Combined pharmaceutical composition and use thereof

Abstract

The present invention provides a combined pharmaceutical composition of a compound of formula (I) and a PARP inhibitor, and use thereof in preparing a drug for treating USP1-mediated diseases. In particular, the combined use of the compound of formula (I) and the PARP inhibitor shows a good synergistic effect in both a cell proliferation inhibition experiment and an in-vivo tumor model.

Description

A combined pharmaceutical composition and its use Technical Field

[0001] The present disclosure relates to the field of biomedicine, and specifically to a pharmaceutical composition of a combination of a USP1 inhibitor and a PARP inhibitor, and its use in preparing drugs for USP1-mediated related diseases, including cellular inflammatory diseases, neurodegenerative diseases, and cancer. Background Art

[0002] Ubiquitin is a small, highly conserved protein of 76 amino acids that is post-translationally conjugated to substrate proteins, including itself, through a three-step enzymatic reaction. The initial covalent attachment occurs primarily between the C-terminal glycine of ubiquitin and the ε-amino group of a lysine residue of the target protein. Additional ubiquitin molecules can be attached to one of the seven internal lysines of ubiquitin, resulting in different ubiquitin chain topologies. Similar to other types of post-translational modifications, ubiquitination is a reversible process counter-regulated by enzymes called deubiquitinating enzymes (DUBs), which catalyze the removal of ubiquitin from modified proteins.

[0003] The ubiquitin-proteasome system offers additional opportunities for therapeutic intervention, with the potential for increased specificity and improved clinical efficacy. The most obvious targets include enzymes involved in ubiquitin conjugation and deconjugation (i.e., ubiquitin ligases and DUBs), upstream processes of proteasome-mediated protein degradation. Among DUBs, ubiquitin-specific protease 1 (USP1) has emerged as an attractive anticancer target due to its involvement in regulating DNA damage response pathways. USP1 associates with UAF1 (USP1-associated factor 1) to generate the heterodimeric USP1 / UAF1 complex required for deubiquitinase activity. The USP1 / UAF1 complex has been shown to regulate tolerance to DNA cross-linker-induced DNA damage through deubiquitination of PCNA (proliferating cell nuclear antigen) and FANCD2 (Fanconi anemia complementation group D2), proteins that function in translesion synthesis and the Fanconi anemia pathway, respectively.

[0004] The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a role in DNA repair and genomic integrity. PARP is crucial for 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. DNA strand breaks activate this poly (ADP-ribosyl)ation ("PARylation") activity, thereby adding Par to PARP itself and other DNA repair enzymes. PARP is crucial for the recruitment of DNA repair proteins to the site of damage. Homologous recombination is a DNA repair process that is crucial for the precise repair of DNA damage. The BRCA1 / 2 genes and other Fanconi anemia pathway genes (e.g., RAD51D, NBN, ATM) are components of homologous recombination-mediated DNA repair. Mutations in genes encoding homologous recombination factors play a role in the development of certain cancers. PARP inhibitors prevent the repair of single-strand DNA breaks and promote the conversion of single-strand breaks into double-strand breaks, which leads to synthetic lethality in cancer cells that lack double-strand break repair mechanisms (such as homologous recombination).

[0005] Combination therapy of USP1 inhibitors and PARP inhibitors. There is currently no combination drug on the market, and this more effective cancer treatment therapy has unmet medical needs.

[0006] PARP inhibitors disclosed in the prior art:

[0007] Patent PCT / CN2023 / 116528 describes a small molecule USP1 inhibitor, the structure of which is shown in Formula (I). This small molecule inhibitor exhibits excellent USP1 kinase inhibitory activity and cell proliferation inhibition activity, as well as good pharmacokinetic properties and in vivo efficacy, and is expected to be developed into a clinical drug.

[0008] There are no reports in the prior art on the combined use of the compound of formula (I) and a PARP inhibitor, and it is not possible to predict whether the combination will have clinical benefits. The present disclosure aims to provide a pharmaceutical composition for combined use of the compound of formula (I) and a PARP inhibitor. Summary of the Invention

[0009] The purpose of the present disclosure is to provide a pharmaceutical composition of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a PARP inhibitor, and its use in preparing drugs for USP1 target-mediated related diseases including cellular inflammatory diseases, neurodegenerative diseases, and cancer.

[0010] Specifically,

[0011] The present disclosure provides a use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a PARP inhibitor in the preparation of a drug for treating diseases mediated by the USP1 target.

[0012] In some embodiments of the present disclosure, the PARP inhibitor is selected from olaparib, niraparib, talazoparib, rucaparib, and AZD5305.

[0013] In some embodiments of the present disclosure, the PARP inhibitor is selected from olaparib.

[0014] The present disclosure provides a combined pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a PARP inhibitor.

[0015] In some embodiments of the present disclosure, the compound (I) or a pharmaceutically acceptable salt thereof is included.

[0016] Any combination with the above-mentioned PARP inhibitor, wherein the PARP inhibitor is selected from olaparib, niraparib, talazoparib, rucaparib, and AZD5305.

[0017] The present disclosure also provides use of the above-mentioned combined pharmaceutical composition in the preparation of a drug for treating diseases mediated by the USP1 target.

[0018] In some embodiments of the present disclosure, the USP1 target-mediated related diseases described in the above uses include cellular inflammatory diseases, neurodegenerative diseases, and cancer.

[0019] In some embodiments of the present disclosure, the cancer described in the above use is selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, and colon cancer; preferably breast cancer and ovarian cancer.

[0020] In some embodiments of the present disclosure, the compound of formula (I) and the PARP inhibitor in the above-mentioned use can be administered simultaneously, sequentially or intermittently.

[0021] The present disclosure also provides a method for treating diseases mediated by USP1 targets, comprising administering to the subject an effective therapeutic amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a PARP inhibitor.

[0022] In some embodiments of the present disclosure, the above-mentioned treatment method comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof

[0023] Any combination with a PARP inhibitor, wherein the PARP inhibitor is selected from olaparib, niraparib, talazoparib, rucaparib, AZD5305; preferably, the PARP inhibitor is selected from olaparib.

[0024] In some embodiments of the present disclosure, the USP1 target-mediated related diseases in the above-mentioned treatment methods include cellular inflammatory diseases, neurodegenerative diseases, and cancer.

[0025] In some embodiments of the present disclosure, the cancer described in the above-mentioned treatment methods is selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, and colon cancer; preferably breast cancer and ovarian cancer.

[0026] In some embodiments of the present disclosure, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the PARP inhibitor in the above-mentioned treatment method can be administered simultaneously, sequentially or intermittently.

[0027] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2.5 mg / kg to about 75 mg / kg (calculated by weight of the compound of formula (I)), for example, about 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 2 3mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 3 4mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg, 50mg / kg, 51mg / kg, 52mg / kg, 53mg / kg, 54mg / kg, 55mg / kg, or 75 mg / kg; or a dose range consisting of any of the foregoing values.

