Crystal form of bipyrimidine compound, preparation method therefor and use thereof

Abstract

The present invention relates to a crystal form of a bipyrimidine compound and a preparation method therefor and a use thereof. In particular, the present invention relates to a crystal form I of 4'-cyclopropyl-5,6'-dimethoxy-N-((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclic[2.2.2]oct-1-yl)methyl)-[2,5'-bipyrimidine]-4-amine, and the X-ray powder diffraction pattern of the crystal form I comprises characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2°, and 22.200±0.2°. The crystal form I has high stability, high solubility, high plasma concentration, high bioavailability, and excellent pharmacological action, and is therefore suitable for pharmaceutical preparations.

Description

Crystal form of bipyrimidine compound, preparation method and use thereof Technical Field

[0001] The present invention relates to a crystal form of a bipyrimidine compound, a preparation method and an application thereof. Background Art

[0002] Ubiquitination is a dynamic, reversible process involving the deubiquitinating enzyme (DUB) family. Approximately 100 DUBs exist in humans, classified as cysteine ​​proteases and metalloproteases. The cysteine ​​protease family primarily includes ubiquitin-specific proteases (USPs), ubiquitin carboxyl-terminal hydrolases (UCHs), Machado-Josephin domain proteases (MJDs), MINDY proteases (MINDYs), and ovarian oncodomain proteases (OTUs). The USP family is the largest known family of deubiquitinating enzymes, with over 50 encoded by human genes. These enzymes play a role in various physiological functions, including cell cycle regulation, signal transduction, DNA damage repair, chromosomal translocations, and gene transcription, by regulating substrate proteases.

[0003] USP1 belongs to the USP subfamily of DUBs. While inactive on its own, it acquires full enzymatic activity upon binding to UAF1 to form a heterodimeric complex (USP1 / UAF1), regulating cellular targets in multiple cancer-related pathways. For example, the USP1 / UAF1 complex deubiquitinates proliferating cell nuclear antigen (PCNA), a key protein in the translesion synthesis (TLS) process, to prevent excessive repair. It also deubiquitinates Fanconi anemia complementation group D2 (FAND2), a key protein in the Fanconi anemia (FA) pathway, to block inappropriate TLS repair in cells, thereby ensuring genomic stability. These two DNA damage response (DDR) pathways are key for repairing DNA damage induced by DNA cross-linking agents such as cisplatin, mitomycin, and ultraviolet radiation. USP1 also interacts with proteins such as ID, stabilizing their expression in cells through deubiquitination. For example, in osteosarcoma cells, USP1 can deubiquitinate ID1, ID2, and ID3, promoting cell proliferation. Inhibiting USP1 can increase the sensitivity of osteosarcoma cells to chemotherapy. Furthermore, studies have confirmed that USP1 is closely associated with the development of resistance to various tumor therapeutic drugs. For example, in cisplatin-resistant non-small cell lung cancer (NSCLC) cells, USP1 expression is high, and knocking down USP1 can significantly enhance the cells' sensitivity to cisplatin. In breast cancer cells, USP1 is highly expressed, promoting cell proliferation and being closely associated with a poor prognosis in breast cancer. A report (J. Med. Chem. 2014, 57, 8099-8110, Synthesis and Structure-Activity Relationship Studies of N-Benzyl-2-phenylpyrimidin-4-amine Derivatives as Potent USP1 / UAF1 Deubiquitinase Inhibitors with Anticancer Activity against Nonsmall Cell Lung Cancer) reported that compounds such as the USP1 inhibitor ML323 could be used in non-small cell lung cancer. A report (Cui SZ, Lei ZY, Guan TP, et al. Targeting USP1-dependent KDM4A protein stability as a potential prostate cancer therapy. Cancer Sci. 2020; 00: 1–15.) reported that USP1 inhibitors are potential therapeutic drugs for prostate cancer. In summary, USP1 is expected to become a popular target for the treatment of various cancers and other diseases.

[0004] CN202310473956.2 discloses a series of USP1 inhibitors, particularly compound 104 having the following structure: 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'-bipyrimidine]-4-amine.

[0005] However, there is no report on the crystal form of this compound in the prior art.

[0006] Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a crystalline form of 4'-cyclopropyl-5,6'-dimethoxy-N-((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]oct-1-yl)methyl)-[2,5'-bipyrimidinyl]-4-amine, as well as a preparation method and use thereof. After extensive experimental research, the inventors discovered a preparation method for the crystalline form of 4'-cyclopropyl-5,6'-dimethoxy-N-((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]oct-1-yl)methyl)-[2,5'-bipyrimidinyl]-4-amine. X-ray powder diffraction analysis of the obtained crystalline form revealed a crystalline form I with excellent stability.

[0008] The present invention solves the above technical problems through the following technical solutions:

[0009] The present invention provides a crystalline form I of 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'-bipyrimidine]-4-amine, characterized in that its X-ray powder diffraction pattern comprises characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2° and 22.200±0.2°.

