Crystalline form of RNA m6A regulator as well as preparation method and application thereof

Abstract

The invention provides a preparation method and application of a crystal form of an RNA (Ribonucleic Acid) m6A regulating agent as shown in a formula (I). The crystal form of the RNA m6A regulating agent disclosed by the invention has the beneficial effects of low hygroscopicity and good stability. The preparation method of the crystal form of the RNAm6A regulating agent is simple in process, the crystallization process is easy to control, and the reproducibility is good. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a crystalline form of an RNA m6A regulator, a preparation method thereof, and an application thereof. Background Art

[0002] Hand-foot syndrome (HFS) and hand-foot skin reaction (HFSR) are erythematous skin lesions on the palms and soles, mainly caused by cytotoxic chemotherapy drugs and tumor-targeted drugs. In severe cases, patients may lose the ability to take care of themselves. The main pathological features of hand-foot syndrome and hand-foot skin reaction are vacuolar degeneration of basal keratinocytes, perivascular lymphocyte infiltration in the skin, keratinocyte apoptosis, and skin edema; inflammatory changes, vasodilation, edema, and leukocyte infiltration can be seen under a microscope.

[0003] Drugs that can cause hand-foot syndrome include chemotherapy drugs such as capecitabine, liposomal doxorubicin, cytarabine, docetaxel, vinorelbine, continuous infusion of doxorubicin, gemcitabine, etc.; drugs that can cause hand-foot skin reaction include targeted drugs such as sunitinib (Sutent), sorafenib (Nexavar), imatinib (Gleevec), erlotinib (Tarceva), etc. The World Health Organization (WHO) classifies HFS into 4 grades: grade 1, numbness, paresthesia, or tingling in the hands and feet; grade 2, discomfort when holding objects and walking, painless swelling, or erythema; grade 3, painful erythema, edema of the palms and soles, perionychial erythema, and swelling; grade 4, peeling, ulceration, blistering, and severe pain. Currently, when severe hand-foot syndrome and hand-foot skin reaction occur clinically, the symptoms are often only relieved through topical skin care, and in some cases, the drug needs to be discontinued. The existing treatment methods only relieve the symptoms but do not address the root cause, which severely limits the use of first-line oncology chemotherapy drugs. Therefore, there is a huge unmet medical need. There is a need to develop a drug with good efficacy, few side effects, and low cost for the prevention and treatment of hand-foot syndrome and hand-foot skin reaction to meet the growing global medical needs.

[0004] m6A methylation modification is the most common RNA modification in mammals, which can regulate various signaling pathways and cellular processes (such as growth, development, and diseases, etc.) to play a key biological role (Frye M., et al. Science 2018, 361, 2073 - 2092; Yang C., et al. Cell Death & Disease 2020, 11, 960; Meyer K.D. & Jaffrey S.R. Nature Review Molecular Cell Biology 2014, 15, 313 - 326). The m6A methylation modification of mRNA, miRNA, circRNA, and lncRNA is a dynamic and reversible process. Methyltransferases (such as METTL3, METTL14, and METTL16, etc.) bind methyl groups to RNA, while demethylases (FTO and ALKBH5) can remove the methyl groups on RNA, thus constituting the regulatory basis of m6A. m6A affects processes such as RNA processing, translation, and degradation by recruiting specific binding proteins (such as YTHDF1, YTHDF2, YTHDC1, and IGF2BP, etc.), thereby leading to changes in downstream protein functions and cellular biological behaviors (Hsu P.J., et al. Journal of Biological Chemistry 2019, 294, 19889 - 19895; Yao Y., et al. FASEB Journal 2019, 33, 7529 - 7544).

[0005] There are few currently known direct associations between RNA m6A and skin-related diseases. Mostly, m6A-related methyltransferases or demethylases play roles in skin diseases (especially skin tumors). For example, the mRNA expression levels of METTL3 and ALKBH5 in the tumor tissues of patients with acral melanoma are significantly higher than those in the adjacent tissues, and the mRNA expression level of METTL3 in patients with advanced acral melanoma is significantly higher than that in early-stage patients (Le Zhanghui, Preliminary Study on the Pathogenesis of LncRNA and RNA m6A Methylation in Acral Melanoma, Dissertation, 2019). In the diagnosis of melanoma, the m6A-specific binding proteins YTHDF1 and HNRNPA2B1 can be used as novel biomarkers (Li T.D., et al. Cancer Cell 2020, 20, 239). In terms of keratinocytes, it has only been reported that long-term, low-level arsenic exposure can inhibit selective autophagy caused by m6A demethylase, thereby inducing the occurrence of skin tumors (Cui Y.H., et al. Nature Communications 2021, 12, 2183). In summary, there is currently no method for preventing and treating skin diseases by regulating RNA m6A methylation.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] Problems to be Solved by the Invention

[0008] Based on the fact that the existence form and quantity of polymorphic compounds are unpredictable, the present invention provides a crystalline form of an RNA m6A regulator represented by formula (I), its preparation method and application.

[0009]

[0010] Solutions for Solving the Problems

[0011] The present invention provides a crystalline form A of the compound represented by formula (I),

[0012]

[0013] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 12.673, 18.557, 24.951, 27.029 and 33.461; preferably has characteristic peaks at 6.760, 12.673, 13.507, 18.557, 19.315, 21.594, 21.901, 22.214, 24.951, 27.029 and 33.461; preferably has characteristic peaks at 6.760, 12.673, 13.507, 18.557, 19.315, 20.380, 21.594, 21.901, 22.214, 24.774, 24.951, 26.125, 27.029, 33.461, 35.425 and 35.428; preferably has characteristic peaks at 5.547, 6.760, 12.326, 12.673, 13.507, 14.870, 15.162, 18.557, 19.315, 20.380, 21.183, 21.384, 21.594, 21.901, 22.214, 24.774, 24.951, 26.125, 27.029, 33.461, 35.425 and 35.428; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 1 shown.

[0014] The present invention provides a B crystal form of the compound shown by formula (I),

[0015]

[0016] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.834, 13.544, 14.962, 21.722 and 30.594; preferably has characteristic peaks at 10.834, 13.544, 14.962, 21.722, 22.809, 23.822, 25.341, 26.812, 29.852 and 30.594; preferably has characteristic peaks at 6.766, 10.140, 10.834, 13.544, 14.962, 19.915, 20.580, 21.722, 22.809, 23.822, 25.341, 26.812, 27.231, 29.852 and 30.594; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 4 shown.

[0017] The present invention provides a C crystal form of the compound shown by formula (I),

[0018]

[0019] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.532, 11.778, 16.749, 19.015, and 21.131; preferably has characteristic peaks at 10.532, 11.778, 16.749, 17.300, 19.015, 21.131, 22.677, 23.402, 24.765, 26.993, and 28.564; preferably has characteristic peaks at 7.462, 10.095, 10.532, 11.778, 14.049, 14.686, 16.749, 17.300, 17.845, 19.015, 21.131, 22.677, 22.878, 23.402, 24.765, 26.993, and 28.564; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 7 shown.

[0020] The present invention provides a D crystal form of the compound shown by formula (I),

[0021]

[0022] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.667, 17.319, 19.284, 21.379, and 25.317; preferably has characteristic peaks at 10.667, 15.726, 17.319, 18.045, 19.284, 19.959, 21.379, 25.317, 27.193, and 28.917; preferably has characteristic peaks at 10.667, 15.726, 16.493, 17.319, 18.045, 19.284, 19.959, 21.379, 23.201, 24.135, 25.317, 26.476, 27.108, 27.193, and 28.917; preferably has characteristic peaks at 9.632, 10.295, 10.667, 11.977, 15.726, 16.493, 17.319, 18.045, 18.341, 19.284, 19.959, 21.379, 23.201, 24.135, 25.317, 25.572, 26.476, 27.108, 27.193, and 28.917; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 10 shown.

