Co-crystal form of RNA m6A regulating agent and preparation method and application thereof

Abstract

The invention provides a crystal form of a co-crystal of an RNAm6A regulating agent as shown in a formula (I) as well as a preparation method and application of the crystal form. The crystal form of the eutectic crystal of the RNAm6A regulating agent disclosed by the invention has the beneficial effects of low hygroscopicity and good stability. The preparation method of the eutectic crystal form of the RNA m6A 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 cocrystal form of an RNA m6A regulator, a preparation method thereof, and applications 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, in clinical practice, severe hand-foot syndrome and hand-foot skin reaction are often relieved only by topical skin care or even require drug withdrawal. Existing treatment methods only relieve the symptoms but do not address the root cause, severely limiting 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 multiple 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 erase 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 tumor tissues of patients with acral melanoma are significantly higher than those in 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, m6A-specific binding proteins YTHDF1 and HNRNPA2B1 can be used as novel biomarkers (Li T.D., et al. Cancer Cell 2020, 20, 239). In 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 cocrystal form of an RNA m6A regulator represented by formula (I), its preparation method, and applications.

[0009]

[0010] Solutions for Solving the Problems

[0011] The present invention provides a pharmaceutical cocrystal formed by the combination of a compound represented by formula (I) and a cocrystal former, wherein the cocrystal former is selected from nicotinamide, isonicotinamide, L-proline, and glycolic acid.

[0012]

[0013] The pharmaceutical cocrystal formed by the compound represented by formula (I) and isonicotinamide.

[0014]

[0015] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566; preferably has characteristic peaks at 9.386, 11.847, 13.641, 14.019, 15.097, 18.822, 21.196, 23.762, 24.300, 26.566 and 35.936; preferably has characteristic peaks at 6.139, 9.386, 11.847, 13.641, 14.019, 15.097, 16.746, 17.556, 17.893, 18.822, 21.196, 23.762, 24.300, 26.354, 26.566, 33.296 and 35.936; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 1 shown.

[0016] The present invention provides a pharmaceutical cocrystal formed by the compound shown in formula (I) and glycolic acid,

[0017]

[0018] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125; preferably has characteristic peaks at 10.588, 12.575, 17.645, 18.231, 19.706, 21.232, 21.527, 23.125, 24.776, 25.300 and 27.608; preferably has characteristic peaks at 10.588, 12.575, 14.928, 17.645, 18.231, 18.444, 19.706, 21.232, 21.527, 23.125, 24.776, 25.300, 27.608, 28.937, 30.376 and 33.925; preferably has characteristic peaks at 10.588, 12.575, 14.928, 17.645, 18.231, 18.444, 19.706, 20.417, 21.232, 21.527, 23.125, 24.776, 25.300, 26.840, 27.608, 28.937, 30.116, 30.376, 32.070, 33.925, 35.159, 35.368 and 36.010; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 4 shown.

[0019] The present invention provides a pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline,

[0020]

[0021] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably has characteristic peaks at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.113, 20.643, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 15.763, 16.443, 18.719, 19.762, 20.113, 20.643, 21.217, 21.383, 21.899, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 7 shown.

[0022] The present invention provides a pharmaceutical cocrystal formed by a compound represented by formula (I) and nicotinamide,

[0023]

[0024] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573; preferably has characteristic peaks at 9.860, 13.311, 17.017, 19.752, 20.856, 23.638, 24.147, 26.573, 27.028 and 32.820; preferably has characteristic peaks at 9.860, 13.311, 17.017, 19.752, 20.856, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.820 and 36.741; preferably has characteristic peaks at 9.860, 11.529, 13.311, 14.036, 16.007, 17.017, 17.503, 19.752, 20.856, 21.661, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.284, 32.820 and 36.741; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 10 shown.

[0025] Effect of the invention

[0026] The drug cocrystals formed by the compound shown in formula (I) of the present invention and isoniazide, the drug cocrystals formed by the compound shown in formula (I) and glycolic acid, the drug cocrystals formed by the compound shown in formula (I) and L-proline, and the drug cocrystals formed by the compound shown in formula (I) and nicotinamide have advantages in terms of physical and chemical properties, formulation processing performance and 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, biological effectiveness, etc. The physical and chemical stability of the drug cocrystals formed by combining the compound shown in formula (I) of the present invention and the cocrystal former is good, the crystal form yield obtained by using the same starting material is high, and there are 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 RNAm6A regulator drugs, which has very important significance. Description of the drawings

[0027] Figure 1 It is the XRPD spectrum of the drug cocrystal formed by the compound shown in formula (I) and isoniazide.

