USP1 inhibitor and pharmaceutical use thereof

Abstract

The present invention relates to a USP1 inhibitor and pharmaceutical use thereof. Specifically, the present invention relates to a combination product comprising a USP1 inhibitor and an additional anti-tumor drug other than the USP1 inhibitor, and pharmaceutical use thereof. The USP1 inhibitor has a structure represented by formula I:

Description

USP1 inhibitors and their medical uses

[0001] This application claims priority to Chinese patent application 202410082515.4 filed on January 19, 2024. Technical Field

[0002] The present invention relates to the field of cancer treatment, and in particular to a novel use of USP1 in treating cancer. Background Art

[0003] Ubiquitin-specific protease 1 (USP1) is a well-characterized member of the deubiquitinating enzyme family that cleaves ubiquitin from various target proteins. USP-1 expression is dysregulated in many cancers, where it acts as an oncogenic driver in various DNA damage repair processes, including translesion synthesis and Fanconi anemia pathways. Loss of USP1 leads to reduced cell survival and replication fork degradation, suggesting that USP1 inhibitors may be particularly useful in BRCA-deficient tumors.

[0004] The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a role in DNA repair and genome integrity. PARP is essential for the single-strand break repair and base excision repair pathways. The key enzymatic activity is the addition of ADP-ribose to substrate proteins via cleavage of NAD+ and release of nicotinamide. Breaks in the DNA strand activate this poly(ADP-ribosylation) ("PARylation") activity, thereby adding Par to PARP itself and other DNA repair enzymes. PARP is essential for the recruitment of DNA repair proteins to sites of damage. Although the use of PARP inhibitors (PARPi) has achieved clinical benefit in people with BRCA mutations (BRCAm) or homologous recombination deficiency (HRD+), some patients either do not respond to treatment or develop resistance. USP1 inhibitors may have the potential to address this unmet clinical need. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a combination product comprising a USP1 inhibitor, or a pharmaceutically acceptable salt, hydrate, solvate, isotope-substituted product or stereoisomer thereof; and an anti-tumor drug other than the USP1 inhibitor, wherein the USP1 inhibitor has a structure shown in Formula I:

[0006] wherein Ring A and Ring B are each independently selected from C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8Cycloalkyl and 3-8 membered heterocyclyl, and Ring A and Ring B are each independently optionally substituted by one or more R1;

[0007] L is selected from chemical bonds, -O-, -S-, -C 1-6 Alkylene-, -OC 1-6 Alkylene-, -C 1-6 Alkylene-O-, -SC 1-6 Alkylene- and -C 1-6 Alkylene-S-;

[0008] R a and R b Each independently selected from H atoms, -CN, C 1-6 Alkyl, -OH, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Hydroxyalkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy; or R a and R b Together they form an oxo group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic group;

[0009] R2 is selected from H atoms, -OH, -CN, C 1-6 Alkyl, C 2-6 Alkynyl, -C 1-6 Alkyl-C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 3-6 Cycloalkyl and 5-7 membered heterocyclic groups, wherein the C 1-6 Alkyl or -C 1-6 Alkyl-C 6-10 Aryl is optionally substituted with one or more R1;

[0010] Preferably, R2 is selected from the group consisting of an H atom, -CN, a methyl group, a trideuterated methyl group, an ethynyl group, a propynyl group, a tetrahydrofuranyl group, a cyclopropyl group, a methoxy group, and a hydroxyl group;

[0011] R3 is selected from the group consisting of H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, 5-7 membered heterocyclic group and -C 1-6Alkylene-C(O)-OC 1-6 Alkyl, wherein the C 1-6 The hydroxyalkyl and 5-7 membered heterocyclic groups are each independently optionally substituted with one or more C 1-6 Alkyl substitution;

[0012] Preferably, R3 is selected from H atom, methoxy, trifluoromethyl, Cl atom, -CN, isopropoxy, ethynyl, difluoromethoxy, morpholinyl, -OH, F atoms, hydroxymethyl and

[0013] More preferably, R3 is selected from H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl;

[0014] More preferably, R3 is selected from H atoms, C 1-6 alkyl;

[0015] R4 is selected from the group consisting of H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy and C 1-6 hydroxyalkyl;

[0016] Preferably, R4 is an H atom;

[0017] R5 is selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 6-10 Aryl, C fused to 3-8 membered heterocyclic group 6-10 Aryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, wherein the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8Cycloalkyl and 3-8 membered heterocyclyl are each independently optionally substituted with one or more R1;

[0018] R1 is independently selected at each occurrence from a D atom, -OH, -COOH, -NH2, -CN, an oxo group, a halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, -OC 1-6 Alkylene-OC 1-6 Alkyl, wherein the C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 The cycloalkyl and 3-8 membered heterocyclic groups are each independently optionally selected from D atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy and C 1-6 substituted with one or more substituents of a hydroxyalkyl group, and

[0019] n is an integer between 0 and 8.

[0020] In one embodiment of the combination product of the present invention, wherein

[0021] Ring A and Ring B are each independently selected from phenyl, piperidinyl, cyclohexyl, cyclopropyl, cyclobutyl, pyridinyl, pyrimidinyl, imidazolyl, pyrazolyl, bicyclo[2.2.2]octanyl, 2-oxabicyclo[2.2.2]octanyl, pentacyclooctanyl, isoindolinone, imidazo[1,2-a]pyrazinyl, piperidine-2,6-dione, thienyl, furanyl, cyclopentyl, pyranyl, pyrrolidinyl, piperazinyl, morpholinyl, naphthyl, pyrrolyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, indolyl, isoindolyl, indolinyl, isoindolyl, indolinonyl, pyrido[3,2-d]pyrimidinyl, pteridinyl, pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4-d]pyrimidinyl and cubanyl, and ring A and ring B are each independently optionally substituted with one or more R1, R1 is as defined in claim 1;

[0022] L is selected from chemical bonds, -O-, -OC 1-6 Alkylene- and -C 1-6 Alkylene-O-;

[0023] In particular, Selected from

[0024] and Ring A and Ring B are each independently optionally substituted by one or more R1, R1 being as defined in claim 1;

[0025] More specifically, Selected from

[0026] The combination product according to the present invention, wherein R5 is selected from C 6-10 Aryl, 5-6 membered heteroaryl, C 6-10 Aryl and C fused to 5-6 membered heteroaryl 6-10 Aryl, preferably selected from phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, thiazolyl, indolyl, indolyl and isoxazolyl, said C 6-10 Aryl, 5-6 membered heteroaryl, C 6-10 Aryl and C fused to 5-6 membered heteroaryl 6-10 Each aryl group is independently optionally selected from -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl and C 3-6 The cycloalkyl group is substituted with one or more substituents;

[0027] In particular, R5 is selected from

[0028] More specifically, R5 is

[0029] The combination product according to the present invention, wherein n is 0 or 1;

[0030] R a and R b Each independently selected from H atoms, -CN, C 1-6 Alkyl, -OH and halogen;

[0031] Preferably, R a and R b are each independently a H atom.

[0032] In one embodiment, the present invention provides a combination product comprising:

[0033] USP1 inhibitor, or a pharmaceutically acceptable salt, hydrate, solvate, isotope-substituted substance or stereoisomer thereof; and

[0034] Other anti-tumor drugs besides USP1 inhibitors;

[0035] wherein the USP1 inhibitor is selected from:

[0036] In particular, the present invention provides the use of the combination product of the present invention for treating cancer.

[0037] In specific embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, uterine and peritoneal cancer, and endometrial cancer.

[0038] In another aspect, the present invention provides use of the USP1 inhibitor having the structure shown in Formula I in combination with other anti-tumor drugs other than the USP1 inhibitor in the preparation of a drug for treating cancer.

[0039] In one aspect, the anti-tumor drug other than the USP1 inhibitor described herein is selected from a PARP inhibitor, an antimetabolite anti-tumor drug and a platinum coordination complex;

[0040] Specifically, the PARP inhibitor is selected from niraparib, pamiparib, olaparib, faruzopanib, rucaparib, saruparib, talazoparib;

[0041] Specifically, the antimetabolite anti-tumor drug is selected from gemcitabine, fluorouracil, methotrexate, cytarabine, mercaptopurine and thioguanine; and

[0042] The platinum coordination complex is selected from cisplatin and carboplatin.

[0043] In a specific embodiment, the anti-tumor drug other than the USP1 inhibitor described herein is an anti-angiogenic drug.

[0044] Anti-angiogenic drugs mainly inhibit the formation of new blood vessels in tumors by blocking angiogenesis-related pathways, thereby limiting the nutrient and oxygen supply to the tumor and inhibiting its growth and metastasis.

[0045] In a specific embodiment, the anti-angiogenic drug is selected from VEGF / VEGFR pathway inhibitors and other drugs with anti-angiogenic mechanisms;

[0046] In a specific embodiment, the VEGF / VEGFR pathway inhibitor is selected from monoclonal antibodies, including but not limited to bevacizumab and ramucirumab; and

[0047] Small molecule tyrosine kinase inhibitors (TKIs), including but not limited to sorafenib, sunitinib, apatinib, fruquintinib, regorafenib, cabozantinib, and lenvatinib;

[0048] In a specific embodiment, the other anti-angiogenic mechanism drug is selected from:

[0049] integrin inhibitors, such as ceritinib;

[0050] Platelet-derived growth factor receptor (PDGFR) inhibitors, such as sunitinib and sorafenib;

[0051] HIF (hypoxia-inducible factor) inhibitors, such as Belinostat, and

[0052] Endothelin receptor antagonists, such as Bosentan.

[0053] In another aspect, the present invention provides a novel, effective, and selective USP1 inhibitor for use in the preparation of a medicament for treating cancer:

[0054] 4'-cyclopropyl-5,6'-dimethoxy-N-((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]octan-1-yl)methyl)-[2,5'-bipyrimidinyl]-4-amine, referred to herein as 104, has the structure shown below:

[0055] Our studies confirmed that 104 is a potent and selective USP1 inhibitor with an IC 50 The selectivity of 104 for USP1 is about 9 nM, and it is more than 1000 times that of USP12 and USP46. It effectively inhibits the growth of tumor cell lines in vitro and exerts effective anti-tumor effects in BRCAm and HRD models in vivo. 104 shows effective anti-proliferative activity (IC 50 , 30 nM). The MDA-MB-436CDX model confirmed the dose-dependent single-agent inhibitory activity of 104, with a tumor growth inhibition (TGI) of 78% at 100 mg / kg QD, and greater potency than KSQ4279. This efficacy was consistent with the increase in ub-PCNA.

[0056] On the other hand, we demonstrated that the combination of 104 and olaparib produced more robust and durable antitumor responses, with TGIs of 108% (TNBC BRCA1 mut; 104, 30 mg / kg QD + olaparib, 100 mg / kg QD) and 103% (104, 30 mg / kg QD + olaparib, 100 mg / kg QD) in MX-1 (TNBC, BCRA1 / 2mut), respectively. Notably, in PARPi-resistant CAOV3 (TNBC, BCRA1 / 2mut), the addition of 104 significantly enhanced the antitumor activity of olaparib, suggesting its potential to overcome PARPi resistance. Combination lethality was also observed when 104 was combined with cisplatin, which causes DNA crosslink damage, or gemcitabine, a drug that impairs DNA synthesis and repair. 104 also exhibited very favorable ADME and PK properties, and non-GLP toxicology studies demonstrated an acceptable therapeutic window. These data support the future clinical development of 104 as a potential best-in-class USP1 inhibitor, either as a single agent or in combination with PARPi, for PARPi-resistant / responsive HRD-mutant cancers.

[0057] Based on the above unexpected discovery, in a first aspect, the present invention provides a use of a USP1 inhibitor in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0058] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with olaparib in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0059] In specific embodiments, the USP1 inhibitor is co-administered with olaparib in the same formulation or in different formulations.

[0060] In specific embodiments, the USP1 inhibitor and olaparib are administered to the subject by the same route or different routes.

[0061] In specific embodiments, the USP1 inhibitor and olaparib are administered to the subject parenterally or orally.

[0062] In a specific embodiment, the USP1 inhibitor is used with olaparib to treat breast cancer and / or ovarian cancer.

[0063] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with cisplatin in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0064] In specific embodiments, the USP1 inhibitor is co-administered with cisplatin in the same formulation or in different formulations.

[0065] In specific embodiments, the USP1 inhibitor and cisplatin are administered to the subject by the same route or different routes.

[0066] In specific embodiments, the USP1 inhibitor and cisplatin are administered to the subject parenterally or orally.

[0067] In specific embodiments, the USP1 inhibitor is used with cisplatin to treat breast cancer and / or ovarian cancer.

[0068] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with gemcitabine in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0069] In specific embodiments, the USP1 inhibitor is co-administered with gemcitabine in the same formulation or in different formulations.

[0070] In specific embodiments, the USP1 inhibitor and gemcitabine are administered to the subject by the same route or different routes.

[0071] In specific embodiments, the USP1 inhibitor and gemcitabine are administered to the subject parenterally or orally.

[0072] In a specific embodiment, the USP1 inhibitor is used with gemcitabine to treat breast cancer and / or ovarian cancer.

[0073] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with enzalutamide and dalostatamide in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0074] In specific embodiments, the USP1 inhibitor is co-administered with enzalutamide and dalostatamide in the same formulation or in different formulations.

[0075] In specific embodiments, the USP1 inhibitor is administered to the subject by the same route or a different route as enzalutamide and dalostatamide.

[0076] In specific embodiments, the USP1 inhibitor and enzalutamide and dalostatin are administered to the subject parenterally or orally.

[0077] In a specific embodiment, the USP1 inhibitor is used with enzalutamide and dalostatamide to treat prostate cancer.

[0078] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with docetaxel in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0079] In specific embodiments, the USP1 inhibitor is co-administered with docetaxel in the same formulation or in different formulations.

[0080] In specific embodiments, the USP1 inhibitor and docetaxel are administered to the subject by the same route or different routes.

[0081] In specific embodiments, the USP1 inhibitor and docetaxel are administered to the subject parenterally or orally.

[0082] In a specific embodiment, the USP1 inhibitor is used with docetaxel to treat prostate cancer.

[0083] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with Eliminsertib in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0084] In specific embodiments, the USP1 inhibitor is co-administered with Eliminsertib in the same formulation or in different formulations.

[0085] In specific embodiments, the USP1 inhibitor and Eliminsertib are administered to the subject by the same route or different routes.

[0086] In specific embodiments, the USP1 inhibitor and Eliminsertib are administered to the subject parenterally or orally.

[0087] In a specific embodiment, the USP1 inhibitor is used with Eliminsertib to treat breast cancer.

[0088] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with Avastin (bevacizumab) in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0089] In specific embodiments, the USP1 inhibitor is co-administered with Avastin (bevacizumab) in the same formulation or in different formulations.

[0090] In specific embodiments, the USP1 inhibitor and Avastin (bevacizumab) are administered to the subject via the same route or different routes.

[0091] In specific embodiments, the USP1 inhibitor and Avastin (bevacizumab) are administered to the subject parenterally or orally.

[0092] In a specific embodiment, the USP1 inhibitor is used with Avastin (bevacizumab) to treat breast cancer and / or ovarian cancer.

[0093] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with olaparib in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0094] In specific embodiments, the USP1 inhibitor is co-administered with olaparib in the same formulation or in different formulations.

[0095] In specific embodiments, the USP1 inhibitor and olaparib are administered to the subject by the same route or different routes.

[0096] In specific embodiments, the USP1 inhibitor and olaparib are administered to the subject parenterally or orally.

[0097] In a specific embodiment, the USP1 inhibitor is used with olaparib to treat breast cancer and / or ovarian cancer.

[0098] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with carboplatin in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0099] In specific embodiments, the USP1 inhibitor is co-administered with carboplatin in the same formulation or in different formulations.

