Salt of thiazolopyrazine compound or crystal thereof and use thereof

Abstract

Provided are a pharmaceutically acceptable salt of a thiazopyrazine compound represented by formula (I) or a solvate (including a hydrate) of the pharmaceutically acceptable salt, a crystal form of the pharmaceutically acceptable salt or a crystal form of the solvate (including a hydrate) of the pharmaceutically acceptable salt, a crystal form of the compound of formula (I), a preparation method therefor, a pharmaceutical composition thereof, and the medical use thereof.

Description

Salt of thiazolopyrazine compound or crystal thereof and application thereof

[0001] Cross-reference to Related Applications

[0002] This disclosure claims priority to and the benefit of Chinese Patent Application No. 202310852344.4, filed on July 12, 2023, with the China National Intellectual Property Office. The entire contents of the aforementioned patent application are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of medicinal chemistry, in particular, to a crystal form of a thiazolopyrazine compound, a pharmaceutically acceptable salt of a thiazolopyrazine compound or a solvate (including a hydrate) of a pharmaceutically acceptable salt, a crystal form of the pharmaceutically acceptable salt or the solvate (including the hydrate) of the pharmaceutically acceptable salt, a preparation method thereof, a pharmaceutical composition comprising the same, and a use thereof. BACKGROUND

[0004] DNA double-strand break repair is essential for maintaining genome stability and cell survival. There are three major repair pathways for DNA double-strand breaks: homologous recombination (HR), non-homologous end joining (NHEJ), and non-canonical non-homologous end joining (alt-NHEJ). Microhomology-mediated end joining (MMEJ) is the most common non-canonical non-homologous end joining. Homologous recombination is a high-fidelity, error-free repair mechanism that can maintain genome stability and avoid inducing cancer. Non-homologous end joining and microhomology-mediated end joining belong to error-prone repair pathways, which can cause mutations at the repair site.

[0005] Unlike normal cells, the survival of tumor cells often depends on the misregulation of DNA double-strand break repair. At the same time, abnormal DNA double-strand break repair can make tumor cells more sensitive to specific types of DNA damage. Therefore, DNA double-strand break repair defects can be used to develop targeted tumor therapies. Tumor cells with impaired homologous recombination or non-homologous end joining repair will rely more on microhomology-mediated end joining repair. Multiple lines of genetic, cellular biological, and biochemical evidence suggest that DNA polymerase theta (POLQ or POLθ) is a key protein in the process of microhomology-mediated end joining repair (Kent et al. Nature Structural & Molecular Biology (2015), 22(3), 230-237, Mateos-Gomez et al. Nature (2015), 518(7538), 254-257).

[0006] POLQ is a multifunctional enzyme consisting of an N-terminal helicase domain (SF2 HEL308-type) and a C-terminal low-fidelity DNA polymerase domain (A-type) (Wood & Doublie DNA Repair (2016), 44, 22-32). The helicase domain mediates the removal of RPA protein from single-stranded DNA and facilitates annealing, the polymerase domain can extend single-stranded DNA ends and fill gaps, both domains work together in the process of microhomology-mediated end joining repair.

[0007] Studies have shown that POLQ is essential for cells with homologous recombination deficiency (e.g. synthetic lethal with FA / BRCA deficiency), and POLQ is upregulated at the protein level in tumor cells with homologous recombination deficiency (Ceccaldi et al. Nature (2015), 518(7538), 258-262). In vivo studies have also shown that POLQ is overexpressed in a series of homologous recombination-deficient and poor-prognosis ovarian cancer, uterine cancer and breast cancer (Higgins et al. Oncotarget (2010), 1, 175-184, Lemee et al. PNAS (2010), 107(30), 13390-13395, Ceccaldi et al. (2015), supra). More importantly, the expression of POLQ in normal tissues is inhibited compared with tumor tissues (Kawamura et al. International Journal of Cancer (2004), 109(1), 9-16).

[0008] In summary, POLQ is essential for cells with homologous recombination deficiency, and there is an unmet market demand for the treatment of homologous recombination-deficient tumors. Inhibiting the function of POLQ can inhibit the microhomology-mediated end joining repair of cells, and the development of POLQ function inhibitors can provide a new strategy for the targeted treatment of homologous recombination-deficient tumors.

[0009] SUMMARY

[0010] In one aspect, the present disclosure provides a pharmaceutically acceptable salt of a compound of Formula I (4-(5-chloro-2-methoxyphenyl)-N-[6-(4-cyanophenyl)thiazolo[4,5-b]pyrazin-2-yl]-6- methyl nicotinamide) as a POLQ inhibitor, wherein the pharmaceutically acceptable salt is selected from a sodium salt or a potassium salt

[0011] In some embodiments, the present disclosure provides a pharmaceutically acceptable salt of a compound of Formula I, wherein the pharmaceutically acceptable salt is a sodium salt

[0012] In some embodiments, the pharmaceutically acceptable salt of the compound of Formula I has a molar ratio of the compound of Formula I to sodium ion or potassium ion of about 1 : 1.

[0013] In some embodiments, the pharmaceutically acceptable salt of the compound of Formula I has a molar ratio of the compound of Formula I to sodium ion or potassium ion of 1 : 1.

[0014] In some embodiments, the present disclosure provides a sodium salt of the compound of Formula I, wherein the molar ratio of the compound of Formula I to sodium ion is about 1 : 1.

[0015] In some embodiments, the present disclosure provides a sodium salt of the compound of Formula I, wherein the molar ratio of the compound of Formula I to sodium ion is 1 : 1.

[0016] In some embodiments, the present disclosure provides a potassium salt of the compound of Formula I, wherein the molar ratio of the compound of Formula I to potassium ion is about 1 : 1.

[0017] In some embodiments, the present disclosure provides a potassium salt of the compound of Formula I, wherein the molar ratio of the compound of Formula I to potassium ion is 1 : 1.

[0018] In some embodiments, the pharmaceutically acceptable salt of the compound of Formula I can exist in an unsolvated or solvated form.

[0019] In another aspect, the present disclosure also provides a solid form of the pharmaceutically acceptable salt of the compound of Formula I.

[0020] In some embodiments, the solid form of the pharmaceutically acceptable salt of the compound of Formula I is selected from an amorphous or crystalline form.

[0021] In another aspect, the present disclosure also provides a solid form of the sodium salt of the compound of Formula I.

[0022] In some embodiments, the solid form of the sodium salt of the compound of Formula I is selected from an amorphous or crystalline form.

[0023] In another aspect, the present disclosure also provides a solid form of the potassium salt of the compound of Formula I.

[0024] In some embodiments, the solid form of the potassium salt of the compound of Formula I is selected from an amorphous or crystalline form.

[0025] In another aspect, the present disclosure also provides a method of preparing the sodium salt of the compound of Formula I, comprising the step of salifying the compound of Formula I with a sodium-containing base.

[0026] In some embodiments, the method of preparing a sodium salt of the compound of Formula I comprises reacting the compound of Formula I with a sodium-containing base in a solvent selected from one or more of an alcoholic solvent, DMSO, NMP, DMF, or DMA.

[0027] In some embodiments, the solvent is selected from one or more of ethanol, DMSO, NMP, DMF, or DMA.

[0028] In some embodiments, the sodium-containing base is selected from sodium tert-butoxide, sodium ethoxide, sodium methoxide, or sodium hydroxide.

[0029] In some embodiments, the sodium-containing base is selected from sodium ethoxide, sodium methoxide, or sodium hydroxide.

[0030] In some embodiments, the sodium-containing base is selected from sodium ethoxide or sodium hydroxide.

[0031] In some embodiments, the method of preparing a potassium salt of the compound of Formula I comprises reacting the compound of Formula I with a potassium-containing base in a solvent selected from one or more of DMF, DMA, or NMP.

[0032] In some embodiments, the method of preparing a potassium salt of the compound of Formula I comprises reacting the compound of Formula I with a potassium-containing base in a solvent selected from one or more of DMF, DMA, or NMP.

[0033] In some embodiments, the potassium-containing base is potassium hydroxide.

[0034] In some embodiments, the method of preparing a potassium salt of the compound of Formula I comprises reacting the compound of Formula I with a potassium-containing base in a solvent selected from one or more of DMF, DMA, or NMP.

[0035] In some embodiments, the solvent of the solvate of the sodium salt of the compound of Formula I is water.

[0036] In some embodiments, the molar ratio of the sodium salt of the compound of Formula I to the solvent in the solvate of the sodium salt of the compound of Formula I is about 1 : 1.

[0037] In some embodiments, the molar ratio of the sodium salt of the compound of Formula I to the solvent in the solvate of the sodium salt of the compound of Formula I is 1 : 1.

[0038] In some embodiments, the method of preparing a potassium salt of the compound of Formula I comprises reacting the compound of Formula I with a potassium-containing base in a solvent selected from one or more of DMF, DMA, or NMP.

[0039] In some embodiments, the solid form of the solvate of the sodium salt of the compound of Formula I is selected from an amorphous or crystalline form.

[0040] In some embodiments, the solvate of the sodium salt of the compound of Formula I is a hydrate.

[0041] In some embodiments, the solvate of the sodium salt of the compound of Formula I is a monohydrate.

[0042] In some embodiments, the solvate of the sodium salt of the compound of Formula I is an ethanol solvate.

[0043] In some embodiments, the solvate of the sodium salt of the compound of Formula I is a monoethanol solvate.

[0044] In some embodiments, the solvate of the potassium salt of the compound of Formula I is a hydrate.

[0045] In some embodiments, the solvate of the potassium salt of the compound of Formula I is a monohydrate.

[0046] In some embodiments, the solvate of the potassium salt of the compound of Formula I is a monohydrate.

[0047] In some embodiments, the molar ratio of the potassium salt of the compound of Formula I to the solvent in the solvate of the potassium salt of the compound of Formula I is about 1 : 1.

[0048] In some embodiments, the molar ratio of the potassium salt of the compound of Formula I to the solvent in the solvate of the potassium salt of the compound of Formula I is 1 : 1.

[0049] The present disclosure also provides a solid form of the solvate of the potassium salt of the compound of Formula I.

[0050] In some embodiments, the solid form of the solvate of the potassium salt of the compound of Formula I is selected from an amorphous or crystalline form.

[0051] In another aspect, the present disclosure provides a crystalline form of the sodium salt of the compound of Formula I.

[0052] In some embodiments, the present disclosure also provides a crystalline Form A of the sodium salt of the compound of Formula I, which has an X-ray powder diffraction pattern, expressed in terms of the diffraction angles 2 theta, having diffraction peaks at 6.39 ± 0.20°, 7.84 ± 0.20°, 13.61 ± 0.20°, and 25.46 ± 0.20°.

[0053] In some embodiments, the crystalline Form A of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, expressed in terms of the diffraction angles 2 theta, having diffraction peaks at 6.39 ± 0.20°, 7.84 ± 0.20°, 13.61 ± 0.20°, 15.95 ± 0.20°, 17.16 ± 0.20°, 20.90 ± 0.20°, 21.81 ± 0.20°, 25.46 ± 0.20°, and 30.79 ± 0.20°.

[0054] In some embodiments, the crystalline Form A of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, with diffraction peaks at 6.13±0.20°, 6.39±0.20°, 6.87±0.20°, 7.84±0.20°, 10.80±0.20°, 13.61±0.20°, 14.10±0.20°, 15.95±0.20°, 16.44±0.20°, 17.16±0.20°, 17.74±0.20°, 19.09±0.20°, 20.90±0.20°, 21.81±0.20°, 22.01±0.20°, 22.73±0.20°, 22.93±0.20°, 25.46±0.20°, and 30.79±0.20°.