[0010] In some embodiments, the X-ray powder diffraction pattern of the crystalline Form I further comprises at least one characteristic peak at diffraction angles (2θ) of 11.938±0.2°, 23.761±0.2°, 25.101±0.2°, and 28.700±0.2°.

[0011] In some embodiments, the X-ray powder diffraction pattern of the crystalline Form I comprises characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 11.938±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2°, 22.200±0.2°, 23.761±0.2°, 25.101±0.2° and 28.700±0.2°.

[0012] In some embodiments, the X-ray powder diffraction pattern of the crystalline Form I further comprises at least one characteristic peak at diffraction angles (2θ) of 10.239±0.2°, 12.400±0.2°, 14.882±0.2°, 20.420±0.2°, and 25.980±0.2°.

[0013] In some embodiments, the X-ray powder diffraction pattern of Form I comprises characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 10.239±0.2°, 11.938±0.2°, 12.400±0.2°, 14.882±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2°, 20.420±0.2°, 22.200±0.2°, 23.761±0.2°, 25.101±0.2°, 25.980±0.2°, and 28.700±0.2°.

[0014] In some embodiments, the X-ray powder diffraction pattern of the crystalline form I is substantially as shown in Figure 1.

[0015] In some embodiments, the DSC spectrum of the crystalline form I shows an endothermic peak at 219.25±2°C.

[0016] In some embodiments, the TGA spectrum of the crystalline Form I shows a weight loss of 0.10% from 20°C to 120°C and a weight loss of 0.37% from 120°C to 250°C.

[0017] The present invention also provides a method for preparing Form I, comprising:

[0018] Mixing 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 with an organic solvent, and performing solid-liquid separation on the resulting suspension to collect the solid;

[0019] The organic solvent is selected from one or more of petroleum ether, C4-C6 ether and C2-C6 nitrile.

[0020] In some embodiments, the method comprises:

[0021] Slurrying 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 with an organic solvent at 0 to 30° C. for 10 minutes to 2 hours, preferably at 25° C. for 30 minutes, and collecting a solid by solid-liquid separation of the resulting suspension, which is optionally further dried;

[0022] The organic solvent is selected from one or more of petroleum ether, methyl tert-butyl ether, tetrahydrofuran and acetonitrile, preferably a mixture of petroleum ether and methyl tert-butyl ether;

[0023] Advantageously, the ratio (W / V) of 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 to the organic solvent is 1:5 to 1:1, preferably 1:3.

[0024] The present invention also provides a method for preparing Form I, comprising:

[0025] 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 was mixed with a solvent, and the resulting suspension was subjected to solid-liquid separation to collect the solid.

[0026] In some embodiments, the method comprises any of the following methods:

[0027] Method (1) comprises the following steps:

[0028] 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 was mixed with a solvent and slurried, and the resulting suspension was subjected to solid-liquid separation to collect the solid;

[0029] The solvent is selected from one or more of water, petroleum ether, C1-C6 alcohol, C4-C8 alkane, C4-C6 ether, C3-C6 ketone, C3-C8 ester, toluene and C2-C6 nitrile;

[0030] Method (2) comprises the following steps:

[0031] 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 is mixed and dissolved with a good solvent, then mixed with an anti-solvent, and crystallized to obtain the crystalline form I;

[0032] The good solvent is selected from one or more of C2-C6 nitrile, methanol, benzyl alcohol, C3-C6 ketone, N-methylpyrrolidone, ethyl acetate, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran;

[0033] The anti-solvent is selected from one or more of petroleum ether, C4-C6 ether and C2-C6 alcohol;

[0034] Method (3) comprises the following steps:

[0035] Mixing 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 with a solvent, cooling, and crystallizing to obtain the crystalline form I;

[0036] The solvent is selected from one or more of C2-C6 nitrile, C1-C3 alcohol, benzyl alcohol, C3-C6 ketone, N-methylpyrrolidone, C3-C8 ester, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, methyltetrahydrofuran and 1,4-dioxane.

[0037] In some embodiments, the method (1) comprises the following steps:

[0038] Slurrying 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 with a solvent at 0 to 50° C. for 10 minutes to 7 days, preferably 25 to 30° C. for 30 minutes to 4 days, and collecting a solid by solid-liquid separation of the resulting suspension, which is optionally further dried;

[0039] The solvent is selected from one or more of water, methanol, n-heptane, ethyl acetate, ethylene glycol, isopropyl acetate, methyl isobutyl ketone, toluene, petroleum ether, methyl tert-butyl ether, tetrahydrofuran and acetonitrile,

[0040] Preferably, the solvent is a mixture of petroleum ether and methyl tert-butyl ether, a mixture of water and ethanol, a mixture of n-heptane and ethyl acetate, a mixture of ethylene glycol and methanol, isopropyl acetate, methyl isobutyl ketone, or toluene.