[0023] The present invention provides an E crystal form of the compound shown by formula (I),

[0024]

[0025] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 11.989, 16.248, 19.659, 19.973 and 23.014; preferably has characteristic peaks at 9.992, 11.989, 16.248, 19.414, 19.659, 19.973, 21.912, 22.448, 22.632, 23.014, 24.190 and 30.862; preferably has characteristic peaks at 9.992, 11.989, 16.248, 19.414, 19.659, 19.973, 21.175, 21.912, 22.448, 22.632, 23.014, 24.190, 25.095, 26.375, 30.862, 32.739, 34.159 and 37.010; preferably has characteristic peaks at 9.992, 11.989, 14.378, 16.248, 19.414, 19.659, 19.973, 21.175, 21.912, 22.448, 22.632, 23.014, 23.880, 24.190, 25.095, 26.375, 28.530, 30.862, 31.163, 32.174, 32.739, 34.159 and 37.010; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 13 shown.

[0026] The present invention provides an F crystal form of the compound shown by formula (I),

[0027]

[0028] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 16.465, 19.824, 21.955, 27.310 and 32.709; preferably has characteristic peaks at 14.843, 16.465, 19.824, 21.955, 24.365, 27.310, 27.740, 28.921, 30.428 and 32.709; preferably has characteristic peaks at 11.546, 13.195, 14.843, 16.465, 19.824, 21.955, 22.478, 23.209, 24.365, 27.310, 27.740, 28.921, 29.397, 30.428 and 32.709; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 16 shown.

[0029] The present invention further provides a G crystal form of the compound shown by formula (I),

[0030]

[0031] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 16.482, 19.844, 21.972, 27.762, 30.447 and 32.731; preferably has characteristic peaks at 14.858, 16.482, 19.844, 21.972, 24.388, 24.992, 27.333, 27.762, 28.938, 30.447 and 32.731; preferably has characteristic peaks at 11.539, 13.207, 14.858, 15.780, 16.482, 19.844, 21.972, 22.490, 24.388, 24.992, 27.333, 27.762, 28.938, 29.412, 30.447 and 32.731; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 19 shown.

[0032] Effects of the Invention

[0033] The A crystal form, B crystal form, C crystal form, D crystal form, E crystal form, F crystal form, and G crystal form of the compound shown in formula (I) provided by the present invention have advantages in terms of physicochemical properties, formulation processing performance, bioavailability, etc. For example, there are advantages in at least one of melting point, solubility, hygroscopicity, purification effect, stability, adhesiveness, compressibility, fluidity, in vitro and in vivo dissolution, bioavailability, etc. The free crystal form of the compound shown in formula (I) of the present invention has good physical and chemical stability, high crystal form yield prepared from the same starting material, and obvious advantages in terms of solubility, hygroscopicity, stability, mechanical stability, fluidity, compressibility, and adhesiveness, etc., providing a new and better choice for the development of RNA m6A regulation drugs, and having very important significance. Description of the Drawings

[0034] Figure 1 It is the XRPD spectrum of the A crystal form of the compound shown in formula (I).

[0035] Figure 2 It is the TGA spectrum of the A crystal form of the compound shown in formula (I).

[0036] Figure 3 It is the DSC spectrum of the A crystal form of the compound shown in formula (I).

[0037] Figure 4 It is the XRPD spectrum of the B crystal form of the compound shown in formula (I).

[0038] Figure 5 It is the TGA spectrum of the B crystal form of the compound shown in formula (I).

[0039] Figure 6DSC spectrum of Form B of the compound shown in formula (I).

[0040] Figure 7 XRPD spectrum of Form C of the compound shown in formula (I).

[0041] Figure 8 TGA spectrum of Form C of the compound shown in formula (I).

[0042] Figure 9 DSC spectrum of Form C of the compound shown in formula (I).

[0043] Figure 10 XRPD spectrum of Form D of the compound shown in formula (I).

[0044] Figure 11 TGA spectrum of Form D of the compound shown in formula (I).

[0045] Figure 12 DSC spectrum of Form D of the compound shown in formula (I).

[0046] Figure 13 XRPD spectrum of Form E of the compound shown in formula (I).

[0047] Figure 14 TGA spectrum of Form E of the compound shown in formula (I).

[0048] Figure 15 DSC spectrum of Form E of the compound shown in formula (I).

[0049] Figure 16 XRPD spectrum of Form F of the compound shown in formula (I).

[0050] Figure 17 TGA spectrum of Form F of the compound shown in formula (I).

[0051] Figure 18 DSC spectrum of Form F of the compound shown in formula (I).

[0052] Figure 19 XRPD spectrum of Form G of the compound shown in formula (I).

[0053] Figure 20 TGA spectrum of Form G of the compound shown in formula (I).

[0054] Figure 21 DSC spectrum of Form G of the compound shown in formula (I).

[0055] Figure 22Exemplary results of the mRNA expression levels of the glial cell differentiation markers KRT1, KRT10, Loricrin, and Involucrin measured by RT-PCR 24 hours after administering human recombinant HBEGF protein and the compound represented by formula (I) to keratinocytes HaCaT.

[0056] Figure 23 Exemplary results of the measurement of the stratum corneum thickness of the plantar skin of the rat paw in Example 4.

[0057] Figure 24 Exemplary histopathological results of the epithelial blisters, inflammatory cell infiltration, and skin tissue congestion in the plantar skin of the rat paw after tissue staining in Example 4.

[0058] Figure 25 Exemplary results of the histopathological scoring of the epithelial blisters, inflammatory cell infiltration, and skin tissue congestion in the plantar skin of the rat paw after tissue staining in Example 4. Detailed Description of the Invention

[0059] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following provides a detailed description by way of listing specific examples. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0060] The present invention provides an A crystal form of the compound represented by formula (I),

[0061]

[0062] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 12.673, 18.557, 24.951, 27.029, and 33.461; preferably has characteristic peaks at 6.760, 12.673, 13.507, 18.557, 19.315, 21.594, 21.901, 22.214, 24.951, 27.029, and 33.461; preferably has characteristic peaks at 6.760, 12.673, 13.507, 18.557, 19.315, 20.380, 21.594, 21.901, 22.214, 24.774, 24.951, 26.125, 27.029, 33.461, 35.425, and 35.428; preferably has characteristic peaks at 5.547, 6.760, 12.326, 12.673, 13.507, 14.870, 15.162, 18.557, 19.315, 20.380, 21.183, 21.384, 21.594, 21.901, 22.214, 24.774, 24.951, 26.125, 27.029, 33.461, 35.425, and 35.428; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 1 shown.

[0063] The present invention further provides a method for preparing the A crystal form of the compound shown in formula (I), comprising the steps of: mixing the compound shown in formula (I) with solvent 1, stirring, and filtering.

[0064] In certain embodiments, the solvent 1 is selected from alcohol solvents.

[0065] In certain embodiments, the solvent 1 is selected from C 1-4 alcohol.

[0066] In certain embodiments, the solvent 1 is selected from one or more of methanol, ethanol, and isopropanol.

[0067] In certain embodiments, the solvent 1 is selected from ethanol.

[0068] In certain embodiments, the preparation method of the present invention further includes steps such as centrifugation, washing, or drying.

[0069] The present invention further provides a B crystal form of the compound shown in formula (I),

[0070]

[0071] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 10.834, 13.544, 14.962, 21.722 and 30.594; preferably has characteristic peaks at 10.834, 13.544, 14.962, 21.722, 22.809, 23.822, 25.341, 26.812, 29.852 and 30.594; preferably has characteristic peaks at 6.766, 10.140, 10.834, 13.544, 14.962, 19.915, 20.580, 21.722, 22.809, 23.822, 25.341, 26.812, 27.231, 29.852 and 30.594; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 4 shown.

[0072] The present invention further provides a method for preparing the B crystal form of the compound represented by formula (I), comprising the steps of: dissolving the compound represented by formula (I) in solvent 2, adding antisolvent 3, and precipitating crystals.

[0073] In certain embodiments, the solvent 2 is selected from alcohol solvents.

[0074] In certain embodiments, the solvent 2 is selected from C 1-4 alcohol.