[0028] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2.5 mg / kg to about 75 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0029] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2.5 mg / kg to about 50 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0030] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2.5 mg / kg to about 30 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0031] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2.5 mg / kg to about 20 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0032] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 50 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0033] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 30 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0034] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 20 mg / kg (calculated by weight of the compound of formula (I)) per administration.

[0035] In some embodiments of any of the above aspects, the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 2.5 mg / kg or about 3 mg / kg or about 4 mg / kg or about 5 mg / kg or about 10 mg / kg or about 15 mg / kg or about 20 mg / kg or about 30 mg / kg or about 40 mg / kg or about 50 mg / kg (calculated by weight of the compound of formula (I)) each time.

[0036] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 150 mg / kg (calculated by weight of the compound of formula (I)), for example 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg 0 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg or 150 mg / kg, or a dose range formed by any of the above values.

[0037] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 150 mg / kg (calculated by weight of the compound of formula (I)).

[0038] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0039] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 80 mg / kg (calculated by weight of the compound of formula (I)).

[0040] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 60 mg / kg (calculated by weight of the compound of formula (I)).

[0041] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 5 mg / kg to about 40 mg / kg (calculated by weight of the compound of formula (I)).

[0042] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 10 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0043] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 10 mg / kg to about 80 mg / kg (calculated by weight of the compound of formula (I)).

[0044] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 20 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0045] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 30 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0046] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 40 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0047] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 50 mg / kg to about 100 mg / kg (calculated by weight of the compound of formula (I)).

[0048] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 50 mg to about 10,000 mg (calculated by weight of the compound of formula (I)).

[0049] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 75 mg to about 5000 mg (calculated by weight of the compound of formula (I)).

[0050] In some embodiments of any of the above aspects, the average daily dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is about 75 mg to about 2500 mg (calculated by weight of the compound of formula (I)).

[0051] In some embodiments of any of the aforementioned aspects, the compound of formula (I) or a pharmaceutically acceptable salt thereof can be administered once, twice, or three times a day.

[0052] In some embodiments of any of the foregoing aspects, the dose of the PARP inhibitor (free form, salt) is from about 0.1 mg to about 1000 mg per dose, for example, about 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, or a range of any of the foregoing values.

[0053] In some embodiments of any of the foregoing, the dose of the PARP inhibitor (free form, salt) is from about 0.1 mg to about 1000 mg (calculated by weight in free form) per dose.

[0054] In some embodiments of any of the foregoing aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.1 mg to about 800 mg (calculated by weight in free form) per dose.

[0055] In some embodiments of any of the above aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is about 0.1 mg to about 600 mg (calculated by weight in free form) per dose.

[0056] In some embodiments of any of the foregoing aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is about 0.1 mg to about 500 mg per dose (calculated by weight in free form).

[0057] In some embodiments of any of the above aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is about 0.25 mg to about 1000 mg per dose (calculated by weight in free form).

[0058] In some embodiments of any of the above aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is about 0.5 mg to about 1000 mg per dose (calculated by weight in free form).

[0059] In some embodiments of any of the foregoing aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is about 0.75 mg / kg to about 1000 mg (calculated by weight in free form) per dose.

[0060] In some embodiments of any of the foregoing aspects, the dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 1 mg to about 1000 mg per dose (calculated by weight in free form).

[0061] In some embodiments of any of the foregoing, the average daily dose of the PARP inhibitor (free form, salt) is from about 0.1 mg to about 2000 mg, e.g., about 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 70 0 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, or a range formed by any of the above values.

[0062] In some embodiments of any of the foregoing, the average daily dose of the PARP inhibitor (free form, salt) is from about 0.1 mg to about 2000 mg (calculated by weight in free form).

[0063] In some embodiments of any of the foregoing aspects, the PARP inhibitor (free form, salt), or a pharmaceutically acceptable average daily dose thereof, is from about 0.1 mg to about 1500 mg (calculated by weight in free form).

[0064] In some embodiments of any of the foregoing aspects, the PARP inhibitor (free form, salt), or a pharmaceutically acceptable average daily dose thereof, is from about 0.1 mg to about 1200 mg (calculated by weight in free form).

[0065] In some embodiments of any of the foregoing aspects, the PARP inhibitor (free form, salt), or a pharmaceutically acceptable average daily dose thereof, is from about 0.1 mg to about 1000 mg (calculated by weight in free form).

[0066] In some embodiments of any of the foregoing aspects, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.1 mg to about 800 mg (calculated by weight in free form).

[0067] In some embodiments of any of the foregoing aspects, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.1 mg to about 600 mg (calculated by weight in free form).

[0068] In some embodiments of any of the foregoing, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.1 mg to about 500 mg (calculated by weight in free form).

[0069] In some embodiments of any of the foregoing aspects, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.25 mg to about 1200 mg (calculated by weight in free form).

[0070] In some embodiments of any of the foregoing aspects, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.5 mg to about 1200 mg (calculated by weight in free form).

[0071] In some embodiments of any of the foregoing aspects, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 0.75 mg to about 1200 mg (calculated by weight in free form).

[0072] In some embodiments of any of the foregoing aspects, the average daily dose of the PARP inhibitor (free form, salt) or a pharmaceutically acceptable salt thereof is from about 1 mg to about 1200 mg (calculated by weight in free form).

[0073] In some embodiments of any of the foregoing, the PARP inhibitor can be administered once, twice, or three times daily.

[0074] Technical Effects

[0075] Research data demonstrates that the combination of the disclosed compound of formula (I) or a pharmaceutically acceptable salt thereof with a PARP inhibitor, particularly the combination of the compound of formula (I) or a pharmaceutically acceptable salt thereof with PARP, exhibits a synergistic effect in cell proliferation inhibition experiments and in vivo tumor models. The combination of the compound of formula (I) or a pharmaceutically acceptable salt thereof with a PARP inhibitor demonstrates clinical potential, significantly reducing the dosage of the PARP inhibitor and improving its effectiveness.

[0076] Definition and Description

[0077] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0078] The term "combination pharmaceutical composition" means that two drugs are contained in preparation units of different strengths, which can be administered simultaneously, sequentially or intermittently.

[0079] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which 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, commensurate with a reasonable benefit / risk ratio.

[0080] The term "pharmaceutically acceptable salt" or "pharmaceutically usable salt" refers to salts of the compounds of the present disclosure, which are prepared by reacting the compounds of the present disclosure with relatively nontoxic acids or bases, having specific substituents. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in neat solution or a suitable inert solvent. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or a suitable inert solvent. The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing acid or base groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two.

[0081] The term "therapeutically effective amount" refers to a sufficient amount of a compound or pharmaceutically acceptable salt of the present disclosure to treat a disorder at a reasonable benefit / risk ratio applicable to any medical treatment and / or prevention. However, it should be recognized that the total daily dosage of the pharmaceutically acceptable salts and compositions of the compounds of Formula I disclosed herein must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field. For example, it is practice in the art to start the dose of the compound at a level lower than that required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0082] The term "administering" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including but not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, by inhalation, vaginal, intraocular, topical, subcutaneous, intrafatty, intraarticular, intraperitoneal, and intrathecal. In some specific embodiments, administration is by oral administration.