[0041] More preferably, the volume ratio of petroleum ether to methyl tert-butyl ether is 1:10 to 10:1, preferably 1:1 to 10:1; the volume ratio of water to ethanol is 5:1 to 30:1, preferably 10:1 to 20:1; the volume ratio of n-heptane to ethyl acetate is 5:1 to 30:1, preferably 10:1 to 20:1; the volume ratio of ethylene glycol to methanol is 5:1 to 30:1, preferably 10:1 to 20:1.

[0042] Advantageously, the ratio (W / V) of 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 to the solvent is 1 g:1 mL to 1 g:20 mL, preferably 1 g:3 mL to 1 g:10 mL.

[0043] In some embodiments, the method (ii) comprises the following steps:

[0044] Dissolving 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 in a good solvent at 5 to 45° C., preferably at room temperature, and then mixing with an antisolvent and crystallizing to obtain the crystalline form I;

[0045] The good solvent is selected from one or more of methanol, acetone, ethyl acetate, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran, acetonitrile and benzyl alcohol;

[0046] The anti-solvent is selected from one or more of methyl tert-butyl ether, isopropyl alcohol, 1-butanol and ethylene glycol;

[0047] Advantageously, the volume of the antisolvent is 2 to 20 times, preferably 3 to 10 times, the volume of the good solvent;

[0048] More advantageously, the ratio (W / V) of 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'-bipyrimidine]-4-amine to the antisolvent is 50 mg:1 mL to 1 mg:1 mL, preferably 50 mg:1 mL to 5 mg:1 mL.

[0049] In some embodiments, the method (ii) comprises the following steps:

[0050] 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'-bipyrimidine]-4-amine is mixed and dissolved with a good solvent at 5 to 45° C., preferably at room temperature, to obtain a saturated or nearly saturated solution, which is optionally filtered and then mixed with an antisolvent, preferably with stirring, and crystallized to obtain the crystalline form I.

[0051] In some embodiments, the method (iii) comprises the following steps:

[0052] Mixing 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 with a solvent, cooling, and crystallizing to obtain the crystalline form I;

[0053] The solvent is a good solvent, preferably one or more selected from ethanol, methyl isobutyl ketone, isopropyl acetate, methyltetrahydrofuran and 1,4-dioxane;

[0054] Preferably, the method (iii) comprises the following steps:

[0055] Mixing 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 with a good solvent, dissolving, optionally filtering, and initially cooling until solid precipitates, and then further cooling and crystallizing to obtain the crystalline form I;

[0056] Preferably, the method (iii) comprises the following steps:

[0057] Mixing 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 with a good solvent at 25 to 60° C., preferably at 30 to 50° C., dissolving, optionally filtering, first cooling to 5 to 35° C., preferably to room temperature, then placing in an environment of -5 to 5° C., preferably in an environment of 4° C., and crystallizing to obtain the crystalline form I;

[0058] More preferably, the ratio (W / V) of 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'-bipyrimidine]-4-amine to the solvent is 100 mg:1 mL to 10 mg:1 mL, preferably 80 mg:1 mL to 30 mg:1 mL.

[0059] The present invention also provides a pharmaceutical composition comprising the crystalline form I and one or more pharmaceutically acceptable carriers.

[0060] The present invention relates to use of crystal form I or a pharmaceutical composition containing the same in preparing a medicament for treating or preventing a disease or condition associated with the inhibition of ubiquitin-specific protease 1 (USP1).

[0061] The present invention also relates to the use of the crystalline form I or a pharmaceutical composition comprising the same in the preparation of a drug for treating or preventing cancer.

[0062] The present invention also relates to the crystalline form I or a pharmaceutical composition comprising the same, for use in treating or preventing diseases or conditions associated with the inhibition of ubiquitin-specific protease 1 (USP1).

[0063] The present invention also relates to the crystalline form I or a pharmaceutical composition comprising the same, which is used for treating or preventing cancer.

[0064] The present invention also relates to a method for treating or preventing a disease or condition associated with the inhibition of ubiquitin-specific protease 1 (USP1), comprising administering an effective amount of crystalline form I or a pharmaceutical composition comprising the same to a patient in need thereof.

[0065] The present invention also relates to a method for treating or preventing cancer, which comprises administering an effective amount of Form I or a pharmaceutical composition comprising the same to a patient in need thereof.

[0066] In some embodiments, the cancer is selected from lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer, and breast cancer.

[0067] As used herein, "pharmaceutically acceptable" is useful in preparing pharmaceutical compositions that are generally safe, neither biologically toxic nor otherwise undesirably toxic, and acceptable for veterinary and human pharmaceutical use.

[0068] The "carrier" described in the present disclosure refers to a diluent, adjuvant or excipient administered together with the compound, including but not limited to fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, etc. The composition of the present invention can be prepared by any method known in the art to provide rapid, sustained or slow release of the active ingredient after administration to the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 is an X-ray powder diffraction (XRPD) pattern of Form I.

[0070] FIG2 is a differential scanning calorimetry (DSC) spectrum of Form I.