[0075] In certain embodiments, the solvent 2 is selected from one or more of methanol, ethanol and isopropanol.

[0076] In certain embodiments, the solvent 2 is selected from methanol.

[0077] In certain embodiments, the solvent 3 is selected from water.

[0078] In certain embodiments, the preparation method of the present invention further includes steps such as centrifugation, washing or drying.

[0079] The present invention further provides a C crystal form of the compound represented by formula (I),

[0080]

[0081] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 10.532, 11.778, 16.749, 19.015 and 21.131; preferably has characteristic peaks at 10.532, 11.778, 16.749, 17.300, 19.015, 21.131, 22.677, 23.402, 24.765, 26.993 and 28.564; preferably has characteristic peaks at 7.462, 10.095, 10.532, 11.778, 14.049, 14.686, 16.749, 17.300, 17.845, 19.015, 21.131, 22.677, 22.878, 23.402, 24.765, 26.993 and 28.564; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 7 shown.

[0082] The present invention further provides a method for preparing the C crystal form of the compound shown in formula (I), comprising the steps of: dissolving the compound shown in formula (I) in solvent 4, adding antisolvent 5, and precipitating crystals.

[0083] In certain embodiments, the solvent 4 is selected from ester solvents.

[0084] In certain embodiments, the solvent 4 is selected from one or more of ethyl acetate, methyl acetate, butyl acetate and isobutyl acetate

[0085] In certain embodiments, the solvent 4 is selected from ethyl acetate.

[0086] In certain embodiments, the solvent 5 is selected from alkane solvents.

[0087] In certain embodiments, the solvent 5 is selected from one or more of n-hexane, cyclohexane, pentane, dimethylpentane and n-heptane.

[0088] In certain embodiments, the solvent 5 is selected from n-heptane.

[0089] In certain embodiments, the preparation method of the present invention further includes steps such as centrifugation, washing or drying.

[0090] The present invention further provides a D crystal form of the compound shown in formula (I),

[0091]

[0092] The X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 10.667, 17.319, 19.284, 21.379 and 25.317; preferably has characteristic peaks at 10.667, 15.726, 17.319, 18.045, 19.284, 19.959, 21.379, 25.317, 27.193 and 28.917; preferably has characteristic peaks at 10.667, 15.726, 16.493, 17.319, 18.045, 19.284, 19.959, 21.379, 23.201, 24.135, 25.317, 26.476, 27.108, 27.193 and 28.917; preferably has characteristic peaks at 9.632, 10.295, 10.667, 11.977, 15.726, 16.493, 17.319, 18.045, 18.341, 19.284, 19.959, 21.379, 23.201, 24.135, 25.317, 25.572, 26.476, 27.108, 27.193 and 28.917; most preferably, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is as Figure 10 shown.

[0093] The present invention further provides a method for preparing the D crystal form of the compound represented by formula (I), comprising the steps of: mixing the compound represented by formula (I) with solvent 6, heating to dissolve, cooling, and filtering.

[0094] In certain embodiments, the solvent 6 is selected from ketone solvents.

[0095] In certain embodiments, the solvent 6 is selected from acetone, butanone, pentanone, hexanone and cyclohexanone.

[0096] In certain embodiments, the solvent 6 is selected from acetone.

[0097] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing or drying.

[0098] The present invention further provides an E crystal form of the compound represented by formula (I),

[0099]

[0100] The X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 11.989, 16.248, 19.659, 19.973 and 23.014; preferably has characteristic peaks at 9.992, 11.989, 16.248, 19.414, 19.659, 19.973, 21.912, 22.448, 22.632, 23.014, 24.190 and 30.862; preferably has characteristic peaks at 9.992, 11.989, 16.248, 19.414, 19.659, 19.973, 21.175, 21.912, 22.448, 22.632, 23.014, 24.190, 25.095, 26.375, 30.862, 32.739, 34.159 and 37.010; preferably has characteristic peaks at 9.992, 11.989, 14.378, 16.248, 19.414, 19.659, 19.973, 21.175, 21.912, 22.448, 22.632, 23.014, 23.880, 24.190, 25.095, 26.375, 28.530, 30.862, 31.163, 32.174, 32.739, 34.159 and 37.010; most preferably, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is as Figure 13 shown.

[0101] The present invention further provides a method for preparing the E crystal form of the compound represented by formula (I), comprising the steps of: mixing the compound represented by formula (I) with solvent 7, stirring, and filtering.

[0102] In certain embodiments, the solvent 7 is selected from ether solvents.

[0103] In certain embodiments, the solvent 7 is selected from one or more of diethyl ether, propyl ether, butyl ether, anisole, petroleum ether, isopropyl ether and 1,4-dioxane.

[0104] In certain embodiments, the solvent 7 is selected from 1,4-dioxane.

[0105] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing or drying.

[0106] The present invention further provides an F crystal form of the compound represented by formula (I),

[0107]

[0108] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 16.465, 19.824, 21.955, 27.310, and 32.709; preferably, it has characteristic peaks at 14.843, 16.465, 19.824, 21.955, 24.365, 27.310, 27.740, 28.921, 30.428, and 32.709; preferably, it has characteristic peaks at 11.546, 13.195, 14.843, 16.465, 19.824, 21.955, 22.478, 23.209, 24.365, 27.310, 27.740, 28.921, 29.397, 30.428, and 32.709; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 16 shown.

[0109] The present invention further provides a method for preparing the F crystal form of the compound represented by formula (I), comprising the steps of: mixing the compound represented by formula (I) with solvent 8, heating to dissolve, cooling, and filtering.

[0110] In certain embodiments, the solvent 8 is selected from alcohol solvents.

[0111] In certain embodiments, the solvent 8 is selected from C 1-4 alcohol.

[0112] In certain embodiments, the solvent 8 is selected from methanol, ethanol, and isopropanol.

[0113] In certain embodiments, the solvent 8 is selected from ethanol.

[0114] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing, or drying.

[0115] The present invention further provides a G crystal form of the compound represented by formula (I),

[0116]

[0117] The X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 16.482, 19.844, 21.972, 27.762, 30.447, and 32.731; preferably, it has characteristic peaks at 14.858, 16.482, 19.844, 21.972, 24.388, 24.992, 27.333, 27.762, 28.938, 30.447, and 32.731; preferably, it has characteristic peaks at 11.539, 13.207, 14.858, 15.780, 16.482, 19.844, 21.972, 22.490, 24.388, 24.992, 27.333, 27.762, 28.938, 29.412, 30.447, and 32.731; most preferably, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is as Figure 19 shown.

[0118] The present invention further provides a method for preparing the G crystal form of the compound represented by formula (I), which is characterized by including the steps of: mixing the compound represented by formula (I) with solvent 9 and solvent 10, stirring to dissolve, continuously adding solvent 10 and adding crystal seeds, stirring, continuously adding solvent 10, stirring, and filtering.

[0119] In certain embodiments, the solvent 9 is selected from ketone solvents.

[0120] In certain embodiments, the solvent 9 is selected from one or more of acetone, butanone, pentanone, hexanone, and cyclohexanone.

[0121] In certain embodiments, the solvent 9 is selected from acetone.

[0122] In certain embodiments, the solvent 10 is selected from water.

[0123] In certain embodiments, the preparation method of the present invention further includes steps such as centrifugation, washing, or drying.

[0124] The present invention further provides a pharmaceutical composition prepared from the A crystal form of the compound represented by formula (I) described above, or the B crystal form of the compound represented by formula (I) described above, or the C crystal form of the compound represented by formula (I) described above, or the D crystal form of the compound represented by formula (I) described above, or the E crystal form of the compound represented by formula (I) described above, or the F crystal form of the compound represented by formula (I) described above, or the G crystal form of the compound represented by formula (I) described above.

[0125] The present invention further provides a pharmaceutical composition, comprising the aforementioned crystalline form A of the compound represented by formula (I) or the aforementioned crystalline form B of the compound represented by formula (I) or the aforementioned crystalline form C of the compound represented by formula (I) or the aforementioned crystalline form D of the compound represented by formula (I) or the aforementioned crystalline form E of the compound represented by formula (I) or the aforementioned crystalline form F of the compound represented by formula (I) or the aforementioned crystalline form G of the compound represented by formula (I) and optionally a pharmaceutically acceptable excipient.

