50 .

[0152] The half effective concentration (EC50) of some compounds of the present invention on ovarian cancer cells Caov-3 50 ). “+” indicates EC 50 >100nM; “++” indicates EC 50 ≤100nM.

Claims

1. Compounds of general formula (I), their enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives: in, X is selected from N, CH; Z is selected from O, -N(R 3 )-; R 1 selected from hydrogen, hydroxy, amino, C1-C6 alkyl, C1-C6 alkoxy which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkylamino which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C3-C8 cycloalkyl, C3-C8 cycloalkylamino, C1-C6 alkylthio; in particular, selected from hydrogen, hydroxy, amino, C1-C3 alkyl, C1-C3 alkoxy which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C1-C3 alkylamino which is unsubstituted or substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C3-C6 cycloalkylamino, C1-C3 alkylthio; more particularly, selected from hydrogen, hydroxy, amino, methyl, ethyl, methoxy, trideuteromethoxy, ethoxy, methylamino, trideuteromethylamino, ethylamino, cyclopropyl, cyclopropylamino, cyclobutylamino, methylthio; R 2 is selected from substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-10 membered heteroaryl; in particular, selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl (e.g. 5-6 membered heteroaryl), wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted quinolinyl; wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms Substituted C1-C6 alkoxy, C3-C8 cycloalkyl, amino, amino substituted by 1 or 2 C1-C6 alkyl groups; in particular, the substituents for substitution are each independently selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl, amino, amino substituted by 1 or 2 C1-C3 alkyl groups; for example, the substituents for substitution are each independently selected from 1, 2 or 3 of methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, methylamino, dimethylamino, cyclopropyl, trifluoromethyl, trifluoromethoxy, fluorine and chlorine; R 3 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C1-C6 alkoxy C1-C6 alkyl; in particular, selected from hydrogen, C1-C3 alkyl, C1-C3 alkyl substituted by 1, 2 or 3 deuterium atoms, C3-C6 cycloalkyl, C1-C3 alkoxy C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, trideuteriomethyl, cyclopropyl, methoxymethyl, methoxyethyl; R 4 、R 5 The same or different and independently selected from hydrogen, C1-C6 alkyl; in particular, selected from hydrogen, C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl; R 6 Selected from R 7 Substituted or unsubstituted C6-C12 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl, R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubanes; in particular, selected from R 7 Substituted or unsubstituted C6-C10 aryl, replaced by R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubane, R 7 Substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from R 7 Substituted or unsubstituted phenyl, replaced by R 7 Substituted or unsubstituted pyridyl, R 7 Substituted or unsubstituted quinolinyl, R 7 Substituted or unsubstituted bicyclo[2.2.2]octyl, replaced by R 7 Substituted or unsubstituted cubane, wherein unsubstituted means R 7 does not exist, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-10 membered heteroaryl, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, substituted by 1, 2 or 3 halogen atoms substituted C1-C6 alkyl, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl; in particular, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-7 membered heteroaryl groups, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, trideuteromethyl, pentadeuteroethyl, cyclopropyl, cyclobutyl, cyclopentyl; In particular, X is selected from N, CH; Z is selected from O, -N(R 3 )-; R 1 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, C3-C8 cycloalkylamino; R 2 is selected from substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 5-10 membered heteroaryl; wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl, amino, amino substituted by 1 or 2 C1-C6 alkyl groups; R 3 Selected from hydrogen, C1-C6 alkyl; R 4 、R 5 are the same or different and are independently selected from hydrogen, C1-C6 alkyl; R 6 Selected from R 7 Substituted or unsubstituted C6-C12 aryl, replaced by R 7 Substituted or unsubstituted 5-10 membered heteroaryl; wherein unsubstituted represents R 7 does not exist, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-10 membered heteroaryl, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl.

2. The compound of general formula (I) according to claim 1, its enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, wherein: X is selected from N, CH; Z is selected from O, -N(R 3 )-; R 1 Selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylamino, C3-C6 cycloalkylamino; more particularly, selected from hydrogen, methyl, ethyl, methoxy, ethoxy, methylamino, ethylamino, cyclopropylamino, cyclobutylamino; R 2 is selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, wherein the heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted quinolinyl, wherein the substituents for substitution are each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl, amino , an amino group substituted by 1 or 2 C1-C6 alkyl groups; in particular, the substituents for substitution are each independently selected from C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl, amino, amino substituted by 1 or 2 C1-C3 alkyl groups; for example, the substituents for substitution are each independently selected from 1, 2 or 3 of methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, methylamino, dimethylamino, trifluoromethyl, trifluoromethoxy, fluorine and chlorine; R 3 is selected from hydrogen, C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl; R 4 、R 5 are identical or different and independently selected from hydrogen, C1-C3 alkyl; more particularly, selected from hydrogen, methyl, ethyl, n-propyl, isopropyl; R 6 Selected from R 7 Substituted or unsubstituted C6-C10 aryl, replaced by R 7 a substituted or unsubstituted 5-10 membered heteroaryl group, wherein The heteroatom is 1, 2 or 3 nitrogen atoms; more particularly, selected from R 7 Substituted or unsubstituted phenyl, replaced by R 7 Substituted or unsubstituted pyridyl, R 7 Substituted or unsubstituted quinolinyl, wherein unsubstituted represents R 7 does not exist, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from 5-7 membered heteroaryl groups, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 Represents R 6 1, 2 or 3 substituents are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, cyclopropyl, cyclobutyl, cyclopentyl.