[0100] In specific embodiments, the USP1 inhibitor and carboplatin are administered to the subject by the same route or different routes.

[0101] In specific embodiments, the USP1 inhibitor and carboplatin are administered to the subject parenterally or orally.

[0102] In a specific embodiment, the USP1 inhibitor is used with carboplatin to treat breast cancer and / or ovarian cancer.

[0103] In another aspect, the present invention provides a use of a USP1 inhibitor in combination with fruquintinib in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure as shown in the following formula:

[0104] In specific embodiments, the USP1 inhibitor is co-administered with fruquintinib in the same formulation or in different formulations.

[0105] In specific embodiments, the USP1 inhibitor and fruquintinib are administered to the subject by the same route or different routes.

[0106] In specific embodiments, the USP1 inhibitor and fruquintinib are administered to the subject parenterally or orally.

[0107] In a specific embodiment, the USP1 inhibitor is used with fruquintinib to treat breast cancer and / or ovarian cancer.

[0108] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure shown in Formula I and olaparib.

[0109] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and olaparib.

[0110] In specific embodiments, the USP1 inhibitor is co-administered with olaparib in the same formulation or in different formulations.

[0111] In specific embodiments, the USP1 inhibitor and olaparib are administered to the subject by the same route or different routes.

[0112] In specific embodiments, the USP1 inhibitor and olaparib are administered to the subject parenterally or orally.

[0113] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure represented by Formula I and cisplatin.

[0114] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and cisplatin.

[0115] In specific embodiments, the USP1 inhibitor is co-administered with cisplatin in the same formulation or in different formulations.

[0116] In specific embodiments, the USP1 inhibitor and cisplatin are administered to the subject by the same route or different routes.

[0117] In specific embodiments, the USP1 inhibitor and cisplatin are administered to the subject parenterally or orally.

[0118] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure shown in Formula I and gemcitabine.

[0119] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and gemcitabine.

[0120] In specific embodiments, the USP1 inhibitor is co-administered with gemcitabine in the same formulation or in different formulations.

[0121] In specific embodiments, the USP1 inhibitor and gemcitabine are administered to the subject by the same route or different routes.

[0122] In specific embodiments, the USP1 inhibitor and gemcitabine are administered to the subject parenterally or orally.

[0123] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure shown in Formula I, enzalutamide, and dalostatamide.

[0124] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I, enzalutamide, and dalostatamide.

[0125] In specific embodiments, the USP1 inhibitor is co-administered with enzalutamide and dalostatamide in the same formulation or in different formulations.

[0126] In specific embodiments, the USP1 inhibitor is administered to the subject by the same route or a different route as enzalutamide and dalostatamide.

[0127] In specific embodiments, the USP1 inhibitor and enzalutamide and dalostatin are administered to the subject parenterally or orally.

[0128] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure shown in Formula I and docetaxel.

[0129] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and docetaxel.

[0130] In specific embodiments, the USP1 inhibitor is co-administered with docetaxel in the same formulation or in different formulations.

[0131] In specific embodiments, the USP1 inhibitor and docetaxel are administered to the subject by the same route or different routes.

[0132] In specific embodiments, the USP1 inhibitor and docetaxel are administered to the subject parenterally or orally.

[0133] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure shown in Formula I and Eliminsertib.

[0134] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and Eliminsertib.

[0135] In specific embodiments, the USP1 inhibitor is co-administered with Eliminsertib in the same formulation or in different formulations.

[0136] In specific embodiments, the USP1 inhibitor and Eliminsertib are administered to the subject by the same route or different routes.

[0137] In specific embodiments, the USP1 inhibitor and Eliminsertib are administered to the subject parenterally or orally.

[0138] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure represented by Formula I and Avastin (bevacizumab).

[0139] In a specific embodiment, the present invention provides a pharmaceutical combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and Avastin (bevacizumab).

[0140] In specific embodiments, the USP1 inhibitor is co-administered with Avastin (bevacizumab) in the same formulation or in different formulations.

[0141] In specific embodiments, the USP1 inhibitor and Avastin (bevacizumab) are administered to the subject via the same route or different routes.

[0142] In specific embodiments, the USP1 inhibitor and Avastin (bevacizumab) are administered to the subject parenterally or orally.

[0143] In another aspect, the present invention provides a pharmaceutical combination comprising a USP1 inhibitor having a structure shown in Formula I and fruquintinib.

[0144] In a specific embodiment, the present invention provides a drug combination for treating cancer, comprising a USP1 inhibitor having a structure shown in Formula I and fruquintinib.

[0145] In specific embodiments, the USP1 inhibitor is co-administered with fruquintinib in the same formulation or in different formulations.

[0146] In specific embodiments, the USP1 inhibitor and fruquintinib are administered to the subject by the same route or different routes.

[0147] In specific embodiments, the USP1 inhibitor and fruquintinib are administered to the subject parenterally or orally.

[0148] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and olaparib.

[0149] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and cisplatin.

[0150] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and gemcitabine.

[0151] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I, enzalutamide, and dalostatamide.

[0152] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and docetaxel.

[0153] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and Eliminsertib.

[0154] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and Avastin (bevacizumab).

[0155] In another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a USP1 inhibitor having the structure shown in Formula I or a pharmaceutical combination comprising a USP1 inhibitor having the structure shown in Formula I and fruquintinib.

[0156] In a specific embodiment, the cancer of the present invention is selected from the group consisting of breast cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, uterine cancer, peritoneal cancer, and endometrial cancer.

[0157] In specific embodiments, the cancer is breast cancer.

[0158] In specific embodiments, the cancer is triple-negative breast cancer (TNBC).

[0159] In specific embodiments, the breast cancer is a BRCA1 mutant cancer, a BRCA2 mutant cancer, or a BRCA1 mutant and BRCA2 mutant cancer.

[0160] In specific embodiments, the cancer is ovarian cancer.

[0161] In specific embodiments, the ovarian cancer is a BRCA1 mutant cancer, a BRCA2 mutant cancer, a BRCA wild-type and CCNE1 amplified cancer, or a p53 mutant cancer.

[0162] In specific embodiments, the ovarian cancer is a BRCA1 mutant cancer and a p53 mutant cancer.

[0163] In specific embodiments, the ovarian cancer is a BRCA1 and BRCA2 mutant cancer.

[0164] In specific embodiments, the ovarian cancer is a BRCA2 mutant cancer.

[0165] In specific embodiments, the ovarian cancer is prostate cancer.

[0166] In specific embodiments, the cancer is a PARP inhibitor-resistant cancer.

[0167] In specific embodiments, the cancer is an olaparib-resistant cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0168] Figure 1 shows that 104 inhibits subcutaneous tumors in mice (triple-negative breast cancer cell line, BRCA1 mutation);

[0169] Figure 2 shows that the combination of 104 and olaparib inhibits the growth of subcutaneous tumors in mice (MDA-MB-436 (BRCA1 mutation));

[0170] Figure 3 shows that the combination of 104 and olaparib inhibits subcutaneous tumor MX-1 (human breast cancer cell line) in mice;

[0171] FIG4 shows that the combination of 104 and olaparib inhibits subcutaneous tumors caov3 (human ovarian cancer cell line) in mice;

[0172] Figure 5 shows that the combination of 104 and gemcitabine inhibits subcutaneous tumor MDA-MB-436 in mice;

[0173] Figure 6 shows a USP1 biochemical assay using ubiquitin rhodamine;

[0174] Figure 7 shows the in vitro inhibitory effect of a USP1 inhibitor in combination with a PARPi compound on tumor cells; Figure 7A: Inhibitory effect of the combination of olaparib and 104 on MX-1; Figure 7B: Inhibitory effect of the combination of saruparib and 104 on MX-1; Figure 7C: Inhibitory effect of the combination of olaparib and 104 on UWB1.289 (human ovarian cancer cell line, BRCA1 mutation); Figure 7D: Inhibitory effect of the combination of saruparib and 104 on UWB1.289;

[0175] FIG8A shows the levels of downstream substrates detected by Western blotting after MDA-MB-436 cells were treated with different doses of 104;

[0176] FIG8B shows the levels of downstream substrates detected by Western blotting after 104 treatment of MDA-MB-436 cells for different time periods.

[0177] Figure 9A shows the in vitro inhibitory effect of olaparib (2.45 nM) in combination with 104 (10 nM) on MDA-MB-436;

[0178] FIG9B shows the in vitro inhibitory effect of olaparib (2.45 nM) in combination with 104 (15 nM) on MDA-MB-436.

[0179] Figure 10A shows the in vitro inhibitory effect of cisplatin (2000 nM) combined with 104 (150 nM) on ES-2 (ovarian cell line);

[0180] FIG10B shows the in vitro inhibitory effect of cisplatin (2000 nM) combined with 104 (300 nM) on ES-2.

[0181] Figure 11A shows the in vitro inhibitory effect of cisplatin (30 nM) combined with 104 (5000 nM) on TOV21G (ovarian cell line);

[0182] FIG11B shows the in vitro inhibitory effect of cisplatin (60 nM) combined with 104 (5000 nM) on TOV21G.

[0183] FIG12A shows the in vitro inhibitory effect of cisplatin (100 nM) combined with 104 (300 nM) on OVCAR3 (ovarian cell line).

[0184] Figure 12B shows the in vitro inhibitory effect of cisplatin (100 nM) combined with 104 (4000 nM) on OVCAR3;

[0185] FIG12C shows the in vitro inhibitory effect of cisplatin (100 nM) combined with 104 (8000 nM) on OVCAR3.

[0186] FIG13 shows the in vitro inhibitory effect of gemcitabine (2.4 nM) in combination with 104 (300 nM) on OVCAR3.

[0187] FIG14A shows the in vitro inhibitory effect of gemcitabine (2.3 nM) in combination with 104 (5 nM) on MDA-MB-436.

[0188] Figure 14B shows the in vitro inhibitory effect of gemcitabine (2.3 nM) in combination with 104 (10 nM) on MDA-MB-436;

[0189] FIG14C shows the in vitro inhibitory effect of gemcitabine (2.3 nM) in combination with 104 (15 nM) on MDA-MB-436.

[0190] Figure 15A shows the in vitro inhibitory effect of gemcitabine (0.76 nM) combined with 104 (40 nM) on Caov3;

[0191] Figure 15B shows the in vitro inhibitory effect of gemcitabine (0.76 nM) combined with 104 (80 nM) on Caov3;

[0192] FIG15C shows the in vitro inhibitory effect of gemcitabine (0.76 nM) in combination with 104 (160 nM) on Caov3.

[0193] FIG16A shows the combined effects of enzalutamide (10 μM), dalostatamide (15 μM) and 104 (2 μM) on 22RV1 (AR +AR-V7 prostate cancer cell line) in vitro;

[0194] Figure 16B shows the combined effects of enzalutamide (10 μM), dalostatin (15 μM) and 104 (6 μM) on 22RV1 (AR + AR-V7 prostate cancer cell line) in vitro;

[0195] Figure 16C shows the combined effects of enzalutamide (10 μM), dalostatin (15 μM) and 104 (10 μM) on 22RV1 (AR + In vitro inhibition of AR-V7 in prostate cancer cell lines.

[0196] FIG17A shows the combined effects of enzalutamide (300 nM), dalostatin (2 μM) and 104 (2 μM) on LNCaP (AR + T878A prostate cancer cell line) in vitro;

[0197] Figure 17B shows the combined effects of enzalutamide (300 nM), dalostatin (2 μM) and 104 (4 μM) on LNCaP (AR + T878A prostate cancer cell line) in vitro;

[0198] Figure 17C shows the combined effects of enzalutamide (300 nM), dalostatin (2 μM) and 104 (8 μM) on LNCaP (AR + T878A prostate cancer cell line) in vitro.

[0199] Figure 18A shows the in vitro inhibitory effect of docetaxel (1.6 nM) in combination with 104 (2000 nM) on 22RV1;

[0200] Figure 18B shows the in vitro inhibitory effect of docetaxel (1.6 nM) in combination with 104 (6000 nM) on 22RV1;

[0201] FIG18C shows the in vitro inhibitory effect of docetaxel (1.6 nM) in combination with 104 (10000 nM) on 22RV1.

[0202] Figure 19A shows the in vitro inhibitory effect of docetaxel (0.32 nM) combined with 104 (4000 nM) on Lncap;

[0203] FIG19B shows the in vitro inhibitory effect of docetaxel (0.32 nM) in combination with 104 (8000 nM) on Lncap.

[0204] Figure 20A shows the in vitro inhibitory effect of Elimusertib (1.6 nM) in combination with 104 (15 nM) on MDA-MB-436;

[0205] Figure 20B shows the in vitro inhibitory effect of Elimusertib (8 nM) in combination with 104 (15 nM) on MDA-MB-436;

[0206] Figure 20C shows the in vitro inhibitory effect of Elimusertib (40 nM) in combination with 104 (15 nM) on MDA-MB-436;

[0207] FIG20D shows the in vitro inhibitory effect of Elimiusertib (200 nM) in combination with 104 (15 nM) on MDA-MB-436.

[0208] Figure 21A shows the tumor inhibitory effect of 215 in combination with carboplatin, fruquintinib, and olaparib in the Caov3 subcutaneous tumor model;

[0209] FIG21B shows the changes in body weight of mice in the caov3 subcutaneous tumor model.

[0210] Figure 22A shows the tumor inhibitory effect of 104 combined with olaparib in the OVCAR3 subcutaneous tumor model;

[0211] FIG22B shows the changes in body weight of mice in the OVCAR3 subcutaneous tumor model. DETAILED DESCRIPTION

[0212] In a specific embodiment, the present invention provides a combination product comprising a USP1 inhibitor, or a pharmaceutically acceptable salt, hydrate, solvate, isotope-substituted product or stereoisomer thereof; and an anti-tumor drug other than the USP1 inhibitor. The USP1 inhibitor has a structure shown in Formula I:

[0213] wherein Ring A and Ring B are each independently selected from C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclyl, and Ring A and Ring B are each independently optionally substituted by one or more R1;

[0214] L is selected from chemical bonds, -O-, -S-, -C 1-6 Alkylene-, -OC 1-6 Alkylene-, -C 1-6 Alkylene-O-, -SC 1-6 Alkylene- and -C 1-6 Alkylene-S-;

[0215] R a and R b Each independently selected from H atoms, -CN, C 1-6Alkyl, -OH, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Hydroxyalkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy; or R a and R b Together they form an oxo group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic group;

[0216] R2 is selected from H atoms, -OH, -CN, C 1-6 Alkyl, C 2-6 Alkynyl, -C 1-6 Alkyl-C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 3-6 Cycloalkyl and 5-7 membered heterocyclic groups, wherein the C 1-6 Alkyl or -C 1-6 Alkyl-C 6-10 Aryl is optionally substituted with one or more R1;

[0217] Preferably, R2 is selected from the group consisting of an H atom, -CN, a methyl group, a trideuterated methyl group, an ethynyl group, a propynyl group, a tetrahydrofuranyl group, a cyclopropyl group, a methoxy group, and a hydroxyl group;

[0218] R3 is selected from the group consisting of H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, 5-7 membered heterocyclic group and -C 1-6 Alkylene-C(O)-OC 1-6 Alkyl, wherein the C 1-6 The hydroxyalkyl and 5-7 membered heterocyclic groups are each independently optionally substituted with one or more C 1-6 Alkyl substitution;

[0219] Preferably, R3 is selected from H atom, methoxy, trifluoromethyl, Cl atom, -CN, isopropoxy, ethynyl, difluoromethoxy, morpholinyl, -OH, F atoms, hydroxymethyl and

[0220] More preferably, R3 is selected from H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl;

[0221] More preferably, R3 is selected from H atoms, C 1-6 alkyl;

[0222] R4 is selected from the group consisting of H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy and C 1-6 hydroxyalkyl;

[0223] Preferably, R4 is an H atom;

[0224] R5 is selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 6-10 Aryl, C fused to 3-8 membered heterocyclic group 6-10 Aryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, wherein the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclyl are each independently optionally substituted with one or more R1;

[0225] R1 is independently selected at each occurrence from a D atom, -OH, -COOH, -NH2, -CN, an oxo group, a halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, -OC1-6 Alkylene-OC 1-6 Alkyl, wherein the C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 The cycloalkyl and 3-8 membered heterocyclic groups are each independently optionally selected from D atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy and C 1-6 substituted with one or more substituents of the hydroxyalkyl group; and

[0226] n is an integer between 0 and 8.

