Salt or crystal of thiazolopyrazine compound and application of salt or crystal

Abstract

Provided are a pharmaceutically acceptable salt of a thiazolopyrazine 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 the hydrate) of the pharmaceutically acceptable salt, a crystal form of the compound represented by formula (I), and preparation methods thereof, pharmaceutical compositions thereof and their medical uses.

Description

Salt of thiazolopyrazine compound or its crystal 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 with the State Intellectual Property Office of China on July 12, 2023. The entire text of the above patent application is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of pharmaceutical chemistry, and specifically to crystalline forms of thiazolopyrazine compounds, pharmaceutically acceptable salts of thiazolopyrazine compounds or solvates (including hydrates) of pharmaceutically acceptable salts, crystalline forms of the pharmaceutically acceptable salts or solvates (including hydrates) of pharmaceutically acceptable salts, and methods for preparing the same, pharmaceutical compositions containing the same, and uses thereof. Background Art

[0004] DNA double-strand break repair is crucial for maintaining genomic stability and cell survival. There are three main repair pathways for DNA double-strand breaks: homologous recombination (HR), non-homologous end joining (NHEJ), and alternative non-homologous end joining (alt-NHEJ). Microhomology-mediated end joining (MMEJ) is the most common alternative non-homologous end joining. Homologous recombination is a high-fidelity, accurate repair mechanism that maintains genomic stability and avoids inducing cancer. However, non-homologous end joining and microhomology-mediated end joining are error-prone repair pathways that can lead to 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, defects in DNA double-strand break repair can be used to develop targeted tumor treatments. Tumor cells with impaired homologous recombination or non-homologous end-joining repair are more dependent on microhomology-mediated end-joining repair. Multiple lines of evidence from genetics, cell biology, and biochemistry indicate that DNA polymerase θ (POLQ or POLθ) is a key protein in the microhomology-mediated end-joining repair process (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 composed 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 the RPA protein from single-stranded DNA and promotes annealing, while the polymerase domain can extend the ends of single-stranded DNA and fill the gaps. These two domains work together to function in the microhomology-mediated end-joining repair process.

[0007] Studies have shown that POLQ is essential for cells with homologous recombination defects (e.g., synthetic lethality with FA / BRCA defects), and that POLQ protein levels are upregulated in homologous recombination-deficient tumor cells (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 ovarian, uterine, and breast cancers with poor prognosis (Higgins et al. Oncotarget (2010), 1, 175-184, Lemee et al. PNAS (2010), 107(30), 13390-13395, Ceccaldi et al. (2015), supra). More importantly, POLQ expression is suppressed in normal tissues compared with tumor tissues (Kawamura et al. International Journal of Cancer (2004), 109(1), 9-16).

[0008] In summary, POLQ is crucial for cells with homologous recombination defects, and there is currently an unmet need for the treatment of homologous recombination-deficient tumors. Inhibiting POLQ function can inhibit microhomology-mediated end-joining repair in cells. The development of POLQ inhibitors may provide a novel strategy for the targeted treatment of homologous recombination-deficient tumors.

[0009] Summary of the Invention

[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-methylnicotinamide) 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, in the pharmaceutically acceptable salt of the compound of Formula I, the molar ratio of the compound of Formula I to sodium ion or potassium ion is about 1:1.

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

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

[0016] In some embodiments, the present disclosure provides a potassium salt of a 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 a 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 salts of the compounds of Formula I may exist in unsolvated or solvated forms.

[0019] In another aspect, the present disclosure also provides solid forms of pharmaceutically acceptable salts of the compounds 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 for preparing the sodium salt of the compound of formula I, comprising the step of forming a salt from the compound of formula I and a sodium-containing base.

[0026] In some embodiments, the method for preparing the sodium salt of the compound of formula I comprises reacting the compound of formula I with a sodium-containing base in a solvent to form a salt, wherein the solvent is selected from one or more of an alcohol 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 another aspect, the present disclosure further provides a method for preparing the potassium salt of the compound I, comprising the step of forming a salt from the compound I with a potassium-containing base.

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

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

[0034] In yet another aspect, the present disclosure provides a solvate of the sodium salt of the compound of Formula I.

[0035] In some embodiments, the solvate of the sodium salt of the compound of Formula I is selected from a hydrate or an ethanolate.

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

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

[0038] The present disclosure also provides a solid form of a solvate of the sodium salt of the compound of Formula I.

[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 ethanolate.

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

[0044] In yet another aspect, the present disclosure provides a solvate of the potassium salt of the compound of Formula I.

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

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

[0047] In some embodiments, 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 in the solvate is about 1:1.

[0048] In some embodiments, 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 in the solvate is 1:1.

[0049] The present disclosure also provides a solid form of a 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 yet 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 further provides Form A of the sodium salt of the compound of Formula I, wherein the Form A has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ 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 as a diffraction angle 2θ 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 X-ray powder diffraction pattern of Form A of the sodium salt of the compound of Formula I expressed at a diffraction angle 2θ is 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°, There are diffraction peaks at 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 X-ray powder diffraction pattern of Form A of the sodium salt of the compound of Formula I, expressed in terms of a diffraction angle 2θ, is substantially as shown in FIG1 .

[0056] In some embodiments, the crystalline form A of the sodium salt of the compound of formula I has a DSC spectrum with a peak at 342.84±5.0° C. In some embodiments, the DSC spectrum of the crystalline form A of the sodium salt of the compound of formula I is shown in FIG2 .

[0057] In another aspect, the present disclosure further provides a method for preparing 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 of preparing 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 Form A of the sodium salt of the compound of Formula I, the volume (mL) of anhydrous ethanol in step (1) is 20 to 45 times or 35 to 45 times the mass (g) of the compound of Formula I.

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

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

[0062] In some embodiments, the present disclosure further provides Form B of the compound of Formula I, wherein the Form B has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ having 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 as a diffraction angle 2θ 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 as a diffraction angle 2θ 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 X-ray powder diffraction pattern of Form B of the compound of Formula I expressed at a diffraction angle 2θ is substantially as shown in FIG4 .

[0066] In some embodiments, the crystalline Form B of the compound of Formula I has a DSC spectrum with a peak at 343.98±5.0° C. In some embodiments, the DSC spectrum of the crystalline Form B of the compound of Formula I is shown in FIG5 .