3. The compound of general formula (I) according to claim 1 or 2, its enantiomer, diastereomer, tautomer, salt, crystal form, solvate and / or isotope-substituted derivative, wherein: Z is selected from -N(R 3 )-; and / or R 2 Selected from: and / or R 3 Selected from: hydrogen, methyl, ethyl; and / or R 4 、R 5 are the same or different and independently selected from hydrogen, methyl; and / or Selected from:

4. The compound of general formula (I) according to any one of claims 1 to 3, its enantiomer, diastereomer, tautomer, salt, crystal form, solvate and / or isotope-substituted derivative, wherein: The general formula (I) is represented by the following general formula (IA), where R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 , X are respectively as described in any one of claims 1 to 3.

5. The compound of general formula (I) according to any one of claims 1 to 3, its enantiomer, diastereomer, tautomer, salt, crystal form, solvate and / or isotope-substituted derivative, wherein: The general formula (I) is represented by the following general formula (IB), where R 1 、R 2 、R 3 、R 4 、R 5 , X are as described in any one of claims 1 to 3, R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from 5-10 membered heteroaryl groups, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl; in particular, R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from a 5-7 membered heteroaryl group, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkyl substituted by 1, 2 or 3 deuterium atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 represents 1, 2 or 3 substituents on the benzene ring and are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, trideuteromethyl, pentadeuteroethyl, cyclopropyl, cyclobutyl, cyclopentyl; or R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from 5-10 membered heteroaryl groups, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C6 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C8 cycloalkyl; in particular, R 7 represents 1, 2 or 3 substituents on the benzene ring and are each independently selected from a 5-7 membered heteroaryl group, wherein the heteroatom is 1, 2 or 3 nitrogen atoms, wherein R 7 Optionally substituted by 1, 2 or 3 of the following groups: C1-C3 alkyl, C1-C3 alkoxy, halogen, C1-C3 alkyl substituted by 1, 2 or 3 halogen atoms, C1-C3 alkoxy substituted by 1, 2 or 3 halogen atoms, C3-C6 cycloalkyl; more particularly, R 7 represents 1, 2 or 3 substituents on the benzene ring and are independently selected from pyrazole, imidazole, triazole, wherein R 7 Optionally substituted with 1, 2 or 3 of the following groups: methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, halogen, CF3, CHF2, cyclopropyl, cyclobutyl, cyclopentyl; Or two adjacent R 7 They connect to each other and the benzene ring to form benzopyridine.

6. The compound of general formula (I) according to claim 1, its enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, wherein: The compound is selected from:

7. A pharmaceutical composition comprising one or more of the compound of general formula (I) according to any one of claims 1 to 6, its enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, and optionally a pharmaceutically acceptable carrier, excipient or diluent.

8. Use of a compound of formula (I) according to any one of claims 1 to 6, its enantiomers, diastereomers, tautomers, salts, crystal forms, solvates and / or isotope-substituted derivatives, or the pharmaceutical composition according to claim 7 for the preparation of a medicament for treating and / or preventing diseases or disorders caused by the activity of ubiquitin-specific protease 1 (USP1).

9. The use according to claim 8, wherein the disease or disorder is selected from the group consisting of cancer; preferably, the cancer is breast cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, liver cancer, esophageal cancer, bladder cancer, kidney cancer, prostate cancer, renal pelvis and ureter cancer, testicular cancer, urethral cancer, Wilms' tumor, thyroid cancer, head and neck cancer, nasopharyngeal cancer, acute lymphoblastic leukemia, Blood cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, melanoma, skin cancer, cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, chondrosarcoma, Ewing's sarcoma, osteosarcoma and malignant fibrous histiocytoma of bone, childhood rhabdomyosarcoma, soft tissue sarcoma, brain tumors, astrocytoma, brain stem glioma, neuroblastoma, pituitary tumors.

10. The use according to claim 9, wherein The cancers include breast cancer and ovarian cancer.

Images