[0227] In the specific embodiment of formula I shown in the present invention, wherein ring A and ring B are each independently selected from phenyl, piperidinyl, cyclohexyl, cyclopropyl, cyclobutyl, pyridinyl, pyrimidinyl, imidazolyl, pyrazolyl, bicyclo[2.2.2]octanyl, 2-oxabicyclo[2.2.2]octanyl, pentacyclooctanyl, isoindolinone, imidazo[1,2-a]pyrazinyl, piperidine-2,6-dione, thienyl, furanyl, cyclopentyl, pyranyl alkyl, pyrrolidinyl, piperazinyl, morpholinyl, naphthyl, pyrrolyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, indolyl, isoindolyl, indolinyl, isoindolyl, indolinonyl, pyrido[3,2-d]pyrimidinyl, pteridinyl, pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4-d]pyrimidinyl and cubanyl, and Ring A and Ring B are each independently optionally substituted with one or more R1, R1 being as defined above;

[0228] L is selected from chemical bonds, -O-, -OC 1-6 Alkylene- and -C 1-6 Alkylene-O-;

[0229] In particular, Selected from

[0230] and Ring A and Ring B are each independently optionally substituted by one or more R1, R1 being as defined above;

[0231] More specifically, Selected from

[0232] In the specific embodiment of formula I shown in the present invention, wherein R5 is selected from C 6-10 Aryl, 5-6 membered heteroaryl, C6-10 Aryl and C fused to 5-6 membered heteroaryl 6-10 Aryl, preferably selected from phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, thiazolyl, indolyl, indolyl and isoxazolyl, said C 6-10 Aryl, 5-6 membered heteroaryl, C 6-10 Aryl and C fused to 5-6 membered heteroaryl 6-10 Each aryl group is independently optionally selected from -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl and C 3-6 The cycloalkyl group is substituted with one or more substituents;

[0233] In particular, R5 is selected from

[0234] In a specific embodiment of formula I shown in the present invention, wherein n is 0 or 1;

[0235] R a and R b Each independently selected from H atoms, -CN, C 1-6 Alkyl, -OH and halogen;

[0236] Preferably, R a and R b Each is independently a H atom or -CN.

[0237] In a specific embodiment, the present invention provides a combination product comprising a USP1 inhibitor, or a pharmaceutically acceptable salt, hydrate, solvate, isotope-substituted product or stereoisomer thereof; and an anti-tumor drug other than the USP1 inhibitor, wherein the USP1 inhibitor is selected from:

[0238] In one embodiment, the present invention provides a use of a USP1 inhibitor in combination with an anti-tumor drug other than a USP1 inhibitor in the preparation of a medicament for treating cancer, wherein the USP1 inhibitor has a structure shown in Formula I:

[0239] wherein Ring A and Ring B are each independently selected from C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclyl, and Ring A and Ring B are each independently optionally substituted by one or more R1;

[0240] L is selected from chemical bonds, -O-, -S-, -C 1-6 Alkylene-, -OC 1-6 Alkylene-, -C 1-6 Alkylene-O-, -SC 1-6 Alkylene- and -C 1-6 Alkylene-S-;

[0241] R a and R b Each independently selected from H atoms, -CN, C 1-6 Alkyl, -OH, halogen, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Hydroxyalkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy; or R a and R b Together they form an oxo group, C 3-8 Cycloalkyl or 3-8 membered heterocyclic group;

[0242] R2 is selected from H atoms, -OH, -CN, C 1-6 Alkyl, C 2-6 Alkynyl, -C 1-6 Alkyl-C 6-10 Aryl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 3-6 Cycloalkyl and 5-7 membered heterocyclic groups, wherein the C 1-6 Alkyl or -C 1-6 Alkyl-C 6-10 Aryl is optionally substituted with one or more R1, R1 is as defined above;

[0243] Preferably, R2 is selected from the group consisting of an H atom, -CN, a methyl group, a trideuterated methyl group, an ethynyl group, a propynyl group, a tetrahydrofuranyl group, a cyclopropyl group, a methoxy group, and a hydroxyl group;

[0244] R3 is selected from the group consisting of H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, 5-7 membered heterocyclic group and -C 1-6 Alkylene-C(O)-OC 1-6 Alkyl, wherein the C 1-6The hydroxyalkyl and 5-7 membered heterocyclic groups are each independently optionally substituted with one or more C 1-6 Alkyl substitution;

[0245] Preferably, R3 is selected from H atom, methoxy, trifluoromethyl, Cl atom, -CN, isopropoxy, ethynyl, difluoromethoxy, morpholinyl, -OH, F atoms, hydroxymethyl and

[0246] R4 is selected from the group consisting of H atoms, -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Haloalkoxy and C 1-6 hydroxyalkyl;

[0247] Preferably, R4 is an H atom;

[0248] R5 is selected from C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 6-10 Aryl, C fused to 3-8 membered heterocyclic group 6-10 Aryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclic groups, wherein the C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 6-10 Aryl, 5-10 membered heteroaryl, C 3-8 Cycloalkyl and 3-8 membered heterocyclyl are each independently optionally substituted with one or more R1; and

[0249] n is an integer between 0 and 8.

[0250] In the specific embodiment of formula I shown in the present invention, wherein ring A and ring B are each independently selected from phenyl, piperidinyl, cyclohexyl, cyclopropyl, cyclobutyl, pyridinyl, pyrimidinyl, imidazolyl, pyrazolyl, bicyclo[2.2.2]octanyl, 2-oxabicyclo[2.2.2]octanyl, pentacyclooctanyl, isoindolinone, imidazo[1,2-a]pyrazinyl, piperidine-2,6-dione, thienyl, furanyl, cyclopentyl, pyranyl alkyl, pyrrolidinyl, piperazinyl, morpholinyl, naphthyl, pyrrolyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, indolyl, isoindolyl, indolinyl, isoindolyl, indolinonyl, pyrido[3,2-d]pyrimidinyl, pteridinyl, pyrazolo[4,3-c]pyridinyl, pyrazolo[3,4-d]pyrimidinyl and cubanyl, and Ring A and Ring B are each independently optionally substituted with one or more R1, R1 being as defined above;

[0251] L is selected from chemical bonds, -O-, -OC 1-6 Alkylene- and -C 1-6 Alkylene-O-;

[0252] In particular, Selected from

[0253] and Ring A and Ring B are each independently optionally substituted by one or more R1, R1 being as defined above;

[0254] More specifically, Selected from

[0255] In the specific embodiment of formula I shown in the present invention, wherein R5 is selected from C 6-10 Aryl, 5-6 membered heteroaryl, C 6-10 Aryl and C fused to 5-6 membered heteroaryl 6-10 Aryl, preferably selected from phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, thiazolyl, indolyl, indolyl and isoxazolyl, said C 6-10 Aryl, 5-6 membered heteroaryl, C 6-10 Aryl and C fused to 5-6 membered heteroaryl 6-10 Each aryl group is independently optionally selected from -OH, -COOH, -NH2, -CN, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl and C3-6 The cycloalkyl group is substituted with one or more substituents;

[0256] In particular, R5 is selected from

[0257] In a specific embodiment of formula I shown in the present invention, wherein n is 0 or 1;

[0258] R a and R b Each independently selected from H atoms, -CN, C 1-6 Alkyl, -OH and halogen;

[0259] Preferably, R a and R b Each is independently a H atom or -CN.

[0260] In a specific embodiment, the present invention provides a use of a USP1 inhibitor selected from the following in combination with other anti-tumor drugs other than USP1 inhibitors in the preparation of a medicament for treating cancer:

[0261] In a specific embodiment of the invention, the cancer is selected from the group consisting of breast cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, uterine cancer, peritoneal cancer, and endometrial cancer.

[0262] In a specific embodiment of the present invention, the cancer is selected from: BRCA1 mutant breast cancer, BRCA2 mutant breast cancer, BRCA1 mutant and BRCA2 mutant breast cancer, BRCA1 mutant ovarian cancer, BRCA2 mutant ovarian cancer or p53 mutant ovarian cancer.

[0263] In a specific embodiment of the present invention, the other anti-tumor drugs other than the USP1 inhibitor are selected from PARP inhibitors, antimetabolite anti-tumor drugs, taxane anti-tumor drugs, ATR kinase inhibitors, estrogen receptor modulators, androgen receptor inhibitors, and platinum coordination complexes, immunotherapy drugs, anti-angiogenesis drugs and platinum coordination complexes;

[0264] Preferably, the PARP inhibitor is selected from niraparib, pamiparib, olaparib, faruzopanib, rucaparib, saruparib, talazoparib;

[0265] The antimetabolite anti-tumor drug is selected from gemcitabine, fluorouracil, methotrexate, cytarabine, mercaptopurine and thioguanine;

[0266] The taxane anti-tumor drug is selected from docetaxel, paclitaxel, liposome paclitaxel, and albumin-bound paclitaxel;

[0267] The ATR kinase inhibitor is selected from Elimiusertib;

[0268] The estrogen receptor modulator is enzalutamide;

[0269] The androgen receptor inhibitor is dalostatamide; and

[0270] The platinum coordination complex is selected from cisplatin and carboplatin.

[0271] In a specific embodiment, the anti-tumor drug other than the USP1 inhibitor of the present invention is selected from olaparib, gemcitabine, niraparib, pamiparib, faruzopanib, rucaparib, saruparib, talazoparib, cisplatin and carboplatin.

[0272] In a specific embodiment, the anti-tumor drug other than the USP1 inhibitor of the present invention is selected from anti-angiogenic drugs;

[0273] The anti-angiogenic drug is selected from VEGF / VEGFR pathway inhibitors, integrin inhibitors, platelet-derived growth factor receptor inhibitors, HIF inhibitors and endothelin receptor antagonists;

[0274] Specifically, the anti-angiogenic drug is selected from monoclonal antibodies and small molecule tyrosine kinase inhibitors;

[0275] More specifically, the anti-angiogenic drug is selected from fruquintinib, bevacizumab, ramucirumab, sorafenib, sunitinib, apatinib, regorafenib, cabozantinib, and lenvatinib.

[0276] definition

[0277] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0278] As used herein, the term "combination product" or "composition" refers to a combination of products that are provided in a single dosage unit form or as a kit of parts for combined administration. The combination product may be used simultaneously, separately or sequentially.

[0279] As used herein, "simultaneous" administration refers to administration of the USP1 inhibitor and other anti-tumor drugs other than the USP1 inhibitor via the same route and at the same time.

[0280] The term "separately" as used herein means that the USP1 inhibitor and the anti-tumor drug other than the USP1 inhibitor are administered separately from each other at the same time or substantially the same time via different routes.

[0281] The term "sequential" as used herein refers to administering the USP1 inhibitor and the other anti-tumor drug other than the USP1 inhibitor separately at different times via the same route or different routes.

[0282] The term "alkyl" refers to a saturated, linear or branched, monovalent hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20) carbon atoms, preferably C 1-10 Alkyl, more preferably C 1-6 Alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 2,2-dimethylpropyl, 2-methylbutyl, n-hexyl, 2,2-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-3-ethylhexyl, n-decyl, and 3,3-diethylhexyl.

[0283] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon group having 2 to 6 (e.g., 2, 3, 4, 5, and 6) carbon atoms and at least one carbon-carbon double bond, wherein the carbon-carbon double bond may be located at any position within the alkenyl group, preferably C 2-5 Alkenyl. Examples of alkenyl groups include, but are not limited to, -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CH-CH2-CH3, -CH2-CH=CH-CH3, -CH=CH-CH=CH2, -CH=C(CH3)-CH3, and -CH2-C(CH3)=CH2.

[0284] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon group having 2 to 6 (e.g., 2, 3, 4, 5, and 6) carbon atoms and at least one carbon-carbon triple bond, wherein the carbon-carbon triple bond may be located at any position within the alkynyl group, preferably C 2-5Alkynyl. Examples of alkynyl groups include, but are not limited to, -C≡CH, -C≡C-CH3, -CH2-C≡CH, -C≡C-CH2-CH3, -CH2-CH2-C≡CH, -CH(CH3)C≡CH, and -CH2-C≡C-CH3.

[0285] The term "cycloalkyl" includes two types, one is a conventional cycloalkyl group and the other is a heterostructure cycloalkyl group.

[0286] Conventional cycloalkyl refers to an aliphatic, saturated or partially unsaturated monovalent cyclic hydrocarbon group having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20) carbon atoms, preferably C 3-12 Conventional cycloalkyl, more preferably C 3-10 Conventional cycloalkyl, more preferably C 3-8 Conventional cycloalkyl, most preferably C 3-6 Conventional cycloalkyl groups. Conventional cycloalkyl groups optionally contain one or more double or triple bonds.

[0287] Conventional cycloalkyl groups can be monocyclic alkyl groups, and examples of monocyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl. Conventional cycloalkyl groups can also be polycyclic alkyl groups (e.g., bicyclic alkyl groups, tricyclic alkyl groups, tetracyclic alkyl groups, and pentacyclic alkyl groups), and polycyclic alkyl groups include spirocyclic alkyl groups, fused cyclic alkyl groups, and bridged cyclic alkyl groups.

[0288] The term "spirocycloalkyl" refers to a 5-20 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 membered) spirocycloalkyl, preferably a 6-14 membered spirocycloalkyl, more preferably a 7-10 membered spirocycloalkyl. The spirocycloalkyl may be a monospirocycloalkyl, a dispirocycloalkyl, or a polyspirocycloalkyl, preferably a monospirocycloalkyl, more preferably a 4 membered / 4 membered, 4 membered / 5 membered, 4 membered / 6 membered, 5 membered / 5 membered, or 5 membered / 6 membered monospirocycloalkyl. Examples of spirocycloalkyl include, but are not limited to:

[0289] The term "fused cycloalkyl" refers to a 5-20 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 membered) fused cycloalkyl, preferably a 6-14 membered fused cycloalkyl, more preferably a 7-10 membered fused cycloalkyl. The fused cycloalkyl may be a bicyclic, tricyclic, tetracyclic, or pentacyclic or higher ring fused cycloalkyl, preferably a bicyclic or tricyclic fused cycloalkyl, more preferably a 5-membered / 5-membered or 5-membered / 6-membered fused cycloalkyl. Examples of fused cycloalkyls include, but are not limited to:

[0290] The term "bridged cycloalkyl" refers to a 5-20 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 membered) bridged cycloalkyl group, preferably a 6-14 membered bridged cycloalkyl group, more preferably a 7-10 membered bridged cycloalkyl group. The bridged cycloalkyl group may be a bicyclic, tricyclic, tetracyclic, or pentacyclic or higher bridged cycloalkyl group, preferably a bicyclic, tricyclic, or tetracyclic bridged cycloalkyl group, more preferably a bicyclic or tricyclic bridged cycloalkyl group. Examples of bridged cycloalkyl groups include, but are not limited to:

[0291] The term "heterocyclic alkyl" includes monocyclic alkyl, spirocyclic alkyl, fused cyclic alkyl and bridged cyclic alkyl fused to any one of conventional aryl, conventional heteroaryl and conventional heterocyclic groups, and the connection point is located on the corresponding conventional cycloalkyl (referring to monocyclic alkyl, spirocyclic alkyl, fused cyclic alkyl or bridged cycloalkyl). Examples of heterocyclic alkyl groups include, but are not limited to:

[0292] The term "heterocyclic group" includes two types, one is a conventional heterocyclic group and the other is a heterostructure heterocyclic group.