[0071] FIG3 is a thermogravimetric analysis (TGA) spectrum of Form I.

[0072] Figure 4 is a PLM photograph of Form I (10X10X).

[0073] Figure 5 is a PLM photograph of Form I (10X40X).

[0074] FIG6 is the DVS result of Form I.

[0075] Figure 7 shows the XRPD patterns of Form I before and after DVS testing. From top to bottom in the figure are the XRPD patterns after DVS and before DVS. DETAILED DESCRIPTION

[0076] The present invention will be described in more detail below with reference to the embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the essence and scope of the present invention.

[0077] Purity analysis was performed using Kinetex EVO C18 (50 × 4.6 mm, 5 μm, ) column, acetonitrile-water was used as the mobile phase for gradient elution, the flow rate was 1.5 mL / min, and the detection wavelength was 220 nm.

[0078] MS was measured using an LC (Agilent 1260 Infinity II) / MS (G6125B single quadrupole) mass spectrometer (manufacturer: Agilent) (Photodiode Array Detector).

[0079] The structure of the compound was confirmed by hydrogen spectrum using WNMR-I-400MHz equipment.

[0080] Preparative liquid chromatography was performed using an Agilent 1260 Infinity II high performance liquid chromatograph (manufacturer: Agilent), with a Daisogel C18 10 μm 100A column (30 mm × 250 mm) and acetonitrile / water as the mobile phase.

[0081] Thin layer chromatography (TLC) used Qingdao Ocean Chemical GF254 silica gel plates. The silica gel plates used for reaction monitoring were of a size of 0.20 mm to 0.25 mm, and the silica gel plates used for separation and purification were of a size of 0.5 mm.

[0082] Silica gel column chromatography method uses Qingdao marine silica gel 100-200 mesh, 200-300 mesh and 300-400 mesh silica gel as the carrier.

[0083] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from online shopping malls, Beijing Coupling, Sigma, Bailingwei, Yishiming, Shanghai Shuya, Shanghai Yinuokai, Anaiji Chemical, Shanghai Bid, etc.

[0084] Unless otherwise specified in the examples, all reactions were carried out under a nitrogen atmosphere.

[0085] Nitrogen atmosphere means that the reaction bottle is connected to a nitrogen balloon with a capacity of about 1 L.

[0086] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0087] The chemical reactions described in the present invention are generally carried out under normal pressure. Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0088] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention.

[0089] Unless otherwise specified, the mixing ratios of different solvents are by volume.

[0090] X-ray powder diffraction (XRPD) method

[0091] The instrument is Shimadzu XRD-6000, and the sample is scanned according to the following parameters:

[0092] The ray source is Cu~Kα target

[0093] The minimum operating voltage and current of the fluorescent tube are 40kV and 30mA respectively.

[0094] The sample scanning range of 2θ values ​​was from 2° to 40°, and the scanning speed was 4° / min.

[0095] Thermogravimetric analysis (TGA) method

[0096] The instrument was a PerkinElmer Pyris 1TGA. The temperature was increased from 20°C to 350°C at a rate of 10°C / min, and maintained at 350°C for 1 min.

[0097] Differential Scanning Calorimetry (DSC) method

[0098] The instrument was a METTLER DSC3. Differential thermal scanning was performed from 30°C to 300°C, with the temperature held at 300°C for 1 minute. The heating rate was 10°C / min.

[0099] Polarized Light Microscopy (PLM) Method

[0100] The testing instrument for PLM is XPV-203E from Shanghai Changfang Optical Instrument Co., Ltd.

[0101] The samples were observed using a 10X eyepiece and a 10X or 40X objective lens, and the images were recorded using a camera computer system.

[0102] Dynamic Water Sorption (DVS) Method

[0103] The test instrument for DVS is SMSDVS Intrinsic.

[0104] The moisture absorption / desorption characteristics were tested in a water environment at 25°C under a relative humidity (RH) cycle of 0% to 90% to 0%.

[0105] HPLC method

[0106] The HPLC method for solubility and stability was performed using a Phenomenex Luna C18(2), 3μm, 2X50 mm column.

[0107] Example 1: Synthesis of 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'-bipyrimidine]-4-amine

[0108] 1.1 Synthesis of General Intermediate A2-7 ((4-cyclopropyl-6-methoxypyrimidin-5-yl)boronic acid)

[0109] Step 1: Synthesis of compound A2-3 (6-cyclopropylpyrimidin-4-ol)

[0110] Compound A2-1 (200.0 g, 1.4 mol, 1.00 eq) and compound A2-2 (292.0 g, 2.81 mol, 2.00 eq) were added to methanol (1.2 L). Sodium methoxide methanol solution (5.4 M, 1.3 L, 5.00 eq) was added at 0°C. After the addition, the temperature was raised to 20°C with stirring. Glacial acetic acid was added at 0°C to adjust the pH to 7-8. The mixture was concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-1 / 1) to obtain compound A2-3 (90.3 g). LC-MS: m / z = 137.1 (M+H) + .