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

[0068] In some embodiments, the present disclosure further provides Form C of the monoethanolate of the sodium salt of the compound of Formula I, wherein the Form C has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ having diffraction peaks at 17.14±0.20°, 21.85±0.20°, 25.46±0.20°, and 25.74±0.20°.

[0069] In some embodiments, the crystalline form C of the monoethanolate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern expressed at a diffraction angle 2θ 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, the crystalline form C of the monoethanolate of the sodium salt of the compound of Formula I has an X-ray powder diffraction pattern expressed at a diffraction angle 2θ 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°, 20.30±0.20°, 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°.

[0071] In some embodiments, the X-ray powder diffraction pattern of Form C of the monoethanolate of the sodium salt of the compound of Formula I expressed at a diffraction angle 2θ is substantially as shown in FIG. 7 .

[0072] In some embodiments, the crystalline Form C of the sodium salt of the compound of Formula I has a DSC spectrum with a peak at 340.70±5.0° C. In some embodiments, the DSC spectrum of the crystalline Form C of the sodium salt of the compound of Formula I is shown in FIG8 .

[0073] In yet 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 further provides Form D of a monohydrate of the sodium salt of the compound of Formula I, wherein the Form D has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ having 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 expressed as a diffraction angle 2θ 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 expressed as a diffraction angle 2θ 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 X-ray powder diffraction pattern of the monohydrate form D of the sodium salt of the compound of formula I expressed in terms of diffraction angle 2θ is 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 There are diffraction peaks at .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 X-ray powder diffraction pattern of Form D of the monohydrate of the sodium salt of the compound of Formula I, expressed at a diffraction angle 2θ, is substantially as shown in FIG. 10 .

[0079] In some embodiments, the crystalline form D of the sodium salt monohydrate of the compound of formula I has a DSC spectrum with a peak at 338.88±5.0° C. In some embodiments, the DSC spectrum of the crystalline form D of the sodium salt monohydrate of the compound of formula I is shown in FIG11 .

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

[0081] In some embodiments, in the method for preparing 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 the mass (mg) of the amorphous form of the sodium salt of the compound of Formula I.

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

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

[0084] In another aspect, the present disclosure further provides Form E of the sodium salt of the compound of Formula I, wherein the Form E has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ having 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 crystalline form E of the sodium salt of the compound of formula I has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ 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 sodium salt of the crystalline form E of the compound of formula I expressed in terms of diffraction angle 2θ is 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°. , there are diffraction peaks at 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 Form E of the sodium salt of the compound of Formula I, expressed at a diffraction angle 2θ, is substantially as shown in FIG. 13 .

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

[0089] In some embodiments, the DSC spectrum of Form E of the sodium salt of the compound of Formula I is shown in FIG14 .

[0090] In yet 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 amorphous sodium salt of the compound of Formula I has an X-ray powder diffraction pattern, expressed at a diffraction angle of 2θ, substantially as shown in FIG. 16 .

[0092] In some embodiments, the amorphous sodium salt of the compound of Formula I has a DSC spectrum with a peak at 337.04° C.±5.0° C. In some embodiments, the DSC spectrum of the amorphous sodium salt of the compound of Formula I is shown in FIG17 .

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

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

[0095] In another aspect, the present disclosure further provides a crystalline form G of a monohydrate of the potassium salt of the compound of formula I, wherein the crystalline form G has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ having 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 crystalline form G of the monohydrate of the potassium salt of the compound of formula I has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ 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 X-ray powder diffraction pattern of the monohydrate form G of the potassium salt of the compound of formula I expressed in terms of diffraction angle 2θ is 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. There are diffraction peaks at 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 X-ray powder diffraction pattern of Form G of the monohydrate of the potassium salt of the compound of Formula I, expressed at a diffraction angle 2θ, is 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 spectrum with peaks at 110.89°C ± 5.0°C and 336.14 ± 5.0°C. In some embodiments, the DSC spectrum of the crystalline form G of the monohydrate of the potassium salt of the compound of Formula I is shown in Figure 20.

[0100] In another aspect, the present disclosure also provides a method for preparing the monohydrate form G 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 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 for 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 to 20 times or 3 to 10 times the mass (g) of the compound of formula I.

[0103] In some embodiments, in the method for 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°C-60°C or 20°C-40°C.

[0104] In yet another aspect, the present disclosure further provides an amorphous potassium salt of the compound of Formula I, wherein the amorphous potassium salt of the compound of Formula I has an X-ray powder diffraction pattern expressed at a diffraction angle 2θ substantially as shown in FIG. 22 .

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

[0106] The crystallization methods for each crystal form disclosed herein are conventional, such as crystallization by evaporating solvents, crystallization by cooling, or crystallization at room temperature.

[0107] Furthermore, the preparation method of each crystal form disclosed in the present invention also includes steps such as filtration, washing or drying.

[0108] On the other hand, 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 herein, and a pharmaceutically acceptable excipient.

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

[0110] In another aspect, the present disclosure provides a method for preventing or treating a disease or condition mediated by DNA polymerase θ 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.

[0111] In another aspect, the present disclosure provides a method for preventing or treating a disease or condition mediated by DNA polymerase θ 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.

[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, in the preparation of a medicament for preventing or treating a disease or condition mediated by DNA polymerase θ.

[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, for preparing a medicament for preventing or treating a disease or condition mediated by DNA polymerase θ.

[0114] 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, for use in preventing or treating diseases or conditions mediated by DNA polymerase θ.

[0115] 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, for use in preventing or treating diseases or conditions mediated by DNA polymerase θ.

[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, for preventing or treating a disease or condition mediated by DNA polymerase θ.

[0117] In another aspect, the present disclosure provides a crystalline form of a compound of Formula I, 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, for preventing or treating a disease or condition mediated by DNA polymerase θ.

[0118] In some embodiments, the disease or condition mediated by DNA polymerase θ is a disease or condition in which DNA polymerase θ is overexpressed. In some embodiments, the disease or condition mediated by DNA polymerase θ is cancer. In some embodiments, the disease or condition mediated by DNA polymerase θ is a cancer with homologous recombination (HR) deficiency. In some embodiments, the cancer is a cancer in which BRCA gene expression is reduced or absent, BRCA gene deficiency, or BRCA protein function is reduced. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is 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 physicochemical stability, has advantages in physicochemical properties, formulation processing performance, etc., and is suitable for preparation into the desired pharmaceutical composition.

[0120] Definitions and Explanations of Terms

[0121] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[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. Solvate typically has a substantially fixed molar ratio of solute to solvent. This term also includes cage compounds, including cage compounds with water. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, etc. 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 the compound (or salt) is about 1:1.