[0293] Conventional heterocyclic groups refer to aliphatic, saturated or partially unsaturated, monovalent cyclic hydrocarbon groups having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20) ring atoms, wherein one or more ring atoms are substituted by one or more elements selected from nitrogen, oxygen, S, S(O), and S(O)2, and the substitution does not form -OO-, -OS-, or -SS-; preferably C 3-12 Conventional heterocyclic groups, wherein 1 to 4 (e.g., 1, 2, 3, and 4) are heteroatoms; more preferably, C 3-8 Conventional heterocyclic groups, wherein 1 to 3 (e.g. 1, 2 and 3) are heteroatoms; most preferably C 5-7 Conventional heterocyclic groups, wherein 1-2 or 1-3 are heteroatoms.

[0294] Conventional heterocyclic groups can be monocyclic heterocyclic groups. Examples of monocyclic heterocyclic groups include, but are not limited to, oxetanyl, 3-pyrrolinyl, pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and pyranyl, preferably 1,2,5-oxadiazolyl, pyranyl, or morpholinyl. Conventional heterocyclic groups can also be polycyclic heterocyclic groups, including spirocyclic heterocyclic groups, fused-ring heterocyclic groups, and bridged-ring heterocyclic groups.

[0295] The term "spiroheterocyclyl" refers to a 5-20 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 membered) spiroheterocyclyl, preferably a 6-14 membered spiroheterocyclyl, more preferably a 7-10 membered spiroheterocyclyl. The spiroheterocyclyl may be a monospiroheterocyclyl, a bispiroheterocyclyl, or a polyspiroheterocyclyl, preferably a monospiroheterocyclyl or a bispiroheterocyclyl, more preferably a 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl. Examples of spiroheterocyclyls include, but are not limited to:

[0296] The term "fused heterocyclic group" refers to a 5-20 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 membered) fused heterocyclic group, preferably a 6-14 membered fused heterocyclic group, more preferably a 7-10 membered fused heterocyclic group. The fused heterocyclic group may be a bicyclic, tricyclic, tetracyclic, or pentacyclic or higher fused heterocyclic group, preferably a bicyclic or tricyclic fused heterocyclic group, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. Examples of fused heterocyclic groups include, but are not limited to:

[0297] The term "bridged heterocyclic group" refers to a 5-14 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 membered) bridged heterocyclic group, preferably a 6-14 membered bridged heterocyclic group, more preferably a 7-10 membered bridged heterocyclic group. The bridged heterocyclic group may be a bicyclic, tricyclic, tetracyclic, or pentacyclic or higher bridged heterocyclic group, preferably a bicyclic, tricyclic, or tetracyclic bridged heterocyclic group, more preferably a bicyclic or tricyclic bridged heterocyclic group. Examples of bridged heterocyclic groups include, but are not limited to:

[0298] The term "heterocyclic group" includes monocyclic heterocyclic groups, spirocyclic heterocyclic groups, fused heterocyclic groups and bridged heterocyclic groups fused to any one of conventional aryl groups, conventional heteroaryl groups and conventional cycloalkyl groups, and the connection point is located on the corresponding conventional heterocyclic group (referring to monocyclic heterocyclic groups, spirocyclic heterocyclic groups, fused heterocyclic groups or bridged heterocyclic groups). Examples of heterocyclic groups include, but are not limited to:

[0299] The term "aryl" includes two types, one is a conventional aryl group and the other is a heterostructure aryl group.

[0300] Conventional aryl refers to 6-14 membered (e.g., 6, 7, 8, 9, 10, 11, 12, 13 and 14 membered) aromatic hydrocarbon groups, preferably C 6-10 Conventional aryl groups are more preferably phenyl, naphthyl, phenanthrenyl or anthracenyl.

[0301] The term "heteroaryl" includes a conventional aryl fused to any one of conventional heteroaryl, conventional heterocyclyl and conventional cycloalkyl, with the attachment point being located on the conventional aryl. Examples of heteroaryl groups include, but are not limited to:

[0302] The term "heteroaryl" includes two types, one is a conventional heteroaryl and the other is a heterostructural heteroaryl.

[0303] Conventional heteroaryl refers to 1-4 (e.g., 1, 2, 3, and 4) carbon atoms in a 5-14 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 membered) aromatic hydrocarbon group replaced with heteroatoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Preferably, the number of ring atoms is 5-10, wherein 1-3 (e.g., 1, 2, and 3) heteroatoms are contained. More preferably, the number of ring atoms is 5 or 6, wherein 1-2 heteroatoms are contained. Examples of conventional heteroaryl include, but are not limited to, imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, and pyrazinyl, preferably imidazolyl, thiazolyl, pyrazolyl, pyrimidinyl, or thiazolyl, more preferably pyrazolyl or thiazolyl.

[0304] The term "heteroaryl" includes a conventional heteroaryl fused to any one of a conventional aryl, a conventional cycloalkyl, and a conventional heterocyclic group, with the point of attachment being located on the conventional heteroaryl. Examples of heteroaryl groups include, but are not limited to:

[0305] The term "alkoxy" includes -O-alkyl and -O-cycloalkyl, wherein "alkyl" and "cycloalkyl" are as defined above. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0306] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0307] The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

[0308] The term "hydroxy" refers to -OH.

[0309] The term "halogen" refers to -F, -Cl, -Br or -I.

[0310] The term "amino" refers to -NH2.

[0311] The term "cyano" refers to -CN.

[0312] The term "nitro" refers to -NO2.

[0313] The term "oxo" refers to =0.

[0314] The term "carboxyl" refers to -C(=O)OH.

[0315] The term "mercapto" refers to -SH.

[0316] The term "ester group" refers to a -C(=O)O-alkyl group or a -C(=O)O-cycloalkyl group, wherein alkyl and cycloalkyl are as defined above.

[0317] The term "acyl" refers to -C(=O)R, where R is selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0318] The term "hydroxy protecting group" refers to a group that is introduced on a hydroxy group and is easily removed, and is used to block or protect the hydroxy group while reacting on other functional groups of the compound. Non-limiting examples include trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, p-nitrobenzoyl, etc.

[0319] symbol Refers to the attachment site.

[0320] As used herein, other anti-tumor drugs besides USP1 inhibitors include, but are not limited to, PARP inhibitors, antimetabolite anti-tumor drugs, platinum coordination complexes, and the like.

[0321] As used herein, the terms "PARPi" and "PARP inhibitor" are used interchangeably and refer to agents that can inhibit the enzyme poly (ADP-ribose) polymerase (PARP), including but not limited to niraparib, pamiparib, olaparib, faruzopanib, rucaparib, saruparib and talazoparib.

[0322] Antimetabolite antitumor drugs are antitumor drugs that act on the S phase of the cell cycle (DNA synthesis) by inhibiting DNA synthesis or by inhibiting the synthesis of purine or pyrimidine bases, thereby limiting DNA synthesis. As a result, the S phase cannot continue, and cell death follows. Examples of antimetabolite antitumor drugs include, but are not limited to, fluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine, and gemcitabine.

[0323] Gemcitabine, 2'-deoxy-2',2'-difluorocytidine monohydrochloride (β-isomer), is commercially available as Gemcitabine. Gemcitabine has cell phase specificity in the S-phase by blocking the progression of cells through the G1 / S boundary. Gemcitabine can be combined with cisplatin for the treatment of localized advanced non-small cell lung cancer, and can also be used alone to treat localized advanced pancreatic cancer. Platinum coordination complexes are anticancer agents that interact with DNA. Platinum complexes enter tumor cells, undergo hydration, and form internal and mutual cross-links with DNA, resulting in biological effects that are detrimental to the tumor. Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.

[0324] As used herein, the term "pharmaceutically acceptable" refers to those compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0325] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0326] As used herein, the term "treatment" is a method for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, any one or more of: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease condition, inhibiting the disease or disease progression, inhibiting or slowing the disease or its progression, preventing its development, and alleviating symptoms (whether partial or total). "Treatment" also encompasses reducing the pathological consequences of proliferative diseases. The methods provided herein encompass any one or more of these therapeutic aspects. As described above, the term treatment does not require 100% removal of all aspects of the disease.

[0327] In the context of cancer, the term "treating" includes, but is not limited to, inhibiting cancer cell growth, inhibiting cancer cell replication, reducing overall tumor burden, and delaying, halting or slowing tumor growth, progression or metastasis.

[0328] As used herein, "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, blood-borne tumors (e.g., multiple myeloma, lymphoma, and leukemia) and solid tumors. Non-limiting examples of blood cancers include non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia, and non-limiting examples of solid tumors include breast cancer (particularly, triple-negative breast cancer), gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer.

[0329] As used herein, "patient," "subject," and "subject" refer to an animal, such as a mammal. In certain embodiments, the patient is a human. In other embodiments, the patient is a non-human animal, such as a dog, cat, livestock (e.g., a horse, pig, or donkey), chimpanzee, or monkey.

[0330] The anticancer effect of an anticancer drug can be inferred from specific data, for example, by the tumor growth inhibition rate (TGI). The tumor growth inhibition rate is calculated using the following formula:

[0331] TGI=100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TV D0对照组 )*100 to calculate the tumor growth inhibition rate (TGI%).

[0332] In specific embodiments, the tumor growth inhibition rate of 104 as a monotherapy or the combination of 104 as a monotherapy and olaparib of the present invention is 30% or higher, preferably, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 100% or higher.

[0333] Example

[0334] Preparation Example 1. Preparation of Compound 4'-cyclopropyl-5,6'-dimethoxy-N-((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]octan-1-yl)methyl)-[2,5'-bipyrimidinyl]-4-amine (herein referred to as 104)

[0335] The preparation method of the compound is as described in the preparation of compound 104 in CN116496252A, the entire contents of which are incorporated herein by reference. Among them, the intermediate compounds BB2C35 and compound A2-7 are the same as those in CN116496252A.

[0336] Briefly, the intermediate compound BB2C35 (125.0 mg, 0.36 mmol, 1.00 eq), compound A2-7 (118.1 mg, 0.72 mmol, 2.00 eq), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (XPhos-Pd-G2, 56.58 mg, 0.072 mmol, 0.20 eq), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos, 68.65 mg) and 1,1'-dichloro-2'-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl were reacted in the presence of 1% paraffin. g, 0.144 mmol, 0.40 eq) and potassium phosphate (229.3 mg, 1.08 mmol, 3.00 eq) were added to dioxane (2.5 mL) and water (0.5 mL), and the mixture was replaced with nitrogen three times. The mixture was stirred at 95°C for 16 hours, and water (15.0 mL) was added. The mixture was extracted three times with ethyl acetate (30.0 mL). The organic layers were combined, washed twice with saturated brine (20.0 mL), and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain compound 104 as a white solid:

[0337] Preparation Example 2. Synthesis of Compound (4'-cyclopropyl-5,6'-dimethoxy-N-(4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)cuban-1-yl)methyl)-[2,5'-bipyrimidine]-4-amine) (herein referred to as Compound 215)

[0338] For details on the synthesis of the compound, refer to International Patent Application PCT / CN2023 / 091270; the entire content of which is incorporated herein by reference.

[0339] In brief, the synthesis steps of compound 215 are as follows:

[0340] Compound 207-5 (500 mg, 1.61 mmol, 1.0 eq) and 7M ammonia methanol (40.0 mL, 173 eq) were used as starting materials to prepare compound 215 (7.77 mg, 14.0 μM, 11.9% yield) as a white solid. LC-MS: m / z = 538.3 (M+H) + .

[0341] In vivo testing example 1.104 single agent effect in the MDA-MB-436 (BRCA1 mutation) subcutaneous tumor model

[0342] This method was used to evaluate the efficacy of 104 monotherapy in the MDA-MB-436 (BRCA1 mutation) subcutaneous tumor model.

[0343] Experimental methods:

[0344] 1. Cell Preparation

[0345] 1.1 Cell culture

[0346] MDA-MB-436: Adherent human breast cancer cells (BRCA1 mutation) purchased from the American Type Culture Collection (ATCC), catalog number HTB-130. The complete culture medium consisted of DMEM + 10% FBS + 1% ITS-G + 1% PS. The cells were assayed during the logarithmic growth phase.

[0347] 1.2 Preparation of cell suspension:

[0348] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 5×10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0349] 2. Experimental Animal Preparation

[0350] 2.1 80 female BALB / c nude mice aged 6-8 weeks were purchased from Zhejiang Weitonglihua for use in the experiment.

[0351] 3. Experimental Design

[0352] After the tumor volume grew to a certain size after inoculation, the grouping and administration of the drug began. The average tumor volume of the experimental group was 196mm 3 , grouping and dosing schedule are as follows.

[0353] Table 1 Pharmacodynamic experimental design

[0354] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0355] po: Oral administration.

[0356] QD: Once a day.

[0357] Blank control: 0.5% CMC-Na + 0.5% Tween-80

[0358] 4. Preparation of test compounds

[0359] 4.1 Compound Information

[0360] Compound name and batch number: 104 / 20220057-05-P1

[0361] Purity: 99.2%

[0362] Calculation formula: Volume = Weight × Purity / Concentration

[0363] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0364] Compound name and batch number: KSQ-4279 / 20210081-024-1, for detailed structure, see Website, catalog number: HY-145471

[0365] Purity: 99.0%

[0366] Calculation formula: Volume = Weight × Purity / Concentration

[0367] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0368] 4.2 Compound preparation

[0369] Table 2. Compound formulations

[0370] 5. Measurement of Tumor and Mouse Weight

[0371] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0372] 6. Data Analysis

[0373] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TV D0对照组 )×100 to calculate tumor growth inhibition rate (TGI%). TGI: tumor inhibition rate. TV Dn给药组： Average tumor volume of the drug-treated group on the day of tumor measurement; TV D0给药组 : Average tumor volume of the drug-treated group on the day of grouping. TV Dn对照组 : Average tumor volume of the blank control group on the day of tumor measurement. TV D0对照组 : Average tumor volume of the blank control group on the day of grouping.

[0374] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0375] 7. Experimental Results

[0376] The experiment concluded on day 21. The 104 monotherapy group demonstrated good efficacy at 100 mpk (100 mpk), with a TGI% of 78%, significantly different from the blank control group. However, no efficacy was observed at 30 mpk (4%) and 10 mpk (10 mpk), with TGI%s of 18% and 4% respectively. The results are shown in Figure 1.

[0377] In vivo testing of the effect of Example 2.104 combined with Olaparib on the MDA-MB-436 (BRCA1 mutation) subcutaneous tumor model

[0378] This method evaluated the efficacy of 104 alone and in combination with olaparib in the MDA-MB-436 (BRCA1 mutation) subcutaneous tumor model.

[0379] Experimental methods:

[0380] 1. Cell Preparation

[0381] 1.1 Cell culture

[0382] MDA-MB-436: Human breast cancer cells (BRCA1 mutation) are adherent cells. The complete culture medium is DMEM + 10% FBS + 1% ITS-G + 1% PS. The cells are tested in the logarithmic growth phase.

[0383] 1.2 Preparation of cell suspension:

[0384] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 4 × 10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0385] 2. Experimental Animal Preparation

[0386] 2.1 80 female BALB / c nude mice aged 6-8 weeks were purchased from Zhejiang Weitonglihua for use in the experiment.

[0387] 3. Experimental Design

[0388] After the tumor volume grows to a certain size after inoculation, the grouping and administration of the drug are started. The average tumor volume of the experimental group is 175mm 3 , grouping and dosing schedule are as follows.