[0055] In some embodiments, the crystalline Form A of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, substantially as shown in FIG. 1.

[0056] In some embodiments, the crystalline Form A of the sodium salt of the compound of Formula I has a DSC pattern with a peak at 342.84±5.0°C. In some embodiments, the DSC pattern of the crystalline Form A of the sodium salt of the compound of Formula I is as shown in FIG. 2.

[0057] In another aspect, the present disclosure provides a method for preparing the crystalline Form A of the sodium salt of the compound of Formula I, comprising: (1) mixing the compound of Formula I with anhydrous ethanol, (2) adding a mixed solution of sodium hydroxide and anhydrous ethanol, stirring, and separating.

[0058] In some embodiments, in the method for preparing the crystalline Form A of the sodium salt of the compound of Formula I, the molar ratio of the compound of Formula I to sodium hydroxide is 1:(0.5-1.5), 1:(0.9-1.2), or about 1:1.

[0059] In some embodiments, in the method for preparing the crystalline Form A of the sodium salt of the compound of Formula I, the volume (mL) of anhydrous ethanol in step (1) is 20-45 times or 35-45 times the mass (g) of the compound of Formula I.

[0060] In some embodiments, in the method for preparing the crystalline Form A of the sodium salt of the compound of Formula I, the stirring temperature in step (2) is 20-60°C or 20-30°C.

[0061] In another aspect, the present disclosure provides a crystalline form of the compound of Formula I.

[0062] In some embodiments, the present disclosure also provides a crystalline Form B of the compound of Formula I having an X-ray powder diffraction pattern, expressed in terms of diffraction angles 2θ, with diffraction peaks at 12.10 ± 0.20°, 17.79 ± 0.20°, 20.13 ± 0.20°, and 25.47 ± 0.20°.

[0063] In some embodiments, the crystalline Form B of the compound of Formula I has an X-ray powder diffraction pattern, expressed in terms of diffraction angles 2θ, with diffraction peaks at 7.98 ± 0.20°, 12.10 ± 0.20°, 14.59 ± 0.20°, 17.80 ± 0.20°, 20.13 ± 0.20°, 22.51 ± 0.20°, 23.61 ± 0.20°, 25.47 ± 0.20°, 26.74 ± 0.20°, and 27.66 ± 0.20°.

[0064] In some embodiments, the crystalline Form B of the compound of Formula I has an X-ray powder diffraction pattern, expressed in terms of diffraction angles 2θ, with diffraction peaks at 7.98 ± 0.20°, 12.10 ± 0.20°, 14.59 ± 0.20°, 15.97 ± 0.20°, 17.31 ± 0.20°, 17.79 ± 0.20°, 18.23 ± 0.20°, 20.13 ± 0.20°, 22.51 ± 0.20°, 23.31 ± 0.20°, 23.61 ± 0.20°, 23.98 ± 0.20°, 24.56 ± 0.20°, 25.47 ± 0.20°, 26.74 ± 0.20°, and 27.66 ± 0.20°.

[0065] In some embodiments, the crystalline Form B of the compound of Formula I has an X-ray powder diffraction pattern, expressed in terms of diffraction angles 2θ, with diffraction peaks at 7.98 ± 0.20°, 12.10 ± 0.20°, 14.59 ± 0.20°, 17.80 ± 0.20°, 20.13 ± 0.20°, 22.51 ± 0.20°, 23.61 ± 0.20°, 25.47 ± 0.20°, 26.74 ± 0.20°, and 27.66 ± 0.20°.

[0066] In some embodiments, the crystalline Form B of the compound of Formula I has a DSC profile with a peak at 343.98 ± 5.0 °C. In some embodiments, the DSC profile of the crystalline Form B of the compound of Formula I is as shown in FIG. 5.

[0067] In another aspect, the present disclosure provides a crystalline form of a monoethanolamine solvate of a sodium salt of a compound of Formula I.

[0068] In some embodiments, the present disclosure also provides a crystalline Form C of a monoethanolamine solvate of a sodium salt of a compound of Formula I having an X-ray powder diffraction pattern, expressed in terms of diffraction angles 2θ, with diffraction peaks at 17.14 ± 0.20°, 21.85 ± 0.20°, 25.46 ± 0.20°, and 25.74 ± 0.20°.

[0069] In some embodiments, Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, with diffraction peaks at 12.37 ± 0.20°, 17.14 ± 0.20°, 20.07 ± 0.20°, 21.40 ± 0.20°, 21.85 ± 0.20°, 23.39 ± 0.20°, 24.51 ± 0.20°, 24.99 ± 0.20°, 25.46 ± 0.20°, and 25.74 ± 0.20°.

[0070] In some embodiments, Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, with diffraction peaks at 12.37 ± 0.20°, 17.14 ± 0.20°, 20.07 ± 0.20°, 21.40 ± 0.20°, 21.85 ± 0.20°, 23.39 ± 0.20°, 24.51 ± 0.20°, 24.99 ± 0.20°, 25.46 ± 0.20°, and 25.74 ± 0.20°.

[0071] In some embodiments, Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern substantially as shown in FIG. 7.

[0072] In some embodiments, Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I has a DSC profile with a peak at 340.70 ± 5.0 °C. In some embodiments, the DSC profile of Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I is as shown in FIG. 8.

[0073] In another aspect, the present disclosure provides a crystalline form of a monohydrate of the sodium salt of the compound of Formula I.

[0074] In some embodiments, the present disclosure also provides Form D of a monohydrate of the sodium salt of the compound of Formula I, which has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, with diffraction peaks at 8.28 ± 0.20°, 10.08 ± 0.20°, 20.05 ± 0.20°, and 23.47 ± 0.20°.

[0075] In some embodiments, the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2Q, with diffraction peaks at 8.28 ± 0.20°, 10.08 ± 0.20°, 14.46 ± 0.20°, 16.79 ± 0.20°, 20.05 ± 0.20°, 22.07 ± 0.20°, 22.89 ± 0.20°, and 23.47 ± 0.20°.

[0076] In some embodiments, the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2Q, with diffraction peaks at 8.28 ± 0.20°, 10.08 ± 0.20°, 14.46 ± 0.20°, 16.67 ± 0.20°, 16.79 ± 0.20°, 17.28 ± 0.20°, 20.05 ± 0.20°, 22.07 ± 0.20°, 22.89 ± 0.20°, 23.47 ± 0.20°, and 26.19 ± 0.20°.

[0077] In some embodiments, the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2Q, with diffraction peaks at 8.28 ± 0.20°, 10.08 ± 0.20°, 12.30 ± 0.20°, 14.01 ± 0.20°, 14.46 ± 0.20°, 16.67 ± 0.20°, 16.79 ± 0.20°, 17.17 ± 0.20°, 17.28 ± 0.20°, 17.45 ± 0.20°, 18.15 ± 0.20°, 20.05 ± 0.20°, 22.07 ± 0.20°, 22.42 ± 0.20°, 22.89 ± 0.20°, 23.47 ± 0.20°, 26.19 ± 0.20°, and 26.46 ± 0.20°.

[0078] In some embodiments, the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2Q, with diffraction peaks at 8.28 ± 0.20°, 10.08 ± 0.20°, 14.46 ± 0.20°, 16.79 ± 0.20°, 20.05 ± 0.20°, 22.07 ± 0.20°, 22.89 ± 0.20°, and 23.47 ± 0.20°.

[0079] In some embodiments, the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I has a DSC pattern with a peak at 338.88 ± 5.0 °C. In some embodiments, the DSC pattern of the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I is as shown in FIG. 11.

[0080] In yet another aspect, the present disclosure also provides a method of preparing the crystalline Form D of the monohydrate of the sodium salt of the compound of Formula I, comprising: mixing the amorphous of the sodium salt of the compound of Formula I with a mixed solvent of water and a first solvent selected from one or more of toluene, methyl tert-butyl ether, isopropyl ether, or dichloromethane, stirring, isolating the solid.

[0081] In some embodiments, in the method of preparing the crystalline form D of the monohydrate of the sodium salt of the compound of Formula I, the volume (uL) of the mixed solvent of water and the first solvent is 1-30 times or 5-15 times of the mass (mg) of the amorphous of the sodium salt of the compound of Formula I.

[0082] In some embodiments, the first solvent is selected from toluene. In some embodiments, in the mixed solvent of water and toluene, the content of water is greater than 0 and less than or equal to 0.5wt%; or greater than 0 and less than or equal to 0.1wt%.

[0083] In some embodiments, in the method of preparing the crystalline form D of the monohydrate of the sodium salt of the compound of Formula I, the stirring temperature is 0-60°C or 15-30°C.

[0084] In another aspect, the present disclosure also provides a crystalline form E of the sodium salt of the compound of Formula I, wherein the X-ray powder diffraction pattern of the crystalline form E, which is expressed by diffraction angle 2θ, has diffraction peaks at 20.40±0.20°, 23.54±0.20°, 24.10±0.20° and 28.67±0.20°.

[0085] In some embodiments, the X-ray powder diffraction pattern of the crystalline form E of the sodium salt of the compound of Formula I, which is expressed by diffraction angle 2θ, has diffraction peaks at 10.09±0.20°, 17.53±0.20°, 20.40±0.20°, 22.84±0.20°, 23.54±0.20°, 24.10±0.20°, 28.67±0.20° and 30.58±0.20°.

[0086] In some embodiments, the X-ray powder diffraction pattern of the crystalline form E of the sodium salt of the compound of Formula I, which is expressed by diffraction angle 2θ, has diffraction peaks at 7.95±0.20°, 10.09±0.20°, 11.60±0.20°, 13.93±0.20°, 14.98±0.20°, 16.90±0.20°, 17.53±0.20°, 19.14±0.20°, 19.76±0.20°, 20.40±0.20°, 21.55±0.20°, 22.84±0.20°, 23.54±0.20°, 24.10±0.20°, 25.47±0.20°, 25.97±0.20°, 27.27±0.20°, 28.67±0.20° and 30.58±0.20°.

[0087] In some embodiments, the X-ray powder diffraction pattern of the crystalline form E of the sodium salt of the compound of Formula I, which is expressed by diffraction angle 2θ, is substantially as shown in Figure 13.

[0088] In some embodiments, the crystalline Form E of the sodium salt of the compound of Formula I has a DSC pattern with a peak at 338.17±5.0 °C.

[0089] In some embodiments, the DSC pattern of the crystalline Form E of the sodium salt of the compound of Formula I is as shown in FIG. 14.

[0090] In another aspect, the present disclosure also provides an amorphous form of the sodium salt of the compound of Formula I.

[0091] In some embodiments, the X-ray powder diffraction pattern of the amorphous form of the sodium salt of the compound of Formula I, in terms of diffraction angles 2θ, is substantially as shown in FIG. 16.

[0092] In some embodiments, the amorphous form of the sodium salt of the compound of Formula I has a DSC pattern with a peak at 337.04±5.0 °C. In some embodiments, the DSC pattern of the amorphous form of the sodium salt of the compound of Formula I is as shown in FIG. 17.

[0093] In another aspect, the present disclosure provides a crystalline form of the potassium salt of the compound of Formula I.