[0124] The term "about" is used in this disclosure to mean approximately, around, roughly, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower limits of the numerical range stated. Unless otherwise indicated, the term "about" is used herein to modify the upper and lower limits of a numerical value stated by a deviation of 10%.

[0125] Unless otherwise indicated, the terms "comprise," "comprise," or "comprises" and variations thereof, such as comprises or comprising, should be construed in an open, non-exclusive sense, ie, "including but not limited to."

[0126] References to "alternative embodiments" or "embodiments" in this disclosure mean that at least one embodiment includes the specific referenced elements, structures, or features described in connection with that embodiment. Thus, the phrases "alternative embodiments" or "embodiments" appearing in different places in this disclosure do not necessarily all refer to the same embodiment. Furthermore, the specific elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0127] The room temperature described in this disclosure refers to 25±5.0°C.

[0128] The range "m-n" described in this disclosure is an abbreviation for any combination of real numbers 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 are listed herein; "1-5" means that 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 are listed herein, and "1-5" is merely an abbreviation for these numerical combinations.

[0129] The "X-ray powder diffraction pattern or XRPD pattern" described in the present disclosure refers to the Bragg formula 2d Sinθ = nλ (wherein, d is the interplanar spacing, θ is the diffraction angle, λ is the wavelength of the incident X-ray, and the diffraction order n is any positive integer, generally the first-order diffraction peak is taken, n = 1). When the X-ray is incident on an atomic plane with a lattice plane spacing d in a crystal or a partial crystal sample at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle), the Bragg equation is satisfied, thereby measuring this set of X-ray powder diffraction patterns.

[0130] For the same crystalline form of the same compound, the peak positions of their XRPD patterns are generally similar, but the relative intensity errors may be large. It should also be noted that in the identification of mixtures, some diffraction lines may be missing due to factors such as a decrease in content. In this case, it is not necessary to rely on all diffraction peaks observed in a high-purity sample; even a single diffraction peak may be characteristic for a given crystal.

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

[0132] Those skilled in the art will appreciate that the molar ratios of the disclosed compounds to the acid / base molecules, and the compounds to the solvent molecules in solvates, often have a certain degree of error due to factors such as crystal defects and measurement errors. Generally, a ±10% ratio is considered within a reasonable error range. While there may be a certain degree of error depending on the context in which the molar ratios are used, this error should not exceed ±10%, preferably ±5%.

[0133] The term "therapeutically effective amount" refers to an amount of a compound of the present disclosure that (i) treats a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a specific disease, condition, or disorder as described herein. The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their knowledge and this disclosure.

[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 problems or complications, commensurate with a reasonable benefit / risk ratio.

[0135] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-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 the compounds of the present disclosure or their pharmaceutically acceptable salts or solvates of their pharmaceutically acceptable salts with suitable pharmaceutically acceptable excipients. For example, they can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0137] Typical routes of administration of the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates of their pharmaceutically acceptable salts, or pharmaceutical compositions comprising the same, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0138] The pharmaceutical composition of the present disclosure can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, and the like.

[0139] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the disclosed compounds, or pharmaceutically acceptable salts thereof, or solvates of pharmaceutically acceptable salts thereof, to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, and the like for oral administration to a patient.

[0140] Solid oral compositions can be prepared by conventional mixing, filling, or tableting methods. For example, they can be prepared by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients as needed, and then granulating the mixture to obtain a tablet or dragee core. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

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

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

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

[0144] Those skilled in the art recognize that the measured data of XRPD peak positions and / or intensities for a given crystalline form of the same compound will vary within a range of error. The 2θ values ​​in this disclosure encompass an appropriate range of error, which is typically represented by "±". For example, a 2θ value represented in this disclosure as a specific angle value of ±0.20° represents that the specific angle value has an error range of ±0.20°, i.e., 5.92±0.20°2θ represents a 2θ range of 6.12 to 5.72. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., those skilled in the art recognize that an appropriate error range for XRPD diffraction angles may be ±0.20°, ±0.15°, ±0.10°, ±0.05° or less, and that some variability in peak intensity is allowed. The terms "substantially the same" or "substantially as shown" when used to describe an XRPD pattern refer to a pattern that includes diffraction peaks that have 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 angles within a standard deviation of ±0.2° 2θ.

[0145] As those skilled in the art recognize that the measured data of the DSC spectra for a given crystalline form of the same compound will vary within an error tolerance. A suitable error range is allowed for the single peak value (expressed in degrees Celsius). Typically, the error range is represented by "±". For the same crystal form of the same compound, in consecutive analyses, the thermal transition temperature and melting point errors are typically within ±5.0°C. For example, a peak value of "140.96 ± 5.0" is represented as being within the range of 145.96 to 135.96. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., those skilled in the art recognize that the appropriate error range for the single peak value may be ±5.0, ±4.0, ±3.0, ±2.0 or less.

[0146] The salt forms and / or crystal forms of the present disclosure may also be isotopically labeled. The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature. Examples of isotopes that may be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, 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 et al.

[0147] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0148] In addition, the use of heavier isotopes such as deuterium (i.e. 2 H)) substitution may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances, wherein deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is replaced by deuterium.

[0149] The compounds of the present invention or their pharmaceutically acceptable salts or solvates of their pharmaceutically acceptable salts can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples disclosed herein.

[0150] The chemical reactions described in the embodiments of the present disclosure are carried out in suitable solvents that are compatible with the chemical transformations described herein and the reagents and materials required. To obtain the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates thereof, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0151] Test conditions of the instruments used in this public experiment:

[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] Scanning mode: θ / 2θ, scanning range: 3-40° (2θ angle)

[0159] Dwell time (seconds): 0.12

[0160] Scan step (°2θ): 0.01

[0161] Voltage: 40kV

[0162] Current: 40mA

[0163] 2. Differential Scanning Calorimeter

[0164] Instrument model: Discovery DSC 2500

[0165] Purge gas: nitrogen

[0166] Sample pan: aluminum pan, non-sealed cover

[0167] Method: Linear temperature ramp

[0168] Heating rate: 10℃ / min

[0169] Temperature range: 30℃~400℃

[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] Heating rate: Starting from the initial temperature of 30℃, heating to 400℃ at a rate of 10℃ per minute

[0176] Temperature range: 30℃~400℃

[0177] 4. Dynamic moisture adsorption instrument

[0178] Instrument model: DVS Intrinsic.