[0389] Table 3 Pharmacodynamic experimental design

[0390] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0391] po: Oral administration.

[0392] QD: Once a day.

[0393] Blank control: 0.5% CMC-Na + 0.5% Tween-80

[0394] 4. Preparation of test compounds

[0395] 4.1 Compound Information

[0396] Compound name and batch number: 104 / 20220057-05-P1

[0397] Purity: 99.2%

[0398] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0399] Compound name and batch number: Olaparib / MCE (MedChemExpress) 136925

[0400] Purity: 99.34%

[0401] Solvent: 10% DMSO+90% 10% HP-β-CD

[0402] Calculation formula: Volume = Weight × Purity / Concentration

[0403] 4.2 Compound preparation

[0404] Table 4. Compound formulation

[0405] 5. Measurement of Tumor and Mouse Weight

[0406] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0407] 6. Data Analysis

[0408] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TVD0对照组 )×100 to calculate tumor growth inhibition rate (TGI%). TGI: tumor inhibition rate. TV Dn给药组： Average tumor volume of the drug-treated group on the day of tumor measurement; TV D0给药组 : Average tumor volume of the drug-treated group on the day of grouping. TV Dn对照组 : Average tumor volume of the blank control group on the day of tumor measurement. TV D0对照组 : Average tumor volume of the blank control group on the day of grouping.

[0409] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0410] 7. Experimental Results

[0411] At the end of the 24-day study, the 104 monotherapy group showed no efficacy at both 10 and 30 mpk, with TGI% of 21% and 22%, respectively. The 104 monotherapy group, administered at 10 and 30 mpk, combined with olaparib (30 mpk), demonstrated good efficacy, with TGI% of 56% and 85%, respectively, significantly different from the blank control group. The results are shown in Figure 2.

[0412] In vivo test Example 3.104 combined with olaparib in the MX-1 subcutaneous tumor model

[0413] This method was used to evaluate the efficacy of 104 alone and in combination with olaparib in the MX-1 subcutaneous tumor model.

[0414] Experimental methods:

[0415] 1. Cell Preparation

[0416] 1.1 Cell culture

[0417] MX-1: Human breast cancer cells (semi-adherent, semi-suspension cells) purchased from Nanjing Kebai (Cat. No. CBP60640) (BRCA1 / 2 deleted). The complete culture medium consisted of 1640 mL / minus 10% FBS and 1% PS. The cells were grown in the logarithmic phase.

[0418] 1.2 Preparation of cell suspension:

[0419] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 4 × 10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0420] 2. Experimental Animal Preparation

[0421] 2.1 100 female BALB / c nude mice aged 6-8 weeks were purchased from Zhejiang Weitonglihua for use in the experiment.

[0422] 3. Experimental Design

[0423] After the tumor volume grew to a certain size after inoculation, the grouping and administration of the drug began. The average tumor volume of the experimental group was 139mm 3 , grouping and dosing schedule are as follows.

[0424] Table 5. Pharmacodynamic experimental design

[0425] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0426] 4. Preparation of test compounds

[0427] 4.1 Compound Information

[0428] Compound name and batch number: 104 / 20220057-05-P1

[0429] Purity: 99.2%

[0430] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0431] Compound name and batch number: Olaparib / 136925

[0432] Purity: 99.34%

[0433] Solvent: 10% DMSO+90% 10% HP-β-CD

[0434] Calculation formula: Volume = Weight × Purity / Concentration

[0435] 4.2 Compound preparation

[0436] Table 6. Compound formulation

[0437] 5. Measurement of Tumor and Mouse Weight

[0438] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0439] 6. Data Analysis

[0440] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组) / (TV Dn对照组 -TV D0对照组 )×100 to calculate the tumor growth inhibition rate (TGI%).

[0441] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0442] 7. Experimental Results

[0443] The study concluded on day 21. The 104 monotherapy group showed no efficacy at 30, 100, and 300 mpk, with TGIs of 27%, -14%, and 2%, respectively. However, 104 demonstrated excellent efficacy in combination with olaparib (100 mpk) at 30, 100, and 300 mpk, respectively, demonstrating TGIs of 89%, 103%, and 109%, respectively, significantly different from the blank control group. The results are shown in Figure 3.

[0444] In vivo test Example 4.104 combined with olaparib on the caov3 subcutaneous tumor model

[0445] This method evaluates the efficacy of cisplatin, 104 alone and in combination with olaparib in a caov3 (HRD+, homologous recombination defect) subcutaneous tumor model.

[0446] Experimental methods:

[0447] 1. Cell Preparation

[0448] 1.1 Cell culture

[0449] Caov3: Human ovarian cancer cells were adherent cells purchased from the American Type Culture Collection (ATCC), catalog number HTB-75. The complete culture medium consisted of DMEM + 10% FBS + 1% PS. The cells were assayed during the logarithmic growth phase.

[0450] 1.2 Preparation of cell suspension:

[0451] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 5×10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0452] 2. Experimental Animal Preparation

[0453] 2.1 Sixty-nine female NCG mice aged 6-8 weeks were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd. for use in the experiment.

[0454] 3. Experimental Design

[0455] After the tumor volume grew to a certain size after inoculation, the grouping and administration of the drug began. The average tumor volume of the experimental group was 163mm 3 , grouping and dosing schedule are as follows.

[0456] Table 7. Pharmacodynamic experimental design

[0457] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0458] 4. Preparation of test compounds

[0459] 4.1 Compound Information

[0460] Compound name and batch number: 104 / 20220057-05-P1

[0461] Purity: 99.2%

[0462] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0463] Compound name and batch number: Olaparib / 136925

[0464] Purity: 99.34%

[0465] Solvent: 10% DMSO+90% 10% HP-β-CD

[0466] 4.2 Compound preparation

[0467] Table 8. Compound formulation

[0468] 5. Measurement of Tumor and Mouse Weight

[0469] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0470] 6. Data Analysis

[0471] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TV D0对照组 )×100 to calculate the tumor growth inhibition rate (TGI%).

[0472] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0473] 7. Experimental Results

[0474] The experiment concluded on day 24. The 104 group, administered at 300 mpk and 30 mpk of olaparib, demonstrated efficacy, with a TGI of 74%, significantly different from the blank control group. However, one mouse died on day 24. Neither the 104 monotherapy group at 300 mpk nor the 100 mpk combined with olaparib group demonstrated efficacy, with TGIs of 45% and 38%, respectively. The results are shown in Figure 4.

[0475] In vivo testing of the effect of Example 5.104 combined with gemcitabine on the MDA-MB-436 (BRCA1 mutation) subcutaneous tumor model

[0476] This method evaluated the efficacy of 104 alone and in combination with gemcitabine in the MDA-MB-436 (BRCA1 mutation) subcutaneous tumor model.

[0477] Experimental methods:

[0478] 1. Cell Preparation

[0479] 1.1 Cell culture

[0480] MDA-MB-436: Human breast cancer cells (BRCA1 mutation) are adherent cells. The complete culture medium is DMEM + 10% FBS + 1% ITS-G + 1% PS. The cells are tested in the logarithmic growth phase.

[0481] 1.2 Preparation of cell suspension:

[0482] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 4 × 10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0483] 2. Experimental Animal Preparation

[0484] 2.1 120 female BALB / c nude mice aged 6-8 weeks were purchased from Zhejiang Weitonglihua for use in the experiment.

[0485] 3. Experimental Design

[0486] After the tumor volume grows to a certain size after inoculation, the grouping and administration of the drug are started. The average tumor volume of the experimental group is 175mm 3 , grouping and dosing schedule are as follows.

[0487] Table 9 Pharmacodynamic experimental design

[0488] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0489] po: Oral administration.

[0490] QD: Once a day.

[0491] ip: intraperitoneal injection.

[0492] BIW: Twice a week.

[0493] Blank control: 0.5% CMC-Na + 0.5% Tween-80

[0494] 4. Preparation of test compounds

[0495] 4.1 Compound Information

[0496] Compound name and batch number: 104 / 20220057-05-P1

[0497] Purity: 99.2%

[0498] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0499] Compound name and batch number: Gemcitabine / MCE (MedChemExpress) 18110

[0500] Purity: 98.8%

[0501] Vehicle: 100% saline

[0502] Calculation formula: Volume = Weight × Purity / Concentration

[0503] 4.2 Compound preparation

[0504] Table 10. Compound formulation

[0505] 5. Measurement of Tumor and Mouse Weight

[0506] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0507] 6. Data Analysis

[0508] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TV D0对照组 )×100 to calculate tumor growth inhibition rate (TGI%). TGI: tumor inhibition rate. TV Dn给药组： Average tumor volume of the drug-treated group on the day of tumor measurement; TV D0给药组 : Average tumor volume of the drug-treated group on the day of grouping. TV Dn对照组 : Average tumor volume of the blank control group on the day of tumor measurement. TV D0对照组 : Average tumor volume of the blank control group on the day of grouping.

[0509] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0510] 7. Experimental Results

[0511] The experiment ended on day 21. The 104 monotherapy group (50 mpk) showed no efficacy, with a TGI% of 1.44%. The 104 50 mpk combined with gemcitabine 20 mpk group showed good efficacy, with a TGI% of 106%, which was significantly different from the blank control group. The results are shown in Figure 5.

[0512] In vivo test example 6. Effect of 215 combined with carboplatin, fruquintinib, and olaparib on the caov3 subcutaneous tumor model

[0513] This method evaluates the efficacy of compound 215 alone and in combination with carboplatin, furiquintinib, and olaparib in a caov3 (HRD+, homologous recombination defect) subcutaneous tumor model.

[0514] Experimental methods:

[0515] 1. Cell Preparation

[0516] 1.1 Cell culture

[0517] Caov3: Human ovarian cancer cells were adherent cells purchased from the American Type Culture Collection (ATCC), catalog number HTB-75. The complete culture medium consisted of DMEM + 10% FBS + 1% PS. The cells were assayed during the logarithmic growth phase.

[0518] 1.2 Preparation of cell suspension:

[0519] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 5×10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0520] 2. Experimental Animal Preparation

[0521] 2.1 155 female NOG mice aged 6-8 weeks were purchased from Zhejiang Weitonglihua for use in the experiment.

[0522] 3. Experimental Design

[0523] After the tumor volume grew to a certain size after inoculation, the grouping and administration of the drug began. The average tumor volume of the experimental group was 176mm 3 , grouping and dosing schedule are as follows.

[0524] Table 11. Pharmacodynamic experimental design

[0525] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0526] 4. Preparation of test compounds

[0527] 4.1 Compound Information

[0528] Compound name and batch number: 215 / EW5066731-P1 / DC230025-0329

[0529] Purity: 98.80%

[0530] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0531] Compound name and batch number: Olaparib / 279339

[0532] Purity: 99.61%

[0533] Solvent: 10% DMSO+90% 10% HP-β-CD

[0534] Compound name and batch number: Fruquintinib / 95759

[0535] Purity: 99.09%

[0536] Solvent: 10% DMSO+90% 20% SBE-β-CD

[0537] Compound name and batch number: Carboplatin / 3M131CA4

[0538] purity: /

[0539] Media: saline

[0540] 4.2 Compound preparation

[0541] Table 12. Compound formulation

[0542] 5. Measurement of Tumor and Mouse Weight

[0543] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0544] 6. Data Analysis

[0545] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TV D0对照组 )×100 to calculate the tumor growth inhibition rate (TGI%).

[0546] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0547] 7. Experimental Results

[0548] The experiment ended on day 32. The combination of 215 at 30 mpk (D1-D5) and 50 mpk (D6-D32) with carboplatin 30 mpk demonstrated efficacy, with a TGI% of 75.80%, significantly better than the 215 and carboplatin 30 mpk monotherapy groups (TGIs of 23.4% and 40.11%, respectively). The combination of 215 at 30 mpk (D1-D5) and 50 mpk (D6-D32) with fruquintinib 3 mpk demonstrated efficacy, with a TGI% of 74.59%, significantly better than the 215 and fruquintinib 3 mpk groups (TGIs of 23.4% and 35.92%, respectively).

[0549] The results are shown in FIG21A and Table 13.

[0550] Table 13 *p<0.05**p<0.01***p<0.001****p<0.0001,versus Vehicle,ANOVA

[0551] In vivo testing of the effect of Example 7.104 combined with olaparib on the OVCAR3 subcutaneous tumor model

[0552] This method evaluated the efficacy of 104 alone and in combination with olaparib in the OVCAR3 (BRCA wild-type and CCNE1 amplified) subcutaneous tumor model.

[0553] Experimental methods:

[0554] 1. Cell Preparation

[0555] 1.1 Cell culture

[0556] OVCAR3: Human ovarian cancer cells, adherent cells, purchased from the American Type Culture Collection (ATCC), catalog number HTB-161. The complete culture medium consisted of RPMI-1640 supplemented with 10% FBS, 1% insulin, and 1% polysaccharide. The cells were assayed during the logarithmic growth phase.

[0557] 1.2 Preparation of cell suspension:

[0558] Harvest cells in the logarithmic growth phase and count them using a Thermo Countess 2. Ensure that the cell viability is above 95%. Adjust the cell suspension concentration to 5×10 7 Each mouse was inoculated with 0.2 mL of cell suspension and Matrigel (1:1).

[0559] 2. Experimental Animal Preparation

[0560] 2.1 100 female BALB / c nude mice aged 6-8 weeks were purchased from Zhejiang Weitonglihua for use in the experiment.

[0561] 3. Experimental Design

[0562] After the tumor volume grew to a certain size after inoculation, the grouping and administration of the drug began. The average tumor volume of the experimental group was 158mm 3 , grouping and dosing schedule are as follows.

[0563] Table 14. Pharmacodynamic experimental design

[0564] Note: Dosing volume: 10 mL / kg based on mouse body weight.

[0565] 4. Preparation of test compounds

[0566] 4.1 Compound Information

[0567] Compound name and batch number: 104 / 231102R

[0568] Purity: 98%

[0569] Solvent: 0.5% CMC-Na+0.5% Tween-80

[0570] Compound name and batch number: Olaparib / 279339

[0571] Purity: 99.61%

[0572] Solvent: 10% DMSO+90% 10% HP-β-CD

[0573] 4.2 Compound preparation

[0574] Table 15. Formulation of compounds

[0575] 5. Measurement of Tumor and Mouse Weight

[0576] Tumor volume was measured twice a week using a vernier caliper and calculated using the formula V = 0.5a × b 2 , where a is the long diameter of the tumor (mm) and b is the short diameter of the tumor (mm). Body weight was measured twice a week using a scale.

[0577] 6. Data Analysis

[0578] 6.1 According to the formula TGI = 100-(TV Dn给药组 -TV D0给药组 ) / (TV Dn对照组 -TV D0对照组 )×100 to calculate the tumor growth inhibition rate (TGI%).

[0579] 6.2 Two-way ANOVA was used to analyze the statistical differences in tumor volume among the various tumor-bearing mice after grouping. A P value less than 0.05 was considered to indicate a significant difference.

[0580] 7. Experimental Results

[0581] The experiment concluded on day 21. The combination of 104 at 300 mpk and olaparib at 100 mpk demonstrated efficacy, with a TGI of 94.99%, significantly better than the 104 and olaparib monotherapy groups. The olaparib monotherapy group demonstrated partial efficacy, with a TGI of 46.85%, while the 104 monotherapy group showed no efficacy, with a TGI of 9.89%.

[0582] The results are shown in Figure 22A, Figure 22B and Table 16.

[0583] Table 16 *p<0.05**p<0.01***p<0.001****p<0.0001,versus Vehicle,ANOVA

[0584] In addition, in the MX-1 animal model experiment, excellent combination results of 104 and Tondawi were observed.