[0126] "Pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, mixture or solvent that is relevant to the dosage form or consistency of a pharmaceutical composition. Suitable pharmaceutically acceptable excipients will vary depending on the dosage form selected. In addition, pharmaceutically acceptable excipients may be selected based on their specific function in the composition.

[0127] In certain embodiments, the pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, penetration enhancers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity enhancers, antioxidants, preservatives, stabilizers, surfactants and buffers.

[0128] In certain embodiments, the pharmaceutical composition is a solid dosage form.

[0129] In certain embodiments, the pharmaceutical composition is a film or coating.

[0130] In certain embodiments, the pharmaceutical composition is an ointment.

[0131] In certain embodiments, the pharmaceutical combination is a plaster.

[0132] In certain embodiments, the pharmaceutical combination is a cream.

[0133] In certain embodiments, the solid dosage form is a capsule.

[0134] In certain embodiments, the pharmaceutical combination is a tablet.

[0135] In certain embodiments, the ointment contains the compound represented by formula (I) in an amount of 0 to 50% by weight.

[0136] The present invention further provides a method for preparing a pharmaceutical composition, which includes the step of mixing the A crystal form of the compound represented by the foregoing formula (I), or the B crystal form of the compound represented by the foregoing formula (I), or the C crystal form of the compound represented by the foregoing formula (I), or the D crystal form of the compound represented by the foregoing formula (I), or the E crystal form of the compound represented by the foregoing formula (I), or the F crystal form of the compound represented by the foregoing formula (I), or the G crystal form of the compound represented by the foregoing formula (I) with a pharmaceutically acceptable excipient.

[0137] The present invention further provides the use of the A crystal form of the compound represented by the foregoing formula (I), or the B crystal form of the compound represented by the foregoing formula (I), or the C crystal form of the compound represented by the foregoing formula (I), or the D crystal form of the compound represented by the foregoing formula (I), or the E crystal form of the compound represented by the foregoing formula (I), or the F crystal form of the compound represented by the foregoing formula (I), or the G crystal form of the compound represented by the foregoing formula (I), or the foregoing composition, or the composition prepared by the foregoing method in the preparation of a drug for treating and / or preventing diseases related to RNA m6A regulation.

[0138] In certain embodiments, the diseases related to RNA m6A regulation are endocrine and metabolic diseases, nervous system diseases, tumors, cardiovascular diseases, infections, immune system diseases, urogenital system diseases, skin and musculoskeletal diseases, respiratory system diseases, genetic diseases and deformities, digestive system diseases, oral and maxillofacial diseases, diseases related to blood vessels and lymphatic systems or pain.

[0139] In certain embodiments, the diseases related to RNA m6A regulation are hand-foot syndrome, hand-foot skin reaction, cancer, dermatological diseases.

[0140] The "2θ or 2θ angle" as used in the present invention refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.2 (including the case after rounding for numbers with more than 1 decimal place), and it can be -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0141] The precipitation methods as used in the present invention include but are not limited to stirring, cooling, volatilization, trituration, precipitation.

[0142] "Slurrying" is a commonly used term in the field of drug preparation, which generally refers to the mechanical or fluidized treatment of solid drug raw materials to disperse or suspend the solid drug in a solvent.

[0143] In some embodiments, the time for slurrying is 5h - 30h.

[0144] According to the description of the hygroscopicity characteristics and the definition of the hygroscopic weight gain in the "Guideline for Drug Hygroscopicity" in Part IV of the Chinese Pharmacopoeia 2020 Edition,

[0145] Deliquescence: Absorbing sufficient moisture to form a liquid;

[0146] Highly hygroscopic: The hygroscopic weight gain is not less than 15%;

[0147] Hygroscopic: The hygroscopic weight gain is less than 15% but not less than 2%;

[0148] Slightly hygroscopic: The hygroscopic weight gain is less than 2% but not less than 0.2%;

[0149] Non - hygroscopic or almost non - hygroscopic: The hygroscopic weight gain is less than 0.2%.

[0150] The "differential scanning calorimetry or DSC" described in the present invention refers to measuring the temperature difference and heat flow difference between the sample and the reference during the heating or constant - temperature process of the sample to characterize all physical and chemical changes related to thermal effects and obtain the phase - change information of the sample.

[0151] The drying temperature in the present invention is generally 25°C - 100°C, preferably 40°C - 70°C, and it can be dried at atmospheric pressure or under reduced pressure.

[0152] The method of the present invention will be described below through specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited by the scope of the following examples; the implementation conditions adopted in the examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually those in conventional experiments.

[0153] The explanations of the abbreviations used in the present invention are as follows:

[0154] XRPD: X - ray powder diffraction

[0155] DSC: Differential scanning calorimetry

[0156] TGA: Thermogravimetric analysis

[0157] DVS: Dynamic vapor sorption

[0158] HPLC: High - performance liquid chromatography

[0159] Detection instruments and methods

[0160] X-ray powder diffraction (XRPD)

[0161] The crystal form of the sample was analyzed using a Bruker D8 ADVANCE X-ray diffractometer. The 2θ scanning angle of the sample was 3° to 40°, the scanning step was 0.02°, and the scanning time per step was 0.12 s / step. The tube voltage and current were 40 kV and 40 mA, respectively. When preparing the sample, an appropriate amount of the sample was placed on the sample holder and flattened with tools such as a glass slide to ensure its surface was smooth and flat.

[0162] Thermogravimetric analysis (TGA)

[0163] The sample was analyzed using a TA Instruments TGA Discovery 5500. The sample was placed in an aluminum pan with the tare weight removed, and the system automatically weighed it. Then, under the protection of nitrogen, the sample was heated to the specified temperature at a rate of 10 °C / min.

[0164] Differential scanning calorimetry (DSC)

[0165] The sample was analyzed using a TA Instruments Discovery 2500. Weigh 0.5 - 1.5 mg of the sample and place it in the sample holder. Under the protection of nitrogen (50 ml / min), the sample was heated to the specified temperature at a rate of 10 °C / min.

[0166] Dynamic vapor sorption analysis (DVS)

[0167] The sample was analyzed using a ProUmid SPSx-1μAdvance. The test sample amount was approximately 5 - 50 mg. The temperature in the test chamber was controlled between 25 ± 1 °C, the relative humidity cycle was 40 - 95 - 0 - 95 - 40%, the step was 10%, the equilibrium was 240 minutes for each step, and the mass data was recorded every 20 s.

[0168] High performance liquid chromatography (HPLC)

[0169] The solubility and stability tests were carried out using an Agilent 1260 infinityII Binary Pump.

[0170] Example 1: Preparation of the compound shown in formula (I)

[0171]

[0172] Ethyl 3-bromo-2-oxocyclohexane-1-carboxylate (20 mg, 0.08 mmol) and 4-chloroaniline (25 mg, 0.2 mmol) were mixed and then heated to 150 °C. After reacting for 3 hours, the reaction solution was cooled to room temperature, diluted with 100 mL of dichloromethane, washed 3 times with 100 mL of 1N HCl and once with 100 mL of saturated NaHCO 3 washed, and the organic layer was dried over anhydrous Na 2 SO 4 and concentrated in vacuo. Purification by silica gel column chromatography gave ethyl 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylate (16 mg). 10 mg of ethyl 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylate (0.036 mmol) was dissolved in 10 mL of ethanol, 2 mL of 2M LiOH solution was added, and the reaction was stirred at room temperature for 1 hour. After rotary evaporation, it was diluted with 20 mL of water, the pH was adjusted to 2, and then extracted 3 times with 50 mL of dichloromethane. The combined organic phases were dried and concentrated, ammonia water was added, and the reaction was carried out at 60 °C for 24 hours. Purification by silica gel column chromatography gave a white solid, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (Compound 1, 7.6 mg). The (S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazolecarboxamide (the compound shown in formula (I)) was obtained by separation using a Chiralpak AD chiral column. The LCMS [M+H] + of the compound shown in formula (I): 249. 1 H-NMR (400 MHz, DMSO-d 6 ): δ 10.79 (s, 1H), 7.37 - 7.39 (m, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.08 (s, 1H), 6.98 - 7.01 (m, 1H), 3.64 - 3.67 (m, 1H), 2.58 - 2.61 (m, 2H), 1.66 - 2.04 (m, 4H).