[0083] The term "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In certain embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably in certain contexts herein.

[0084] "Synergy" or "synergistic effect" means that when two drugs are used together, their total effect is greater than the sum of the effects of each drug when used alone, and their actions are in the same direction, achieving a mutually reinforcing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] FIG1 is a diagram showing the in vivo pharmacodynamic synergy of the compound of formula (I) in combination with Olaparib in an MDA-MB-436 transplanted tumor model.

[0086] Figure 2 is a graph showing the in vivo pharmacodynamic synergy of the compound of formula (I) in combination with Olaparib in a human breast cancer BR-05-0042 subcutaneous xenograft tumor model.

[0087] Figure 3 is a graph showing the in vivo pharmacodynamic synergy of the compound of formula (I) in combination with Olaparib in a subcutaneous xenograft tumor model of human ovarian cancer OV-10-0073.

[0088] FIG4 shows the tumor growth of mice in groups G1 to G4 in Test Example 4 of Example 6 of the present disclosure.

[0089] FIG5 shows the weight changes of mice in groups G1 to G4 in Test Example 4 of Example 6 of the present disclosure. DETAILED DESCRIPTION

[0090] The present disclosure is described in detail below by way of examples, but these examples are intended to illustrate and do not limit the scope of the present disclosure. Similarly, the present disclosure is not limited to any specific preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions made to the technical features of the present disclosure, or corresponding improvements, still fall within the scope of protection of the present disclosure.

[0091] Example 1: In vivo pharmacodynamic study of the compound of formula (I) combined with Olaparib in an MDA-MB-436 transplanted tumor model

[0092] 1. Purpose of the experiment

[0093] The in vivo efficacy of the compound of formula (I) in combination with Olaparib was evaluated in a subcutaneous xenograft tumor mouse model of human breast cancer MDA-MB-436 cells.

[0094] 2. Experimental Materials

[0095] 2.1 Experimental animals and breeding environment

[0096] Species: Mouse

[0097] Strain: BALB / c nude mice

[0098] Arrival age: 6-8 weeks

[0099] Gender: Female

[0100] Weight: 18-22 grams

[0101] Supplier: Shanghai Lingchang Biotechnology Co., Ltd.

[0102] Animal Certificate: 2018-0003 20180003032257

[0103] 2.2 Compound Information

[0104] Table 1 Compound information

[0105] 3. Experimental methods and steps

[0106] 3.1 Cell culture

[0107] MDA-MB-436 (human breast cancer cells, ATCC, HTB-130) were cultured in L15 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 100% O₂. Cells were digested and passaged twice weekly using trypsin-EDTA. When cells reached the exponential growth phase, they were harvested, counted, and resuspended to the appropriate concentration.

[0108] 3.2 Tumor cell inoculation and grouping

[0109] Will contain 1.0*10 7 0.2 mL of MDA-MB-436 cell suspension (cells suspended in culture medium L15:Matrigel = 1:1) was subcutaneously inoculated on the right back of each mouse. On day 37 after inoculation, animals with uniform and appropriate tumor size were selected for inclusion in the group. The average tumor volume reached 139.19 mm 3 (Range 84.80mm 3 -198.60mm 3 ), the patients were divided into groups according to tumor volume using a randomized block design (Table 1), and drug administration was started.

[0110] Table 2 Experimental animal groups and dosing regimen

[0111] Note:

[0112] 1. N: number of mice in each group;

[0113] 2. Dosing volume parameters: 10 μL / g based on mouse body weight;

[0114] 3.3 Preparation of test substances

[0115] Table 3 Preparation method of test substances

[0116] Note: Prepare the solution freshly before use and ensure that the solution is homogeneous before administration.

[0117] 3.4 Daily observation of experimental animals

[0118] The formulation of this experimental protocol was carried out in accordance with the evaluation criteria of the "Institutional Animal Care and Use Committee (IACUC)" of Shanghai Roche Pharmaceuticals Co., Ltd. The use and welfare of experimental animals were carried out in accordance with the regulations of the "Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)". The health status and mortality of animals were monitored daily. Routine examinations included observing the effects of the test substance and drugs on the daily behavior of animals, such as behavioral activities, food and water intake, body weight changes, external signs or other abnormal conditions. The number of animal deaths and side effects within each group were recorded based on the number of animals in each group.

[0119] 3.5 Tumor measurement and experimental indicators

[0120] The tumor diameter was measured twice a week using a vernier caliper. The formula for calculating the tumor volume was: V = 0.5a × b2, where a and b represent the long diameter and short diameter of the tumor, respectively.

[0121] The antitumor efficacy of the compound was evaluated by the tumor growth inhibition rate TGI (%). Calculation of TGI (%): TGI (%) = [1 - (average tumor volume at the end of drug administration in a certain treatment group - average tumor volume at the start of drug administration in that treatment group) / (average tumor volume at the end of treatment in the vehicle control group - average tumor volume at the start of treatment in the vehicle control group)] × 100%.

[0122] 4. Experimental results and analysis

[0123] During the treatment period, all treatment groups had good tolerance and no obvious weight loss.

[0124] Table 4 Tumor growth inhibition rate

[0125] Note: King's formula Q value = TGI AB / 100 / (TGI A / 100 + TGI B / 100 - TGI A / 100 * TGI B / 100). When Q ≤ 0.85, the combination of the two drugs shows an antagonistic effect; when 0.85 < Q < 1.15, the combination of the two drugs shows an additive effect; when Q ≥ 1.15, the combination of the two drugs shows a synergistic effect.

[0126] The experimental results showed that the compound of formula (I) had an obvious synergistic effect when combined with Olaparib.

[0127] Example 2: In vivo pharmacodynamic study of the combination of the compound of formula (I) and Olaparib on a human breast cancer BR-05-0042 subcutaneous xenograft tumor model

[0128] 1. Experimental purpose

[0129] The in vivo efficacy of the compound of formula (I) in combination with Olaparib was evaluated in a subcutaneous xenograft tumor mouse model of human breast cancer BR-05-0042 cells.

[0130] 2. Experimental Materials

[0131] 2.1 Experimental animals and breeding environment

[0132] Species: Mouse

[0133] Strain: BALB / c nude mice

[0134] Arrival age: 6-8 weeks

[0135] Gender: Female

[0136] Weight: 18-22 grams

[0137] Supplier: Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0138] Animal Certificate: 20230921Abzz0619000555

[0139] 2.2 Compound Information

[0140] Table 5 Compound information

[0141] 3. Experimental methods and steps

[0142] 3.1 PDX model establishment

[0143] The BR-05-0042 human triple-negative breast cancer model was originally derived from a tumor sample removed during clinical surgery. Specimen collection and use strictly adhered to national, hospital, and company-specific ethical regulations. The model development process adhered strictly to the company's internal SOPs. The passage naming convention is that tumor samples inoculated into nude mice are designated as P0, further passages are designated as P1, and so on. Resuscitated specimens are designated as FP. The tumor tissue used in this experiment was FP5.