[0028] Figure 2 It is the TGA spectrum of the drug cocrystal formed by the compound shown in formula (I) and isoniazide.

[0029] Figure 3 The DSC spectrum of the drug cocrystal formed by the compound shown in formula (I) and isonicotinamide.

[0030] Figure 4 The XRPD spectrum of the drug cocrystal formed by the compound shown in formula (I) and glycolic acid.

[0031] Figure 5 The TGA spectrum of the drug cocrystal formed by the compound shown in formula (I) and glycolic acid.

[0032] Figure 6 The DSC spectrum of the drug cocrystal formed by the compound shown in formula (I) and glycolic acid.

[0033] Figure 7 The XRPD spectrum of the drug cocrystal formed by the compound shown in formula (I) and L-proline.

[0034] Figure 8 The TGA spectrum of the drug cocrystal formed by the compound shown in formula (I) and L-proline.

[0035] Figure 9 The DSC spectrum of the drug cocrystal formed by the compound shown in formula (I) and L-proline.

[0036] Figure 10 The XRPD spectrum of the drug cocrystal formed by the compound shown in formula (I) and nicotinamide.

[0037] Figure 11 The TGA spectrum of the drug cocrystal formed by the compound shown in formula (I) and nicotinamide.

[0038] Figure 12 The DSC spectrum of the drug cocrystal formed by the compound shown in formula (I) and nicotinamide.

[0039] Figure 13 Exemplary 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 shown in formula (I) to keratinocytes HaCaT.

[0040] Figure 14 Exemplary results of the measurement of the stratum corneum thickness of the plantar skin of the rat paw in Example 4.

[0041] Figure 15 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.

[0042] Figure 16Exemplary results of histopathological scoring of epithelial blisters, inflammatory cell infiltration, and skin tissue congestion in the plantar skin of rat paws after tissue staining in Example 4. Detailed Description of the Invention

[0043] 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 enumerating 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 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.

[0044] The present invention provides a pharmaceutical cocrystal formed by a compound represented by formula (I) and isoniazide.

[0045]

[0046] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196, and 26.566; preferably has characteristic peaks at 9.386, 11.847, 13.641, 14.019, 15.097, 18.822, 21.196, 23.762, 24.300, 26.566, and 35.936; preferably has characteristic peaks at 6.139, 9.386, 11.847, 13.641, 14.019, 15.097, 16.746, 17.556, 17.893, 18.822, 21.196, 23.762, 24.300, 26.354, 26.566, 33.296, and 35.936; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 1 shown.

[0047] The present invention further provides a preparation method of a pharmaceutical cocrystal formed by a compound represented by formula (I) and isoniazide, including the steps of: mixing the compound represented by formula (I) and isoniazide, adding solvent 1, stirring, and filtering.

[0048] In some embodiments, the solvent 1 is selected from alcohol solvents.

[0049] In some embodiments, the solvent 1 is selected from C 1-4 alcohol.

[0050] In some embodiments, the solvent 1 is selected from one or more of methanol, ethanol, and isopropanol.

[0051] In some embodiments, the solvent 1 is selected from ethanol.

[0052] In certain embodiments, the preparation method of the present invention further includes steps such as centrifugation, washing, or drying.

[0053] The present invention further provides a pharmaceutical cocrystal formed by the compound shown in formula (I) and glycolic acid,

[0054]

[0055] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.588, 17.645, 21.232, 21.527, and 23.125; preferably has characteristic peaks at 10.588, 12.575, 17.645, 18.231, 19.706, 21.232, 21.527, 23.125, 24.776, 25.300, and 27.608; preferably has characteristic peaks at 10.588, 12.575, 14.928, 17.645, 18.231, 18.444, 19.706, 21.232, 21.527, 23.125, 24.776, 25.300, 27.608, 28.937, 30.376, and 33.925; preferably has characteristic peaks at 10.588, 12.575, 14.928, 17.645, 18.231, 18.444, 19.706, 20.417, 21.232, 21.527, 23.125, 24.776, 25.300, 26.840, 27.608, 28.937, 30.116, 30.376, 32.070, 33.925, 35.159, 35.368, and 36.010; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 4 shown.