[0173] Example 2: Effect of the compound shown in formula (I) on the mRNA methylation level of THP-1 cells

[0174] 8×10 6THP-1 cells were placed in a culture dish and added with DMEM medium, an equal volume of DMSO was added to the control group, 2 μM UZH1a (Shanghai Haoyuan Pharmaceutical Co., Ltd.) was added to the positive reference group 2, and 5 nM, 100 nM and 2 μM of the compound represented by formula (I) were added to sample groups A, B and C, respectively. They were placed in an incubator containing 5% carbon dioxide and cultured at 37°C for 24 hours. After washing with PBS, they were centrifuged at high speed, and the supernatant was discarded and RLT lysis buffer (QIAGEN) was added. After adding 400 μL of 70% ethanol to dilute the sample, 700 μL of the sample was taken out and total RNA was extracted with Trizol reagent (Sigma Aldrich), and the RNA sample was purified with Oligo (dT) magnetic beads. Take 200 ng of the purified mRNA sample, add 20 μL of nuclease P1 mixture (1 unit nuclease P1, 0.4 μL of 2M NaCl, 2 μL of 0.1M ZnCl 2 , 17.1 μL of PCR-grade water) at 37°C for 2 hours, and then 2 μL of NH 4 HCO 3 The solution and 1 unit of alkaline phosphatase were reacted at 37°C for 2 hours, 1.2M HCl was added for neutralization and centrifugation, 20 μL of supernatant was collected and the ion peak area (150.000549 / 150.000549) was detected by LC-MS. The inhibition rate of m6A was calculated as follows, and the results are shown in Table 1:

[0175] m6A inhibition rate = (peak area (control group) – peak area (sample group)) / peak area (control group) * 100%

[0176] Table 1. mA inhibition rate of control group and sample group AC.

[0177]

[0178]

[0179] UZH1a is a nanomolar inhibitor of the selective m6A methylase METTL3 (Moroz-Omori EV et al. ChemMedChem 2021, 2021, 16(19): 3035-3043). In THP-1 cells, the inhibition rate of UZH1a for RNA m6A is about 41.5%. The compound represented by formula (I) inhibits m6A in a dose-dependent manner, and the inhibition rate of RNA m6A is higher (56.8%) at the same concentration as UZH1a.

[0180] Example 3: Inhibitory effect of the compound represented by formula (I) on differentiation of keratinocytes HaCaT

[0181] Human keratinocytes HaCaT were cultured in DMEM medium containing 10% fetal bovine serum (Gibco, 10099141), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were seeded into 96-well plates at a density of 2×10 6 cells per square centimeter and incubated in an incubator containing 5% carbon dioxide at 37 °C for 24 hours. In control well 1, an equal volume of DMSO was added. In control well 2, 2.5 ng / mL human recombinant HBEGF protein (Abcam, ab205523) was added. In the sample wells, 2.5 ng / mL human recombinant HBEGF protein and the compound shown in formula (I) at the corresponding concentration were added. After continuous incubation for 24 hours, the cells were collected by high-speed centrifugation after washing with PBS. Total RNA was extracted using Trizol reagent (SigmaAldrich), and 1 μg of RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, AT311-03). 1.25 μL of primers (primer sequences are shown in Table 2), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, 172-5125), and an appropriate amount of DEPC ultrapure water were added to prepare a 20 μL reaction solution for RT-PCR reaction. After the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression levels of the keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin.

[0182] Table 2. Information on partial primer sequences

[0183]

[0184] As Figure 22 shown, after induction with human recombinant HBEGF protein, the mRNA expression levels of the HaCaT differentiation markers KRT1, KRT10, Loricrin, and Involucrin increased significantly. However, after adding the compound shown in formula (I), the mRNA expression levels of the HaCaT differentiation markers KRT1, KRT10, Loricrin, and Involucrin were significantly inhibited.

[0185] Example 4: Inhibitory effect of the compound shown in formula (I) on keratinocyte differentiation in a rat hand-foot skin reaction model

[0186] After SD rats (weighing approximately 200 g) were acclimated for one week, they were evenly grouped by weight, with 12 rats in each group. The modeling drugs (sorafenib, erlotinib, afatinib, and osimertinib) were separately dissolved in a solution containing 5% DMSO, 45% PEG400, and 50% H2 In the solution of O, the modeling drug was made up to the required concentration, and the rats were gavaged once a day with the dose shown in Table 3. One hour after gavage, an ointment containing the compound shown in formula (I) with different mass ratios of 0.05 g was evenly applied to the left paw of the rats, while the right paw was used as a self-control and smeared with the blank matrix, and the rats in the blank control group were not smeared with the drug. After applying the drug, the four limbs were fixed for another 2 hours, then the residual drug was wiped off with water, the fixation was released and the rats were allowed to move freely again. The modeling drug and the test compound ointment were both administered once a day. After continuous administration for 30 days, the rats were euthanized, and the skin tissue of the paw plantar was taken, fixed in 10% neutral formaldehyde and cut into 5-μm sections, dehydrated and embedded in paraffin.

[0187] Tissue staining: After dewaxing and rehydrating the skin tissue sections of the rat paw plantar mentioned above, they were stained in Hematoxylin solution for several minutes, washed and then soaked in 1% acidic alcohol until the sections faded to light blue-red. After washing with running water for 5 minutes, they were stained with Eosin for 2 - 3 minutes, washed to remove the excess dye, dehydrated, then made transparent with xylene for several minutes, and sealed with neutral resin. The stratum corneum was observed under an optical microscope, and the thickness of the epidermal stratum corneum was measured using Dmetrix software.

[0188] Immunohistochemical staining: After dewaxing and hydrating the skin tissue sections of the rat paw plantar mentioned above, they were incubated with 3% H 2 O 2 at room temperature for 30 minutes. After antigen retrieval, they were blocked with 10% goat serum for 30 minutes. The KRT1 antibody (Abcam, ab93652) and Loricrin antibody (Abcam, ab183646) were added dropwise and incubated overnight at 4°C. The HRP secondary antibody (ZSGB-BIO, PV-6001) and DAB kit (ZSG-BIO, ZLI9017) were added for color development, counterstained with hematoxylin, washed and sealed with neutral resin. The expression levels of KRT1 and Loricrin were observed under an optical microscope.

[0189] The criteria for determining the successful establishment of the rat model as described above are: (i) symptoms such as erythema, swelling, desquamation, ulcer or blister appear at the paw site; and / or (ii) the thickness of the stratum corneum in tissue staining is significantly higher than that of normal rats; and / or (iii) the markers such as KRT1, KRT5 and Loricrin are significantly increased. The incidence rate is calculated as the proportion of animals in each group that meet the criteria for successful establishment of the model, that is, incidence rate = (number of rats with successful modeling / total number of rats in the group) * 100%.

[0190] As described above, the pathological scoring criteria for tissue staining of rats are as follows: no blisters, score 0; 1 - 3 blisters, score 1; 4 - 6 blisters, score 2; 7 - 9 blisters, score 3; more than 10 blisters, score 4. If the inflammatory area accounts for less than 10% of the total area of the section, score 0; if the inflammatory area accounts for 10 - 25% of the total area of the section, score 1; if the inflammatory area accounts for 25 - 50% of the total area of the section, score 2; if the inflammatory area accounts for 50 - 75% of the total area of the section, score 3; if the inflammatory area accounts for more than 75% of the total area of the section, score 4. If the congested area accounts for less than 10% of the total area of the section, score 0; if the congested area accounts for 10 - 25% of the total area of the section, score 1; if the congested area accounts for 25 - 50% of the total area of the section, score 2; if the congested area accounts for 50 - 75% of the total area of the section, score 3; if the congested area accounts for more than 75% of the total area of the section, score 4. The scoring uses a double - blind scoring system. After the statistics of each group of data are completed, it is presented in the form of Mean + SEM (N = 12).