[0585] In vitro experimental example 1

[0586] In vitro enzymatic activity of the present invention

[0587] Experimental reagents:

[0588] Recombinant human His6-USP1 / His6-UAF1 Complex Protein (R&D, Cat. NO. E-568-050)

[0589] Ubiquitin Rhodamine 110 Protein, CF (Ub-Rho) (R&D, Cat. NO. U-555-050)

[0590] Experimental consumables

[0591] 384-well plate (Perkin Elmer, Cat. No. 6007279)

[0592] Experimental methods:

[0593] Compound management: The compounds of the present invention were prepared in DMSO to 10 mM as test stock solutions.

[0594] Experimental methods

[0595] 1 Prepare 1× detection buffer: Prepare 1× analysis buffer (modified Tris buffer).

[0596] 2. Compound serial dilution: Transfer the compound to the assay plate using an Echo. The final DMSO content is 1%.

[0597] 3. Prepare enzyme solution: Prepare enzyme solution with 1× assay buffer.

[0598] 4. Prepare substrate solution: Add Ubiquitin Rhodamine 110 Protein, CF (Ub-Rho) to 1× assay buffer to prepare substrate solution.

[0599] 5. Transfer 10 μL of enzyme solution to the assay plate, or 10 μL of 1× assay buffer for low concentration controls.

[0600] 6 Incubate at room temperature for 1 hour.

[0601] 7. Add 10 μL of substrate solution to each well to start the reaction, centrifuge for 30 seconds, and shake for 30 seconds.

[0602] 8. Read the plate on an Envision or Paradigm for 30 minutes with excitation at 480 nm and emission at 540 nm.

[0603] 9. Collect data on Envision or Paradigm.

[0604] Data Analysis:

[0605] Curve Fitting

[0606] The data were fitted in Excel using formula (1) to obtain the inhibition value

[0607] Formula (1): Percent inhibition rate = (maximum value - signal value) / (maximum value - minimum value) × 100

[0608] The data were fitted in XL-Fit using equation (2) to obtain IC 50 value

[0609] Formula (2): 50% inhibitory concentration formula = minimum concentration + (maximum concentration - minimum concentration) / (1 + (50% inhibitory concentration / compound concentration) 斜率

[0610] 104 Enzyme activity: IC 50 =8.6±1.1 nM (see the results in Figure 6).

[0611] In vitro experimental example 2

[0612] In vitro cytological activity of the compounds of the present invention

[0613] The cell lines used in the following experiments are as follows: MDA-MB-436: human breast cancer cells (BRCA1 mutation), purchased from American Type Culture Collection (ATCC), catalog number: HTB-130.

[0614] Experimental method: (CelltiterGlo assay)

[0615] 1. Cell Preparation

[0616] 1.1 Cell culture

[0617] All cells were adherent cells. The complete culture medium was DMEM+10% FBS+1% ITS-G. The cells were tested in the logarithmic growth phase.

[0618] 1.2 Preparation of cell suspension:

[0619] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1500 cells into a 96-well plate, using 190 μL of cell suspension per well.

[0620] 2. Preparation of test compounds

[0621] 2.1 Prepare DMSO stock solutions of the test compounds. The concentration of the stock solutions of the test compounds was 10 mM.

[0622] 2.2 Preparation of Test Compound Working Stock Solution: For a 10 mM test compound stock solution, add 2 μL of the compound stock solution to 98 μL of complete medium without DMSO. Serially dilute the solution 5-fold into complete medium containing 2% DMSO for a total of 10 concentrations. This is the test compound working stock solution (compound concentration is 20 times the final concentration, with a maximum concentration of 200 μM).

[0623] 2.3 Compound treatment

[0624] 10 μL of compound working stock solution (20-fold diluted DMSO with a final concentration of 0.1%) was added to each well of a 96-well plate seeded with cells.

[0625] The final concentrations of the test compounds were: 10000.00 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.0256 nM, 0.00512 nM.

[0626] 2.4 Control well setting

[0627] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0628] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator for 7 days

[0629] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0630] 4. Data Processing

[0631] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0632] 2) Draw a graph, obtain a curve and calculate IC 50 The specific data are as follows:

[0633] In vitro cytological activity of the compounds of the present invention

[0634] The cell lines used in the following experiments are as follows: UWB1.289 / UWB1.289+BRCA1: human ovarian cancer cells (BRCA1 mutation) were purchased from American Type Culture Collection (ATCC), catalog number: CRL-2945 / CRL-2946.

[0635] Experimental method: (CelltiterGlo assay)

[0636] 1. Cell Preparation

[0637] 1.1 Cell culture

[0638] All cells were adherent and cultured in 50% ATCC-prepared RPMI-1640 medium plus 50% MEGM (Mammary Epithelial Growth Medium from Clonetics / Lonza (MEGM Bullet Kit; CC-3150), consisting of MEBM basal medium and SingleQuot supplements (ATCC did not use gentamicin-amphotericin B). Cells were assayed in the logarithmic growth phase.

[0639] 1.2 Preparation of cell suspension:

[0640] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1000 cells into a 96-well plate, with 190 μL of cell suspension per well.

[0641] 2. Preparation of test compounds

[0642] 2.1 Prepare DMSO stock solutions of test compounds. The stock solution concentration of each test compound is 10 mM.

[0643] 2.2 Preparation of Test Compound Working Stock Solution: For a 10 mM test compound stock solution, add 2 μL of the compound stock solution to 98 μL of complete medium without DMSO. Serially dilute the solution 5-fold into complete medium containing 2% DMSO for a total of 10 concentrations. This is the test compound working stock solution (compound concentration is 20 times the final concentration, with a maximum concentration of 200 μM).

[0644] 2.3 Compound treatment

[0645] 10 μL of compound working stock solution (20-fold diluted DMSO with a final concentration of 0.1%) was added to each well of a 96-well plate seeded with cells.

[0646] The final concentrations of the test compounds were: 10000.00 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.0256 nM, 0.00512 nM.

[0647] 2.4 Control well setting

[0648] Solvent control: 0.1% DMSO. Blank control: 96-well plate detection reading at 0h after drug addition

[0649] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days

[0650] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 5 μL of CTG reagent to 10 μL of PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Mix by shaking on an oscillator for 10 minutes (protect from light). Equilibrate at room temperature for 5 minutes (protect from light and read the light signal value using a multi-function microplate reader).

[0651] 4. Data Processing

[0652] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0653] 2) Draw a graph, obtain a curve and calculate IC 50 The specific data are as follows:

[0654] In vitro cytological activity of the compounds of the present invention

[0655] In vitro combination testing - PARPi + USP1i (Niraparib, Pamiparib, Olaparib, Fluzoparib, Rucaparib, Saruparib, Talazoparib) + 104 25 / 30 nM or none.

[0656] The cell lines used in the following experiments are as follows: MX-1 human breast cancer cells, purchased from Nanjing Kebai, catalog number: CBP60640; human breast cancer cells (BRCA1 / 2 deletion) UWB1.289 were purchased from American Type Culture Collection (ATCC), catalog number: CRL-2945.

[0657] Experimental method: (CelltiterGlo assay)

[0658] 1. Cell Preparation

[0659] 1.1 Cell culture

[0660] All cells were adherent cells and the culture medium was 1640+10% FBS. The cells were tested in the logarithmic growth phase.

[0661] 1.2 Preparation of cell suspension:

[0662] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 2,000 cells into a 96-well plate.

[0663] 2. Preparation of test compounds

[0664] 2.1 Prepare DMSO stock solutions of test PARPi compounds. The stock solution concentration of each test compound is 10 mM.

[0665] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0666] 2.3 Compound treatment

[0667] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0668] The final concentrations of the test compounds (except Saruparib and Talazoparib) were: 10000.00 nM, 2500 nM, 625 nM, 156.2 nM, 39.1 nM, 9.8 nM, 2.4 nM, and 0.6 nM.

[0669] The final concentrations of Saruparib and Talazoparib compounds were 200nM, 50nM, 12.5nM, 3.125nM, 0.78nM, 0.195nM, 0.049nM, and 0.012nM.

[0670] 2.4 Control well setting

[0671] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0672] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 14 days. Discard the culture medium and add the drug again during the treatment.

[0673] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0674] 4. Data Processing

[0675] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0676] 2) Draw a graph, obtain a curve and calculate IC 50 The specific data is shown in Figure 7.

[0677] Conclusion: 104 can effectively enhance the killing activity of PARPi inhibitors in vitro.

[0678] In vitro experimental example 3

[0679] In vitro PD maker test (I) Compound concentration point test.

[0680] The cell lines used in the following experiments are as follows: MDA-MB-436: human breast cancer cells (BRCA1 mutation), purchased from American Type Culture Collection (ATCC), catalog number: HTB-130.

[0681] Experimental method: (Western blot assay)

[0682] 1. Cell Preparation

[0683] 1.1 Cell culture

[0684] All cells were adherent cells, and the culture medium was DMEM+10% FBS+1% ITS-G. The cells were tested in the logarithmic growth phase.

[0685] 1.2 Preparation of cell suspension:

[0686] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 250,000 cells into a 12-well plate.

[0687] 2. Preparation of test compounds

[0688] 2.1 Prepare DMSO stock solutions of test compounds. The stock solution concentration of each test compound is 10 mM.

[0689] 2.2 Preparation of Test Compound Working Stock Solution: 10 mM test compound stock solution was prepared by adding 1 μL of the compound stock solution to 65.7 μL of complete medium without DMSO. Serial 3-fold dilutions were performed in 10 concentrations into complete medium supplemented with 1.4% DMSO. This was the test compound working stock solution (compound concentration was 50 times the final concentration, with a maximum concentration of 150 μM).

[0690] 2.3 Compound treatment

[0691] 20 μL of compound working stock solution (10-fold diluted DMSO with a final concentration of 0.1%) was added to each well of a 96-well plate seeded with cells.

[0692] The final concentrations of the test compounds were: 3000.00 nM, 1000 nM, 333 nM, 111 nM, 37 nM, 12.3 nM, 4.1 nM, 1.4 nM, 0.5 nM, 0.2 nM, 0.05 nM.

[0693] 2.4 Control well setting

[0694] Blank control: 0, 0.1% DMSO

[0695] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 6 hours.

[0696] 3. Add immunoblotting loading buffer to lyse the sample.

[0697] 4. Immunoblotting to detect Ub-PCNA, Ub-FANCD2 and other indicators

[0698] Specific results are shown in FIG8A , which showed that 104 enhanced the levels of downstream substrates in a dose-dependent manner.

[0699] In vitro PD maker test (II) Compound time point test.

[0700] The cell lines used in the following experiments are as follows: MDA-MB-436: purchased from American Type Culture Collection (ATCC), catalog number: HTB-130. Human breast cancer cells (BRCA1 mutation)

[0701] Experimental method: (Western blot assay)

[0702] 1. Cell Preparation

[0703] 1.1 Cell culture

[0704] All cells were adherent cells, and the culture medium was DMEM+10% FBS+1% ITS-G. The cells were tested in the logarithmic growth phase.

[0705] 1.2 Preparation of cell suspension:

[0706] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 250,000 cells into a 12-well plate.

[0707] 2. Preparation of test compounds

[0708] 2.1 Prepare DMSO stock solutions of test compounds. The stock solution concentration of each test compound is 10 mM.

[0709] 2.2 Compound treatment

[0710] The corresponding volume of compound working stock solution (final DMSO concentration of 0.1%) was added to each well of a 96-well plate seeded with cells using a Tecan D300e ultra-micro dispensing instrument.

[0711] The final concentration of the test compound was: 500 nM.

[0712] 2.4 Control well setting

[0713] Blank control: 0, 0.1% DMSO

[0714] 2.5 Place the 12-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 4, 6, 24, 48, 72, 96, 120, 144, and 168 hours.

[0715] 3. Add immunoblotting loading buffer to lyse the sample.

[0716] 4. Immunoblotting to detect Ub-PCNA, Ub-FANCD2, H2Ax and other indicators

[0717] Specific results are shown in FIG8B , which indicate that 104 enhances the levels of downstream substrates in a time-dependent manner.

[0718] In vitro experimental example 4

[0719] In vitro combination drug test - PARPi + USP1i Olaparib + 104 10 / 15nM or none.

[0720] The cell lines used in the following experiments are as follows: MDA-MB-436: human breast cancer cells (BRCA1 mutation), purchased from American Type Culture Collection (ATCC), catalog number: HTB-130.

[0721] Experimental method: (CelltiterGlo assay)

[0722] 1. Cell Preparation

[0723] 1.1 Cell culture

[0724] All cells were adherent and cultured in 50% ATCC-prepared RPMI-1640 medium plus 50% MEGM (Mammary Epithelial Growth Medium from Clonetics / Lonza (MEGM Bullet Kit; CC-3150), consisting of MEBM basal medium and SingleQuot supplements (ATCC did not use gentamicin-amphotericin B). Cells were assayed in the logarithmic growth phase.

[0725] 1.2 Preparation of cell suspension:

[0726] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1500 cells into a 96-well plate.

[0727] 2. Preparation of test compounds

[0728] 2.1 Prepare DMSO stock solutions of test PARPi compounds. The stock solution concentration of each test compound is 10 mM.

[0729] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0730] 2.3 Compound treatment

[0731] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0732] Olaparib 2.46nM.

[0733] 2.4 Control well setting

[0734] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0735] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0736] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 5 μL of CTG reagent to 10 μL of PBS. Add 150 μL of diluted CTG reagent (Celltiter Glo assay kit) to each well. Mix by shaking on an oscillator for 10 minutes (protect from light). Equilibrate at room temperature for 5 minutes (protect from light. Read the light signal value using a multi-function microplate reader).

[0737] 4. Data Processing

[0738] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0739] 2) Draw a graph, obtain a curve and calculate IC 50 The specific data are shown in Figures 9A and 9B.

[0740] Conclusion: 104 can effectively enhance the killing activity of PARPi inhibitors in vitro.

[0741] In vitro experimental example 5

[0742] In vitro combination drug test USP1i+Cisplatin (cisplatin) 104 150 / 300nM or none.

[0743] The cell lines used in the following experiments are as follows: ES-2 (BRCA1 / 2WT & HRD-ovarian cancer), purchased from American Type Culture Collection (ATCC), catalog number: CRL-1978.

[0744] Experimental method: (CelltiterGlo assay)

[0745] 1. Cell Preparation

[0746] 1.1 Cell culture

[0747] ES-2 cells are adherent cells. The culture medium is 90% ATCC-prepared McCoy's 5a Medium Modified Medium (Cat. No. 30-2007) + 10% FBS. The cells were tested in the logarithmic growth phase.

[0748] 1.2 Preparation of cell suspension:

[0749] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 300 cells into a 96-well plate.

[0750] 2. Preparation of test compounds

[0751] 2.1 Preparation of test cisplatin stock solution, the concentration of each test compound stock solution is 10mM

[0752] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0753] 2.3 Compound treatment

[0754] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0755] The final concentration of the test compound was: 2000 nM

[0756] 2.4 Control well setting

[0757] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0758] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0759] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0760] 4. Data Processing

[0761] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0762] 2) Plot the graph and calculate the inhibition rate. The specific data are shown in Figures 10A and 10B.

[0763] Conclusion: 104 can effectively enhance the killing activity of cisplatin in vitro.

[0764] In vitro experimental example 6

[0765] In vitro combination drug test USP1i+Cisplatin (cisplatin) 104 5000nM or none.

[0766] The cell lines used in the following experiments are as follows:

[0767] TOV21G (BRCA1 / 2WT & HRD-ovarian cancer) was purchased from American Type Culture Collection (ATCC), catalog number: CRL-3577.

[0768] Experimental method: (CelltiterGlo assay)

[0769] 1. Cell Preparation

[0770] 1.1 Cell culture

[0771] TOV21G cells are adherent cells, and the culture medium is 50% MCDB 105 medium (1.5 g / L sodium bicarbonate) + 50% Medium 199 (2.2 g / L sodium bicarbonate). The final culture medium contains 15% FBS.