[0179] DVS parameters:

[0180] Temperature: 25℃;

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

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

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

[0184] 5. Ion chromatography

[0185] Instrument model: Thermo Fisher Integrion, No. IC001

[0186] Detection method: conductivity (ELSD)

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

[0188] Eluent: 20 mM methanesulfonic acid solution

[0189] Flow rate: 1.0ml / min

[0190] This disclosure uses the following abbreviations:

[0191] BRIEF DESCRIPTION OF THE DRAWINGS

[0192] FIG1 is an XRPD pattern of Form A of the sodium salt of the compound of Formula I;

[0193] FIG2 is a DSC spectrum of Form A of the sodium salt of the compound of Formula I;

[0194] FIG3 is a TGA spectrum of Form A of the sodium salt of the compound of Formula I;

[0195] FIG4 is an XRPD pattern of Form B of the compound of Formula I;

[0196] FIG5 is a DSC spectrum of Form B of the compound of Formula I;

[0197] FIG6 is a TGA spectrum of Form B of the compound of Formula I;

[0198] FIG7 is an XRPD pattern of Form C of the monoethanolate of the sodium salt of the compound of Formula I;

[0199] FIG8 is a DSC spectrum of Form C of the monoethanolate of the sodium salt of the compound of Formula I;

[0200] FIG9 is a TGA spectrum of Form C of the monoethanolate of the sodium salt of the compound of Formula I;

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

[0202] FIG11 is a DSC spectrum of Form D of the monohydrate of the sodium salt of the compound of Formula I;

[0203] FIG12 is a TGA spectrum of Form D of the monohydrate of the sodium salt of the compound of Formula I;

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

[0205] FIG14 is a DSC spectrum of Form E of the sodium salt of the compound of Formula I;

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

[0207] Figure 16 is an XRPD pattern of the amorphous sodium salt of the compound of formula I;

[0208] Figure 17 is a DSC spectrum of the amorphous sodium salt of the compound of formula I;

[0209] Figure 18 is a TGA spectrum of the amorphous sodium salt of the compound of formula I;

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

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

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

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

[0214] Figure 23 is a DSC spectrum of the amorphous potassium salt of the compound of formula I;

[0215] FIG24 is a TGA spectrum of the amorphous potassium salt of the compound of formula I. Example

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

[0217] Unless otherwise specified, the ratios expressed for mixed solvents are volume ratios.

[0218] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0219] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 (ppm). The solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC 50 ” refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved.

[0220] In the following purification methods using high performance liquid chromatography, unless otherwise specified, the "%" of the acid or base used in mobile phase A refers to the volume fraction. For example, "water (0.05% formic acid)" means that the volume of formic acid is 0.05% of the total volume of formic acid and water. B% represents the ratio of the volume of mobile phase B to the total volume of mobile phases A and B during gradient elution. "B%: 50%-70%" means that the volume of mobile phase B to the total volume of mobile phases A and B varies from 50% to 70% during gradient elution.

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

[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), and DMAP (41.77 mg) were dissolved in dichloromethane (10 mL). (Boc)2O (820.78 mg) was added dropwise to the reaction mixture and stirred at room temperature for 16 h. The reaction mixture was then washed with saturated brine, 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), and potassium phosphate (1.28 g) were added to dioxane (10 mL) and water (2 mL) and stirred at 80°C for 4 h. The reaction solution was poured into water and extracted with ethyl acetate (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was slurried with ethyl acetate, filtered, and the filter cake was dried to obtain 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) and stirred at room temperature for 1 h. Ethyl acetate was added to the reaction solution, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 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 a nitrogen atmosphere, intermediate 6 (1 g) was dissolved in dioxane (20 mL) and water (5 mL), and intermediate 5 (995.75 mg), Pd (dppf) Cl2 (349.65 mg) and potassium carbonate (1.48 g) were added to the reaction solution. Subsequently, the reaction solution was stirred at 90 ° C for 2 hours under a nitrogen atmosphere. After the reaction was completed, water (50 mL) was added to the reaction solution, and ethyl acetate was used for extraction (50 mL * 3 times). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the organic phase was concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography ( 20g Silica Flash column chromatography, gradient 0-50% ethyl acetate / petroleum ether, flow rate 20 mL / min) to obtain 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 to tetrahydrofuran (8 mL) and water (4 mL). Lithium hydroxide (157.13 mg) was added to the reaction solution, and the reaction solution was stirred at 25°C for 16 hours. After the reaction, the pH of the reaction solution was adjusted to 3 and the solvent was removed by concentration under reduced pressure. The residue was washed with a dichloromethane / methanol mixture (10 / 1, 20 mL), filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 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 (compound of formula I)

[0233] Under a nitrogen atmosphere, intermediate 8 (300 mg) was added to N,N-dimethylformamide (5 mL). To the reaction solution were added intermediate 4 (273.62 mg), HATU (410.76 mg), and N,N-diisopropylethylamine (279.24 mg). The reaction solution was then stirred at 25°C under a nitrogen atmosphere for 2 hours. After completion of the reaction, the reaction solution was purified by preparative HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 48%-68%, 11 minutes) to obtain the title compound (51.96 mg). MS m / z (ESI): 513.1 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ13.40(brs,1H),9.27(s,1H),8.84(s,1H),8.36(d,J=8.4Hz,2H),8.00 (d,J=8.3Hz,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 Form A of the Sodium Salt of the Compound of Formula I

[0235] Suspend 9.5 g of the compound of Formula I in 190 mL of anhydrous ethanol, add a solution of sodium hydroxide (742 mg) in anhydrous ethanol (190 mL), and stir at 25°C for 3 h. Filter the reaction mixture, wash the filter cake with anhydrous ethanol (40 mL), and collect the filter cake to obtain the title compound. Dry the filter cake in vacuo at 50°C for 12 h to obtain Form A of the sodium salt of the compound of Formula I. 1H NMR (400MHz, DMSO-d6) δ9.02(s,1H),8.96(s,1H),8.28(d,J=8.5Hz,2H),7.92(d,J=8.5Hz,2H),7 .37(m,1H),7.24(d,J=2.7Hz,1H),7.13(s,1H),6.99(d,J=8.9Hz,1H),3.52(s,3H),2.53(s,3H).