[0094] In another aspect, the present disclosure provides a crystalline form of the monohydrate of the potassium salt of the compound of Formula I.

[0095] In another aspect, the present disclosure also provides a crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I, which has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, with diffraction peaks at 17.33±0.20°, 20.48±0.20°, 24.07±0.20° and 25.06±0.20°.

[0096] In some embodiments, the X-ray powder diffraction pattern of the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I, in terms of diffraction angles 2θ, has diffraction peaks at 9.06±0.20°, 10.75±0.20°, 15.22±0.20°, 17.33±0.20°, 20.48±0.20°, 22.84±0.20°, 23.20±0.20°, 24.07±0.20°, 25.06±0.20°, 28.03±0.20° and 30.18±0.20°.

[0097] In some embodiments, the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, with diffraction peaks at 9.06±0.20°, 10.75±0.20°, 12.87±0.20°, 15.22±0.20°, 16.33±0.20°, 17.33±0.20°, 19.09±0.20°, 20.48±0.20°, 21.61±0.20°, 22.84±0.20°, 23.20±0.20°, 24.07±0.20°, 25.06±0.20°, 26.01±0.20°, 28.03±0.20°, 28.72±0.20°, 30.18±0.20°, 30.89±0.20°, 33.04±0.20°, 33.80±0.20°, and 37.12±0.20°.

[0098] In some embodiments, the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I has an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, substantially as shown in FIG. 19.

[0099] In some embodiments, the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I has a DSC profile with peaks at 110.89 °C ± 5.0 °C and 336.14 ± 5.0 °C. In some embodiments, the DSC profile of the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I is as shown in FIG. 20.

[0100] In another aspect, the present disclosure also provides a method of preparing the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I, comprising: (1) mixing the compound of Formula I with potassium hydroxide in DMF, (2) adding ethyl acetate, stirring, and separating.

[0101] In some embodiments, in the method of preparing the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I, the molar ratio of the compound of Formula I to potassium hydroxide is 1:(0.5-1.5), 1:(0.9-1.2), or about 1:1.

[0102] In some embodiments, in the method of preparing the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I, the volume (mL) of DMF in step (1) is 2-20 times or 3-10 times the mass (g) of the compound of Formula I.

[0103] In some embodiments, in the method of preparing the crystalline Form G of the monohydrate of the potassium salt of the compound of Formula I, the stirring temperature in step (2) is 20-60 °C or 20-40 °C.

[0104] In still another aspect, the present disclosure also provides an amorphous form of a potassium salt of a compound of Formula I, having an X-ray powder diffraction pattern, in terms of diffraction angles 2θ, substantially as shown in FIG. 22.

[0105] In some embodiments, the amorphous form of a potassium salt of a compound of Formula I has a DSC profile with a peak at 338.12 °C ± 5.0 °C. In some embodiments, the DSC profile of the amorphous form of a potassium salt of a compound of Formula I is as shown in FIG. 23.

[0106] The crystallization methods of each crystalline form in the present disclosure are conventional, such as solvent evaporation crystallization, temperature reduction crystallization or room temperature crystallization.

[0107] Further, the preparation methods of each crystalline form in the present disclosure further comprise steps of filtration, washing or drying, etc.

[0108] In another aspect, the present disclosure provides a pharmaceutical composition comprising a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a potassium salt of a compound of Formula I, or a crystalline form of a solvate of a potassium salt of a compound of Formula I, as described in the present disclosure, and a pharmaceutically acceptable excipient.

[0109] In another aspect, the present disclosure provides a pharmaceutical composition comprising a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, and a pharmaceutically acceptable excipient.

[0110] In another aspect, the present disclosure provides a method for preventing or treating a DNA polymerase theta-mediated disease or disorder in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a potassium salt of a compound of Formula I, or a crystalline form of a solvate of a potassium salt of a compound of Formula I, or a pharmaceutical composition thereof, as described in the present disclosure.

[0111] In another aspect, the present disclosure provides a method for preventing or treating a DNA polymerase theta-mediated disease or disorder in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a pharmaceutical composition thereof, as described in the present disclosure.

[0112] In another aspect, the present disclosure provides use of a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a potassium salt of a compound of Formula I, or a crystalline form of a solvate of a potassium salt of a compound of Formula I, or a pharmaceutical composition thereof described herein in the manufacture of a medicament for preventing or treating a DNA polymerase theta mediated disease or disorder.

[0113] In another aspect, the present disclosure provides use of a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a pharmaceutical composition thereof described herein in the manufacture of a medicament for preventing or treating a DNA polymerase theta mediated disease or disorder.

[0114] In another aspect, the present disclosure provides use of a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a potassium salt of a compound of Formula I, or a crystalline form of a solvate of a potassium salt of a compound of Formula I, or a pharmaceutical composition thereof in the prevention or treatment of a DNA polymerase theta mediated disease or disorder.

[0115] In another aspect, the present disclosure provides use of a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a pharmaceutical composition thereof in the prevention or treatment of a DNA polymerase theta mediated disease or disorder.

[0116] In another aspect, the present disclosure provides a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a potassium salt of a compound of Formula I, or a crystalline form of a solvate of a potassium salt of a compound of Formula I, or a pharmaceutical composition thereof described herein for use in preventing or treating a DNA polymerase theta mediated disease or disorder.

[0117] In another aspect, the present disclosure provides a crystalline form of a compound of Formula I, or a sodium salt of a compound of Formula I, or a crystalline form of a solvate of a sodium salt of a compound of Formula I, or a pharmaceutical composition thereof described herein for use in preventing or treating a DNA polymerase theta mediated disease or disorder.

[0118] In some embodiments, the DNA polymerase theta-mediated disease or disorder is a disease or disorder of DNA polymerase theta overexpression. In some embodiments, the DNA polymerase theta-mediated disease or disorder is a cancer. In some embodiments, the DNA polymerase theta-mediated disease or disorder is a homologous recombination (HR) deficient cancer. In some embodiments, the cancer is a cancer in which BRCA gene expression is reduced or absent, a deficiency in BRCA gene, or a reduction in BRCA protein function. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is a colorectal adenocarcinoma.

[0119] The sodium salt of the compound of Formula I or the solvate of the sodium salt of the compound of Formula I described in the present disclosure, including its crystalline form, has good physical and chemical stability, has advantages in physicochemical properties, formulation processing performance, etc., and is suitable for preparation into a desired pharmaceutical composition.

[0120] Definitions and explanations of terms

[0121] Unless otherwise indicated, the terms used in the present disclosure have the following meanings. The definitions of the groups and terms described in the present disclosure, including the definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in examples, etc., can be combined and integrated with each other arbitrarily. A particular term should not be considered indefinite or unclear without a specific definition, but should be understood according to the ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or active ingredient thereof.

[0122] The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute and one or more molecules of a solvent. Solvates generally have a substantially fixed molar ratio of solute to solvent. This term also includes clathrate compounds, including clathrate compounds with water. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.

[0123] The term "monohydrate" refers to a hydrate in which the molar ratio of water to compound (or salt) is about 1 : 1.

[0124] The term "about" is used in the present disclosure to mean approximately, around, in the region of, in the vicinity of, or the like. When the term "about" is used in conjunction with a range of numbers, the range is modified to allow the upper and lower limits of the stated range to be variable with the value of the modifier. Unless otherwise stated, the term "about" is used in this document to modify the upper and lower limits of a stated value with a ten percent deviation of the stated value.

[0125] The terms "comprise", "comprise" or "comprise" and variations thereof such as "comprises" or "comprising", unless otherwise specified, are to be construed as open-ended, non-exclusive, i.e. "including but not limited to".

[0126] "Optional implementation" or "implementation" mentioned in the present disclosure refers to including the specific reference elements, structures or features related to the implementation described in the implementation in at least one implementation. Therefore, the phrases "optional implementation" or "implementation" appearing in different places in the present disclosure do not necessarily refer to the same implementation. In addition, specific elements, structures or features can be combined in one or more implementations in any appropriate manner.

[0127] The room temperature mentioned in the present disclosure refers to 25±5.0℃.

[0128] The range "m~n" mentioned in the present disclosure represents a shorthand representation of any real number combination between m and n, where m and n are both real numbers. For example, the numerical range "5~10" means that 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 have been listed herein; "1~5" means that 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 have been listed herein, and "1~5" is only a shorthand representation of these numerical combinations.

[0129] The "X-ray powder diffraction pattern or XRPD pattern" mentioned in the present disclosure refers to the X-ray powder diffraction pattern obtained when X-rays are incident on a certain atomic plane with a d lattice plane spacing of a crystal or part of a crystal sample at a grazing angle θ (complementary angle of incidence angle, also known as Bragg angle), which satisfies the Bragg equation, and thus the X-ray powder diffraction pattern is measured.

[0130] For the same crystal form of the same compound, the peak position of the XRPD pattern has overall similarity, and the relative intensity error can be large. It should also be pointed out that in the identification of mixtures, some diffraction lines may be missing due to factors such as content reduction, at this time, it is not necessary to rely on all the diffraction peaks observed in high-purity samples, and even one diffraction peak can be characteristic for a given crystal.

[0131] The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle θ, which is the Bragg angle, and the unit is ° or degree.

[0132] For those skilled in the art, due to factors such as crystal defects, measurement errors, etc., there is often a certain degree of error in the molar ratio of the compounds of the present disclosure to acid / base molecules, compounds to solvent molecules in solvates. Generally speaking, ±10% is within a reasonable error range. There is a certain degree of error variation depending on the context in which it is used, which does not exceed ±10%, preferably ±5%.

[0133] The term "therapeutically effective amount" refers to the amount of a compound of the present disclosure that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays onset of one or more symptoms of the particular disease, condition, or disorder described herein. The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by the skilled practitioner by a consideration of the factors.

[0134] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0135] The term "pharmaceutically acceptable excipient" refers to those excipients that are not biologically or otherwise undesirable, and that do not interfere with the biological activity of the active compound. Suitable excipients are well known to those skilled in the art, e.g., carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0136] The pharmaceutical compositions of the present disclosure can be prepared by combining a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a solvate of a pharmaceutically acceptable salt thereof, with a suitable pharmaceutically acceptable excipient, and formulating the combination into a solid, semi-solid, liquid, or gaseous dosage form, such as tablets, pills, capsules, powders, granules, creams, emulsions, suspensions, suppositories, injections, inhalers, gels, microspheres, aerosols, and the like.

[0137] Typical routes of administering a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

[0138] The pharmaceutical compositions of the present disclosure can be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, emulsifying, lyophilizing processes, and the like, into pharmaceutically acceptable dosage forms.

[0139] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These excipients can make the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates of the pharmaceutically acceptable salts thereof, be formulated into tablets, pills, dragees, sugar-coated tablets, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to a patient.

[0140] Solid oral compositions can be prepared by conventional mixing or compaction techniques. For example, the active compounds can be mixed with a solid excipient, optionally ground, and if necessary, with other suitable excipients, and then processed into granules, which are filled into tablets or sugar-coated tablets. Suitable excipients include, but are not limited to, binding agents, diluents, disintegrating agents, lubricants, flow-inducing agents, sweetening agents or flavoring agents.

[0141] The pharmaceutical composition can also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

[0142] The therapeutically effective amount of the crystalline form of the compound of Formula I, the sodium salt of the compound of Formula I, or the solvate of the sodium salt of the compound of Formula I contained in the pharmaceutical composition of the present disclosure is selected from 0.001 mg / kg to 1000 mg / kg, in the form of a single or separate doses.