[0191] Table 3. Drug dosage and experimental results in the rat model

[0192]

[0193]

[0194] As shown in Table 3, sorafenib, erlotinib, afatinib, and osimertinib were respectively administered by gavage once a day to SD rats at doses of 100 mg / kg, 70 mg / kg, 50 mg / kg, and 60 mg / kg, and the success rates of inducing hand - foot skin reaction models were 75%, 83.3%, 66.7%, and 75% respectively. The compound shown in formula (I) can significantly reduce the incidence of hand - foot skin reaction at the drug - applied site at low doses (for example, the mass content of the compound in the ointment is 1%), and can significantly reduce the incidence of hand - foot skin reaction in all paw pads of rats at high mass contents (for example, the mass content of the compound in the ointment is 3% and 10%).

[0195] As Figure 23 shown in the measurement results of the stratum corneum thickness, in the rats successfully modeled with sorafenib, erlotinib, afatinib, and osimertinib, the stratum corneum thickness of the plantar skin of the paw is significantly higher than that of normal rats. The compound shown in formula (I) can effectively reduce the stratum corneum thickness of the plantar skin of the paw in the modeled rats.

[0196] As Figure 24As shown in the tissue staining results, the skin toxicity caused by the modeling drug can form blisters under the subcutaneous and epidermal layers of the rat paw sole, with inflammatory cell infiltration in the dermis and hyperemia symptoms in the skin tissue. The compound shown in formula (I) can effectively improve the formation of blisters under the subcutaneous and epidermal layers caused by the above-mentioned modeling drug, inhibit the inflammatory cell infiltration in the dermis, and significantly improve the skin hyperemia phenomenon.

[0197] As Figure 25 shown in the pathological scoring results, the modeling drug can cause blisters to form under the subcutaneous and epidermal layers of the rat paw sole, with inflammatory cell infiltration in the dermis and hyperemia symptoms in the skin tissue. The compound shown in formula (I) can effectively improve the formation of blisters under the subcutaneous and epidermal layers caused by the above-mentioned modeling drug, inhibit the inflammatory cell infiltration in the dermis, and significantly improve the skin hyperemia phenomenon.

[0198] The immunohistochemical staining results also show that in rats successfully modeled with sorafenib, erlotinib, afatinib, and osimertinib, KRT1 and Loricrin are significantly increased in the tissue. However, with the addition of the compound shown in formula (I), KRT1 and Loricrin are significantly decreased in the tissue. In summary, the compound shown in formula (I) can effectively inhibit the differentiation of keratinocytes in vitro and in vivo and has the potential to treat hyperkeratosis-related diseases.

[0199] Example 5: Preparation of the A crystal form of the compound shown in formula (I)

[0200] Weigh 50 mg of the compound shown in formula (I) and add ethanol (0.4 mL). Suspend it at 25 °C with a magnetic stirrer at a rate of 300 - 400 rpm for 1 week. Centrifuge and filter the obtained suspension through a 0.45 μm nylon membrane centrifuge tube at 14,000 rpm to obtain a solid. After X-ray powder diffraction detection, this product is the A crystal form, and the XRPD spectrum is as Figure 1 shown, and the characteristic peak positions are shown in Table 4. The DSC spectrum shows endothermic peaks at T onset 155.80 °C, 163.89 °C, and 164.69 °C.

[0201] Table 4: XRPD diffraction peak data of the A crystal form of the compound shown in formula (I)

[0202]

[0203] Example 6: Preparation of the B crystal form of the compound shown in formula (I)

[0204] Weigh 50 mg of the compound shown in formula (I) and add it to methanol (0.4 mL). Dissolve it thoroughly at ambient temperature. The resulting solution or suspension is filtered through a 0.45-μm nylon membrane syringe filter to obtain a clear solution. Slowly add water (1.6 mL) to the obtained clear solution. Centrifuge and filter the resulting suspension through a 0.45-μm nylon membrane centrifuge tube at 14,000 rpm to obtain a solid. After X-ray powder diffraction detection, this product is in Form B, and the XRPD pattern is as shown in Figure 4 shown, and the characteristic peak positions are shown in Table 5. The DSC pattern shows that the melting point T onset is 166.07 °C; the endothermic peak T onset is 171.93 °C.

[0205] Table 5: XRPD diffraction peak data of Form B of the compound shown in formula (I)

[0206]

[0207] Example 7: Preparation of Form C of the compound shown in formula (I)

[0208] Weigh 50 mg of the compound shown in formula (I) and add it to ethyl acetate (0.8 mL). Dissolve it thoroughly at ambient temperature. The resulting solution or suspension is filtered through a 0.45-μm nylon membrane syringe filter to obtain a clear solution. Slowly add n-heptane (4 mL) to the obtained clear solution. Centrifuge and filter the resulting suspension through a 0.45-μm nylon membrane centrifuge tube at 14,000 rpm to obtain a solid. After X-ray powder diffraction detection, this product is in Form C, and the XRPD pattern is as shown in Figure 7 shown, and the characteristic peak positions are shown in Table 6. The DSC pattern shows that the solvent is removed at about 53 °C; there are two unresolved melting peaks, and the melting point T onset is 150.71 °C.

[0209] Table 6: XRPD diffraction peak data of Form C of the compound shown in formula (I)

[0210]

[0211]

[0212] Example 8: Preparation of Form D of the compound shown in formula (I)

[0213] Weigh about 50 mg of the compound shown in formula (I), add acetone (0.6 mL), and fully dissolve it at 50 °C. The resulting solution or thin suspension is passed through a 0.45 μm nylon membrane syringe filter to obtain a clear solution. The obtained clear solution is cooled to 5 °C at a cooling rate of 0.1 °C / min. The resulting suspension is centrifugally filtered through a 0.45 μm nylon membrane centrifuge tube at 14,000 rpm to obtain a solid. After X-ray powder diffraction detection, the product is in D crystal form, and the XRPD pattern is as shown in Figure 10 , and the characteristic peak positions are shown in Table 7. The DSC pattern shows that desolvation occurs between approximately 68 °C and 130 °C (endothermic peak T onset 103.18 °C; exothermic peak T onset 112.36 °C; endothermic peak T onset 117.81 °C); endothermic peak T onset 164.75 °C; exothermic peak T onset 168.22 °C; endothermic peak T onset 171.36 °C).

[0214] Table 7: XRPD diffraction peak data of the D crystal form of the compound shown in formula (I)

[0215]

[0216]

[0217] Example 9: Preparation of the E crystal form of the compound shown in formula (I)

[0218] Weigh 50 mg of the compound shown in formula (I), add 1,4-dioxane (0.4 mL), and suspend it at 25 °C at a rate of 300 - 400 rpm under magnetic stirring for 1 week. The resulting suspension is centrifugally filtered through a 0.45 μm nylon membrane centrifuge tube at 14,000 rpm to obtain a solid. After X-ray powder diffraction detection, the product is in E crystal form, and the XRPD pattern is as shown in Figure 13 , and the characteristic peak positions are shown in Table 8. The DSC pattern shows that desolvation occurs between approximately 53 °C and 130 °C (exothermic peak T onset 97.80 °C; endothermic peak T onset 105.62 °C); endothermic peak T onset 161.98 °C; two unresolved endothermic peaks T onset 169.77 °C.