[0236] Crystalline Form A of the sodium salt of the compound of Formula I was prepared using the method of Example 2. The XRPD pattern of this crystalline sample is shown in Figure 1, its DSC pattern is shown in Figure 2, and its TGA pattern is shown in Figure 3. Ion chromatography determined that the sodium ion content was 4.2%, indicating a molar ratio of the compound of Formula I to sodium of 1:1 (theoretical sodium ion content: 4.3%). The DSC peak was near 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] To the reactor, 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, 8.5 V), DIPEA (10.21 g, 2.00 eq.) were added, and then EDCI (11.35 g, 1.50 eq.) was added. The temperature was controlled at 25±5°C with stirring, and sampling was performed after 15 h of reaction.

[0239] After the reaction is completed, the temperature is lowered to 0-10°C, stirring is continued for 1-2 hours, and the mixture is filtered. The filter cake is rinsed with DMF (1V) and anhydrous ethanol (2V) to obtain a wet filter cake, which is then placed in a vacuum drying oven at 55±5°C and vacuum dried to constant weight to obtain 14.28 g of Form B of the compound of Formula I. 1 HNMR (400MHz, DMSO-d6) δ13.43(s,1H),9.36(s,1H),8.80(s,1H),8.38(d,J=8.5Hz,2H),8.02(d,J=8.5Hz,2 H),7.50(d,J=2.6Hz,1H),7.49–7.44(m,1H),7.42(s,1H),7.02(d,J=8.8Hz,1H),3.52(s,3H),2.61(s,3H).

[0240] The method of Example 3 was used to prepare Form B of the compound of Formula I. The XRPD pattern of the crystalline sample is shown in Figure 4, its DSC pattern is shown in Figure 5, and its TGA pattern is shown in Figure 6. The DSC peak is around 343.98°C, and its XRPD diffraction peak parameters are shown in the following table.

[0241] Example 4: Crystalline Form C of the Monoethanolate of the Sodium Salt of the Compound of Formula I

[0242] Preparation method 1:

[0243] 3 g of the compound of formula I and 15 mL of DMSO were added to a reaction flask and stirred at room temperature. A solution of sodium hydroxide (257 mg) in ethanol (9 mL) was added, the reaction solution became clear, heated to 45° C. in a water bath, 30 mL of ethanol was added, and the temperature was slowly cooled to room temperature. The solution was filtered, and the filter cake was rinsed with anhydrous ethanol and dried in vacuo at 40° C. to obtain Form C of the ethanolate of the sodium salt of the compound of formula I (a crystalline form of the monoethanolate of the sodium salt of the compound of formula I). 1 HNMR(400MHz,DMSO-d6)δ9.02(s,1H),8.95(s,1H),8.27(d,J=8.5Hz,2H),7.91(d,J=8.5Hz,2H),7.4 8–7.30(m,1H),7.23(d,J=2.7Hz,1H),7.12(s,1H),6.98(d,J=8.9Hz,1H),3.51(s,3H),2.53(s,3H).

[0244] The method of Example 4 was used to prepare Form C of the monoethanolate of the sodium salt of the compound of Formula I. The XRPD pattern of the crystalline sample is shown in Figure 7, its DSC pattern is shown in Figure 8, and its TGA pattern is shown in Figure 9. The DSC peak is near 340.70°C, and the XRPD diffraction peak positions are shown in the following table.

[0245] Preparation method 2:

[0246] 3 g of the compound of formula I and 15 mL of NMP, DMF or DMA were added to a reaction flask and stirred at room temperature. A solution of sodium hydroxide (257 mg) in ethanol (9 mL) was added. The reaction solution became clear and was heated to 45° C. in a water bath. 30 mL of ethanol was added and the temperature was slowly lowered to 25° C. The filter cake was rinsed with anhydrous ethanol and dried in vacuo at 40° C. to obtain Form C of the monoethanolate of the sodium salt of the compound of formula I.

[0247] Preparation method three:

[0248] 0.5 g of the compound of formula I and 1.8 mL of DMSO were added to a reaction flask and stirred at room temperature. 0.35 g of a 20% aqueous sodium ethoxide solution was added, and the reaction solution became clear. The solution was heated to 45° C. in a water bath, 30 ml of ethanol was added, and the temperature was slowly lowered to 25° C. The solution was filtered, and the filter cake was rinsed with anhydrous ethanol and dried in vacuo at 40° C. to obtain Form C of the monoethanolate of the sodium salt of the compound of formula I.

[0249] Example 5: Crystalline Form D of the Monohydrate of the Sodium Salt of the Compound of Formula I

[0250] 10 mg of the amorphous sodium salt of the compound of formula I was added to a reaction flask, 100 uL of toluene with a water content of 0.08 wt% was added (the solvent can also be methyl tert-butyl ether, isopropyl ether or dichloromethane with a water content of about 0.1 wt%), stirred at 25 ° C overnight, filtered, and the filter cake was dried under vacuum at room temperature to obtain Form D of the monohydrate of the sodium salt of the compound of formula I. 1 H NMR (400MHz, DMSO-d6) δ9.02(s,1H),8.95(s,1H),8.28(d,J=8.6Hz,2H),7.91(d,J=8.6Hz,2H),7 .36(m,1H),7.23(d,J=2.7Hz,1H),7.12(s,1H),6.98(d,J=8.8Hz,1H),3.52(s,3H),2.53(s,3H).

[0251] Form D was prepared using the method of Example 5. The XRPD pattern of the crystalline sample is shown in Figure 10, its DSC pattern is shown in Figure 11, and its TGA pattern is shown in Figure 12. The DSC peak is near 338.88°C, and the XRPD diffraction peak parameters are shown in the following table. Ion chromatography determined that the sodium ion content was 3.7%, and the molar ratio of the compound of Formula I to sodium was 1:1 (theoretical value is 4.2% sodium ion content). Form D has excellent physical stability and is stable under conditions such as light, high temperature, and high humidity, with no crystal transformation.

[0252] Example 6: Crystalline Form E of the Sodium Salt of the Compound of Formula I

[0253] 10 mg of the amorphous sodium salt of the compound of formula I was placed in a reaction flask, 100 uL of ethyl acetate was added, and the mixture was stirred at room temperature overnight. The mixture was filtered and the filter cake was dried under vacuum at room temperature to obtain Form E of the sodium salt of the compound of formula I. 1H NMR (400MHz, DMSO-d6) δ9.01(s,1H),8.98(s,1H),8.28(d,J=8.5Hz,2H),7.92(d,J=8.4Hz,2H),7.4 5–7.33(m,1H),7.25(d,J=2.7Hz,1H),7.14(s,1H),6.99(d,J=8.8Hz,1H),3.52(s,3H),2.54(s,3H).