[0111] Step 2: Synthesis of compound A2-4 (4-chloro-6-cyclopropylpyrimidine)

[0112] Compound A2-3 (40.0 g, 293.0 mmol, 1.00 eq) was added to phosphorus oxychloride (180.0 mL), the temperature was raised to 60°C with stirring, and the reaction was concentrated in vacuo to obtain a crude product. The crude product was dissolved in ethyl acetate (400.0 mL) and water (400.0 mL), extracted with ethyl acetate (400.0 mL), dried, filtered, and concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1-1 / 1) to obtain compound A2-4 (11.0 g). LC-MS: m / z = 155.0 (M+H) + .

[0113] Step 3: Synthesis of compound A2-5 (5-bromo-4-chloro-6-cyclopropylpyrimidine)

[0114] Compound A2-4 (11.0 g, 71.1 mmol, 1.00 eq) was dissolved in methanol (150.0 mL), and bromine (34.1 g, 213.0 mmol, 3.00 eq) was added at -60°C. The temperature was raised to 20°C and the reaction was stirred. Then, saturated sodium bicarbonate solution (200.0 mL) and water (100.0 mL) were added at 0°C, and the mixture was extracted three times with dichloromethane (200.0 mL). The mixture was washed with water, dried, 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 3 / 1) to obtain compound A2-5 (7.8 g). 1 H NMR (400MHz, CDCl3) δ8.61 (s, 1H), 2.56-2.62 (m, 1H), 1.23-1.26 (m, 2H), 1.16-1.21 (m, 2H); LC-MS: m / z=232.9 (M+H) + .

[0115] Step 4: Synthesis of compound A2-6 (5-bromo-4-cyclopropyl-6-methoxypyrimidine)

[0116] Compound A2-5 (7.8 g, 33.4 mmol, 1.00 eq) was dissolved in methanol (240.0 mL). Sodium methoxide (18.0 g, 100.0 mmol, 3.00 eq) was added at 0°C. The mixture was heated to 30°C with stirring and reacted. The mixture was concentrated in vacuo to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1-5 / 1) to obtain compound A2-6 (7.3 g). LC-MS: m / z = 228.9 (M+H) + .

[0117] Step 5: Synthesis of compound A2-7 ((4-cyclopropyl-6-methoxypyrimidin-5-yl)boronic acid)

[0118] Compound A2-6 (10.3 g, 44.9 mmol, 1.00 eq) and triisopropyl borate (11.8 g, 62.9 mmol, 1.40 eq) were dissolved in tetrahydrofuran (30.0 mL) and toluene (90.0 mL). n-Butyl lithium (2.5 M, 25.1 mL, 1.40 eq) was added dropwise at -70°C, and the mixture was stirred at -70°C. Then, 1N hydrochloric acid solution (50.0 mL) was added dropwise at -70°C, and the mixture was heated to 20°C and stirred. Saturated aqueous sodium bicarbonate was added to adjust the pH to 7-8. The mixture was extracted three times with ethyl acetate (100.0 mL). The mixture was washed with water, dried, filtered, and concentrated in vacuo to obtain compound A2-7 (6.9 g). 1 H NMR (400MHz, CDCl3) δ8.52(s,1H),8.45(s,2H),3.84(s,3H),1.88-1.92(m,2H),0.93-1.01(m,4H).

[0119] 1.2 General intermediate BB2 (2-chloro-5-methoxypyrimidin-4-amine)

[0120] The intermediate BB2 was purchased from Leyan Reagent.

[0121] 1.3 Synthesis of General Intermediate C35 ((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]octan-1-yl)methyl-4-methylbenzenesulfonate)

[0122] Step 1: Synthesis of compound C35-2 (1-methyl-4-(trifluoromethyl)-1H imidazole)

[0123] Compound 4-(trifluoromethyl)-1H-imidazole (compound C35-1, 26 g, 0.19 mol) and potassium carbonate (105.5 g, 0.76 mol, 4.0 eq) were added to acetonitrile (390 mL). Methyl iodide (32.6 g, 0.23 mol, 1.2 eq) was added dropwise at 0°C and stirred at room temperature. The mixture was then concentrated in vacuo to obtain a crude product, which was extracted three times with ethyl acetate (200 mL). The product was washed with water, dried, filtered, and concentrated in vacuo to obtain compound C35-2 (26 g, crude product). LC-MS: m / z = 151.0 (M+H) + .