[0143] The therapeutically effective amount of the crystalline form of the potassium salt of the compound of Formula I, or the solvate of the potassium salt of the compound of Formula I contained in the pharmaceutical composition of the present disclosure is selected from 0.001 mg / kg to 1000 mg / kg, in the form of a single or separate doses.

[0144] One skilled in the art will recognize that measured data for XRPD peak position and / or intensity for a given crystalline form of the same compound will vary within a margin of error. The 2-theta values in the present disclosure encompass the appropriate margin of error, which is generally indicated by the “±”. For example, a 2-theta value in the present disclosure indicated with a specific angle value ± 0.20° indicates that the specific angle value therein has a margin of error of ± 0.20°, i.e., 5.92 ± 0.20° 2-theta indicates that the 2-theta is within the range of 6.12 to 5.72. Depending on sample preparation techniques, calibration techniques applied to the instrument, human manipulation bias, etc., one skilled in the art recognizes that the appropriate margin of error for XRPD diffraction angles can be ± 0.20°, ± 0.15°, ± 0.10°, ± 0.05° or less, and that peak intensities allow for some variability. The term “substantially the same” or “substantially as shown” when used to describe an XRPD pattern means a pattern that includes at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the diffraction peaks within a standard deviation of ± 0.2° 2-theta.

[0145] One skilled in the art recognizes that measured data for DSC thermograms for a given crystalline form of the same compound will vary within a margin of error. Single peak peak values (expressed in degrees Celsius) allow for an appropriate margin of error. Generally, the margin of error is indicated by the “±”. For the same crystalline form of the same compound, the thermal transition temperature and melting point error is typically within ± 5.0 °C in successive analyses. For example, a peak value of “140.96 ± 5.0” indicates a range of 145.96 to 135.96. Depending on sample preparation techniques, calibration techniques applied to the instrument, human manipulation bias, etc., one skilled in the art recognizes that the appropriate margin of error for single peak peak values can be ± 5.0, ± 4.0, ± 3.0, ± 2.0 or less.

[0146] The salt forms and / or crystalline forms of the present disclosure can also be isotopically-labeled. The present disclosure also includes isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be present in compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35S、 18 F、 123 I、 125 I and 36 Cl, and the like.

[0147] Certain isotopically-labeled compounds of the present disclosure (for example, those 3 H and 14 C) are useful in compound and / or substrate tissue distribution analysis. Tritiated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as 15 O, 13 N, 11 C, and 18 F are useful in positron emission tomography (PET) studies for measurement of substrate occupancy. Isotopically-labeled compounds of the present disclosure can generally be prepared by substituting a readily available isotopically-labeled reagent for a non-isotopically labeled reagent in a procedure similar to those disclosed in the schemes and / or examples below.

[0148] Moreover, substitution with heavier isotopes such as deuterium (i.e., 2 H) can afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence can be preferred in some circumstances. Deuterium substitutions can be partial or total, with partial deuterium substitution referring to replacement of at least one hydrogen by deuterium.

[0149] The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates of the pharmaceutically acceptable salts thereof, can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining the specific embodiments set forth below with other synthetic methods known in the art, and equivalents thereof as appreciated by those skilled in the art, preferred embodiments including but not limited to the examples of the present disclosure.

[0150] The chemical reactions of the specific embodiments of the present disclosure are performed in solvents appropriate to the reagents and materials employed and suitable for the transformations being effected. Typically, the reactions are conducted under an inert atmosphere, such as nitrogen or argon, in solvents such as water, halogenated aliphatic solvents, aromatic hydrocarbons, acyclic or cyclic ethers or mixtures of these. The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates of the pharmaceutically acceptable salts thereof, can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining the specific embodiments set forth below with other synthetic methods known in the art, and equivalents thereof as appreciated by those skilled in the art, preferred embodiments including but not limited to the examples of the present disclosure.

[0151] Test conditions for instruments used in the experiments of the present disclosure:

[0152] 1. X-ray powder diffraction

[0153] Instrument model: Bruker D8 Focus

[0154] X-ray source: Cu Kα

[0155] Kα1 1.54060;

[0156] Wavelength λ 1.54060

[0157] Slit (°): 2.5

[0158] Scan mode: θ / 2θ, scan range: 3-40° (2θ angle)

[0159] Dwell time (sec): 0.12

[0160] Scan step (°2θ): 0.01

[0161] Voltage: 40 kV

[0162] Current: 40 mA

[0163] 2. Differential scanning calorimeter

[0164] Instrument model: Discovery DSC 2500

[0165] Purge gas: nitrogen

[0166] Sample pan: aluminum pan, non-sealed press lid

[0167] Method: linear temperature ramp

[0168] Temperature ramp rate: 10 °C / min

[0169] Temperature range: 30 °C to 400 °C

[0170] 3. Thermogravimetric analyzer

[0171] Instrument model: Discovery TA 55

[0172] Purge gas: nitrogen

[0173] Sample pan: platinum, open

[0174] Method: linear temperature ramp

[0175] Temperature ramp rate: 10 °C / min from start temperature 30 °C to 400 °C

[0176] Temperature range: 30 °C to 400 °C

[0177] 4. Dynamic vapor sorption

[0178] Instrument model: DVS Intrinsic.

[0179] DVS parameters:

[0180] Temperature: 25 °C;

[0181] Equilibrium: dm / dt = 0.002% / min

[0182] RH (%) test step: 10%

[0183] RH (%) test step range: 0%-90%-0%.

[0184] 5. Ion Chromatography

[0185] Instrument model: Thermo Integrion, no. IC001

[0186] Detection: Conductivity (ELSD)

[0187] Separation column: Dionex Ionpac™ CS12 Guard 4 x 250 mm

[0188] Eluent: 20 mM Methanesulfonic acid solution

[0189] Flow rate: 1.0 ml / min

[0190] The present disclosure employs the following abbreviations:

[0191] BRIEF DESCRIPTION OF THE DRAWINGS

[0192] Figure 1 is an XRPD pattern of Form A of the sodium salt of the compound of Formula I;

[0193] Figure 2 is a DSC pattern of Form A of the sodium salt of the compound of Formula I;

[0194] Figure 3 is a TGA pattern of Form A of the sodium salt of the compound of Formula I;

[0195] Figure 4 is an XRPD pattern of Form B of the compound of Formula I;

[0196] Figure 5 is a DSC pattern of Form B of the compound of Formula I;

[0197] Figure 6 is a TGA pattern of Form B of the compound of Formula I;

[0198] Figure 7 is an XRPD pattern of Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I;

[0199] Figure 8 is a DSC pattern of Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I;

[0200] Figure 9 is a TGA pattern of Form C of the monoethanolamine solvate of the sodium salt of the compound of Formula I;

[0201] Figure 10 is an XRPD pattern of Form D of a monohydrate of a sodium salt of a compound of Formula I;

[0202] Figure 11 is a DSC pattern of Form D of a monohydrate of a sodium salt of a compound of Formula I;

[0203] Figure 12 is a TGA pattern of Form D of a monohydrate of a sodium salt of a compound of Formula I;

[0204] Figure 13 is an XRPD pattern of Form E of a sodium salt of a compound of Formula I;

[0205] Figure 14 is a DSC pattern of Form E of a sodium salt of a compound of Formula I;

[0206] Figure 15 is a TGA pattern of Form E of a sodium salt of a compound of Formula I;

[0207] Figure 16 is an XRPD pattern of an amorphous form of a sodium salt of a compound of Formula I;

[0208] Figure 17 is a DSC pattern of an amorphous form of a sodium salt of a compound of Formula I;

[0209] Figure 18 is a TGA pattern of an amorphous form of a sodium salt of a compound of Formula I;

[0210] Figure 19 is an XRPD pattern of Form G of a monohydrate of a potassium salt of a compound of Formula I;

[0211] Figure 20 is a DSC pattern of Form G of a monohydrate of a potassium salt of a compound of Formula I;

[0212] Figure 21 is a TGA pattern of Form G of a monohydrate of a potassium salt of a compound of Formula I;

[0213] Figure 22 is an XRPD pattern of an amorphous form of a potassium salt of a compound of Formula I;

[0214] Figure 23 is a DSC pattern of an amorphous form of a potassium salt of a compound of Formula I;

[0215] Figure 24 is a TGA pattern of an amorphous form of a potassium salt of a compound of Formula I. Examples

[0216] The present disclosure is described in detail below by way of Examples, but it is not meant to be limited by any of the details of the Examples. The present disclosure has been described in detail by specific embodiments, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and used without further purification.

[0217] Unless otherwise stated, the ratio indicated for mixed solvents is the ratio of the volumes mixed.

[0218] Compounds were named by hand or by software, commercially available compounds used the supplier's catalog name.

[0219] The structure of the compounds was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The unit of NMR shift is 10 -6 (ppm). The solvent for NMR determination was deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard was tetramethylsilane (TMS); "IC 50 " refers to the half maximal inhibitory concentration, which refers to the concentration at which the effect of a substance reaches half of its maximum.

[0220] In the following purification by high performance liquid chromatography, unless otherwise specified, the " % " of the amount of acid or base in the mobile phase A refers to the volume fraction, for example " water (0.05 % formic acid) " refers to the volume of formic acid is 0.05 % of the total volume of formic acid and water. B % indicates the proportion of the volume of mobile phase B to the total volume of mobile phase A and mobile phase B when gradient elution, " B % : 50 % - 70 % " indicates that when gradient elution, the proportion of the volume of mobile phase B to the total volume of mobile phase A and mobile phase B changes from 50 % to 70 %.

[0221] Example 1: Synthesis of 4-(5-chloro-2-methoxyphenyl)-N-[6-(4-cyanophenyl)thiazolo[4,5- b]pyrazin-2-yl]-6-methylnicotinamide (compound of formula I)

[0222] Step 1: Synthesis of tert-butyl (6-bromothiazolo[4,5-b]pyrazin-2-yl)carbamate (intermediate 2)

[0223] The starting material 1 (790 mg), triethylamine (691.90 mg), DMAP (41.77 mg) were dissolved in dichloromethane (10 mL), and (Boc)2O (820.78 mg) was added dropwise to the reaction solution, which was stirred at room temperature for 16 h. Subsequently, the reaction solution was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound (1 g). MS m / z (ESI): 331.2 / 333.2 [M+H] + .

[0224] Step 2: Synthesis of tert-butyl (6-(4-cyanophenyl)thiazolo[4,5-b]pyrazin-2-yl)carbamate (intermediate 3)

[0225] ​Intermediate 2 (1 g), 4-cyanophenylboronic acid (887.34 mg), Pd(dppf)Cl2(220.93 mg), potassium phosphate (1.28 g) were added into dioxane (10 mL) and water (2 mL), stirred at 80 °C for 4 h. The reaction was poured into water, extracted with ethyl acetate (10 mL*3), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated to dryness under reduced pressure, the residue was slurried with ethyl acetate, filtered, the filter cake was dried to give the title compound (0.5 g). MS m / z (ESI): 354.0 [M+H] + .

[0226] Step 3: Synthesis of 4-(2-aminothiazolo[4,5-b]pyrazin-6-yl)benzonitrile (Intermediate 4)

[0227] Intermediate 3 (0.48 g) was dissolved in trifluoroacetic acid (2 mL), stirred at room temperature for 1 h. Ethyl acetate was added to the reaction, filtered, the filtrate was concentrated to dryness under reduced pressure to give the title compound (230 mg). MS m / z (ESI): 254.0 [M+H] + .