[0254] Form E of the sodium salt of the compound of Formula I was prepared using the method of Example 6. The XRPD pattern of this crystalline sample is shown in Figure 13, its DSC pattern is shown in Figure 14, and its TGA pattern is shown in Figure 15. The DSC peak is near 338.17°C, and its XRPD diffraction peak parameters are shown in the table below. Form E exhibits poor physical stability under high humidity (92.5% RH) and accelerated conditions (40°C, 75% RH).

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

[0256] Add 1 g of the monoethanolate of the sodium salt of the compound of formula I, Form C (or any one of the crystalline form A of the sodium salt of the compound of formula I, the monohydrate crystalline form D of the sodium salt of the compound of formula I, and the crystalline form E of the sodium salt of the compound of formula I) to a single-necked bottle, add 60 mL of hexafluoroisopropanol, stir to dissolve, and then spin dry to obtain the amorphous sodium salt of the compound of formula I.

[0257] An amorphous sample of the sodium salt of the compound of formula I was prepared using the method of Example 7. The XRPD pattern of the crystalline sample is shown in Figure 16, its DSC pattern is shown in Figure 17, and its TGA pattern is shown in Figure 18. The DSC peak is near 337.04°C.

[0258] Example 8: Form G of the monohydrate of the potassium salt of the compound of formula I

[0259] 100 mg of the compound of formula I, 0.5 ml of N,N'-dimethylformamide, and 13 mg of potassium hydroxide were added to a centrifuge tube and stirred at room temperature. The reaction solution became clear. 2 ml of ethyl acetate was added and stirred at room temperature. After a large amount of solid precipitated, the reaction solution was filtered and the filter cake was vacuum dried at 45°C to obtain Form G of the monohydrate of the potassium salt of the compound of formula I.

[0260] The method of Example 8 was used to prepare Form G of the monohydrate of the potassium salt of the compound of Formula I. The XRPD spectrum of the crystalline sample is shown in Figure 19, its DSC spectrum is shown in Figure 20, and its TGA spectrum is shown in Figure 21. The DSC peaks are near 110.89°C and 336.14°C. Its XRPD diffraction peak parameters are shown in the following table.

[0261] Example 9: Amorphous Form of the Potassium Salt of the Compound of Formula I

[0262] 100 mg of the compound of formula I, 0.5 ml of N,N'-dimethylformamide, and 13 mg of potassium hydroxide were added to a centrifuge tube and stirred at room temperature. The reaction solution became clear. 2 ml of ethanol was added and stirred at room temperature. After a large amount of solid precipitated, the reaction solution was filtered and the filter cake was dried in vacuo at 45°C to obtain an amorphous potassium salt of the compound of formula I.

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

[0264] Experimental Example 10: Influencing Factors and Accelerated Experiments

[0265] The samples were placed in 2mL screw-capped vials under conditions of high temperature (60°C), high humidity (92.5%), light (7000 lux), or accelerated temperature (40°C, 75% RH) to test their stability. Alternatively, the samples were placed in packaging (two layers of pharmaceutical low-density polyethylene bags plus one layer of polyester / aluminum / polyethylene composite film) under conditions of light (7000 lux) or accelerated temperature (40°C, 75% RH) to test their stability. The results are shown in the table below.

[0266] Experimental results of influencing factors

[0267] Acceleration experiment results

[0268] Experimental Example 11: Solubility Experiment

[0269] The thermodynamic solubility of the disclosed compounds was determined using the following test method.

[0270] 1. Dissolution Medium Configuration

[0271] Purified water: homemade in the laboratory

[0272] 2. Test steps

[0273] To investigate the thermodynamic solubility of the disclosed compounds in water, 1000 μL of water was measured and placed in a 1 mL stoppered transparent glass tube. An appropriate amount of sample was weighed to saturate the solution and then shaken to disperse. The sample was placed on a roller mixer and shaken in water for 24 hours. Afterwards, an appropriate amount of sample was taken, filtered through a microporous membrane, and the content was determined using a high-performance liquid chromatograph using the external standard method. The thermodynamic solubility of the compound in water (μg / mL) was calculated. The solubility results are detailed in the table below.

[0274] Experimental Example 12: Moisture-attracting test

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

[0276] (2) Experimental conditions: Take the samples to be tested and place them in the DVS sample tray for testing.

[0277] (3)DVS parameters:

[0278] Temperature: 25℃;

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

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

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

[0282] Experimental results:

[0283] The weight gain of Form A upon moisture absorption at 80.0% RH was 1.6%.

[0284] The weight gain of Form B upon moisture absorption at 80.0% RH was 1.5%.

[0285] The moisture absorption weight gain of Form C at 80.0% RH was -4.3%, and solvent desorption was observed during the cycle.

[0286] The weight gain of Form E upon moisture absorption at 80.0% RH was 2.5%.

[0287] Biological activity and related properties test examples

[0288] The compounds in the following test examples were all prepared according to the methods of the above embodiments of the present disclosure.

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

[0290] Brief introduction to experimental principle: After the N-terminal active peptide segment (M1-N899) of POLQ with ATPase activity 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-coupled enzymatic reaction, and catalyzes the reaction of NADH to generate NAD. + The decrease in NADH OD at 340 nm was measured using a Perkin Elmer Envision microplate reader to reflect 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 Method: POLQ enzyme was diluted to 100 nM in 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). Test compounds were diluted to various concentrations in dimethyl sulfoxide (DMSO) using an Echo 650 pipetting system and transferred to a 384-well plate. 20 μL / well of 100 nM POLQ was added and incubated at room temperature for 15 minutes. A reaction mixture was prepared containing the following components: 100 μM ATP, 300 nM dT50, 300 μM NADH, 6 mM PEP, 10 U / mL lactate dehydrogenase, and 20 U / mL pyruvate kinase. The enzyme reaction was initiated by adding 20 μL / well of the reaction mixture. The reaction system was diluted from 10 μM to 0.0005 μM using a three-fold serial dilution. The final DMSO concentration was 0.2% v / v. The 384-well plate was incubated at room temperature for 20 minutes, and the OD at 340 nm was read using an Envision microplate reader.