[0772] 1.2 Preparation of cell suspension:

[0773] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1000 cells into a 96-well plate.

[0774] 2. Preparation of test compounds

[0775] 2.1 Preparation of test cisplatin stock solution, the concentration of each test compound stock solution is 10mM

[0776] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0777] 2.3 Compound treatment

[0778] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0779] The final concentration of the test compound was: 30 / 60 nM

[0780] 2.4 Control well setting

[0781] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0782] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0783] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well and shake on a shaker for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0784] 4. Data Processing

[0785] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0786] 2) Draw a graph, obtain a curve and calculate IC 50 The specific data are shown in Figures 11A and 11B.

[0787] Conclusion: 104 can effectively enhance the killing activity of cisplatin in vitro.

[0788] In vitro experimental example 7

[0789] In vitro combination drug test: USP1i+Cisplatin 104 300 / 4000 / 8000nM or none.

[0790] The cell lines used in the following experiments are as follows:

[0791] OVCAR3 (BRCA1 / 2WT&HRD-ovarian cancer) was purchased from American Type Culture Collection (ATCC), catalog number: HTB-161.

[0792] Experimental method: (CelltiterGlo assay)

[0793] 1. Cell Preparation

[0794] 1.1 Cell culture

[0795] OVCAR3 cells are adherent cells and the culture medium is 90% 1640 medium. The final culture medium contains 10% FBS.

[0796] 1.2 Preparation of cell suspension:

[0797] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1000 cells into a 96-well plate.

[0798] 2. Preparation of test compounds

[0799] 2.1 Preparation of test cisplatin stock solution, the concentration of each test compound stock solution is 10mM

[0800] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0801] 2.3 Compound treatment

[0802] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0803] The final concentration of the test compound was: 100 nM

[0804] 2.4 Control well setting

[0805] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0806] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0807] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well and shake on a shaker for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0808] 4. Data Processing

[0809] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0810] 2) Plot the graph and calculate the inhibition ratio. The specific data are shown in Figures 12A to 12C.

[0811] Conclusion: 104 can effectively enhance the killing activity of cisplatin in vitro.

[0812] In vitro experimental example 8

[0813] In vitro combination drug test USP1i+Gemcitabine 104 300nM or none.

[0814] The cell lines used in the following experiments are as follows:

[0815] OVCAR3 (BRCA1 / 2WT&HRD-ovarian cancer) was purchased from American Type Culture Collection (ATCC), catalog number: HTB-161.

[0816] Experimental method: (CelltiterGlo assay)

[0817] 1. Cell Preparation

[0818] 1.1 Cell culture

[0819] OVCAR3 cells are adherent cells and the culture medium is 90% 1640 medium. The final culture medium contains 10% FBS.

[0820] 1.2 Preparation of cell suspension:

[0821] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1000 cells into a 96-well plate.

[0822] 2. Preparation of test compounds

[0823] 2.1 Prepare the test gemcitabine stock solution. The stock solution concentration of each test compound is 10mM

[0824] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0825] 2.3 Compound treatment

[0826] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0827] The final concentration of the test compound was 2.4 nM

[0828] 2.4 Control well setting

[0829] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0830] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0831] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0832] 4. Data Processing

[0833] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0834] 2) Plot the graph and calculate the inhibition ratio. The specific data are shown in Figure 13.

[0835] Conclusion: 104 can effectively enhance the killing activity of gemcitabine in vitro.

[0836] In vitro experimental example 9

[0837] In vitro combination drug test USP1i+Gemcitabine 104 5 / 10 / 15 nM or none.

[0838] The cell lines used in the following experiments are as follows:

[0839] MDA-MB-436: human breast cancer cells (BRCA1 mutation), purchased from American Type Culture Collection (ATCC), catalog number: HTB-130.

[0840] Experimental method: (CelltiterGlo assay)

[0841] 1. Cell Preparation

[0842] 1.1 Cell culture

[0843] All cells were adherent and cultured in 50% ATCC-prepared RPMI-1640 medium plus 50% MEGM (Mammary Epithelial Growth Medium from Clonetics / Lonza (MEGM Bullet Kit; CC-3150), consisting of MEBM basal medium and SingleQuot supplements (ATCC did not use gentamicin-amphotericin B). Cells were assayed in the logarithmic growth phase.

[0844] 1.2 Preparation of cell suspension:

[0845] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1500 cells into a 96-well plate.

[0846] 2. Preparation of test compounds

[0847] 2.1 Prepare the test gemcitabine stock solution. The stock solution concentration of each test compound is 10mM

[0848] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0849] 2.3 Compound treatment

[0850] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0851] The final concentration of the test compound was 2.3 nM

[0852] 2.4 Control well setting

[0853] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0854] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0855] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well and shake on a shaker for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0856] 4. Data Processing

[0857] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0858] 2) Plot the graph and calculate the inhibition ratio. The specific data are shown in Figures 14A to 14C.

[0859] Conclusion: 104 can effectively enhance the killing activity of gemcitabine in vitro.

[0860] In vitro experimental example 10

[0861] In vitro combination drug test USP1i+Gemcitabine 104 40 / 80 / 160 nM or none.

[0862] The cell lines used in the following experiments are as follows:

[0863] Caov3: Human ovarian cancer cells are adherent cells purchased from American Type Culture Collection (ATCC), catalog number: HTB-75.

[0864] Experimental method: (CelltiterGlo assay)

[0865] 1. Cell Preparation

[0866] 1.1 Cell culture

[0867] All cells were adherent cells, the complete culture medium was DMEM + 10% FBS + 1% PS, and the cells were tested in the logarithmic growth phase;

[0868] 1.2 Preparation of cell suspension:

[0869] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 2,000 cells into a 96-well plate.

[0870] 2. Preparation of test compounds

[0871] 2.1 Prepare the test gemcitabine stock solution. The stock solution concentration of each test compound is 10mM

[0872] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0873] 2.3 Compound treatment

[0874] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0875] The final concentration of the test compound was: 0.76 nM

[0876] 2.4 Control well setting

[0877] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0878] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 14 days. Discard the culture medium and add the drug again during the treatment.

[0879] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0880] 4. Data Processing

[0881] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0882] 2) Plot the graph to obtain a curve and calculate the inhibition ratio. The specific data are shown in Figures 15A to 15C.

[0883] Conclusion: 104 can effectively enhance the killing activity of gemcitabine in vitro.

[0884] In vitro experimental example 11

[0885] In vitro combination drug test USP1i+Enzalutamide and Darolutamide 104 2000 / 6000 / 10000nM or none.

[0886] The cell lines used in the following experiments are as follows:

[0887] 22RV1 prostate cancer cells are adherent cells purchased from American Type Culture Collection (ATCC), catalog number: CRL-2505.

[0888] Experimental method: (CelltiterGlo assay)

[0889] 1. Cell Preparation

[0890] 1.1 Cell culture

[0891] All cells were adherent cells, the complete culture medium was 1640 + 10% FBS + 1% PS, and the cells were tested in the logarithmic growth phase;

[0892] 1.2 Preparation of cell suspension:

[0893] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 3,000 cells into a 96-well plate.

[0894] 2. Preparation of test compounds

[0895] 2.1 Prepare the test gemcitabine stock solution. The stock solution concentration of each test compound is 10mM

[0896] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0897] 2.3 Compound treatment

[0898] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0899] The final concentrations of the test compounds were: Enzalutamide 10 μM and Darolutamide 15 μM.

[0900] 2.4 Control well setting

[0901] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0902] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0903] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0904] 4. Data Processing

[0905] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0906] 2) Plot the graph to obtain a curve and calculate the inhibition ratio. The specific data are shown in Figures 16A to 16C.

[0907] Conclusion: 104 can effectively enhance the anti-prostate cancer activity of androgen receptor-related drugs in vitro.

[0908] In vitro experimental example 12

[0909] In vitro combination drug testing USP1i+Enzalutamide and Darolutamide 104 2000 / 4000nM or none.

[0910] The cell lines used in the following experiments are as follows:

[0911] LNCaP prostate cancer cells are adherent cells purchased from American Type Culture Collection (ATCC), catalog number: CRL-1740.

[0912] Experimental method: (CelltiterGlo assay)

[0913] 1. Cell Preparation

[0914] 1.1 Cell culture

[0915] All cells were adherent cells, the complete culture medium was 1640 + 10% FBS + 1% PS, and the cells were tested in the logarithmic growth phase;

[0916] 1.2 Preparation of cell suspension:

[0917] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1500 cells into a 96-well plate.

[0918] 2. Preparation of test compounds

[0919] 2.1 Prepare the test stock solutions of enzalutamide and dalostatin. The stock solution concentration of each test compound is 10 mM.

[0920] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0921] 2.3 Compound treatment

[0922] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0923] The final concentrations of the test compounds were: Enzalutamide 10 μM and Darolutamide 15 μM.

[0924] 2.4 Control well setting

[0925] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0926] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator for 7 days.

[0927] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0928] 4. Data Processing

[0929] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0930] 2) Plot the graph to obtain a curve and calculate the inhibition ratio. The specific data are shown in Figures 17A to 17C.

[0931] Conclusion: 104 can effectively enhance the anti-prostate cancer activity of androgen receptor-related drugs in vitro.

[0932] In vitro experimental example 13

[0933] In vitro combination drug test: USP1i + docetaxel 104 6000 / 10000 nM or none.

[0934] The cell lines used in the following experiments are as follows:

[0935] 22RV1 prostate cancer cells are adherent cells purchased from American Type Culture Collection (ATCC), catalog number: CRL-2505.

[0936] Experimental method: (CelltiterGlo assay)

[0937] 1. Cell Preparation

[0938] 1.1 Cell culture

[0939] All cells were adherent cells, the complete culture medium was 1640 + 10% FBS + 1% PS, and the cells were tested in the logarithmic growth phase;

[0940] 1.2 Preparation of cell suspension:

[0941] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 3,000 cells into a 96-well plate.

[0942] 2. Preparation of test compounds

[0943] 2.1 Preparation of test docetaxel stock solution, the concentration of each test compound stock solution was 10mM

[0944] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0945] 2.3 Compound treatment

[0946] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0947] The final concentration of the test compound was: Docetaxel 1.6 nM

[0948] 2.4 Control well setting

[0949] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0950] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0951] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0952] 4. Data Processing

[0953] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0954] 2) Plot the graph to obtain a curve and calculate the inhibition ratio. The specific data are shown in Figures 18A to 18C.

[0955] Conclusion: 104 can effectively enhance the killing activity of docetaxel against prostate cancer in vitro.

[0956] In vitro experimental example 14

[0957] In vitro combination drug testing: USP1i + docetaxel 104 4000 / 8000 nM or none.

[0958] The cell lines used in the following experiments are as follows:

[0959] LNCaP prostate cancer cells are adherent cells purchased from American Type Culture Collection (ATCC), catalog number: CRL-1740.

[0960] Experimental method: (CelltiterGlo assay)

[0961] 1. Cell Preparation

[0962] 1.1 Cell culture

[0963] All cells were adherent cells, the complete culture medium was 1640 + 10% FBS + 1% PS, and the cells were tested in the logarithmic growth phase;

[0964] 1.2 Preparation of cell suspension:

[0965] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 1500 cells into a 96-well plate.

[0966] 2. Preparation of test compounds

[0967] 2.1 Preparation of Docetaxel stock solution. The concentration of each test compound stock solution was 10 mM.

[0968] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0969] 2.3 Compound treatment

[0970] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0971] The final concentration of the test compound was: Docetaxel 0.32 nM

[0972] 2.4 Control well setting

[0973] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0974] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0975] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[0976] 4. Data Processing

[0977] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[0978] 2) Plot the graph to obtain a curve and calculate the inhibition ratio. The specific data are shown in Figures 19A and 19B.

[0979] Conclusion: 104 can effectively enhance the anti-prostate cancer activity of AR-related drugs (androgen receptor-related drugs) in vitro.

[0980] In vitro experimental example 15

[0981] In vitro combination testing of USP1i+Elimusertib 15nM or none.

[0982] The cell lines used in the following experiments are as follows:

[0983] MDA-MB-436: human breast cancer cells (BRCA1 mutation), purchased from American Type Culture Collection (ATCC), catalog number: HTB-130.

[0984] Experimental method: (CelltiterGlo assay)

[0985] 1. Cell Preparation

[0986] 1.1 Cell culture

[0987] All cells were adherent and cultured in 50% ATCC-prepared RPMI-1640 medium plus 50% MEGM (Mammary Epithelial Growth Medium from Clonetics / Lonza (MEGM Bullet Kit; CC-3150), consisting of MEBM basal medium and SingleQuot supplements (ATCC did not use gentamicin-amphotericin B). Cells were assayed in the logarithmic growth phase.

[0988] 1.2 Preparation of cell suspension:

[0989] Harvest cells in the logarithmic growth phase and count using a Thermo Countess 2. Ensure cell viability is above 90%. Adjust to the appropriate concentration and seed 2,000 cells into a 96-well plate.

[0990] 2. Preparation of test compounds

[0991] 2.1 Preparation of Elimusertib stock solution. The stock solution concentration of each test compound was 10 mM.

[0992] 2.2 Set up the dosing program on the Tecan D300e ultra-micro dispensing instrument.

[0993] 2.3 Compound treatment

[0994] The corresponding volume of compound was added to each well of a 96-well plate seeded with cells using Tecan D300e.

[0995] The final concentrations of the test compounds were: 1.6 / 8 / 40 / 200 nM

[0996] 2.4 Control well setting

[0997] Solvent control: 0.1% DMSO. Blank control: Read the 96-well plate at 0 h after drug addition.

[0998] 2.5 Place the 96-well plate in a 37°C, 5% CO2 cell culture incubator and culture for 7 days.

[0999] 3. Thaw the CTG reagent and equilibrate the 96-well plate to room temperature for 30 minutes. Remove the drug-containing culture medium from the plate and dilute the CTG reagent with PBS. Add 150 μL of the diluted CTG reagent (Celltiter Glo assay kit) to each well. Shake on an oscillator for 10 minutes to mix (protect from light). Equilibrate at room temperature for 5 minutes (protect from light). Read the light signal using a multi-function microplate reader.

[1000] 4. Data Processing

[1001] 1) Inhibition rate (%) = (DMSO solvent control well reading - test substance well reading) / (DMSO solvent control well reading - blank control well reading) × 100%;

[1002] 2) Plot the graph to obtain a curve and calculate the inhibition ratio. The specific data are shown in Figures 20A to 20D.

[1003] Conclusion: 104 can effectively enhance the killing activity of Eliminusertib in vitro.