[0228] Step 4: Synthesis of methyl 4-(5-chloro-2-methoxyphenyl)-6-methylpyridine-3- carboxylate (Intermediate 7)

[0229] Under nitrogen atmosphere, intermediate 6 (1 g) was dissolved in dioxane (20 mL) and water (5 mL), intermediate 5 (995.75 mg), Pd(dppf)Cl2(349.65 mg) and potassium carbonate (1.48 g) were added into the reaction. Then, the reaction was stirred at 90 °C for 2 hours under nitrogen atmosphere. After the reaction was completed, water (50 mL) was added to the reaction, extracted with ethyl acetate (50 mL*3 times), the organic phase was combined and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure to remove the solvent, the residue was purified by silica gel column chromatography (20 g Silica Flash column, gradient 0-50% ethyl acetate / petroleum ether, flow rate 20 mL / min) to give the title compound (870 mg). MS m / z (ESI): 292.1 [M+H] 20g Silica Flash column, gradient 0-50% ethyl acetate / petroleum ether, flow rate 20 mL / min), give the title compound (870 mg). MS m / z (ESI): 292.1 [M+H] + .

[0230] Step 5: Synthesis of 4-(5-chloro-2-methoxyphenyl)-6-methylpyridine-3-carboxylic acid (Intermediate 8)

[0231] Intermediate 7 (870 mg) was added into tetrahydrofuran (8 mL) and water (4 mL), and lithium hydroxide (157.13 mg) was added into the reaction solution, then the reaction solution was stirred at 25 °C for 16 h. After the reaction was completed, the reaction solution was adjusted to pH 3, and the solvent was removed by concentration under reduced pressure. The residue was washed with dichloromethane / methanol mixed solvent (10 / 1, 20 mL), and then filtered. The filtrate was concentrated to dryness under reduced pressure to give the title compound (1.2 g). MS m / z (ESI): 277.9 [M+H] + .

[0232] Step 6: Synthesis of 4-(5-chloro-2-methoxyphenyl)-N-[6-(4-cyanophenyl)thiazolo[4,5- b]pyrazin-2-yl]-6-methylnicotinamide (the compound of Formula I)

[0233] Intermediate 8 (300 mg) was added into N,N-dimethylformamide (5 mL) under a nitrogen atmosphere, and intermediate 4 (273.62 mg), HATU (410.76 mg) and N,N- diisopropylethylamine (279.24 mg) were added into the reaction solution. Then the reaction solution was stirred at 25 °C for 2 h under a nitrogen atmosphere. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 48%-68%, 11 min) to give the title compound (51.96 mg). MS m / z (ESI): 513.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 13.40 (br s, 1H), 9.27 (s, 1H), 8.84 (s, 1H), 8.36 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.3 Hz, 2H), 7.50-7.42 (m, 2H), 7.36 (s, 1H), 7.03-6.99 (m, 1H), 3.52 (s, 3H), 2.59 (s, 3H)

[0234] Example 2: Preparation of crystalline form A of the sodium salt of the compound of Formula I

[0235] The compound of Formula I (9.5 g) was suspended in 190 mL of anhydrous ethanol, and a solution of sodium hydroxide (742 mg) in anhydrous ethanol (190 mL) was added. The reaction solution was stirred at 25 °C for 3 h. The reaction solution was filtered, the filter cake was washed with anhydrous ethanol (40 mL), and the filter cake was collected to give the title compound. The filter cake was dried at 50 °C under vacuum for 12 h to give crystalline form A of the sodium salt of the compound of Formula I. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.96 (s, 1H), 8.28 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.5 Hz, 2H), 7.37 (m, 1H), 7.24 (d, J = 2.7 Hz, 1H), 7.13 (s, 1H), 6.99 (d, J = 8.9 Hz, 1H), 3.52 (s, 3H), 2.53 (s, 3H).

[0236] The crystalline sample of Form A of the sodium salt of the compound of Formula I was prepared by the method of Example 2. The XRPD pattern of the crystalline sample is shown in Figure 1, the DSC pattern is shown in Figure 2, and the TGA pattern is shown in Figure 3. The sodium ion content was determined by ion chromatography to be 4.2%, and the molar ratio of the compound of Formula I to sodium was determined to be 1:1 (the theoretical value is a sodium ion content of 4.3%). The DSC peak is at about 342.84 °C, and the XRPD diffraction peak parameters are shown in the table below.

[0237] Example 3: Form B of the compound of Formula I

[0238] Into a reaction kettle were added intermediate 4 (10.00 g, 1.00 eq.), intermediate 8 (16.45 g, 1.50 eq.), HOBt (8.54 g, 1.60 eq.), DMF (85 ml, 8.5V), DIPEA (10.21 g, 2.00 eq.), and then EDCI (11.35 g, 1.50 eq.), and the temperature was controlled at 25±5°C, and stirring was performed for 15 h, and then a sample was taken for analysis.

[0239] After the reaction was completed, the temperature was lowered to 0-10°C, and stirring was continued for 1-2 h, and then filtration was performed, and the filter cake was rinsed with DMF (1V) and anhydrous ethanol (2V), respectively, to obtain a wet filter cake, which was placed in a vacuum drying oven at 55±5°C for vacuum drying until the weight was constant, to obtain 14.28 g of Form B of the compound of Formula I. 1 H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.96 (s, 1H), 8.28 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.5 Hz, 2H), 7.37 (m, 1H), 7.24 (d, J = 2.7 Hz, 1H), 7.13 (s, 1H), 6.99 (d, J = 8.9 Hz, 1H), 3.52 (s, 3H), 2.53 (s, 3H).

[0240] The crystalline sample of the sodium salt of the compound of formula I monohydrate Form C prepared by the method of Example 4 has an XRPD pattern as shown in Figure 7, a DSC pattern as shown in Figure 8, and a TGA pattern as shown in Figure 9, with a DSC peak at about 340.70 °C, and XRPD peak positions as shown in the table below.

[0241] Example 4: Crystalline Form C of the sodium salt of the compound of formula I monohydrate

[0242] Preparation Method One:

[0243] To a reaction flask was added 3 g of the compound of formula I and 15 mL of DMSO, stirred at room temperature, added a solution of sodium hydroxide (257 mg) in ethanol (9 mL), the reaction solution was clear, heated to 45 °C in a water bath, added 30 mL of ethanol, slowly reduced to room temperature, suction filtered, the filter cake was rinsed with anhydrous ethanol, and dried at 40 °C under vacuum to obtain the crystalline Form C of the sodium salt of the compound of formula I monohydrate (crystalline Form C of the sodium salt of the compound of formula I monohydrate). 1 HNMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.95 (s, 1H), 8.27 (d, J = 8.5 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2H), 7.48 - 7.30 (m, 1H), 7.23 (d, J = 2.7 Hz, 1H), 7.12 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H), 3.51 (s, 3H), 2.53 (s, 3H).

[0244] The crystalline sample of the sodium salt of the compound of formula I monohydrate Form C prepared by the method of Example 4 has an XRPD pattern as shown in Figure 7, a DSC pattern as shown in Figure 8, and a TGA pattern as shown in Figure 9, with a DSC peak at about 340.70 °C, and XRPD peak positions as shown in the table below.

[0245] Preparation Method Two:

[0246] To a reaction flask was added 3 g of the compound of formula I and 15 mL of NMP or DMF or DMA, stirred at room temperature, added a solution of sodium hydroxide (257 mg) in ethanol (9 mL), the reaction solution was clear, heated to 45 °C in a water bath, added 30 mL of ethanol, slowly reduced to 25 °C, suction filtered, the filter cake was rinsed with anhydrous ethanol, and dried at 40 °C under vacuum to obtain the crystalline Form C of the sodium salt of the compound of formula I monohydrate.

[0247] Preparation Method Three:

[0248] Into a reaction bottle was taken 0.5 g of the compound of formula I and 1.8 mL of DMSO, stirred at room temperature, 0.35 g of 20% sodium ethoxide aqueous solution was added, the solution was clear, heated to 45°C in a water bath, 30 mL of ethanol was added, slowly reduced to 25°C, suction filtered, the filter cake was washed with anhydrous ethanol, and dried at 40°C under vacuum to obtain a crystal form C of the monoethanolate of the sodium salt of the compound of formula I.

[0249] Example 5: Crystal form D of the monohydrate of the sodium salt of the compound of formula I

[0250] Into a reaction bottle was taken 10 mg of the amorphous sodium salt of the compound of formula I, 100 uL of toluene with a water content of 0.08 wt% (the solvent can also be methyl tert-butyl ether, isopropyl ether or dichloromethane with a water content of about 0.1 wt%) was added, stirred at 25°C overnight, suction filtered, and the filter cake was dried at room temperature under vacuum to obtain a crystal form D of the monohydrate of the sodium salt of the compound of formula I. 1 H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.95 (s, 1H), 8.28 (d, J = 8.6 Hz, 2H), 7.91 (d, J = 8.6 Hz, 2H), 7.36 (m, 1H), 7.23 (d, J = 2.7 Hz, 1H), 7.12 (s, 1H), 6.98 (d, J = 8.8 Hz, 1H), 3.52 (s, 3H), 2.53 (s, 3H).

[0251] Crystal form D was prepared by the method of Example 5. The XRPD pattern of the crystalline sample is shown in Figure 10, the DSC pattern is shown in Figure 11, and the TGA pattern is shown in Figure 12. The peak of the DSC is at about 338.88°C, and the XRPD diffraction peak parameters of the XRPD diffraction peaks contained are shown in the following table. The sodium ion content determined by ion chromatography was 3.7%, and the molar ratio of the compound of formula I to sodium was 1:1 (the theoretical value is a sodium ion content of 4.2%). Crystal form D has excellent physical stability, and is stable in properties under conditions such as light, high temperature and high humidity, and does not undergo crystal transformation.

[0252] Example 6: Crystal form E of the sodium salt of the compound of formula I

[0253] Into a reaction bottle was taken 10 mg of the amorphous sodium salt of the compound of formula I, 100 uL of toluene with a water content of 0.08 wt% (the solvent can also be methyl tert-butyl ether, isopropyl ether or dichloromethane with a water content of about 0.1 wt%) was added, stirred at 25°C overnight, suction filtered, and the filter cake was dried at room temperature under vacuum to obtain a crystal form D of the monohydrate of the sodium salt of the compound of formula I. 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.98 (s, 1H), 8.28 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.45 - 7.33 (m, 1H), 7.25 (d, J = 2.7 Hz, 1H), 7.14 (s, 1H), 6.99 (d, J = 8.8 Hz, 1H), 3.52 (s, 3H), 2.54 (s, 3H).

[0254] The crystalline sample of Form E of the sodium salt of the compound of Formula I was prepared according to the method of Example 6. The XRPD pattern of the crystalline sample is shown in Figure 13, the DSC pattern is shown in Figure 14, and the TGA pattern is shown in Figure 15. The DSC peak is at about 338.17 °C. The XRPD peaks for Form E are shown in the table below. Form E is less physically stable under high humidity (92.5% RH), accelerated (40 °C, 75% RH) conditions.