[0144] 3.2 Tumor inoculation and grouping

[0145] The volume is about 20-30mm 3 FP4 generation human breast cancer BR-05-0042 tumor tissue was subcutaneously inoculated on the right back of each mouse. In the efficacy experiment, on the 32nd day after tumor inoculation, the average tumor volume reached 153mm 3 The group drug administration was started at 14:00, and the tumor volume range was 119-210 mm. 3 Eighteen mice were enrolled and divided into groups according to tumor volume using a randomized block design (Table 6), and drug administration began.

[0146] Table 6 Experimental animal groups and dosing regimen

[0147] Note:

[0148] aN: number of mice in each group;

[0149] b. Dosing volume: 10 μL / g of drug is administered based on the mouse's body weight. If the body weight decreases >15%, the drug is discontinued. If the body weight decreases ≤10%, dosing is resumed.

[0150] c. Vehicle 1: 10% DMSO + 20% Solutol + 70% purified water, which is the solvent for the compound of formula (I);

[0151] d. Vehicle 2: 10% DMSO + 90% (10% HP-β-CD), the solvent of Olaparib.

[0152] 3.3 Preparation of test substances

[0153] Table 7 Preparation method of test substances

[0154] 3.4 Daily observation of experimental animals

[0155] This experimental protocol was developed in accordance with the evaluation standards of the Institutional Animal Care and Use Committee (IACUC) of Shanghai WuXi AppTec Co., Ltd. The use and welfare of experimental animals were carried out in accordance with the regulations of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observing the effects of the test substances and drugs on the animals' daily behaviors, such as activity, food and water intake, weight changes, physical appearance, or other abnormalities. The number of deaths and adverse reactions within each group was recorded based on the number of animals in each group.

[0156] 3.5 Tumor Measurement and Experimental Indicators

[0157] Tumor diameter was measured twice a week with a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b2, where a and b represent the major and minor diameters of the tumor, respectively.

[0158] The tumor inhibition efficacy of the compound was evaluated using the tumor inhibition rate (TGI) (%). TGI (%) was calculated as follows: TGI (%) = [1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] × 100%.

[0159] 4. Experimental Results and Analysis

[0160] During the treatment period, all treatment groups had good tolerance and no significant weight loss.

[0161] The tumor suppression effect is shown in Table 8 and Figure 2 below.

[0162] Table 8 Tumor inhibition rate

[0163] Note: The Q value of the King formula = TGI AB / 100 / (TGI A / 100 + TGI B / 100 - TGI A / 100 * TGI B / 100). When Q ≤ 0.85, the combination of the two drugs shows an antagonistic effect; when 0.85 < Q < 1.15, the combination of the two drugs shows an additive effect; when Q ≥ 1.15, the combination of the two drugs shows a synergistic effect.

[0164] The experimental results show that the combination of the compound of formula (I) and Olaparib has an obvious synergistic effect.

[0165] Example 3: In vivo pharmacodynamic study of the combination of the compound of formula (I) and Olaparib on a subcutaneous xenograft tumor model of human ovarian cancer OV-10-0073

[0166] 1. Experimental purpose

[0167] Evaluate the in vivo pharmacodynamics of the combination of the compound of formula (I) and Olaparib on a mouse model of subcutaneous xenograft tumors of human ovarian cancer OV-10-0073 cells.

[0168] 2. Experimental materials

[0169] 2.1 Experimental animals and rearing environment

[0170] Species: Mouse

[0171] Strain: BALB / c nude mouse

[0172] Arrival age: 6 - 8 weeks old

[0173] Gender: Female

[0174] Weight: 18 - 22 grams

[0175] Supplier: Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0176] Animal certificate: 20230427Abzz0619000335

[0177] 2.2 Compound information

[0178] Table 9. Compound information

[0179] 3. Experimental methods and steps

[0180] 3.1 PDX model establishment

[0181] The human ovarian cancer model OV-10-0073 was originally derived from a tumor sample removed during clinical surgery. Specimen collection and use strictly adhered to national, hospital, and company-specific ethical regulations. The model establishment process adhered strictly to the company's internal SOPs. The passage naming convention is that tumor samples inoculated into nude mice are designated as P0, further passages are designated as P1, and so on. Resuscitated specimens are designated as FP. The tumor tissue used in this experiment was FP5.

[0182] 3.2 Tumor inoculation and grouping

[0183] The volume is about 20-30mm 3 The FP4 generation human ovarian cancer OV-10-0073 tumor tissue was subcutaneously inoculated on the right back of each mouse. The mice after inoculation were FP5 generation. In the efficacy experiment, on the 34th day after tumor inoculation, the average tumor volume reached 144mm 3 The group drug administration was started at 18:00, and the tumor volume range was 106-182 mm. 3 Eighteen mice were enrolled and divided into groups according to tumor volume using a randomized block design (Table 10), and drug administration was started.

[0184] Table 10 Experimental animal groups and dosing regimen

[0185] Note:

[0186] eN: number of mice in each group;

[0187] 3.3 Preparation of test substances

[0188] Table 11 Preparation method of test substances

[0189] 3.4 Daily observation of experimental animals

[0190] This experimental protocol was developed in accordance with the evaluation standards of the Institutional Animal Care and Use Committee (IACUC) of Shanghai WuXi AppTec Co., Ltd. The use and welfare of experimental animals were carried out in accordance with the regulations of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observing the effects of the test substances and drugs on the animals' daily behaviors, such as activity, food and water intake, weight changes, physical appearance, or other abnormalities. The number of deaths and adverse reactions within each group was recorded based on the number of animals in each group.

[0191] 3.5 Tumor measurement and experimental indicators

[0192] Measure the tumor diameter twice a week with a vernier caliper. The formula for calculating the tumor volume is: V = 0.5a × b2, where a and b represent the long diameter and short diameter of the tumor, respectively.

[0193] The antitumor efficacy of the compound was evaluated by the tumor growth inhibition rate TGI (%). Calculation of TGI (%): TGI (%) = [1 - (average tumor volume at the end of drug administration in a certain treatment group - average tumor volume at the start of drug administration in this treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × 100%.

[0194] 4. Experimental results and analysis

[0195] During the treatment period, all treatment groups had good tolerance and no obvious weight loss.

[0196] See Table 12 and Figure 3 below for the tumor inhibition effect.

[0197] Table 12 Tumor growth inhibition rate

[0198] Note: King's formula Q value = TGI AB / 100 / (TGI A / 100+TGI B / 100-TGI A / 100*TGI B / 100). When Q ≤ 0.85, the combination of the two drugs shows an antagonistic effect; when 0.85 < Q < 1.15, the combination of the two drugs shows an additive effect; when Q ≥ 1.15, the combination of the two drugs shows a synergistic effect.

[0199] The experimental results show that the combination of the compound of formula (I) and Olaparib has an obvious synergistic effect.