[0219] Table 8: XRPD diffraction peak data of the E crystal form of the compound shown in formula (I)

[0220]

[0221]

[0222] Example 10: Preparation of Form F of the Compound Shown by Formula (I)

[0223] Weigh about 50 mg of the compound shown by Formula (I), add ethanol (0.5 mL), and fully dissolve it at 50 °C. The resulting solution or thin suspension is passed through a 0.45 μm nylon membrane syringe filter to obtain a clear solution. The obtained clear solution is cooled to 5 °C at a cooling rate of 0.1 °C / min. The resulting suspension is centrifugally filtered through a 0.45 μm nylon membrane centrifuge tube at 14,000 rpm to obtain a solid. After detection by X-ray powder diffraction, this product is Form F of the compound shown by Formula (I), and the XRPD pattern is as Figure 16 shown, and the characteristic peak positions are shown in Table 9. The DSC pattern shows that the melting point T onset is 187.84 °C

[0224] Table 9: XRPD Diffraction Peak Data of Form F of the Compound Shown by Formula (I)

[0225]

[0226] Example 11: Preparation of Form F of the Compound Shown by Formula (I)

[0227] Weigh about 300 mg of the compound shown by Formula (I), add 7.6 mL of acetonitrile, and fully dissolve it at 50 °C. The resulting solution is passed through a 0.45 μm nylon membrane syringe filter to obtain a clear solution. The obtained clear solution is kept at 50 °C for 30 min, and then slowly cooled from 50 °C to 5 °C at a rate of 0.1 °C / min. After the start of cooling, about 5 mg of Form F crystals are added as seeds to the saturated solution, and after stirring for 2 min, a suspension is obtained. The resulting suspension is kept stirring at 5 °C for one day. The resulting suspension is centrifuged at 5 °C through a 0.45 μm nylon membrane centrifuge tube at 14,000 rpm, placed in an oven at 25 °C, and vacuum dried for 2 h to obtain 141 mg of dry white powder Form F crystals, with a yield of 47%. After detection by X-ray powder diffraction, this product is Form F of the compound shown by Formula (I).

[0228] Example 12: Preparation of Form G of the Compound Shown by Formula (I)

[0229] Take 100 g of the compound shown in formula (I), add 1 L of acetone and 300 mL of water, control the temperature at about 20 - 30 °C, stir to dissolve. After it becomes clear, add 200 mL of water dropwise to the clear solution until the seeding point (the reaction solution becomes turbid), add seeds (crystal form F), and stir at 20 - 30 °C for 1 hour. Subsequently, continue to add 1 L of water, stir for 2 hours, filter, wash the filter cake with 500 mL of water to obtain a wet product, and dry it under vacuum at 40 - 50 °C to obtain the product. After detection by X-ray powder diffraction, this product is the G crystal form of the compound shown in formula (I), and the XRPD spectrum is as Figure 19 shown, and the characteristic peak positions are shown in Table 10. The DSC spectrum shows that the endothermic peak T onset is 144.92 °C; the melting point T onset is 187.21 °C.

[0230] Table 10: XRPD diffraction peak data of the G crystal form of the compound shown in formula (I)

[0231]

[0232] Example 13: Suspension competition test of crystal forms A, B, F, and G of the compound shown in formula (I)

[0233] To study the relative stability relationship among crystal forms A, B, F, and G of the compound shown in formula (I), competitive suspension experiments were carried out in different solvents.

[0234] Weigh about 2 mg of crystal form A, 2 mg of crystal form B, 2 mg of crystal form F, and 2 mg of crystal form G, and add them to 0.2 mL of the saturated solution of the selected solvent. The obtained suspensions were suspended for 3 days at 5 °C, 25 °C, and 50 °C respectively. The obtained suspensions were centrifuged, and the solid part was detected by XRPD.

[0235] Table 11: Results of the suspension competition test of crystal forms A, B, F, and G

[0236]

[0237] Through the suspension competition pulping experiments of crystal forms A, B, F, and G, it was found that they were all converted into crystal form F in isopropanol, isopropyl acetate, and acetonitrile. This indicates that crystal form F is more stable than crystal forms A, B, and G in the range of 5 °C to 50 °C.

[0238] Example 14: Stability of crystal form F of the compound shown in formula (I)

[0239] Weigh a certain amount of the F crystal form of the compound shown in formula (I) and place it in an open container, and leave it in 25°C / 92% RH for 1 week. Weigh a certain amount of the F crystal form of the compound shown in formula (I) and place it in a sealed container, and leave it in 60°C for 1 week. Perform XRPD and HPLC tests on the stability samples under these pressure conditions and observe whether there is a color change in the samples. The results are shown in Table 12.

[0240] Table 12: Results of the stability test of the F crystal form of the compound shown in formula (I)

[0241]

[0242] It can be seen from the results of the stability test shown in Table 12 that in terms of physical stability, the crystal form of the F crystal form did not change under the two test conditions. In terms of chemical stability, the F crystal form hardly degraded under these two conditions. There was no obvious color change in the F crystal form sample during the stability experiment.

[0243] Example 15: Hygroscopicity of the F crystal form of the compound shown in formula (I)

[0244] The water absorption and dehydration behaviors of the F crystal form of the compound shown in formula (I) were studied by DVS test at 25°C. The DVS cycle was 40 - 0 - 95 - 0 - 40% RH. And XRPD test was performed on the sample after DVS test to judge whether there was a crystal form transformation. The results are shown in Table 13.

[0245] Table 13: Results of the water adsorption and desorption experiments of the F crystal form of the compound shown in formula (I)

[0246]

[0247] It should be understood that the above examples are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present invention, various deformations and changes can also be made on the basis of the above examples. Similarly, the technical features of the above examples can also be arbitrarily combined to form other embodiments of the present invention that may not be clearly described. Therefore, the above examples only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. The A crystal form of the compound represented by formula (I), characterized in that, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 12.673, 18.557, 24.951, 27.029 and 33.461; preferably has characteristic peaks at 6.760, 12.673, 13.507, 18.557, 19.315, 21.594, 21.901, 22.214, 24.951, 27.029 and 33.461; preferably has characteristic peaks at 6.760, 12.673, 13.507, 18.557, 19.315, 20.380, 21.594, 21.901, 22.214, 24.774, 24.951, 26.125, 27.029, 33.461, 35.425 and 35.428; preferably has characteristic peaks at 5.547, 6.760, 12.326, 12.673, 13.507, 14.870, 15.162, 18.557, 19.315, 20.380, 21.183, 21.384, 21.594, 21.901, 22.214, 24.774, 24.951, 26.125, 27.029, 33.461, 35.425 and 35.428; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 1.

2. The B crystal form of the compound represented by formula (I), characterized in that, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.834, 13.544, 14.962, 21.722 and 30.594; preferably has characteristic peaks at 10.834, 13.544, 14.962, 21.722, 22.809, 23.822, 25.341, 26.812, 29.852 and 30.594; preferably has characteristic peaks at 6.766, 10.140, 10.834, 13.544, 14.962, 19.915, 20.580, 21.722, 22.809, 23.822, 25.341, 26.812, 27.231, 29.852 and 30.594; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 4.

3. The C crystal form of the compound represented by formula (I), characterized in that, The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.532, 11.778, 16.749, 19.015 and 21.131; preferably has characteristic peaks at 10.532, 11.778, 16.749, 17.300, 19.015, 21.131, 22.677, 23.402, 24.765, 26.993 and 28.564; preferably has characteristic peaks at 7.462, 10.095, 10.532, 11.778, 14.049, 14.686, 16.749, 17.300, 17.845, 19.015, 21.131, 22.677, 22.878, 23.402, 24.765, 26.993 and 28.564; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 7.

4. The D crystal form of the compound represented by formula (I), which is characterized in that the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.667, 17.319, 19.284, 21.379 and 25.317; preferably has characteristic peaks at 10.667, 15.726, 17.319, 18.045, 19.284, 19.959, 21.379, 25.317, 27.193 and 28.917; preferably has characteristic peaks at 10.667, 15.726, 16.493, 17.319, 18.045, 19.284, 19.959, 21.379, 23.201, 24.135, 25.317, 26.476, 27.108, 27.193 and 28.917; preferably has characteristic peaks at 9.632, 10.295, 10.667, 11.977, 15.726, 16.493, 17.319, 18.045, 18.341, 19.284, 19.959, 21.379, 23.201, 24.135, 25.317, 25.572, 26.476, 27.108, 27.193 and 28.917; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 10.