[0255] Example 7: Amorphous Form of the Sodium Salt of the Compound of Formula I

[0256] The crystalline sample of Form E of the sodium salt of the compound of Formula I was prepared according to the method of Example 6. The XRPD pattern of the crystalline sample is shown in Figure 13, the DSC pattern is shown in Figure 14, and the TGA pattern is shown in Figure 15. The DSC peak is at about 338.17 °C. The XRPD peaks for Form E are shown in the table below. Form E is less physically stable under high humidity (92.5% RH), accelerated (40 °C, 75% RH) conditions.

[0257] The crystalline sample of Form E of the sodium salt of the compound of Formula I was prepared according to the method of Example 6. The XRPD pattern of the crystalline sample is shown in Figure 13, the DSC pattern is shown in Figure 14, and the TGA pattern is shown in Figure 15. The DSC peak is at about 338.17 °C. The XRPD peaks for Form E are shown in the table below. Form E is less physically stable under high humidity (92.5% RH), accelerated (40 °C, 75% RH) conditions.

[0258] Example 8: Form G of the Potassium Salt of the Compound of Formula I Monohydrate

[0259] The crystalline sample of Form E of the sodium salt of the compound of Formula I was prepared according to the method of Example 6. The XRPD pattern of the crystalline sample is shown in Figure 13, the DSC pattern is shown in Figure 14, and the TGA pattern is shown in Figure 15. The DSC peak is at about 338.17 °C. The XRPD peaks for Form E are shown in the table below. Form E is less physically stable under high humidity (92.5% RH), accelerated (40 °C, 75% RH) conditions.

[0260] The crystalline sample of Form E of the sodium salt of the compound of Formula I was prepared according to the method of Example 6. The XRPD pattern of the crystalline sample is shown in Figure 13, the DSC pattern is shown in Figure 14, and the TGA pattern is shown in Figure 15. The DSC peak is at about 338.17 °C. The XRPD peaks for Form E are shown in the table below. Form E is less physically stable under high humidity (92.5% RH), accelerated (40 °C, 75% RH) conditions.

[0261] Example 9: Amorphous of potassium salt of compound of formula I

[0262] Take 100 mg of compound of formula I, 0.5 ml of N,N'-dimethylformamide and 13 mg of potassium hydroxide into a centrifuge tube, stir at room temperature, dissolve the reaction solution, add 2 ml of ethanol, stir at room temperature, and after a large amount of solid is precipitated, the reaction solution is filtered under suction, and the filter cake is dried at 45°C under vacuum to obtain the amorphous of potassium salt of compound of formula I.

[0263] The amorphous sample of potassium salt of compound of formula I is prepared by the method of Example 9, the XRPD pattern of the crystalline sample is shown in Figure 22, the DSC pattern is shown in Figure 23, and the TGA pattern is shown in Figure 24, and the peak of DSC is near 338.12°C.

[0264] Experimental Example 10: Influencing factors and accelerated experiment

[0265] The sample to be tested is placed in high temperature (60°C), high humidity (92.5%), light (7000 lux) or accelerated (40°C, 75% RH) conditions using a 2 mL screw cap sample bottle, and is placed open; and is placed in light (7000 lux) or accelerated (40°C, 75% RH) conditions with packaging (2 layers of pharmaceutical low density polyethylene bag + 1 layer of polyester / aluminum / polyethylene composite film bag), and the stability of the sample is investigated. The results are shown in the following table.

[0266] Influencing factor experiment results

[0267] Accelerated experiment results

[0268] Experimental Example 11: Solubility experiment

[0269] The thermodynamic solubility of the compound of the present disclosure is determined by the following test method.

[0270] I. Preparation of dissolution medium

[0271] Purified water: laboratory-made

[0272] II. Test procedure

[0273] The thermodynamic solubility of the compound of the present disclosure in water is investigated, 1000 μL of water is measured and placed in a 1 ml transparent glass tube with a plug, an appropriate amount of sample is weighed to saturate the solution, and is shaken to disperse. The sample is placed on a roller mixer and shaken in water for 24 h, and then an appropriate amount of sample is taken, filtered with a microporous filter membrane, and the content is determined by high performance liquid chromatography using an external standard method, and the thermodynamic solubility of the compound in water (μg / ml) is calculated. The solubility results are shown in the following table.

[0274] Experimental Example 12: Hygroscopicity Experiment

[0275] (1) Experimental instrument: Dynamic water adsorption instrument DVS Intrinsic;

[0276] (2) Experimental conditions: The sample to be tested was placed in the DVS sample disc for testing.

[0277] (3) DVS parameters:

[0278] Temperature: 25°C;

[0279] Equilibrium: dm / dt = 0.002 / min

[0280] RH (%) test steps: 10%

[0281] RH (%) test step range: 0%-90%-0%.

[0282] Experimental results:

[0283] Crystal Form A under 80.0% RH conditions, the moisture absorption weight gain was 1.6%.

[0284] Crystal Form B under 80.0% RH conditions, the moisture absorption weight gain was 1.5%.

[0285] Crystal Form C under 80.0% RH conditions, the moisture absorption weight gain was -4.3%, and solvent desorption was found in the cycle.

[0286] Crystal Form E under 80.0% RH conditions, the moisture absorption weight gain was 2.5%.

[0287] Biological activity and related property test examples

[0288] The compounds in the following test examples were prepared according to the method of the above-mentioned examples of the present disclosure.

[0289] Test Example 1: POLQ enzyme activity inhibition experiment

[0290] Brief introduction of experimental principle: After the N-terminal active peptide segment (M1-N899) with ATPase activity of POLQ is incubated with the compound, it reacts with the substrate dT50 under the action of ATP to generate ADP, participates in the subsequent NADH oxidation coupling enzyme reaction, and catalyzes the NADH reaction to generate NAD + . The reduction of OD value of NADH at 340 nm is measured using the Envision enzyme label instrument of Perkin Elmer company, thereby reflecting the enzyme activity.

[0291] Experimental instruments: Labcyte Echo 650 pipetting system; Perkin Elmer Envision microplate reader; Eppendorf 5810R centrifuge; Boxun BSD-YX3400 constant temperature shaker.

[0292] Experimental materials:

[0293] Experimental Methods: POLQ enzyme was diluted to 100 nM with reaction buffer (20 mM Tris HCl (pH 7.80), 80 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.01% w / v bovine serum albumin, 0.01% v / v Tween-20, 5% v / v glycerol). The test compounds were diluted to different concentrations in dimethyl sulfoxide (DMSO) using an Echo 650 pipette system and transferred to 384-well plates. 20 μL / well of 100 nM POLQ was added, and the plates were incubated at room temperature for 15 minutes. The reaction mixture was prepared with the following concentrations: 100 μM ATP, 300 nM dT50, 300 μM NADH, 6 mM PEP, 10 U / mL lactate dehydrogenase, and 20 U / mL pyruvate kinase. 20 μL / well of the reaction mixture was added to initiate the enzyme reaction. The final concentration of the compound in the reaction system started at 10 μM and was serially diluted 3-fold, ranging from 10 μM to 0.0005 μM. The final concentration of DMSO in the system was 0.2% v / v. After the 384-well plate was incubated at room temperature for 20 minutes, the OD value at 340 nm was read using an Envision microplate reader.

[0294] Data Analysis:

[0295] The inhibition rate was calculated, and the IC50 of the compound was obtained by fitting the data using XLfit software. 50 .

[0296] The experiment included a blank group and a DMSO group. The blank group consisted of 0.2% v / v DMSO and a reaction mixture solution, and the inhibition rate was considered to be 100%. The DMSO group consisted of 0.2% v / v DMSO, POLQ(N) (100 nM) and a reaction mixture solution, and the inhibition rate was considered to be 0%.

[0297] Inhibition rate = (100 - 100 * (OD)) max -OD 化合物 ) / (OD max -OD min ))%

[0298] Among them, OD max OD value refers to the pore size of a pore containing a reactant mixture and 0.2% v / v DMSO.化合物 OD value of the well containing the compound, enzyme and reaction mixture. min OD value of the well containing the enzyme, reaction mixture and 0.2% v / v DMSO.

[0299] The biological activity of the compounds of the present disclosure was determined by the above test, and the IC 50 values are shown in Table 1 below.

[0300] Table 1 IC50values of the example compounds for inhibition of POLQ enzyme activity 50

[0301] In the above table, the meaning of the symbols used to indicate the inhibitory activity is as follows:

[0302] “++++” indicates that the test compound has an inhibitory activity IC 50 in the range of: IC 50 <100 nM.

[0303] “+++” indicates that the test compound has an inhibitory activity IC 50 in the range of: 100 ≤ IC 50 <500 nM.

[0304] “++” indicates that the test compound has an inhibitory activity IC 50 in the range of: 500 ≤ IC 50 <1000 nM.

[0305] Test Example 2: Compound inhibition of tumor cell proliferation experiment

[0306] Brief introduction of the experimental principle: after incubating the compound with tumor cells for 7 days, the ATP in the living cells is quantified using the CTG kit of Promega Company, thereby reflecting the effect of the compound on tumor cell proliferation.

[0307] Experimental instruments: Perkin Elmer Envision enzyme label instrument; Eppendorf 5810R centrifuge, Countstar automatic cell counter.

[0308] Experimental materials:

[0309] Experimental method: DLD-1 parental cells or DLD-1 BRCA2(- / -) cells were diluted with RPMI 1640 medium containing 10% FBS and added to 96-well plates (90 μL / well), with cell numbers of 600 cells / well or 1200 cells / well, respectively, and incubated in a 37°C, 5% CO2 incubator overnight. The test compounds were diluted to different concentrations in dimethyl sulfoxide (DMSO) and added to the 96-well plates, with the final concentration of the compounds in the reaction system starting from 25 μM, 4-fold gradient dilution, the concentration range of the compounds being 25 μM to 0.0004 μM, and the final concentration of DMSO being 0.25% v / v. After incubation for 7 days, 50 μL / well of CTG was added, incubated at room temperature for 10 minutes, and then the light signal value (Lum) was read using an Envision enzyme label instrument, and the inhibition rate and half-inhibitory concentration (IC 50 ) were calculated.

[0310] Data analysis:

[0311] The inhibition rate was calculated, and the IC 50 of the compound was fitted using XLfit software.

[0312] Blank wells and DMSO wells were set up in the experiment, the blank wells being 100 μL of RPMI Medium 1640 medium containing 10% FBS, and the inhibition rate of the compound on tumor cell growth at this time being considered to be 100%; the DMSO wells being wells in which 0.25% v / v DMSO was added to the cell wells, and the inhibition rate of the compound on tumor cell growth at this time being considered to be 0.

[0313] Inhibition rate = 100*(Lum max -Lum 化合物 ) / (Lum max -Lum min )%

[0314] Wherein, Lum max refers to the light signal value of the well containing cells and 0.25% v / v DMSO, Lum 化合物 refers to the light signal value of the well containing the compound and the cells. Lum min refers to the light signal value of the well containing the medium and 0.25% v / v DMSO.

[0315] The growth inhibition of the compounds of the present disclosure on tumor cells was determined by the above experiment, and the IC 50 value was measured.

[0316] Table 2 IC 50 of the example compounds of the present disclosure on tumor cell growth inhibition

[0317] In the above table, the meaning indicated by the symbols for indicating the inhibition activity is as follows:

[0318] “++++” indicates that the tested compound has an IC50 of 0-100 nM for cell inhibition activity. 50 The range of IC50 is: 0-200 nM. 50 <200 nM.