[0200] Example 4: In vivo pharmacodynamic study of the combination of the compound of formula (I) and Olaparib on the LD1-0032-200876 xenograft tumor model

[0201] 1. Experimental purpose

[0202] Evaluate the in vivo pharmacodynamics of the combination of the compound of formula (I) and Olaparib on the human ovarian cancer LD1-0032-200876 PDX model.

[0203] 2. Experimental materials

[0204] 2.1 Experimental animals and rearing environment

[0205] Species: Mouse

[0206] Strain: Nu / Nu mice

[0207] Arrival age: 6-8 weeks

[0208] Gender: Female

[0209] Weight: 18-22 grams

[0210] Supplier: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0211] 2.2 Compound Information

[0212] Table 13 Compound information

[0213] 3. Experimental methods and steps

[0214] 3.1 Tumor inoculation and grouping

[0215] Successfully revived FP2+7 generation LD1-0032-200876 human ovarian cancer xenografts were cut into small pieces approximately 3 mm × 3 mm × 3 mm in size. 20 μL of Matrigel was added and inoculated subcutaneously on the right side of the back of NU / NU mice (the efficacy test generation was FP2+8). The mice were observed and tumor growth was monitored. On day 35 of inoculation, the average tumor volume of the tumor-bearing mice was 147.42 mm. 3 The day of group administration was defined as day 0. Specific group information is shown in Table 14.

[0216] Table 14 Experimental animal groups and dosing regimen

[0217] Note:

[0218] 1. N: number of mice in each group;

[0219] 2. Dosing volume parameters: 10 μL / g based on mouse body weight;

[0220] 3.3 Preparation of test substances

[0221] Table 15 Preparation method of test substances

[0222] Note: Prepare the solution freshly before use and ensure that the solution is homogeneous before administration.

[0223] 3.4 Daily observation of experimental animals

[0224] The use and welfare of the experimental animals in this experimental protocol comply with the regulations of the "Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)". The health status and mortality of the animals are monitored daily. Routine examinations include observing the effects of the test substances and drugs on the daily behavior of the animals, such as behavioral activities, food and water intake, body weight changes, external signs, or other abnormal conditions. Record the number of animal deaths and side effects within each group based on the number of animals in each group.

[0225] 3.5 Tumor measurement and experimental indicators

[0226] Measure the tumor diameter with a vernier caliper twice a week. The calculation formula for tumor volume is: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively.

[0227] The antitumor efficacy of the compound is evaluated by the tumor growth inhibition rate TGI (%). Calculation of TGI (%): TGI (%) = [1 - (average tumor volume at the end of drug administration in a certain treatment group - average tumor volume at the start of drug administration in this treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × 100%.

[0228] 4. Experimental results and analysis

[0229] During the treatment period, all treatment groups had good tolerance and no obvious weight loss.

[0230] Table 16 Tumor growth inhibition rate

[0231] Note: King's formula Q value = TGI AB / 100 / (TGI A / 100 + TGI B / 100 - TGI A / 100 * TGI B / 100). When Q ≤ 0.85, the combination of the two drugs shows antagonistic effect; when 0.85 < Q < 1.15, the combination of the two drugs shows additive effect; when Q ≥ 1.15, the combination of the two drugs shows synergistic effect.

[0232] The experimental results show that the compound of formula (I) has an obvious synergistic effect when combined with Olaparib.

[0233] Example 5: Cell proliferation inhibition test of the compound of formula (I) in combination with a PARP inhibitor

[0234] 1. Experimental purpose

[0235] The CTG (Cell Titer-Glo Luminescent viability assay) method was used to detect the inhibitory effect of the compound of formula (I) in combination with a PARP inhibitor on the proliferation of tumor cell lines, thereby evaluating the combined effect of the two.

[0236] 2. Experimental Materials

[0237] 2.1 Cell Information

[0238] Table 17 Cell information

[0239] 2.2 Sample Information

[0240] Table 18 Sample information

[0241] 2.3 Other reagents and consumables

[0242] Table 19 Other reagents and consumables information

[0243] 3. Experimental Methods

[0244] 3.1 Experimental design

[0245] The checkerboard assay was used to evaluate the inhibitory effects of the compound of formula (I) and KSQ-4279 (USP1 inhibitor) alone and in combination with PARP inhibitors (OLAPARIB, AZD5305, Niraparib, Rucaparib, and Talazoparib) on the proliferation of different tumor cells. The concentrations of the compound of formula (I) and KSQ-4279 ranged from 0.64 to 10,000 nM, and the concentration of the PARP inhibitor ranged from 3.2 to 10,000 nM.

[0246] 3.2 Experimental steps

[0247] 3.2.1 Cell seeding and drug treatment

[0248] Select cells that are in good growth condition and in the logarithmic growth phase, wash with PBS, digest the cells with 0.25% Trypsin-EDTA digestion solution for about 3-10 minutes, terminate the digestion with the corresponding complete cell culture medium, collect the cells, centrifuge the cells at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in complete cell culture medium, count the cells with a cell counter, adjust the cells to the desired density, add 80.0 μL of cell suspension to each well, and incubate the cell plate in a 37°C, 5% CO2 cell culture incubator overnight.

[0249] Remove the cell plate from the incubator and observe the cell attachment status. Once the cells have attached, add 10.00 μL of the compound of formula (I) or KSQ-4279 and 10.00 μL of different PARP inhibitors to each well in a checkerboard pattern. A single-drug group and a DMSO control group are also established. After addition, gently shake well and incubate in a 37°C, 5% CO2 incubator for 7 days.

[0250] 3.2.2 Detection

[0251] After the incubation, remove the culture plate and equilibrate at room temperature for 30 minutes. The reagent was equilibrated at room temperature for 30 minutes and mixed thoroughly. Then, 100 μL of CellTiter-Glo reagent was added to each well and incubated at 22 ± 1°C and 200 rpm for 10 minutes. All the above operations should be performed in the dark. The plate was read using a microplate reader or microplate reader.

[0252] 3.3 Data Analysis

[0253] After obtaining the raw data, the cell inhibition rate was calculated using the formula: Cell inhibition rate / inhibition (%) = (1-RLU 样品 / RLU DMSO对照 )×100%

[0254] SynergyFinder online analysis software (https: / / synergyfinder.fimm.fi / ) was used to evaluate drug synergy, and the ZIP (Zero-inflated Poisson) regression mixed model was used for calculation.

[0255] 4. Experimental Results and Analysis

[0256] Table 20 In vitro study results of combined drug use (CTG)

[0257] Note: ZIP-Most synergistic area score evaluation criteria: When the score is less than -10, the interaction between the two drugs may be antagonistic; when the score is greater than -10 but less than 10, the interaction between the two drugs may be additive; when the score is greater than 10, the interaction between the two drugs may be synergistic.

[0258] The data showed that the combination of the compound of formula (I) and the PARP inhibitor had a significant synergistic effect in inhibiting the proliferation of the above-mentioned tumor cells.