[0056] The present invention further provides a preparation method of a pharmaceutical cocrystal formed by the compound shown in formula (I) and glycolic acid, including the steps of: mixing the compound shown in formula (I) and glycolic acid, adding solvent 2, stirring, and filtering.

[0057] In certain embodiments, the solvent 1 is selected from ester solvents.

[0058] In certain embodiments, the solvent 1 is selected from one or more of ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, and isobutyl acetate.

[0059] In certain embodiments, the solvent 1 is selected from isopropyl acetate.

[0060] In certain embodiments, the preparation method of the present invention further includes steps such as centrifugation, washing, or drying.

[0061] The present invention further provides a pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline,

[0062]

[0063] The X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably has characteristic peaks at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.113, 20.643, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 15.763, 16.443, 18.719, 19.762, 20.113, 20.643, 21.217, 21.383, 21.899, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as Figure 7 shown.

[0064] The present invention further provides a preparation method of a pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline, comprising the steps of: mixing the compound shown in formula (I) and L-proline, adding solvent 3, stirring, and filtering.

[0065] In certain embodiments, the solvent 3 is selected from alcohol solvents.

[0066] In certain embodiments, the solvent 3 is selected from C 1-4 alcohol.

[0067] In certain embodiments, the solvent 3 is selected from one or more of methanol, ethanol and isopropanol.

[0068] In certain embodiments, the solvent 3 is selected from ethanol.

[0069] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing or drying.

[0070] The present invention further provides a pharmaceutical cocrystal formed by the compound shown in formula (I) and nicotinamide,

[0071]

[0072] The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ has characteristic peaks at 13.311, 17.017, 19.752, 23.638, and 26.573; preferably has characteristic peaks at 9.860, 13.311, 17.017, 19.752, 20.856, 23.638, 24.147, 26.573, 27.028, and 32.820; preferably has characteristic peaks at 9.860, 13.311, 17.017, 19.752, 20.856, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.820, and 36.741; preferably has characteristic peaks at 9.860, 11.529, 13.311, 14.036, 16.007, 17.017, 17.503, 19.752, 20.856, 21.661, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.284, 32.820, and 36.741; most preferably, the X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is as Figure 10 shown.

[0073] The present invention further provides a method for preparing a pharmaceutical cocrystal formed by the compound shown in formula (I) and nicotinamide, comprising the steps of: mixing the compound shown in formula (I) and nicotinamide, adding solvent 4, stirring, and filtering.

[0074] In certain embodiments, the solvent 1 is selected from nitrile solvents.

[0075] In certain embodiments, the solvent 1 is selected from one or more of acetonitrile, trimethylacetonitrile, propionitrile, and valeronitrile.

[0076] In certain embodiments, the solvent 1 is selected from acetonitrile.

[0077] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing, or drying.

[0078] The present invention further provides a pharmaceutical cocrystal formed by binding the compound shown in the foregoing formula (I) and a cocrystal formation product, or a pharmaceutical cocrystal formed by the compound shown in the foregoing formula (I) and isoniazid, or a pharmaceutical cocrystal formed by the compound shown in the foregoing formula (I) and glycolic acid, or a pharmaceutical cocrystal formed by the compound shown in the foregoing formula (I) and L-proline, or a pharmaceutical composition prepared from the pharmaceutical cocrystal formed by the compound shown in the foregoing formula (I) and nicotinamide.

[0079] The present invention further provides a pharmaceutical composition, which contains a drug co-crystal formed by combining the compound shown in the foregoing formula (I) with a co-crystal former, or a drug co-crystal formed by the compound shown in the foregoing formula (I) and isoniazid, or a drug co-crystal formed by the compound shown in the foregoing formula (I) and glycolic acid, or a drug co-crystal formed by the compound shown in the foregoing formula (I) and L-proline, or a drug co-crystal formed by the compound shown in the foregoing formula (I) and nicotinamide, and an excipient arbitrarily selected from pharmaceutically acceptable excipients.