[0294] Data Analysis:

[0295] The inhibition rate was calculated and the IC of the compound was obtained using XLfit software. 50 .

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

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

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

[0299] The biological activities of the disclosed compounds were determined by the above tests, and the IC 50 The values ​​are shown in Table 1 below.

[0300] Table 1 IC of the Example compounds against POLQ enzyme activity 50

[0301] In the above table, the symbols used to indicate inhibitory activity mean the following:

[0302] “++++” indicates the IC inhibitory activity of the tested compound on the enzyme 50 Range: IC 50 <100nM.

[0303] “+++” indicates the inhibitory activity of the tested compound on the enzyme IC 50 Range: 100≤IC 50 <500nM.

[0304] “++” indicates the inhibitory activity of the tested compound on the enzyme IC 50 Range: 500≤IC 50 <1000nM.

[0305] Test Example 2: Compound Inhibition of Tumor Cell Proliferation

[0306] Brief introduction to experimental principle: After incubating the compound with tumor cells for 7 days, the ATP in the living cells is quantified using Promega's CTG kit to reflect the effect of the compound on tumor cell proliferation.

[0307] Experimental instruments: Envision microplate reader from Perkin Elmer; 5810R centrifuge from Eppendorf; automatic cell counter from Countstar.

[0308] Experimental Materials:

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

[0310] Data Analysis:

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

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

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

[0314] Among them, 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 compound and cells. min Refers to the light signal value of the well containing culture medium and 0.25% v / v DMSO.

[0315] The inhibition of tumor cell growth by the disclosed compounds was determined by the above test, and the IC 50 value.

[0316] Table 2 IC of the compounds in the examples for inhibition of tumor cell growth 50

[0317] In the above table, the symbols used to indicate inhibitory activity mean the following:

[0318] “++++” indicates the IC value of the test compound against cells 50 Range: IC 50 <200nM.

[0319] “+++” indicates the inhibitory activity of the tested compound on the cell IC 50 Range: 200nM≤IC 50 <500nM.

[0320] “++” indicates the inhibitory activity of the tested compound on the cell IC 50 Range: 500nM≤IC 50 <1000nM.

[0321] “+” indicates the inhibitory activity of the tested compound on the cell IC 50 Range: 1000nM≤IC 50 <10000nM.

[0322] “-” indicates the inhibitory activity of the tested compound on the cell IC 50 Range: IC 50 ≥10000nM.

[0323] After testing, the compound of formula I disclosed herein has a good inhibitory effect on BRCA2 mutated tumor cells and has good selectivity.

[0324] Test Example 3: Compound inhibition experiment on cell MMEJ pathway

[0325] Experimental Principle: POLQ is a key protein in the cellular MMEJ repair process. The NanoLuciferase MMEJ Repair Reporter System is transfected into HEK293T cells. When the MMEJ repair pathway is functioning normally, the NanoLuciferase reporter protein is correctly expressed, and luminescence can be detected. A BMG multi-function microplate reader (BMG LABTECH) is used to measure the decrease in luminescence, reflecting the compound's inhibition of the cellular MMEJ pathway.

[0326] Experimental instruments: Incucyte live cell imaging system from ESCD, Echo 655 pipetting system from Labcyte, BMG multifunctional microplate reader from BMG LABTECH, and Neon transfection system from Invitrogen.

[0327] Experimental Materials:

[0328] Experimental method: The test compound was diluted to different concentrations in dimethyl sulfoxide (DMSO) using the Echo 655 pipetting system and transferred to a 384-well plate. The final concentration of the compound in the reaction system started from 10 μM and was serially diluted 3-fold to a final DMSO concentration of 0.1%. HEK293T cells were collected and the MMEJ luciferase substrate was transfected into the cells using the Neon transfection system. 4000 transfected HEK293T cells / well were diluted with DMEM medium containing 10% FBS and added to a 384-well plate (25 μL / well). After the compound and cells were cultured in a 37°C, 5% CO2 incubator for 24 hours, 40 μL / well NanoGlo substrate buffer was added to measure the inhibitory effect of the compound on tumor cell growth, and the inhibition rate and half inhibitory concentration (IC50) were calculated. 50 ).

[0329] Data Analysis:

[0330] Calculate the compound inhibition rate (Compound inhibition) and use XLfit software to fit the compound IC 50 .

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

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

[0333] After testing, the compound of formula I disclosed in the present invention has strong inhibitory activity on the MMEJ pathway mediated by POLQ in cells. It is expected that the compound can effectively inhibit the target POLQ and the related pathway MMEJ in tumors, thereby exerting corresponding pharmacological effects.

[0334] Test Example 4: Determination of the metabolic stability of compounds in hepatocytes

[0335] The metabolic stability of the disclosed compounds in hepatocytes was determined using the following test method.

[0336] 1. 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(10×)(Gibco by Life Technologies 2060570)

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

[0342] 6.GlutaMAXTM-1(100×)(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

[0349] 2. Test steps

[0350] 1. Prepare hepatocyte recovery medium according to the information in the table below. Mix 49.5mL Williams'Medium E and 0.5mL GlutaMAXTM-1 (100×) as incubation medium. Preheat the hepatocyte recovery medium and incubation medium in a 37°C water bath for at least 15 minutes before use. Take a tube of ultra-low temperature preserved hepatocytes and ensure that the hepatocytes are still in a low-temperature frozen state before recovery. Quickly place the hepatocytes in a 37°C water bath and shake gently until all ice crystals are dispersed, spray with 70% ethanol and transfer to a biosafety cabinet. Pour the contents of the hepatocyte tubule into a centrifuge tube containing 50mL recovery medium and centrifuge it at 100g for 10 minutes. After centrifugation, aspirate the recovery medium and add sufficient incubation medium to obtain a cell density of approximately 1.5×10 6 Cells / mL of cell suspension were prepared. Liver cells were counted and viable cell density was determined using Cellometer Vision. Liver cell viability must be greater than 75%. Liver cell suspension was diluted with incubation medium to a viable cell density of 0.5×10 6 viable 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 an incubator on a vortex for 10 minutes. All samples are incubated in duplicate. Initiate the reaction by adding 2.5 μL of 100 μM test compound or control verapamil to each well and return the plate to the incubator on a vortexer. At 0, 15, 30, 60, 90, and 120 minutes, a 25 μL sample is collected and terminated by adding 125 μL of acetonitrile containing the internal standard. Vortex for 10 minutes and centrifuge at 3220 g and 4°C for 30 minutes. After centrifugation, transfer 100 μL of the supernatant to a sample plate and mix with 150 μL of purified water for LC-MS / MS analysis.