5. The E crystal form of the compound represented by formula (I), which is characterized in that The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 11.989, 16.248, 19.659, 19.973 and 23.014; preferably has characteristic peaks at 9.992, 11.989, 16.248, 19.414, 19.659, 19.973, 21.912, 22.448, 22.632, 23.014, 24.190 and 30.862; preferably has characteristic peaks at 9.992, 11.989, 16.248, 19.414, 19.659, 19.973, 21.175, 21.912, 22.448, 22.632, 23.014, 24.190, 25.095, 26.375, 30.862, 32.739, 34.159 and 37.010; preferably has characteristic peaks at 9.992, 11.989, 14.378, 16.248, 19.414, 19.659, 19.973, 21.175, 21.912, 22.448, 22.632, 23.014, 23.880, 24.190, 25.095, 26.375, 28.530, 30.862, 31.163, 32.174, 32.739, 34.159 and 37.010; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 13.

6. The F crystal form of the compound represented by formula (I), characterized in that, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 16.465, 19.824, 21.955, 27.310 and 32.709; preferably has characteristic peaks at 14.843, 16.465, 19.824, 21.955, 24.365, 27.310, 27.740, 28.921, 30.428 and 32.709; preferably has characteristic peaks at 11.546, 13.195, 14.843, 16.465, 19.824, 21.955, 22.478, 23.209, 24.365, 27.310, 27.740, 28.921, 29.397, 30.428 and 32.709; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 16.

7. The G crystal form of the compound represented by formula (I), characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 16.482, 19.844, 21.972, 27.762, 30.447, and 32.731; preferably has characteristic peaks at 14.858, 16.482, 19.844, 21.972, 24.388, 24.992, 27.333, 27.762, 28.938, 30.447, and 32.731; preferably has characteristic peaks at 11.539, 13.207, 14.858, 15.780, 16.482, 19.844, 21.972, 22.490, 24.388, 24.992, 27.333, 27.762, 28.938, 29.412, 30.447, and 32.731; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as shown in Figure 19.

8. The crystalline form according to any one of claims 1-7, wherein, the error range of the 2θ angle is ±0.

20.

9. The preparation method of the A crystalline form of the compound represented by formula (I) according to claim 1, wherein, it includes the steps of: mixing the compound represented by formula (I) with solvent 1, stirring, and filtering; Preferably, the solvent 1 is selected from alcohol solvents, preferably C 1-4 alcohol, more preferably one or more of methanol, ethanol and isopropanol, and most preferably ethanol.

10. The preparation method of the B crystalline form of the compound represented by formula (I) according to claim 2, wherein, it includes the steps of: dissolving the compound represented by formula (I) with solvent 2, adding antisolvent 3, and precipitating crystals; Preferably, the solvent 2 is selected from alcohol solvents, preferably C 1-4 alcohol, more preferably one or more of methanol, ethanol and isopropanol, and most preferably methanol; Preferably, the solvent 3 is selected from water.

11. The preparation method of the C crystalline form of the compound represented by formula (I) according to claim 3, wherein, it includes the steps of: dissolving the compound represented by formula (I) with solvent 4, adding antisolvent 5, and precipitating crystals; Preferably, the solvent 4 is selected from ester solvents, preferably one or more of ethyl acetate, methyl acetate, butyl acetate, and isobutyl acetate, more preferably ethyl acetate; Preferably, the solvent 5 is selected from alkane solvents, preferably one or more of n-hexane, cyclohexane, pentane, dimethylpentane, and n-heptane, more preferably n-heptane.

12. The preparation method of the D crystalline form of the compound represented by formula (I) according to claim 4, wherein, it includes the steps of: mixing the compound represented by formula (I) with solvent 6, heating to dissolve, cooling, and filtering; Preferably, the solvent 6 is selected from ketone solvents, preferably one or more of acetone, butanone, pentanone, hexanone, and cyclohexanone, more preferably acetone.

13. The preparation method of the E crystalline form of the compound represented by formula (I) according to claim 5, wherein, it includes the steps of: mixing the compound represented by formula (I) with solvent 7, stirring, and filtering; Preferably, the solvent 7 is selected from ether solvents, preferably one or more of diethyl ether, propyl ether, butyl ether, anisole, petroleum ether, isopropyl ether, and 1,4-dioxane, more preferably 1,4-dioxane.

14. The preparation method of the F crystalline form of the compound represented by formula (I) according to claim 6, wherein, it includes the steps of: mixing the compound represented by formula (I) with solvent 8, heating to dissolve, cooling, and filtering; Preferably, the solvent 8 is selected from alcohol solvents, preferably C 1-4 alcohol, more preferably one or more of methanol, ethanol and isopropanol, and most preferably ethanol.

15. A method for preparing the G crystal form of the compound represented by formula (I) according to claim 7, characterized in that, it comprises the steps of: mixing the compound represented by formula (I) with solvent 9 and solvent 10, stirring to dissolve, continuously adding solvent 10 and adding seed crystals, stirring, continuously adding solvent 10, stirring, and filtering; Preferably, the solvent 9 is selected from ketone solvents, preferably one or more of acetone, butanone, pentanone, hexanone and cyclohexanone, more preferably acetone; Preferably, the solvent 10 is selected from water.

16. A pharmaceutical composition prepared from the A crystal form of the compound represented by formula (I) according to claim 1, or the B crystal form of the compound represented by formula (I) according to claim 2, or the C crystal form of the compound represented by formula (I) according to claim 3, or the D crystal form of the compound represented by formula (I) according to claim 4, or the E crystal form of the compound represented by formula (I) according to claim 5, or the F crystal form of the compound represented by formula (I) according to claim 6, or the G crystal form of the compound represented by formula (I) according to claim 7.

17. A pharmaceutical composition comprising the A crystal form of the compound represented by formula (I) according to claim 1, or the B crystal form of the compound represented by formula (I) according to claim 2, or the C crystal form of the compound represented by formula (I) according to claim 3, or the D crystal form of the compound represented by formula (I) according to claim 4, or the E crystal form of the compound represented by formula (I) according to claim 5, or the F crystal form of the compound represented by formula (I) according to claim 6, or the G crystal form of the compound represented by formula (I) according to claim 7 and a pharmaceutically acceptable excipient selected arbitrarily.

18. A method for preparing a pharmaceutical composition, comprising the step of mixing the A crystal form of the compound represented by formula (I) according to claim 1, or the B crystal form of the compound represented by formula (I) according to claim 2, or the C crystal form of the compound represented by formula (I) according to claim 3, or the D crystal form of the compound represented by formula (I) according to claim 4, or the E crystal form of the compound represented by formula (I) according to claim 5, or the F crystal form of the compound represented by formula (I) according to claim 6, or the G crystal form of the compound represented by formula (I) according to claim 7 with a pharmaceutically acceptable excipient.

19. Use of the A crystal form of the compound represented by formula (I) according to claim 1, or the B crystal form of the compound represented by formula (I) according to claim 2, or the C crystal form of the compound represented by formula (I) according to claim 3, or the D crystal form of the compound represented by formula (I) according to claim 4, or the E crystal form of the compound represented by formula (I) according to claim 5, or the F crystal form of the compound represented by formula (I) according to claim 6, or the G crystal form of the compound represented by formula (I) according to claim 7, or the composition according to claim 16 or 17, or the composition prepared by the method according to claim 18 in the preparation of a drug for treating and / or preventing diseases related to RNA m6A regulation.

20. The application according to claim 19, wherein the RNA m6A regulation-related disease is an endocrine and metabolic disease, a nervous system disease, a tumor, a cardiovascular disease, an infection, an immune system disease, a urogenital system disease, a skin and musculoskeletal disease, a respiratory system disease, a genetic disease and malformation, a digestive system disease, an oral and maxillofacial disease, a vascular and lymphatic system-related disease or pain, preferably hand-foot syndrome, hand-foot skin reaction, cancer, a dermatological disease.

Images