[0080] "Pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, mixture or solvent related to the consistency of a dosage form or a pharmaceutical composition. Suitable pharmaceutically acceptable excipients will vary depending on the selected dosage form. In addition, pharmaceutically acceptable excipients can be selected according to their specific functions in the composition.

[0081] 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, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, thickening agents, antioxidants, preservatives, stabilizers, surfactants and buffers.

[0082] In certain embodiments, the pharmaceutical composition is a solid preparation.

[0083] In certain embodiments, the pharmaceutical composition is a film preparation and a film coating preparation.

[0084] In certain embodiments, the pharmaceutical composition is an ointment.

[0085] In certain embodiments, the pharmaceutical composition is a plaster.

[0086] In certain embodiments, the pharmaceutical composition is a cream.

[0087] In certain embodiments, the solid preparation is a capsule.

[0088] In certain embodiments, the pharmaceutical composition is a tablet.

[0089] In certain embodiments, the co-crystal of the compound shown in formula (I) in the ointment is 0 to 50% by weight.

[0090] The present invention further provides a method for preparing a pharmaceutical composition, which includes the step of mixing a pharmaceutical cocrystal formed by combining the compound represented by the foregoing formula (I) and a cocrystal former, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and isoniazid, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and glycolic acid, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and L-proline, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and nicotinamide with a pharmaceutically acceptable excipient.

[0091] The present invention further provides the use of a pharmaceutical cocrystal formed by combining the compound represented by the foregoing formula (I) and a cocrystal former, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and isoniazid, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and glycolic acid, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and L-proline, or a pharmaceutical cocrystal formed by the compound represented by the foregoing formula (I) and nicotinamide, or the foregoing composition, or a composition prepared by the foregoing method in the preparation of a drug for treating and / or preventing diseases related to RNA m6A regulation.

[0092] In certain embodiments, the diseases related to 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.

[0093] In certain embodiments, the diseases related to m6A regulation are hand-foot syndrome, hand-foot skin reaction, cancer, dermatological diseases.

[0094] 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.

[0095] The precipitation methods described in the present invention include but are not limited to stirring, cooling, volatilization, trituration, precipitation.

[0096] "Pulping" is a commonly used term in the field of drug preparation, usually referring to the mechanical or fluidized treatment of solid drug raw materials to disperse or suspend the solid drug in a solvent.

[0097] In some embodiments, the pulping time is 5h - 30h.

[0098] According to the description of hygroscopicity characteristics and the definition of hygroscopic weight gain in "Guideline for Drug Hygroscopicity" in Part IV of the Chinese Pharmacopoeia 2020 Edition,

[0099] Deliquescence: Absorbing sufficient moisture to form a liquid;

[0100] Highly hygroscopic: Hygroscopic weight gain not less than 15%;

[0101] Hygroscopic: Hygroscopic weight gain less than 15% but not less than 2%;

[0102] Slightly hygroscopic: Hygroscopic weight gain less than 2% but not less than 0.2%;

[0103] Non - or almost non - hygroscopic: Hygroscopic weight gain less than 0.2%.

[0104] The "differential scanning calorimetry or DSC" mentioned 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.

[0105] 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.

[0106] 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.

[0107] The explanations of the abbreviations used in the present invention are as follows:

[0108] XRPD: X - ray powder diffraction

[0109] DSC: Differential scanning calorimetry

[0110] TGA: Thermogravimetric analysis

[0111] DVS: Dynamic vapor sorption

[0112] HPLC: High - performance liquid chromatography

[0113] Detection instruments and methods

[0114] X-ray powder diffraction (XRPD)

[0115] The crystal form analysis of the sample was carried out 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 carrier plate and flattened with tools such as a glass slide to ensure its surface was smooth and flat.

[0116] Thermogravimetric analysis (TGA)

[0117] The sample was analyzed using a TA Instruments TGADiscovery 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.

[0118] Differential scanning calorimetry (DSC)

[0119] The sample was analyzed using a TA Instruments Discovery 2500. Weighed 0.5 - 1.5 mg of the sample and placed it in the sample carrier plate. Under the protection of nitrogen (50 ml / min), the sample was heated to the specified temperature at a rate of 10 °C / min.

[0120] Dynamic vapor sorption analysis (DVS)

[0121] The sample was analyzed using a ProUmid SPSx-1μAdvance. The test sample amount was about 5 - 50 mg. The temperature of 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.