Claims

1. A combination product, comprising: a USP1 inhibitor, and an anti-tumor drug other than the USP1 inhibitor; wherein The USP1 inhibitor has the structure shown in Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, isotope-substituted compound or stereoisomer thereof: Among them, Ring A and Ring B are each independently selected from C 6-10 aryl, 5- to 10-membered heteroaryl, C 3-8 cycloalkyl, and 3- to 8-membered heterocyclic group, and Ring A and Ring B are each independently optionally substituted by one or more R1; L is selected from a chemical bond, -O-, -S-, -C 1-6 alkylene-, -O-C 1-6 alkylene-, -C 1-6 alkylene-O-, -S-C 1-6 alkylene- and -C 1-6 alkylene-S-; R a and R b each independently selected from an H atom, -CN, C 1-6 alkyl, -OH, halogen, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 hydroxyalkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy; or R a and R b together form an oxo group, C 3-8 cycloalkyl or a 3- to 8-membered heterocyclic group; R2 is selected from an H atom, -OH, -CN, C 1-6 alkyl, C 2-6 alkynyl, -C 1-6 alkyl-C 6-10 aryl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 3-6 cycloalkyl and a 5- to 7-membered heterocyclic group, wherein the C 1-6 alkyl or -C 1-6 alkyl-C 6-10 aryl is optionally substituted by one or more R1; preferably, R2 is selected from an H atom, -CN, methyl, trideuteriomethyl, ethynyl, propargyl, tetrahydrofuranyl, cyclopropyl, methoxy, and hydroxy; R3 is selected from H atom, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, 5-7 membered heterocyclic group and -C 1-6 alkylene-C(O)-O-C 1-6 alkyl, wherein the C 1-6 hydroxyalkyl and 5-7 membered heterocyclic group are each independently optionally substituted by one or more C 1-6 alkyl; Preferably, R3 is selected from an H atom, a methoxy group, a trifluoromethyl group, a Cl atom, -CN, an isopropoxy group, an ethynyl group, a difluoromethoxy group, a morpholinyl group, F atom, hydroxymethyl and More preferably, R3 is selected from an H atom, -OH, -COOH, -NH2, -CN, a halogen, C 1-6 alkyl; Further preferably, R3 is selected from an H atom, C 1-6 alkyl group; R4 is selected from H atom, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy and C 1-6 hydroxyalkyl; preferably, R4 is an H atom; R5 is selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl, C 6-10 aryl fused to a 3- to 8-membered heterocyclic group, C 6-10 aryl, C 3-8 cycloalkyl and 3- to 8-membered heterocyclic group, wherein the C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl, C 3-8 cycloalkyl and 3- to 8-membered heterocyclic group are each independently optionally substituted by one or more R1; R1 is independently selected from D atom, -OH, -COOH, -NH2, -CN, oxo group, halogen, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyl, 3-8 membered heterocyclic group, -O-C 1-6 alkylene-O-C 1-6 alkyl, wherein the C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyl and 3-8 membered heterocyclic group are each independently optionally substituted with one or more substituents selected from D atom, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy and C 1-6 hydroxyalkyl; and n is an integer between 0 and 8.

2. The combination product according to claim 1, wherein ring A and ring B are each independently selected from phenyl, piperidinyl, cyclohexyl, cyclopropyl, cyclobutyl, pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, bicyclo[2.2.2]octyl, 2-oxabicyclo[2.2.2]octyl, pentacyclooctyl, isoindolinone, imidazo[1,2-a]pyrazine, piperidine-2,6-dione, thienyl, furanyl, cyclopentyl, pyranyl, pyrrolidinyl, piperazinyl, morpholinyl, naphthyl, pyrrolyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, indolinone, pyrido[3,2-d]pyrimidinyl, pteridinyl, pyrazolo[4,3-c]pyridyl, pyrazolo[3,4-d]pyrimidinyl, and cubyl, and ring A and ring B are each independently optionally substituted by one or more R1s, where R1 is as defined in claim 1; L is selected from a chemical bond, -O-, -O-C 1-6 alkylene- and -C 1-6 alkylene-O-; In particular, Selected from and ring A and ring B are each independently optionally substituted by one or more R1s, where R1 is as defined in claim 1; More particularly, Selected from 3. The combination product according to claim 1 or 2, wherein R5 is selected from C 6-10 aryl, 5- to 6-membered heteroaryl, C fused to a 5- to 6-membered heterocyclic group 6-10 aryl and C fused to a 5- to 6-membered heteroaryl 6-10 aryl, preferably selected from phenyl, pyridyl, pyrimidyl, pyrazolyl, imidazolyl, thiazolyl, indolyl, indolinyl and isoxazolyl, the C 6-10 aryl, 5- to 6-membered heteroaryl, C fused to a 5- to 6-membered heterocyclic group 6-10 aryl and C fused to a 5- to 6-membered heteroaryl 6-10 aryl is each independently optionally substituted with one or more substituents selected from -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl and C 3-6 cycloalkyl; In particular, R5 is selected from More particularly, R5 is 4. The combination product according to any one of claims 1-3, wherein n is 0 or 1; R a and R b each independently selected from an H atom, -CN, C 1-6 alkyl, -OH, and a halogen; Preferably, R a and R b are each independently an H atom.

5. The combination product according to any one of claims 1-4, wherein the USP1 inhibitor is selected from:

6. The combined product according to any one of claims 1-5, wherein, the anti-tumor drug other than the USP1 inhibitor is selected from PARP inhibitors, anti-metabolic anti-tumor drugs, taxane anti-tumor drugs, ATR kinase inhibitors, estrogen receptor modulators, androgen receptor inhibitors, platinum coordination complexes, immunotherapy drugs, and anti-angiogenic drugs; preferably, the PARP inhibitors are selected from niraparib, pamiparib, olaparib, farruxoparib, rucaparib, Saruparib, and talazoparib; the anti-metabolic anti-tumor drugs are selected from gemcitabine, fluorouracil, methotrexate, cytarabine, mercaptopurine, and thioguanine; the taxane anti-tumor drugs are selected from docetaxel, paclitaxel, paclitaxel liposome, and albumin-bound paclitaxel; the ATR kinase inhibitor is selected from Elimusertib; the estrogen receptor modulator is enzalutamide; the androgen receptor inhibitor is darolutamide; the platinum coordination complexes are selected from cisplatin and carboplatin; and the anti-angiogenic drugs are selected from VEGF / VEGFR pathway inhibitors, integrin inhibitors, platelet-derived growth factor receptor inhibitors, HIF inhibitors, and endothelin receptor antagonists; preferably, the anti-angiogenic drugs are selected from monoclonal antibodies and small molecule tyrosine kinase inhibitors; more preferably, the anti-angiogenic drugs are selected from fruquintinib, bevacizumab, ramucirumab, sorafenib, sunitinib, apatinib, regorafenib, cabozantinib, and lenvatinib.

7. The combination product according to any one of claims 1-6, for treating cancer.

8. The combination product according to claim 7, wherein the cancer is selected from: breast cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, uterine cancer, peritoneal cancer, and endometrial cancer; Preferably, the cancer is a PARP inhibitor-resistant cancer; Preferably, wherein the cancer is a BRCA1 mutant cancer, a BRCA2 mutant cancer, or a BRCA1 mutant, BRCA2 mutant cancer, BRCA wild-type and CCNE1 amplified tumor, HRD-positive tumor, and DDR gene mutation tumor; Preferably, wherein the cancer is selected from BRCA1 mutant breast cancer, BRCA2 mutant breast cancer, BRCA1 mutant and BRCA2 mutant breast cancer, BRCA1 mutant ovarian cancer, BRCA2 mutant ovarian cancer, BRCA wild-type and CCNE1 amplified ovarian cancer, or p53 mutant ovarian cancer.

9. The combination product according to any one of claims 1-8, wherein the other anti-tumor drugs other than the USP1 inhibitor are selected from olaparib, gemcitabine, niraparib, pamiparib, farruxoparib, rucaparib, Saruparib, talazoparib, docetaxel, Elimusertib, enzalutamide, darolutamide, cisplatin, carboplatin, fruquintinib, bevacizumab, ramucirumab, sorafenib, sunitinib, apatinib, regorafenib, cabozantinib, and lenvatinib.

10. The combination product according to any one of claims 1-9, wherein the USP1 inhibitor and the other anti-tumor drugs other than the USP1 inhibitor are administered simultaneously, separately, or sequentially.

11. Use of a USP1 inhibitor in combination with other anti-tumor drugs other than the USP1 inhibitor in the preparation of a drug for the treatment of cancer, wherein The USP1 inhibitor has the structure shown in Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, isotope-substituted compound or stereoisomer thereof: Among them, Ring A and Ring B are each independently selected from C 6-10 aryl, 5- to 10-membered heteroaryl, C 3-8 cycloalkyl, and 3- to 8-membered heterocyclic group, and Ring A and Ring B are each independently optionally substituted by one or more R1; L is selected from a chemical bond, -O-, -S-, -C 1-6 alkylene-, -O-C 1-6 alkylene-, -C 1-6 alkylene-O-, -S-C 1-6 alkylene- and -C 1-6 alkylene-S-; R a and R b are each independently selected from an H atom, -CN, C 1-6 alkyl, -OH, halogen, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 hydroxyalkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy; or R a and R b together form an oxo group, C 3-8 cycloalkyl or a 3- to 8-membered heterocyclic group; R2 is selected from an H atom, -OH, -CN, C 1-6 alkyl, C 2-6 alkynyl, -C 1-6 alkyl-C 6-10 aryl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 3-6 cycloalkyl and a 5- to 7-membered heterocyclic group, wherein the C 1-6 alkyl or -C 1-6 alkyl-C 6-10 aryl is optionally substituted by one or more R1; Preferably, R2 is selected from an H atom, -CN, methyl, trideuteriomethyl, ethynyl, propargyl, tetrahydrofuranyl, cyclopropyl, methoxy, and hydroxy; R3 is selected from H atom, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, 5-7 membered heterocyclic group and -C 1-6 alkylene-C(O)-O-C 1-6 alkyl, wherein the C 1-6 hydroxyalkyl and 5-7 membered heterocyclic group are each independently optionally substituted by one or more C 1-6 alkyl; More preferably, R3 is selected from an H atom, -OH, -COOH, -NH2, -CN, a halogen, C 1-6 alkyl; More preferably, R3 is selected from an H atom, C 1-6 alkyl; Preferably, R3 is selected from an H atom, a methoxy group, a trifluoromethyl group, a Cl atom, -CN, an isopropoxy group, an ethynyl group, a difluoromethoxy group, a morpholinyl group, -OH, F atom, hydroxymethyl and R4 is selected from H atom, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy and C 1-6 hydroxyalkyl; Preferably, R4 is an H atom; R5 is selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl, C 6-10 aryl fused to a 3- to 8-membered heterocyclic group, C 6-10 aryl, C 3-8 cycloalkyl and 3- to 8-membered heterocyclic group, wherein the C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl, C 3-8 cycloalkyl and 3- to 8-membered heterocyclic group are each independently optionally substituted with one or more R1; R1 is independently selected from D atom, -OH, -COOH, -NH2, -CN, oxo group, halogen, C 1-6 alkyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl, C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyl, 3-8 membered heterocyclic group, -O-C 1-6 alkylene-O-C 1-6 alkyl, wherein the C 6-10 aryl, 5-10 membered heteroaryl, C 3-8 cycloalkyl and 3-8 membered heterocyclic group are each independently optionally substituted with one or more substituents selected from D atom, -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy and C 1-6 hydroxyalkyl, and n is an integer between 0 and 8.

12. The use according to claim 11, wherein Ring A and ring B are each independently selected from phenyl, piperidinyl, cyclohexyl, cyclopropyl, cyclobutyl, pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, bicyclo[2.2.2]octyl, 2-oxabicyclo[2.2.2]octyl, pentacyclooctyl, isoindolinone, imidazo[1,2-a]pyrazine, piperidine-2,6-dione, thiophenyl, furanyl, cyclopentyl, pyranyl, pyrrolidinyl, piperazinyl, morpholinyl, naphthyl, pyrrolyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, indolinone, pyrido[3,2-d]pyrimidinyl, pteridinyl, pyrazolo[4,3-c]pyridyl, pyrazolo[3,4-d]pyrimidinyl, and cubyl, and ring A and ring B are each independently optionally substituted by one or more R1s as defined in claim 11; L is selected from a chemical bond, -O-, -O-C 1-6 alkylene-, and -C 1-6 alkylene-O-; In particular, Selected from And ring A and ring B are each independently optionally substituted by one or more R1, where R1 is as defined in claim 11; More particularly, Selected from 13. The use according to claim 11 or 12, wherein R5 is selected from C 6-10 aryl, 5- to 6-membered heteroaryl, C fused to a 5- to 6-membered heterocyclic group 6-10 aryl and C fused to a 5- to 6-membered heteroaryl 6-10 aryl, preferably selected from phenyl, pyridyl, pyrimidinyl, pyrazolyl, imidazolyl, thiazolyl, indolyl, indolinyl and isoxazolyl, the C 6-10 aryl, 5- to 6-membered heteroaryl, C fused to a 5- to 6-membered heterocyclic group 6-10 aryl and C fused to a 5- to 6-membered heteroaryl 6-10 aryl is each independently optionally substituted with one or more substituents selected from -OH, -COOH, -NH2, -CN, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 1-6 hydroxyalkyl and C 3-6 cycloalkyl; In particular, R5 is selected from More particularly, R5 is 14. The use according to any one of claims 11-13, wherein n is 0 or 1; R a and R b are each independently selected from an H atom, -CN, C 1-6 alkyl, -OH, and a halogen; Preferably, R a and R b are each independently an H atom.

15. Use according to any one of claims 11-14, wherein the USP1 inhibitor is selected from:

16. The use according to any one of claims 11 - 15, wherein, The other anti-tumor drugs other than the USP1 inhibitor are selected from PARP inhibitors, anti-metabolic anti-tumor drugs, taxane anti-tumor drugs, ATR kinase inhibitors, estrogen receptor modulators, androgen receptor inhibitors, platinum coordination complexes, immunotherapy drugs, and anti-angiogenic drugs; Preferably, the PARP inhibitor is selected from niraparib, pamiparib, olaparib, farruxoparib, rucaparib, Saruparib, talazoparib; The anti-metabolic anti-tumor drugs are selected from gemcitabine, fluorouracil, methotrexate, cytarabine, mercaptopurine, and thioguanine; The taxane anti-tumor drugs are selected from docetaxel, paclitaxel, paclitaxel liposome, albumin-bound paclitaxel; The ATR kinase inhibitor is selected from Elimusertib; The estrogen receptor modulator is enzalutamide; The androgen receptor inhibitor is darolutamide; The platinum coordination complexes are selected from cisplatin and carboplatin; and The anti-angiogenic drugs are selected from VEGF / VEGFR pathway inhibitors, integrin inhibitors, platelet-derived growth factor receptor inhibitors, HIF inhibitors, and endothelin receptor antagonists; Preferably, the anti-angiogenic drugs are selected from monoclonal antibodies and small molecule tyrosine kinase inhibitors; More preferably, the anti-angiogenic drugs are selected from fruquintinib, bevacizumab, ramucirumab, sorafenib, sunitinib, apatinib, regorafenib, cabozantinib, and lenvatinib.

17. The use according to any one of claims 11-16, wherein the USP1 inhibitor is co-administered with other anti-tumor drugs other than the USP1 inhibitor in the same formulation or different formulations; Preferably, the USP1 inhibitor is administered simultaneously, separately, or sequentially with other anti-tumor drugs other than the USP1 inhibitor; Preferably, the other anti-tumor drugs other than the USP1 inhibitor are selected from olaparib, gemcitabine, niraparib, pamiparib, farruxoparib, rucaparib, Saruparib, talazoparib, docetaxel, Elimusertib, enzalutamide, darolutamide, cisplatin, carboplatin, fruquintinib, bevacizumab, ramucirumab, sorafenib, sunitinib, apatinib, regorafenib, cabozantinib, and lenvatinib.

18. The use according to any one of claims 11-17, wherein the USP1 inhibitor significantly enhances the anti-tumor activity of other anti-tumor drugs other than the USP1 inhibitor.

19. The use according to any one of claims 11-17, wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, uterine cancer, peritoneal cancer, and endometrial cancer; Preferably, the cancer is a PARP inhibitor-resistant cancer; Preferably, the cancer is BRCA1 mutant cancer, BRCA2 mutant cancer, or BRCA1 mutant, BRCA2 mutant cancer, BRCA wild-type and CCNE1 amplified tumor, HRD-positive tumor, and DDR gene mutant tumor; Preferably, the cancer is selected from BRCA1 mutant breast cancer, BRCA2 mutant breast cancer, BRCA1 mutant and BRCA2 mutant breast cancer, BRCA1 mutant ovarian cancer, BRCA2 mutant ovarian cancer, BRCA wild-type and CCNE1 amplified ovarian cancer, or p53 mutant ovarian cancer.

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