[0124] Step 2: Synthesis of compound C35-3 (methyl 4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]octane-1-carboxylate)

[0125] Compound C35-2 (14.75 g, 98.3 mmol) and bicyclo[2,2,2]octane-1,4-cyclohexanedicarboxylic acid monomethyl ester (25 g, 117.9 mmol, 1.2 eq) were dissolved in dichloromethane (295 mL) and water (295 mL), and silver nitrate (6.0 g, 35.4 mmol, 0.3 eq.) and ammonium persulfate (44.9 g, 78.7 mmol, 2.0 eq.) were added. The mixture was stirred at 25°C and filtered through celite. The mixture was extracted three times with dichloromethane (200 mL). The filtrate was washed with water, dried, and filtered, and concentrated in vacuo to obtain compound C35-3 (35.2 g, crude product) as a yellow oil. 1 H NMR (400MHz, CDCl3)7.09-7.08(m,1H),3.77(s,3H),3.67(s,3H),2.07-2.03(m,6H),1.93-1.89(m,6H); LC-MS: m / z=317.2(M+H) + .

[0126] Step 3: Synthesis of compound C35-4 ((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]octan-1-yl)methanol)

[0127] Compound C35-3 (35.2 g, 111.4 mmol) was dissolved in tetrahydrofuran (180.0 mL). Lithium aluminum tetrahydride (2.5 M, 111.4 mL, 278.5 mmol, 2.5 eq) was added at 0°C and the mixture was heated to 20°C with stirring. Ice water (50.0 mL) was then added at 0°C and the mixture was extracted three times with ethyl acetate (500.0 mL). The filtrate was dried, filtered, and concentrated in vacuo to afford compound C35-4 (25.1 g, crude product) as a yellow oil. LC-MS: m / z = 289.1 (M+H) + .

[0128] Step 4: Synthesis of compound C35

[0129] Compound C35-4 (25.1 g, 87.2 mmol), p-toluenesulfonyl chloride (33.2 g, 174.4 mmol, 2.0 eq) and 4-dimethylaminopyridine (32.0 g, 261.6 mmol, 3.0 eq) were added to dichloromethane (251 mL) in sequence, and the mixture was stirred at 25°C. The mixture was washed with water and dried, and the filtrate was filtered to obtain the filtrate, which was concentrated in vacuo to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether / dichloromethane = 1 / 0-0 / 1) to obtain white solid intermediate C35 (10.0 g). 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=8.2Hz,2H),7.67(s,1H),7.52(d,J=8.0Hz,2H),3.77(s,3 H),3.72(s,2H),2.46(s,3H),1.95–1.86(m,6H),1.49–1.37(m,6H); LC-MS: m / z=443.3(M+H) + .

[0130] 1.4 Synthesis of Intermediate BB2C35 (2-chloro-5-methoxy-N-((4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl)bicyclo[2.2.2]octan-1-yl)methyl)pyrimidin-4-amine)

[0131] Compound BB2 (1.0 g, 6.29 mmol, 1.00 eq), compound C35 (2.78 g, 6.29 mmol, 1.0 eq), and cesium carbonate (10.2 g, 31.45 mmol, 5.0 eq) were added to N,N-dimethylformamide (20.0 mL) in sequence. The mixture was stirred at 130°C and cooled to 25°C. Dichloromethane (100.0 mL) was added and the filtrate was filtered. The filtrate was washed with water, dried, and concentrated in vacuo to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether / dichloromethane = 1 / 0 to 0 / 1) to obtain the intermediate BB2C35 (1.03 g, 2.41 mmol, 38.3% yield) as a white solid. LC-MS: m / z = 430.1 (M+H) + .

[0132] 1.5 Synthesis of 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

[0133] Compound BB2C35 (85.8 g, 0.2 mol), compound A2-7 (48.5 g, 0.25 mol, 1.25 eq.), ligand XPhos (37.4 g, 0.08 mol, 0.4 eq.), potassium phosphate (169.8 g, 0.8 mol, 4 eq.), and catalyst XPhos-Pd-G2 (31.5 g, 0.04 mol, 0.2 eq.) were added to 1,4-dioxane (1026 mL) / water (171.6 mL) in sequence under nitrogen protection. The temperature was raised to 85°C with stirring to react, and the mixture was concentrated under reduced pressure. The temperature was lowered to 25°C, dissolved in water, and then extracted with ethyl acetate (1026 mL), dried, and concentrated under reduced pressure to obtain about 200 g of the crude title compound.

[0134] Wherein: XPhos is 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; XPhos-Pd-G2 is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II).

[0135] Example 2: Preparation of Form I of 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'-bipyrimidine]-4-amine

[0136] About 200 g of the crude product 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'-bipyrimidine]-4-amine obtained in Example 1 was dissolved in 1 L (5V) of ethyl acetate, and then silica gel was added and concentrated in vacuo to a powder. The mixture was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 100:5) to obtain about 91 g of a light yellow solid. The solid was then slurried with 275 ml of a solvent (petroleum ether: methyl tert-butyl ether = 10:1) at 25°C for 30 min, filtered, and dried in vacuo at 50°C to obtain 85.0 g of a white powder solid with a yield of 78% and a purity of 99.2%.