[0259] Example 5: Preparation of compound of formula (I):

[0260] Step A: Dissolve 5-bromo-2-chloropyrimidin-4-amine (108 g, 518 mmol) in 1,4-dioxane / water (2119 mL / 235 mL) at room temperature under nitrogen. Then, add 3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (150 g, 570 mmol), sodium carbonate (110 g, 1036 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (38 g, 46.6 mmol) sequentially to the solution. The reaction mixture is stirred at 90°C for 16 hours.

[0261] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered, the filtrate was collected, and it was concentrated under reduced pressure. The resulting residue was slurried with ethyl acetate / petroleum ether, and the resulting filter cake was slurried again with acetonitrile / water to obtain 94.6 g of a crude product of 4-(4-amino-2-chloropyrimidin-5-yl)-3-chlorobenzonitrile.

[0262] MS (ESI) M / Z: 265.0 [M+H] + .

[0263] Step B: 4-(4-amino-2-chloropyrimidin-5-yl)-3-chlorobenzonitrile (94.6 g, 358 mmol) was dissolved in 1,4-dioxane / water (1394 mL / 232 mL) at room temperature under nitrogen. (4-cyclopropyl-6-methoxypyrimidin-5-yl)boronic acid (69.5 g, 358 mmol), cesium carbonate (233 g, 716 mmol), and chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (36.5 g, 46.5 mmol) were then added to the solution. The reaction was stirred at 90°C for 16 hours.

[0264] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was filtered and concentrated under reduced pressure. The resulting residue was slurried with ethyl acetate / petroleum ether, and the filter cake was rinsed with water and dried to obtain 118 g of crude 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9H-pyrimido[4,5-b]indole-7-carbonitrile.

[0265] MS (ESI) M / Z: 343.2 [M+H] + .

[0266] Step C: Dissolve 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9H-pyrimido[4,5-b]indole-7-carbonitrile (53.8 g, 157.2 mmol) in N,N-dimethylformamide (436 mL) at room temperature. Cesium carbonate (114 g, 349.2 mmol) and 9-(chloromethyl)-2-(trifluoromethyl)-6,7-dihydro-5H-benzo[c]imidazo[1,2-a]azepine (26.2 g, 87.3 mmol) were then added to the reaction. The reaction was stirred at 45°C for 16 hours.

[0267] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was added to water (900 mL) to precipitate a solid, which was purified by silica gel column chromatography to give 20.42 g of 2-(4-cyclopropyl-6-methoxypyrimidin-5-yl)-9-((2-(trifluoromethyl)-6,7-dihydro-5H-benzo[c]imidazo[1,2-a]azepin-9-yl)methyl)-9H-pyrimido[4,5-b]indole-7-carbonitrile.

[0268] MS (ESI) M / Z: 607.2 [M+H] + .

[0269] 1 H NMR (400MHz, DMSO-d6) δ9.82 (s, 1H), 8.72 (s, 1H), 8.59 (d, J = 8.4Hz, 1H), 8.50 (s, 1H), 7.9 6(d,J=0.8Hz,1H),7.86(dd,J=8.0,1.2Hz,1H),7.62(d,J=8.0Hz,1H),7.41(d,J=0.8Hz,1 H),7.33(dd,J=8.0,1.6Hz,1H),5.81(s,2H),3.95(t,J=6.8Hz,2H),3.87(s,3H),2.60(t, J=7.0Hz,2H),2.27-2.15(m,2H),1.77-1.67(m,1H),1.10-1.02(m,2H),0.88-0.77(m,2H).

[0270] Referring to the preparation method in WO2020132269, the reference compound KSQ-4279 was prepared:

[0271] Referring to the preparation method in WO2022228399, comparative compound 1 and comparative compound 2 were prepared:

[0272] Example 6: Biological test evaluation:

[0273] Test Example 1 USP1 enzyme activity test method

[0274] 1. Experimental plan:

[0275] USP1 enzyme activity assay was used to screen USP1i compounds.

[0276] 1.1 Experimental Materials: Recombinant human His6-USP1 / His6-UAF1 complex protein (R&D, catalog number E-568-050); Ubiquitin Rhodamine 110 (Ub-Rho) (R&D, catalog number U-555-050); Fluorescence 384-well plate (Perkin Elmer, catalog number 6007279).

[0277] 1.2 Test sample: Compound of formula (I), whose structural formula and preparation method are shown in the above examples.

[0278] 1.3 Experimental process:

[0279] (1) Prepare 1× assay buffer (modified Tris buffer, components: 50 mM Tris-HCl (pH 7.8) (Sigma, Catalog No.: T2569-1L), 0.01% Tween-20 (Sigma, Catalog No.: P2287-100ML), 1 mM DTT (Sigma, Catalog No.: D0632-10G), 0.01% BSA (Sigma, Catalog No.: B2064-100G), 0.5 mM EDTA (Invitrogen, Catalog No.: 15575020)).

[0280] (2) Compound dilution: Use dimethyl sulfoxide (DMSO, 100% purity) to prepare a 10 mM (mol / L) solution of the test compound; dilute the test compound solution 3-fold to 10 concentrations, with the highest concentration being 10 mM; transfer the diluted test compound solution to a fluorescent 384-well plate using an Echo acoustic pipetting system, with two replicate wells set for each concentration, and the final DMSO concentration is 1 vol%; the final concentrations of the test compound solution are 10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM, 13.7 nM, 4.6 nM, 1.5 nM, and 0.5 nM.

[0281] (3) Prepare enzyme solution: Prepare enzyme solution in 1× assay buffer.

[0282] (4) Prepare substrate solution: Add ubiquitin rhodamine 110 (Ub-Rho) to 1× detection buffer to form a substrate solution.

[0283] (5) Transfer 10 μL of the enzyme solution prepared in step (3) to a fluorescent 384-well plate.

[0284] (6) Incubate at room temperature for 1 hour.

[0285] (7) Add 10 μL of the substrate solution prepared in step (4) to each well to start the reaction. The final reaction system is 200 nL of the test compound + 10 uL of the enzyme solution + 10 uL of the substrate solution. The final concentration of the enzyme is 0.05 nM, and the final concentration of the substrate solution is 300 nM. Centrifuge for 30 s and shake for 30 s.

[0286] (8) The plate was read on a SpectraMax Paradigm multifunctional microplate reader for 30 minutes with an excitation wavelength of 480 nm and an emission wavelength of 540 nm.

[0287] (9) Collect data on SpectraMax Paradigm.

[0288] (10) Curve fitting:

[0289] The data were fitted in Excel using equation (I) to obtain inhibition values;

[0290] Equation (I): Inhibition rate % = (maximum signal value - target signal value) / (maximum signal value - minimum signal value) × 100;

[0291] Among them, the maximum signal value represents the luminescent signal intensity of the positive control well without the compound of the present application; the minimum signal value represents the luminescent signal intensity of the negative control well without the enzyme; and the target signal value represents the luminescent signal intensity of the test compound.

[0292] Fit the data in XL-Fit using equation (II) to obtain IC 50 value;

[0293] Equation (II): Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)×HillSlope)

[0294] Where Y is the inhibition percentage, X is the compound concentration; Bottom is the lowest inhibition rate; Top is the highest inhibition rate; HillSlope is the slope.