[0319] “+++” indicates that the tested compound has an IC50 of 200-500 nM for cell inhibition activity. 50 The range of IC50 is: 200-500 nM. 50 <500 nM.

[0320] “++” indicates that the tested compound has an IC50 of 500-1000 nM for cell inhibition activity. 50 The range of IC50 is: 500-1000 nM. 50 <1000 nM.

[0321] “+” indicates that the tested compound has an IC50 of 1000-10000 nM for cell inhibition activity. 50 The range of IC50 is: 1000-10000 nM. 50 <10000 nM.

[0322] “-” indicates that the tested compound has an IC50 of >10000 nM for cell inhibition activity. 50 The range of IC50 is: >10000 nM. 50

[0323] It is found that the compound of the present disclosure has a good inhibitory effect on tumor cells with BRCA2 mutation, and has good selectivity.

[0324] Test Example 3: Compound Inhibition Experiment on Cell MMEJ Pathway

[0325] Brief introduction of experimental principle: POLQ is a key protein in the MMEJ repair process of cells. The NanoLuciferase MMEJ repair reporter system is transferred into HEK293T cells, and when the MMEJ repair pathway in the cells is normally carried out, the NanoLuciferase reporter protein is correctly expressed, and the cell luminescence signal can be detected. The reduction of cell luminescence is measured using the BMG multifunctional enzyme label instrument of BMG LABTECH company, so as to reflect the inhibition of the compound on the cell MMEJ pathway.

[0326] Experimental instruments: Incucyte live cell imaging system of ESCD company, Echo 655 pipetting system of Labcyte company, BMG multifunctional enzyme label instrument of BMG LABTECH company, Neon transfection system of Invitrogen company.

[0327] Experimental materials:

[0328] ​Experimental method: The test compound was diluted in dimethyl sulfoxide (DMSO) to different concentrations by using Echo 655 pipetting system, and then transferred to a 384-well plate, so that the final concentration of the compound in the reaction system was 10 μM, with 3-fold gradient dilution, and the final concentration of DMSO was 0.1%. HEK293T cells were collected, and MMEJ luciferase substrate was transferred into the cells by using Neon transfection system, and then 4000 cells per well of transfected HEK293T cells were diluted in DMEM medium containing 10% FBS and added to a 384-well plate (25 μL / well). After incubation of the compound and cells in a 37°C, 5% CO2 incubator for 24 hours, 40 μL / well of NanoGlo substarte buffer was added to measure the growth inhibition of the compound on tumor cells, and the inhibition rate and half inhibitory concentration (IC 50 ) of the compound were calculated.

[0329] Data analysis:

[0330] The compound inhibition rate was calculated, and the IC 50 of the compound was fitted by using XLfit software.

[0331] Blank wells and DMSO wells were set up, 10 μM of positive compound ART558 (doi: 10.1038 / s41467-021-23463-8) was added to the blank wells, and it was considered that the compound inhibition rate at this time was 100%; 0.1% DMSO was added to the DMSO wells, and it was considered that the compound inhibition rate at this time was 0.

[0332] Compound inhibition rate (%) = (100 * (DMSO well - test compound well) / (DMSO well - blank well))%

[0333] It was found that the compound of formula I of the present disclosure had strong inhibitory activity on the POLQ-mediated MMEJ pathway in cells, and it was expected that the compound could effectively inhibit the target POLQ and related pathway MMEJ in tumors, thereby exerting corresponding pharmacological effects.

[0334] Test example 4: determination of metabolic stability of the compound in liver cells

[0335] The metabolic stability of the compound of the present disclosure in liver cells was determined by using the following test method.

[0336] I. Test materials and instruments

[0337] 1. Caucasian human hepatocytes (Biopredic BQHPCH10), cynomolgus monkey hepatocytes (RILD HP-SXH-02M), beagle dog hepatocytes (BioIVT M00205), SD rat hepatocytes (BioIVT M00005) and CD-1 mouse hepatocytes (BioIVT M00505)

[0338] 2. AOPI stain (Nexcelom 200710-01-01)

[0339] 3. Dexamethasone (NIFDC 100129-201506)

[0340] 4. DPBS (10x) (Gibco by Life Technologies 2060570)

[0341] 5. Fetal bovine serum (FBS) (Corning 35081001)

[0342] 6. GlutaMAX™-1 (100x) (Gibco by Life Technologies 2186980)

[0343] 7. HEPES (Sigma RNBJ1276)

[0344] 8. Human recombinant insulin (Gibco by Life Technologies 2090407)

[0345] 9. Isotonic Percoll (GE Healthcare 10288259)

[0346] 10. Verapamil (Sigma MKBV4993V)

[0347] 11. Williams’ Medium E (Sigma RNBJ3314)

[0348] 12. AB Sciex API4000 LC-MS-MS

[0349] II. Experimental Procedures

[0350] 1. Prepare hepatocyte resuscitation medium according to the information in the table below. Mix 49.5 mL Williams' Medium E and 0.5 mL GlutaMAX™-1 (100×) as incubation medium. Preheat the hepatocyte resuscitation medium and incubation medium in a 37°C water bath for at least 15 minutes before use. Take a tube of cryopreserved hepatocytes, ensuring that the hepatocytes are still frozen before resuscitation. Quickly place the hepatocytes in a 37°C water bath and gently shake until all ice crystals are dispersed. Spray with 70% ethanol and transfer to a biosafety cabinet. Pour the contents of the hepatocyte tube into a centrifuge tube containing 50 mL of resuscitation medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the resuscitation medium and add sufficient incubation medium to obtain a cell density of approximately 1.5 × 10⁻⁶ cells / mL. 6 A cell suspension of [number] cells / mL was prepared. Hepatocytes were counted and viable cell density was determined using Cellometer Vision; hepatocyte viability must be greater than 75%. The hepatocyte suspension was diluted with incubation medium to a viable cell density of 0.5 × 10⁶ cells / mL. 6 live cells / mL.

[0351] 2. Transfer 247.5 μL of live cell suspension or culture medium to a 96-well deep-well plate and preheat the plate in a vortex incubator for 10 minutes. All samples were incubated in duplicate. Add 2.5 μL of 100 μM analyte or control drug verapamil to each well to start the reaction, and place the deep-well plate back on the vortex incubator. Take 25 μL of incubated sample at 0, 15, 30, 60, 90, and 120 minutes, and add 125 μL of acetonitrile containing internal standard to terminate the reaction. Vortex for 10 minutes, centrifuge at 3220 g, 4 °C for 30 minutes, and after centrifugation, transfer 100 μL of supernatant to the sample plate, add 150 μL of pure water and mix well for LC-MS / MS analysis.

[0352] All data were calculated using Microsoft Excel software. Peak areas were detected by extracting ion spectra, and the in vitro half-life (t0.05) of the compound was determined by linearly fitting the natural logarithm of the elimination percentage of the compound against time. 1 / 2 ).

[0353] in vitro half-life (t) 1 / 2 ) Calculated by slope:

[0354] in vitro t 1 / 2 =0.693 / k

[0355] In vitro intrinsic clearance rate (unit: μL / min / mg protein) is calculated using the following formula:

[0356] in vitro CL int = k x volume of incubation (mL) / amount of proteins (mg)

[0357] CL int = k x volume of incubation (mL) / amount of proteins (mg)

[0358] It is tested that the compound of formula I of the present disclosure is metabolically stable in various species of hepatocytes, and is expected to be relatively stable in vivo in liver metabolism, and is relatively less affected by the liver first-pass effect.

[0359] The above describes the embodiments of the present disclosure. However, the present disclosure is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A sodium salt of a compound of formula I having the following structure, 2. The sodium salt according to claim 1, wherein, The molar ratio of the compound of Formula I to sodium ions is approximately 1:1; or, the molar ratio of the compound of Formula I to sodium ions is 1:

1.

3. The sodium salt according to any one of claims 1-2, wherein, The sodium salt is in an amorphous or crystalline form; or, the sodium salt is in a crystalline form; or the sodium salt is amorphous.

4. The sodium salt according to claim 3, wherein, The sodium salt is crystal form A of the sodium salt of compound of formula I, and the X-ray powder diffraction pattern of crystal form A, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 6.39±0.20°, 7.84±0.20°, 13.61±0.20°, and 25.46±0.20°; or, The X-ray powder diffraction pattern of crystal form A, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 6.39±0.20°, 7.84±0.20°, 13.61±0.20°, 15.95±0.20°, 17.16±0.20°, 20.90±0.20°, 21.81±0.20°, 25.46±0.20°, and 30.79±0.20°; or, The X-ray powder diffraction pattern of crystal form A, expressed as a diffraction angle 2θ, is at 6.13±0.20°, 6.39±0.20°, 6.87±0.20°, 7.84±0.20°, 10.80±0.20°, 13.61±0.20°, 14.10±0.20°, 15.95±0.20°, and 16.44±0.20°. Diffraction peaks are observed at 17.16±0.20°, 17.74±0.20°, 19.09±0.20°, 20.90±0.20°, 21.81±0.20°, 22.01±0.20°, 22.73±0.20°, 22.93±0.20°, 25.46±0.20°, and 30.79±0.20°; or The X-ray powder diffraction pattern of crystal form A, expressed in terms of diffraction angle 2θ, is basically as shown in Figure 1.

5. The sodium salt according to claim 4, wherein, The crystal form A has a DSC spectrum with a peak at 342.84±5.0℃.

6. The sodium salt according to claim 3, wherein, The sodium salt is crystal form E of the sodium salt of the compound of formula I, and the X-ray powder diffraction pattern of crystal form E, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 20.40±0.20°, 23.54±0.20°, 24.10±0.20°, and 28.67±0.20°; or The X-ray powder diffraction pattern of crystal form E, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 10.09±0.20°, 17.53±0.20°, 20.40±0.20°, 22.84±0.20°, 23.54±0.20°, 24.10±0.20°, 28.67±0.20°, and 30.58±0.20°; or The X-ray powder diffraction pattern of crystal form E, expressed as a diffraction angle 2θ, is at 7.95±0.20°, 10.09±0.20°, 11.60±0.20°, 13.93±0.20°, 14.98±0.20°, 16.90±0.20°, 17.53±0.20°, 19.14±0.20°, and 19.76±0.20°. Diffraction peaks are observed at 0°, 20.40±0.20°, 21.55±0.20°, 22.84±0.20°, 23.54±0.20°, 24.10±0.20°, 25.47±0.20°, 25.97±0.20°, 27.27±0.20°, 28.67±0.20°, and 30.58±0.20°; or The X-ray powder diffraction pattern of crystal form E, expressed in terms of diffraction angle 2θ, is basically as shown in Figure 13.

7. The sodium salt according to claim 6, wherein, The crystal form E has a DSC spectrum with a peak at 338.17±5.0℃.

8. The sodium salt according to claim 3, wherein, The sodium salt is an amorphous form of the sodium salt of the compound of formula I; the X-ray powder diffraction pattern of the amorphous form, expressed in terms of diffraction angle 2θ, is essentially as shown in Figure 16.

9. The sodium salt according to claim 8, wherein, The amorphous material has a DSC spectrum with a peak at 337.04℃ ± 5.0℃.

10. The sodium salt according to any one of claims 1-2, wherein, The sodium salt is a solvate of the sodium salt of the compound of formula I; or the sodium salt is a hydrate or ethanolic compound of the sodium salt of the compound of formula I.