[0137] LC-MS: m / z=544.3 (M+H) + ;

[0138] 1H NMR (400MHz, DMSO-d6) δ8.62(s,1H),7.90(s,1H),7.65(d,J=1.5Hz,1H),6.99(t,J=6.5Hz,1H),3.93(s,3H),3.84(s, 3H), 3.77 (s, 3H), 3.21 (d, J = 6.5Hz, 2H), 1.90 (m, 6H), 1.74 (m, 1H), 1.48 (m, 6H), 1.06–1.01 (m, 2H), 0.92–0.85 (m, 2H).

[0139] The X-ray powder diffraction analysis showed that the crystal form was defined as Form I. The X-ray powder diffraction data are shown in Table 1, and the X-ray powder diffraction pattern is shown in FIG1 .

[0140] The DSC spectrum of Form I is shown in Figure 2, which shows an obvious endothermic peak at 219.25±2°C.

[0141] The TGA spectrum of Form I is shown in Figure 3, which shows a weight loss of 0.10% from 20°C to 120°C and a weight loss of 0.37% from 120°C to 250°C.

[0142] The PLM results of Form I are shown in Figures 4 and 5.

[0143] The DVS results of Form I are shown in Figure 6, which show that the water absorption rate from 0% RH to 95% RH is 0.02%, indicating that Form I is not hygroscopic. The XRPD pattern of the form after the DVS test is shown in Figure 7, which shows that the form does not change after the DVS test.

[0144] Table 1

[0145] Example 3-Example 10: Preparation of Form I (Anti-Solvent Method)

[0146] Weigh approximately 50 mg of Form I and dissolve it in an appropriate volume of a good solvent listed in Table 2 at room temperature to obtain a saturated or nearly saturated solution. Filter the solution. Slowly add an antisolvent (3 to 10 times the volume of the good solvent) listed in Table 2 under magnetic stirring. Stir until sufficient solid precipitates, then collect the solid by centrifugation. Dry under reduced pressure and vacuum (40°C) to obtain a solid.

[0147] Table 2

[0148] X-ray powder diffraction analysis showed that the crystal forms obtained in Examples 3 to 10 were all Form I.

[0149] Example 11-Example 16: Preparation of Form I (suspension beating method)

[0150] About 50 mg of Form I was weighed, and an appropriate volume of the solvent shown in Table 3 was added. After magnetic stirring (400 rpm) at 50° C. for 4 days, the solid was collected by centrifugation.

[0151] Table 3

[0152] X-ray powder diffraction analysis showed that the crystal forms obtained in Examples 11 to 16 were all Form I.

[0153] Example 17-Example 21: Preparation of Form I (cooling method)

[0154] Weigh approximately 50 mg of Form I and dissolve it in an appropriate volume of a good solvent listed in Table 4 at 50°C. Filter the mixture, cool to room temperature until solid precipitates, and then place it in a refrigerator at 4°C. Once sufficient solid precipitates, filter the solid and dry it under reduced pressure (40°C) to obtain a solid.

[0155] Table 4

[0156] X-ray powder diffraction analysis showed that the crystal forms obtained in Examples 17 to 21 were all Form I.

[0157] Example 22: Grinding study of Form I

[0158] Using the conditions shown in Table 5, approximately 50 mg of Form I was manually ground in a mortar and then tested by XRPD to investigate the crystalline form of the ground solid.

[0159] Table 5

[0160] The results showed that the crystal forms obtained through the above grinding studies were all Form I. Form I had good grinding stability.

[0161] Example 23: Solubility Study of Form I

[0162] 2 mg of Form I was sampled and 50 μL or 100 μL of the solvent listed in Table 6 was gradually added. The mixture was shaken, vortexed, or sonicated. No further solvent was added when the concentration fell below 1 mg / mL. The solubility results were calculated based on the sample size and the total amount of solvent added. The results are shown in Table 6.

[0163] Table 6

[0164] Note: * is the solubility result of Form I in pH 7.4 phosphate buffer at 37°C for 24 hours.

[0165] The results showed that Form I had good solubility in organic solvents; good solubility in low pH solution (pH 1.2), ranging from 8.45 to 16.9 mg / mL; weak solubility in high pH buffer (pH 7.4 phosphate buffer), with a solubility of 0.31 mg / mL at 37°C for 24 hours.

[0166] Example 24: Stability Study of Form I

[0167] (1) Forced degradation stability

[0168] 3 mg of Form I was weighed into 3 mL of 90% acetonitrile to prepare a 1 mg / mL stock solution. Stability studies were performed using oxidative stress (3% H2O2 at 40°C for 1.5 h) and alkaline stress (0.1 N NaOH at 40°C for 2 h).

[0169] The results showed that Form I was only slightly degraded when heated in 0.1NaOH at 40°C for 2 h and in 3% H2O2 at 40°C for 1.5 h, indicating good stability.

[0170] (2) Temperature and humidity stability

[0171] 1-3 mg of Form 1 solid powder was weighed and placed at 60° C. and 60° C. / 75% RH for 30 days. Samples were taken for LC detection and analysis.

[0172] The results showed that the main component content of Form I changed by less than 0.2% under the conditions of 60°C and 75% RH, and was relatively stable for 30 days.