[0295] 2. Experimental results:

[0296] The results show that the compound of formula (I) has a good inhibitory effect on USP1. The results are shown in Table 20.

[0297] Table 21 Enzyme inhibition results

[0298] 3. Conclusion:

[0299] It can be seen from the above experimental results that the compound of formula (I) has a good inhibitory effect on USP1 and is an effective USP1 inhibitor.

[0300] Test Example 2 USP1CTG Test Method

[0301] 1. Experimental Plan

[0302] The in vitro biological activity of USP1i compounds was verified using the NCI-H1693 cell killing assay.

[0303] 1.1 Experimental Materials:

[0304] NCI-H1693 cells were purchased from ATCC (Cat. No. CRL-5866) and cultured in a 37°C, 5% CO2 (containing 5% CO2 and 95% air) cell culture incubator; 1640 complete medium: 94wt% RPMI-1640 liquid medium (Gibco Cat. No. 11875-093), 5wt% FBS (Gibco Cat. No. 10099-141), 1wt% Pen Strep (Gibco Cat. No. 15070-063); fluorescent 384-well plates (Perkin Elmer, Cat. No. 6007279); trypsin (Gibco Cat. No. 25200056); CTG buffer ( 2.0 Cell Viability Assay, Promega, Catalog No.: G9241).

[0305] 1.2 Test sample:

[0306] The compound of formula (I), its structural formula and preparation method are shown in the above examples;

[0307] The structural formula of comparative compound 1 is:

[0308] The structural formula of comparative compound 2 is:

[0309] 1.3 Experimental process:

[0310] (1) 75cm 2 NCI-H1693 cells in a culture flask were digested with 2 mL of trypsin for 2-3 minutes, then neutralized with 2 mL of 1640 complete medium. Centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and the cell pellet was resuspended in 4 mL of 1640 complete medium. 500 μL of the cell suspension was collected and counted using a Vi-CELL-XR cell counter.

[0311] (2) Using a Multidrop instrument, 500 NCI-H1693 cells were seeded per well in a fluorescent 384-well plate (each well contained 40 μL of 1640 complete medium), and drugs were added after 24 hours.

[0312] (3) Using an ultra-micropipette, the test sample (concentration: 3.33 mM, dissolved in DMSO) was diluted with 10 uM as the highest starting concentration, and then diluted with 9 gradients (10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM, 13.7 nM, 4.6 nM, 1.5 nM) in a 1:3 ratio, and the drug was added. Two replicate wells were set for each concentration; 9 wells were set for positive control and blank control, with the positive control being the replicate wells of 10 μM positive compound and the blank control being the replicate wells of DMSO.

[0313] (4) After drug addition, the cells were placed in a 37°C, 5% CO2 (containing 5% CO2 by volume and the rest being air) incubator and cultured for 6 days. After 6 days, 25 μL of CTG buffer was added to each well for CTG detection, and the plate was read and analyzed using a microplate reader.

[0314] (5) Analyze the CTG readings:

[0315] The average inhibition rate of the positive control replicates was set as 100%;

[0316] The average value of negative control replicates was set as the relative inhibition rate of 0%.

[0317] Convert the CTG readings to relative inhibition rates and calculate the inhibition rate (inhibition%) of each compound concentration on cells using the following formula: Inhibition% = (bx) / (ba × 100%); a = CTG value (highest concentration well) b = CTG value (blank control well) x = CTG value (x nM)

[0318] (6) Using GraphPad PRISM 8 to analyze IC 50 Perform calculations.

[0319] The concentrations and inhibition rates corresponding to 10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM, 13.7 nM, 4.6 nM, and 1.5 nM were statistically analyzed and calculated using Log10 (compound concentration).

[0320] Input the data into GraphPad PRISM 8, select "Analysis", select "Nonlinear regression (curve fit)", select "Log (inhibitor) vs. response-Variable slope", select the calculation formula, and calculate it according to the following formula: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X)×HillSlope))

[0321] X:Log10(compound concentration)

[0322] Y: corresponding response

[0323] Top and Bottom: corresponding responses with the same units as Y

[0324] Notice:

[0325] - If X is not already the logarithm of dose, go back and transform the data.

[0326] - If any base response is subtracted, clamp Bottom to a constant value of 0.0.

[0327] Fit the data to obtain IC 50 value.

[0328] Adjust the fitting conditions according to the specific data conditions. Make appropriate constraint adjustments to Bottom, Top, and HillSlope to achieve the curve that best fits the actual situation.

[0329] 2. Experimental results:

[0330] The inhibition results of the compounds in the examples of the present application on NCI-H1693 cells are shown in Table 22.

[0331] Table 22 NCI-H1693 cell inhibition results

[0332] 3. Conclusion:

[0333] It can be seen from the above experimental results that the compound of formula (I) of the present application has obvious cell inhibitory activity.

[0334] Test Example 3 Pharmacokinetics of the Compound of the Application in Male Beagle Dogs

[0335] 1. Experimental plan:

[0336] Male Beagle dogs were used as test animals to study the pharmacokinetic characteristics of the compound of the present application in dog plasma after intravenous and oral administration. Plasma samples were collected at specific time points, and the compound concentration in plasma was detected by LC-MS / MS. DMPK parameters were calculated and the pharmacokinetic characteristics of the compound of the present application in dog plasma were evaluated.

[0337] 1.1 Experimental drugs:

[0338] The compound of formula (I), whose structural formula and preparation method are shown in the above examples; KSQ-4279.

[0339] 1.2 Experimental Animals

[0340] Male Beagle dogs, weighing 9-13kg, supplied by Beijing Mas Biotechnology Co., Ltd.

[0341] 1.3 Administration:

[0342] Dosing information for the compound of this application: 3 dogs each received IV injection (0.2 mg / kg in a 0.5 mL / kg volume) and PO administration (3 mg / kg in a 3 mL / kg volume). The dosing vehicle consisted of 5 vol% DMSO, 10 vol% Solutol (Kolliphor HS15), and 85 vol% saline.

[0343] 1.4 Sample collection:

[0344] After administration to the dogs, 0.5 mL of blood was collected from the cephalic vein at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours for the IV group and at 0.25, 0.5, 1, 2, 4, 8, and 24 hours for the PO group. The blood was placed in EDTA-K2 tubes and centrifuged at 2000 g for 10 minutes at 2-8°C to separate plasma. The plasma was then stored at -80°C until sample analysis.

[0345] 1.5 Sample processing:

[0346] Canine plasma sample processing:

[0347] 1) 50 μL of plasma sample was added to 200 μL of acetonitrile for precipitation, vortexed for 30 seconds, and centrifuged at 3900 rpm at 4°C for 15 minutes.

[0348] 2) The supernatant after treatment was diluted 3-fold with water, and the concentration of the test compound was analyzed by LC / MS / MS.

[0349] 2. Experimental results:

[0350] Table 23

[0351] Note: In Table 19, “CL” is total clearance; “AUC last ” is the area under the plasma drug concentration-time curve from time 0 to the final quantifiable time point.