11. The sodium salt according to claim 10, wherein, The molar ratio of the sodium salt of the compound of formula I in the solvate to the solvent is approximately 1:1; or, the molar ratio of the sodium salt of the compound of formula I in the solvate to the solvent is 1:

1.

12. The sodium salt according to any one of claims 10-11, wherein, The solvate is a monohydrate of the sodium salt of the compound of formula I; or, the solvate is crystal form D of the monohydrate of the sodium salt of the compound of formula I, wherein the X-ray powder diffraction pattern of crystal form D, expressed in terms of diffraction angle 2θ, has diffraction peaks at 8.28±0.20°, 10.08±0.20°, 20.05±0.20°, and 23.47±0.20°; or, the X-ray powder diffraction pattern of crystal form D, expressed in terms of diffraction angle 2θ, has diffraction peaks at 8.28±0.20°, 10.08±0.20°, 10.08±0.20°, 20.05±0.20°, and 23.47±0.20°. Diffraction peaks are observed at ±0.20°, 14.46±0.20°, 16.79±0.20°, 20.05±0.20°, 22.07±0.20°, 22.89±0.20°, and 23.47±0.20°; or, the X-ray powder diffraction pattern of crystal form D, expressed in terms of diffraction angle 2θ, shows peaks at 8.28±0.20°, 10.08±0.20°, 14.46±0.20°, 16.67±0.20°, 16.79±0.20°, and 17.28±0.20°. The diffraction peaks are observed at 0.20°, 20.05±0.20°, 22.07±0.20°, 22.89±0.20°, 23.47±0.20°, and 26.19±0.20°; or, the X-ray powder diffraction pattern of the crystal form D, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 8.28±0.20°, 10.08±0.20°, 12.30±0.20°, 14.01±0.20°, 14.46±0.20°, 16.67±0.20°, and 16.79±0.20°. Diffraction peaks are observed at 20°, 17.17±0.20°, 17.28±0.20°, 17.45±0.20°, 18.15±0.20°, 20.05±0.20°, 22.07±0.20°, 22.42±0.20°, 22.89±0.20°, 23.47±0.20°, 26.19±0.20°, and 26.46±0.20°; or, the X-ray powder diffraction pattern of the crystal form D, expressed in terms of diffraction angle 2θ, is essentially as shown in Figure 10.

13. The sodium salt according to claim 12, wherein, The crystal form D has a DSC spectrum with a peak at 338.88±5.0℃.

14. The sodium salt according to any one of claims 10-11, wherein, The solvate is a monoethanolate of the sodium salt of the compound of formula I; or, the solvate is crystal form C of the monoethanolate of the sodium salt of the compound of formula I, wherein the X-ray powder diffraction pattern of crystal form C, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 17.14±0.20°, 21.85±0.20°, 25.46±0.20°, and 25.74±0.20°; or, The X-ray powder diffraction pattern of crystal form C, expressed as a diffraction angle 2θ, is at 12.37±0.20°, 17.14±0.20°, 20.07±0.20°, 21.40±0.20°, 21.85±0.20°, 23.39±0.20°, 24.51±0.20°, 24.99±0.20°, 25.46±0.20°, and 25.7°. A diffraction peak is observed at 4±0.20°; or, the X-ray powder diffraction pattern of crystal form C, expressed as a diffraction angle 2θ, shows peaks at 12.37±0.20°, 12.70±0.20°, 15.47±0.20°, 17.14±0.20°, 17.96±0.20°, 19.76±0.20°, 20.07±0.20°, and 20.30±0.20°. Diffraction peaks are observed at 21.40±0.20°, 21.85±0.20°, 22.29±0.20°, 23.39±0.20°, 24.51±0.20°, 24.99±0.20°, 25.46±0.20°, and 25.74±0.20°; or, the X-ray powder diffraction pattern of crystal form C, expressed in terms of diffraction angle 2θ, is essentially as shown in Figure 7.

15. The sodium salt according to claim 14, wherein, The crystal form C has a DSC spectrum with a peak at 340.70±5.0℃.

16. A method for preparing crystal form A of the sodium salt of the compound of formula I according to claim 4, comprising: (1) Mix the compound of Formula I with anhydrous ethanol, and (2) add a mixed solution of sodium hydroxide and anhydrous ethanol, stir, and separate.

17. The method according to claim 16, wherein, The molar ratio of the compound of formula I to sodium hydroxide is 1:(0.5-1.5), 1:(0.9-1.2), or about 1:1; and / or, in step (1), the volume (mL) of anhydrous ethanol is 20 to 45 times or 35 to 45 times the mass (g) of the compound of formula I; and / or, in step (2), stirring is carried out at a temperature of 20-60°C or 20-30°C.

18. A method for preparing crystal form D of the monohydrate of the sodium salt of the compound of formula I according to claim 12, comprising: The amorphous form of the sodium salt of the compound of formula I is mixed with a mixed solvent of water and toluene, stirred, and the solid is separated.

19. The method according to claim 18, wherein, The volume (µL) of the mixed solvent of water and toluene is 1-30 times or 5-15 times the mass (mg) of the amorphous sodium salt of the compound of formula I, and / or, the mixture is stirred at a temperature of 0-60°C or 15-30°C.

20. Crystallization of compound I:

21. The crystallization of the compound of formula I according to claim 20, wherein, The crystal is crystal form B of the compound of formula I, and the X-ray powder diffraction pattern of crystal form B, expressed in terms of diffraction angle 2θ, has diffraction peaks at 12.10±0.20°, 17.79±0.20°, 20.13±0.20°, and 25.47±0.20°; or, the X-ray powder diffraction pattern of crystal form B, expressed in terms of diffraction angle 2θ, has diffraction peaks at 7.98±0.20°, 12.10±0.20°, 14.59±0.20°, 17.80±0.20°, 20.13±0.20°, 22.51±0.20°, 23.61±0.20°, 25.47±0.20°, 26.74±0.20°, and 27.66±0.20°; or, the crystal... The X-ray powder diffraction pattern of type B, expressed in terms of diffraction angle 2θ, shows diffraction peaks at 7.98±0.20°, 12.10±0.20°, 14.59±0.20°, 15.97±0.20°, 17.31±0.20°, 17.79±0.20°, 18.23±0.20°, 20.13±0.20°, 22.51±0.20°, 23.31±0.20°, 23.61±0.20°, 23.98±0.20°, 24.56±0.20°, 25.47±0.20°, 26.74±0.20°, and 27.66±0.20°; or, the X-ray powder diffraction pattern of crystal type B, expressed in terms of diffraction angle 2θ, is essentially as shown in Figure 4.

22. The crystallization of the compound of formula I according to claim 21, wherein, The crystal form B has a DSC spectrum with a peak at 343.98±5.0℃.

23. Potassium salts of compounds of formula I having the following structures, 24. The potassium salt according to claim 23, wherein, The molar ratio of the compound of Formula I to potassium ions is approximately 1:1; or, the molar ratio of the compound of Formula I to potassium ions is 1:

1.

25. The potassium salt according to any one of claims 23-24, wherein, The potassium salt is in amorphous or crystalline form; or, the potassium salt is in crystalline form.

26. The potassium salt according to any one of claims 23-25, wherein, The potassium salt is a solvate of the potassium salt of the compound of formula I; or, the potassium salt is a hydrate or ethanolic compound of the potassium salt of the compound of formula I.

27. The potassium salt according to claim 26, wherein, In the solvate of the potassium salt of the compound of formula I, the molar ratio of the potassium salt of the compound of formula I to the solvent is approximately 1:1; or, the molar ratio of the potassium salt of the compound of formula I to the solvent is 1:

1.

28. The potassium salt according to claim 27, wherein, The solvate is a monohydrate of the potassium salt of the compound of formula I; or, the solvate is crystal form G of the monohydrate of the potassium salt of the compound of formula I, wherein the X-ray powder diffraction pattern of crystal form G, expressed in terms of diffraction angle 2θ, has diffraction peaks at 17.33±0.20°, 20.48±0.20°, 24.07±0.20°, and 25.06±0.20°; or, the X-ray powder diffraction pattern of crystal form G, expressed in terms of diffraction angle 2θ, has diffraction peaks at 17.33±0.20°, 20.48±0.20°, 24.07±0.20°, and 25.06±0.20°. The final diffraction pattern shows diffraction peaks at 9.06±0.20°, 10.75±0.20°, 15.22±0.20°, 17.33±0.20°, 20.48±0.20°, 22.84±0.20°, 23.20±0.20°, 24.07±0.20°, 25.06±0.20°, 28.03±0.20°, and 30.18±0.20°; or, the crystal form G shows diffraction peaks at diffraction angles... The X-ray powder diffraction patterns represented by 2θ are at 9.06±0.20°, 10.75±0.20°, 12.87±0.20°, 15.22±0.20°, 16.33±0.20°, 17.33±0.20°, 19.09±0.20°, 20.48±0.20°, 21.61±0.20°, 22.84±0.20°, 23.20±0.20°, and 24.07±0.20°. Diffraction peaks are observed at 20°, 25.06±0.20°, 26.01±0.20°, 28.03±0.20°, 28.72±0.20°, 30.18±0.20°, 30.89±0.20°, 33.04±0.20°, 33.80±0.20°, and 37.12±0.20°; or, the X-ray powder diffraction pattern of the crystal form G, expressed in terms of diffraction angle 2θ, is essentially as shown in Figure 19.

29. The potassium salt according to claim 28, wherein, The crystal form G has a DSC spectrum with peaks at 110.89℃±5.0℃ and 336.14±5.0℃.

30. The potassium salt according to claim 25, wherein, The potassium salt is an amorphous form of the potassium salt of the compound of formula I; the X-ray powder diffraction pattern of the amorphous form, expressed in terms of diffraction angle 2θ, is essentially as shown in Figure 22.

31. The potassium salt according to claim 30, wherein, The amorphous material has a DSC spectrum with a peak at 338.12℃ ± 5.0℃.

32. A pharmaceutical composition comprising a sodium salt of any one of claims 1-15, a crystal of a compound of formula I of any one of claims 20-22, or a potassium salt of any one of claims 23-31, and a pharmaceutically acceptable excipient.

33. A method for preventing or treating a disease mediated by DNA polymerase θ in mammals, comprising administering to a mammal in need, preferably a human, a therapeutically effective amount of the sodium salt of any one of claims 1-15, a crystal of the compound of formula I of any one of claims 20-22, or a potassium salt of any one of claims 23-31, or the pharmaceutical composition of claim 32.

34. Use of the sodium salt of any one of claims 1-15, the crystallization of the compound of formula I of any one of claims 20-22, or the potassium salt of any one of claims 23-31, or the pharmaceutical composition of claim 32 in the preparation of a medicament for the prevention or treatment of DNA polymerase θ-mediated diseases.

35. Use of the sodium salt of any one of claims 1-15, the crystallization of the compound of formula I of any one of claims 20-22, or the potassium salt of any one of claims 23-31, or the pharmaceutical composition of claim 32 in the prevention or treatment of DNA polymerase θ-mediated diseases or conditions.

36. A sodium salt of any one of claims 1-15, a crystal of the compound of formula I of any one of claims 20-22, or a potassium salt of any one of claims 23-31, or a pharmaceutical composition of claim 32, for the prevention or treatment of DNA polymerase θ-mediated diseases or conditions.

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