[0352] All data were calculated using Microsoft Excel software. The peak areas of the extracted ion chromatograms were detected, and the in vitro half-life (t 1 / 2 ).

[0353] In vitro half-life (t 1 / 2 ) is calculated by slope:

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

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

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

[0357] CL int is the intrinsic clearance rate; k is the elimination rate constant; volume of incubation is the incubation volume (μL); amount of proteins is the protein amount (mg)

[0358] According to tests, the compound of formula I disclosed herein is metabolically stable in hepatocytes of various species and is expected to be relatively stable in vivo through liver metabolism and relatively less affected by the first-pass effect of the liver.

[0359] The above describes the embodiments of the present disclosure. However, the present disclosure is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. The sodium salt of a compound of formula I having the structure 2. The sodium salt according to claim 1, wherein The molar ratio of the compound of formula I to the sodium ion is about 1:1; alternatively, the molar ratio of the compound of formula I to the sodium ion is 1:

1.

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

4. The sodium salt according to claim 3, wherein The sodium salt is the crystalline form A of the sodium salt of the compound of formula I, and the X-ray powder diffraction pattern of the crystalline form A expressed by the diffraction angle 2θ has 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 the crystalline form A expressed by the diffraction angle 2θ has 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 the crystalline form A expressed by the diffraction angle 2θ is 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°. There are diffraction peaks 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 the crystalline form A represented by the diffraction angle 2θ is substantially as shown in FIG1 .

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

6. The sodium salt according to claim 3, wherein The sodium salt is the crystalline form E of the sodium salt of the compound of formula I, and the X-ray powder diffraction pattern of the crystalline form E represented by the diffraction angle 2θ has 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 the crystalline form E represented by the 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°; or The X-ray powder diffraction pattern of the crystalline form E represented by the diffraction angle 2θ is 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±0.2 Diffraction peaks are present 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 the crystalline form E represented by the diffraction angle 2θ is substantially as shown in FIG. 13 .

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

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 at a diffraction angle of 2θ is substantially as shown in FIG. 16 .

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

10. The sodium salt according to any one of claims 1 to 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 ethanolate 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 to the solvent in the solvate is about 1:1; or, the molar ratio of the sodium salt of the compound of formula I to the solvent in the solvate 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 a crystalline form D of the monohydrate of the sodium salt of the compound of formula I, and the X-ray powder diffraction pattern of the crystalline form D represented by a 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 the crystalline form D represented by a diffraction angle 2θ has diffraction peaks at 8.28±0.20°, 10.08±0.20°, 20.05±0.20° and 23.47±0.20°. The crystalline form D has 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°, 18.87±0.20°, 19.70±0.20°, 21.91±0.20°, 23.50±0.20°, 24.47±0.20°, 26.89±0.20°, 27.91±0.20°, 28.89±0.20°, 29.91±0.20°, 30.91±0.20°, 31.71±0.20°, 32.47±0.20°, 33.47±0.20°, 34. .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 crystalline form D represented by the diffraction angle 2θ is 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.

10. The present invention relates to a crystalline form D having diffraction peaks 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 crystalline form D represented by the diffraction angle 2θ is substantially as shown in Figure 10.

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

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 a crystalline form C of the monoethanolate of the sodium salt of the compound of formula I, and the X-ray powder diffraction pattern of the crystalline form C expressed by the diffraction angle 2θ has 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 the crystalline form C expressed by the diffraction angle 2θ is 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 or the X-ray powder diffraction pattern of the crystalline form C represented by the diffraction angle 2θ is 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°, 20.30±0.20°, There are diffraction peaks 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 the crystalline form C represented by the diffraction angle 2θ is substantially as shown in Figure 7.

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

16. A method for preparing the crystalline form A of the sodium salt of the compound of formula I according to claim 4, comprising: (1) mixing the compound of formula I with anhydrous ethanol, and (2) adding a mixed solution of sodium hydroxide and anhydrous ethanol, stirring, and separating.

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, the volume (mL) of anhydrous ethanol in step (1) is 20 to 45 times or 35 to 45 times the mass (g) of the compound of formula I; and / or, stirring is performed at a temperature of 20-60°C or 20 to 30°C in step (2).

18. A method for preparing the crystalline 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 (uL) 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 stirring is carried out at a temperature of 0-60°C or 15-30°C.

20. Crystallization of the compound of formula I:

21. The crystal of the compound of formula (I) according to claim 20, wherein The crystal is the crystal form B of the compound of formula I, and the X-ray powder diffraction pattern of the crystal form B represented by the 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 the crystal form B represented by the 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 X-ray powder diffraction pattern of Form B represented by a diffraction angle 2θ has 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 Form B represented by a diffraction angle 2θ is substantially as shown in Figure 4.

22. The crystal of the compound of formula (I) according to claim 21, wherein The crystalline form B has a DSC spectrum with a peak at 343.98±5.0°C.

23. The potassium salt of a compound of formula I having the structure 24. The potassium salt according to claim 23, wherein The molar ratio of the compound of formula I to potassium ions is about 1:1; alternatively, 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; alternatively, the potassium salt is in crystalline form.

26. The potassium salt according to any one of claims 23 to 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 ethanolate 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 about 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 a crystalline form G of the monohydrate of the potassium salt of the compound of formula I, and the X-ray powder diffraction pattern of the crystalline form G represented by a 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 the crystalline form G represented by a 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 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°; or, the crystalline form G has diffraction peaks at diffraction angles The X-ray powder diffraction pattern expressed in 2θ is 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. There are diffraction peaks 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 crystalline form G expressed by the diffraction angle 2θ is substantially as shown in Figure 19.

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

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 at a diffraction angle of 2θ is substantially as shown in FIG. 22 .

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

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

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

34. Use of the sodium salt of any one of claims 1-15, the crystal 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 preventing or treating a disease mediated by DNA polymerase theta.

35. Use of the sodium salt of any one of claims 1-15, the crystal 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 preventing or treating diseases or disorders mediated by DNA polymerase θ.

36. The sodium salt of any one of claims 1 to 15, the crystal of the compound of formula I of any one of claims 20 to 22, or the potassium salt of any one of claims 23 to 31, or the pharmaceutical composition of claim 32 for use in preventing or treating a disease or condition mediated by DNA polymerase theta.

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