[0352] 3. Conclusion:

[0353] It can be seen from the above experimental results that the compound of formula (I) of the present application has obvious advantages in clearance rate level and drug exposure compared with the control compound.

[0354] Test Example 4: OV0589 transplant tumor model (PDX) efficacy experiment

[0355] 1. Experimental plan:

[0356] Female BALB / c nude mice were used as test animals. After oral administration of the compounds of the present application, tumors were measured and weighed regularly to investigate tumor growth inhibition and tolerance in different dosing groups. Plasma and tumor samples were collected at specific time points after the study for pharmacokinetic / pharmacodynamic (PK / PD) analysis.

[0357] 1.1 Experimental drugs:

[0358] The compound of formula (I), whose structural formula and preparation method are shown in the above examples; compound KSQ4279.

[0359] 1.2 Experimental Materials:

[0360] Tumor-bearing mouse tumor (R10P8) was purchased from Sino-US Crown Biotechnology (Beijing) Co., Ltd.; Ubiquityl-PCNA (Lys164) (D5C7P) mAb antibody was purchased from Cell Signaling, catalog number: 13439S; PCNA (PC10) antibody was purchased from Santa Cruz, catalog number: sc-56.

[0361] 1.3 Experimental Animals

[0362] Female BALB / c nude mice, weighing 21-25 g, supplier: Beijing Ankai Yibo Biotechnology Co., Ltd.

[0363] 2. Experimental steps:

[0364] Under the conditions of temperature 20-26℃, humidity 30-70%, and alternating light and day, the tumor (R10P8) of the tumor-bearing mice grew to a diameter of about 1 cm (the tumor size reached 50-800 mm). 3 ) when the tumor was collected and cut into pieces of about 2 to 3 mm 3The tumor mass (R10P8) was inoculated subcutaneously in the right anterior scapula of female BALB / c nude mice. The tumor growth in the mice was regularly observed. 35 days after inoculation, when the tumor (R10P9) grew to an average volume of approximately 150 mm 3 (Inclusion range 86.54mm 3 ~262.96mm 3 ) According to the tumor size and mouse weight, the mice were randomly divided into groups using StudyDirector™ (version 3.1.399.19, supplier: Studylog System, Inc., S. San Francisco, CA, USA), and the drugs were administered on the next day after grouping.

[0365] Twelve mice were randomly divided into four groups, namely G1 to G4. Group G1 was the vehicle control group, with the vehicle consisting of 10 vol% DMSO + 20 vol% Solutol + 70 vol% Water + 5 vol% DMSO + 10 vol% Solutol + 85 vol% Saline; Group G2 was orally administered with KSQ4279 at a dose of 100 mg / kg (mpk) in a vehicle consisting of 5 vol% DMSO + 10 vol% Solutol + 85 vol% Saline; Group G3 was orally administered with the compound of formula (I) at a dose of 30 mg / kg in a vehicle consisting of 10 vol% DMSO + 20 vol% Solutol + 70 vol% Water; and Group G4 was orally administered with the compound of formula (I) at a dose of 100 mg / kg in a vehicle consisting of 10 vol% DMSO + 20 vol% Solutol + 70 vol% Water.

[0366] The growth of tumors in mice was observed regularly, and the tumor volume was measured (see Figure 4 for the results). The weight changes of mice were also observed regularly (see Figure 5 for the results). After 36 days of administration, plasma and tumor samples were collected for PK / PD analysis.

[0367] PK assay The pharmacokinetic assay in mice was performed as described in the previous test example 3.

[0368] PD detection: Tumor samples were collected 8 hours after the last administration and divided into tumor samples weighing 50-100 mg. 1× tissue lysis buffer was prepared using ddH2O, and then 20uL of tissue lysis buffer (Cell Signaling Cell Lysis Buffer (10×) Catalog Number: #9803) was added per mg of sample. The tumor samples were ground using a tissue grinder. The ground tumor sample lysate was placed on ice for 30 minutes. Subsequently, centrifuged at 12000rpm and 4°C for 15 minutes. The sample supernatant was retained. The PD marker (Ubiquityl-PCNA / PCNA) in the sample was detected using a protein blotting assay.

[0369] 3. Experimental results:

[0370] Figure 4 shows the tumor growth of mice in groups G1 to G4. As can be seen from Figure 4, the administration of the compound of formula (I) (dosage of 100 mg / kg) has the best inhibitory effect on tumor growth, followed by the administration of the compound of formula (I) (dosage of 30 mg / kg). The inhibitory effect of the administration of KSQ4279 (dosage of 100 mg / kg) on ​​tumor growth is significantly inferior to that of the administration of the compound of formula (I) (dosage of 100 mg / kg) and the administration of the compound of formula (I) (dosage of 30 mg / kg).

[0371] FIG5 shows the changes in body weight of mice in groups G1 to G4. As can be seen from FIG5 , the changes in body weight of mice in groups G1-G4 were all within the normal range and were well tolerated.

[0372] Table 24 shows the statistical results of tumor growth inhibition rate of mice in groups G2 to G4. As can be seen from Table 20, the administration of compound of formula (I) (dosage of 100 mg / kg) has the best inhibitory effect on tumor growth, followed by the administration of compound of formula (I) (dosage of 30 mg / kg). The inhibitory effect of administration of KSQ4279 (dosage of 100 mg / kg) on ​​tumor growth is significantly inferior to that of administration of compound of formula (I) (dosage of 100 mg / kg) and administration of compound of formula (I) (dosage of 30 mg / kg).

[0373] Table 24 Statistical results of tumor growth inhibition rate

[0374] Note: "TGI%" is tumor growth inhibition rate; TGI% = [1-ΔT / C] × 100%, ΔT / C = (mean(T)-mean(T0)) / (mean(C)-mean(C0)), T and C are the average tumor volumes of the drug-treated group and the vehicle control group on the 38th day, respectively, and T0 and C0 are the average tumor volumes of the drug-treated group and the vehicle control group on the 0th day, respectively.

[0375] 4. Conclusion:

[0376] From the above data, it can be seen that compared with the control compound, the compound of formula (I) has obvious advantages in inhibiting tumor growth, and the dosage is significantly lower than that of the control compound.

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a PARP inhibitor in the preparation of a drug for treating USP1-mediated diseases 2. The use according to claim 1, characterized in that The PARP inhibitor is selected from olaparib, niraparib, talazoparib, rucaparib, and AZD5305.

3. The use according to claim 1, characterized in that The PARP inhibitor is selected from olaparib.

4. The use according to claims 1 to 3, characterized in that The related diseases mediated by the USP1 target include cellular inflammatory diseases, neurodegenerative diseases, and cancer.

5. The use according to claim 4, characterized in that The cancer is selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, and colon cancer; preferably breast cancer and ovarian cancer.

6. The use according to claim 4, characterized in that In the above use, the compound of formula (I) or a pharmaceutically acceptable salt thereof and the PARP inhibitor can be administered simultaneously, sequentially or intermittently.

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