[0173] In addition, an appropriate amount of Form 1 solid powder was weighed and placed under stability conditions (40° C. / 75% RH, 60° C. / 75% RH) for 30 days, and samples were taken for XRPD analysis.

[0174] The results showed that Form 1 did not undergo any crystal form change when placed under 40°C / 75% RH and 60°C / 75% RH conditions for 30 days.

[0175] (3) Light stability

[0176] Weigh an appropriate amount of Form 1 solid powder and place it in a light stabilization box. Use ICH light intensity conditions (at least greater than 1.2*10^6Lux*h, illumination 4500±500Lux, and 200w*hr / m 2 Samples were taken for LC detection and result analysis.

[0177] The results showed that the content of the main component of Form I changed by less than 0.2% and was stable to light.

[0178] Example 25: Pharmacokinetic Study of Oral Gavage Suspension

[0179] Form I was used in a pharmacokinetic study in rats. Twelve male Sprague-Dawley rats were weighed before dosing and housed using standard household practices. The animals were housed in standard cages. Food and water were available ad libitum throughout the experiment. The rats were orally gavaged with a suspension of Form I. At each sampling point, 40 μl of blood was collected in an EDTA-containing anticoagulant tube. After centrifugation, 20 μl of plasma was obtained. All samples were frozen at -80°C immediately after collection.

[0180] The results showed that the suspension of Form I administered orally had high blood drug concentration, high exposure, and high bioavailability in SD rats, and had obvious pharmacokinetic advantages.

Claims

1. Crystalline Form I of 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'-bipyrimidine]-4-amine, characterized in that: Its X-ray powder diffraction pattern includes characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2° and 22.200±0.2°.

2. The crystalline form I according to claim 1, characterized in that Its X-ray powder diffraction pattern further comprises at least one characteristic peak at diffraction angles (2θ) of 11.938±0.2°, 23.761±0.2°, 25.101±0.2° and 28.700±0.2°.

3. The crystalline form I according to claim 1 or 2, characterized in that: Its X-ray powder diffraction pattern contains characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 11.938±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2°, 22.200±0.2°, 23.761±0.2°, 25.101±0.2° and 28.700±0.2°.

4. The crystalline form I according to any one of claims 1 to 3, characterized in that Its X-ray powder diffraction pattern further comprises at least one characteristic peak at diffraction angles (2θ) of 10.239±0.2°, 12.400±0.2°, 14.882±0.2°, 20.420±0.2° and 25.980±0.2°.

5. The crystalline form I according to any one of claims 1 to 4, characterized in that Its X-ray powder diffraction pattern contains characteristic peaks at diffraction angles (2θ) of 6.378±0.2°, 9.521±0.2°, 10.239±0.2°, 11.938±0.2°, 12.400±0.2°, 14.882±0.2°, 15.721±0.2°, 16.379±0.2°, 16.981±0.2°, 17.560±0.2°, 19.080±0.2°, 20.420±0.2°, 22.200±0.2°, 23.761±0.2°, 25.101±0.2°, 25.980±0.2° and 28.700±0.2°.

6. The crystalline form I according to any one of claims 1 to 5, characterized in that Its DSC spectrum showed an endothermic peak at 219.25±2℃.

7. The crystalline form I according to any one of claims 1 to 6, characterized in that Its TGA spectrum showed a weight loss of 0.10% from 20°C to 120°C and a weight loss of 0.37% from 120°C to 250°C.

8. A method for preparing the crystalline form I according to any one of claims 1 to 7, comprising: Mixing 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'-bipyrimidine]-4-amine with an organic solvent, and performing solid-liquid separation on the obtained suspension to collect the solid; The organic solvent is selected from one or more of petroleum ether, C4-C6 ether and C2-C6 nitrile.

9. The method according to claim 8, comprising: Slurrying 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'-bipyrimidine]-4-amine with an organic solvent at 0 to 30° C. for 10 minutes to 2 hours, preferably slurrying at 25° C. for 30 minutes, and collecting a solid by solid-liquid separation of the obtained suspension, which is optionally further dried; The organic solvent is selected from one or more of petroleum ether, methyl tert-butyl ether, tetrahydrofuran and acetonitrile, and is a mixture of petroleum ether and methyl tert-butyl ether; Advantageously, the ratio (W / V) of 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 to the organic solvent is 1:5 to 1:1, preferably 1:

3.

10. A pharmaceutical composition comprising the crystalline form I according to any one of claims 1 to 7 and one or more pharmaceutically acceptable excipients.

11. Use of the crystalline form I according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating or preventing a disease or condition associated with the inhibition of ubiquitin-specific protease 1 (USP1).

12. Use of the crystalline form I according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 10 in the preparation of a drug for treating or preventing cancer, In particular, the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer (NSCLC), colon cancer, bladder cancer, osteosarcoma, ovarian cancer, skin cancer and breast cancer.

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