[0122] High performance liquid chromatography (HPLC)

[0123] The solubility and stability tests were carried out using an Agilent 1260 infinityII Binary Pump.

[0124] Example 1: Preparation of the compound shown in formula (I)

[0125]

[0126] 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 washing, 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 mixture 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). (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).

[0127] Example 2: Effect of the compound shown in formula (I) on the mRNA methylation level of THP-1 cells

[0128] 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 solution (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 (SigmaAldrich), 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:

[0129] m6A inhibition rate = (peak area (control group) – peak area (sample group)) / peak area (control group) * 100%

[0130] Table 1. mA inhibition rate of control group and sample group AC.

[0131]

[0132]

[0133] 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 RNAm6A is about 41.5%. The compound represented by formula (I) inhibits m6A in a dose-dependent manner, and the inhibition rate of RNAm6A is higher (56.8%) at the same concentration as UZH1a.

[0134] Example 3: Inhibitory effect of the compound represented by formula (I) on differentiation of keratinocytes HaCaT

[0135] 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 in a 96-well plate at a density of 2*10 6 cells per square centimeter and incubated in a 5% carbon dioxide incubator at 37 °C for 24 hours. DMSO of equal volume was added to control well 1, and 2.5 ng / mL human recombinant HBEGF protein (Abcam, ab205523) was added to control well 2. The test compound of formula (I) at the corresponding concentration and 2.5 ng / mL human recombinant HBEGF protein were added to the sample wells. 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). A 20 μL reaction solution was prepared by adding 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, and RT-PCR was performed. 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.

[0136] Table 2. Information on partial primer sequences

[0137]

[0138] As Figure 13 shown, after induction with human recombinant HBEGF protein, the mRNA expression levels of the differentiation markers KRT1, KRT10, Loricrin, and Involucrin in HaCaT increased significantly. However, after adding the compound of formula (I), the mRNA expression levels of the differentiation markers KRT1, KRT10, Loricrin, and Involucrin in HaCaT were significantly inhibited.

[0139] Example 4: Inhibitory effect of the compound of formula (I) on keratinocyte differentiation in a rat hand-foot skin reaction model After one week of acclimation, Sprague-Dawley rats (body weight approximately 200 g) were evenly grouped by body weight, with 12 rats in each group. The modeling drugs (sorafenib, erlotinib, afatinib, and osimertinib) were dissolved in a solution containing 5% DMSO, 45% PEG400, and 50% H 2In the solution of O, the modeling drug was made up to the required concentration and administered by gavage once a day at the doses shown in Table 3. One hour after gavage administration, an ointment containing the compound shown in formula (I) with different mass ratios was evenly applied to the left paw of the rats, while the right paw was used as a self-control and applied with a blank matrix, and the rats in the blank control group were not medicated. 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 removed 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 sole was taken, fixed in 10% neutral formaldehyde and cut into 5-μm sections, dehydrated and embedded in paraffin.

[0140] Tissue staining: After dewaxing and rehydrating the skin tissue sections of the rat paw soles mentioned above, they were stained in a 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.

[0141] Immunohistochemical staining: After dewaxing and hydrating the skin tissue sections of the rat paw soles mentioned above, they were incubated with 3% H 2 O 2 for 30 minutes at room temperature. After antigen retrieval, they were blocked with 10% goat serum for 30 minutes. The KRT1 antibody (Abcam, ab93652) and the loricrin antibody (Abcam, ab183646) were added dropwise and incubated overnight at 4°C. Then, the HRP secondary antibody (ZSGB-BIO, PV-6001) and the 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.

[0142] The criteria for determining the successful establishment of the above-mentioned rat model were as follows: (i) symptoms such as erythema, swelling, desquamation, ulcer or blister appeared at the paw site; and / or (ii) the thickness of the stratum corneum was significantly higher than that of normal rats in tissue staining; and / or (iii) the markers such as KRT1, KRT5 and loricrin were significantly increased. The incidence was calculated as the proportion of animals in each group that met the above criteria for successful modeling, that is, incidence = (number of successfully modeled rats / total number of rats in the group) * 100%.

[0143] As described above, the pathological scoring criteria for tissue staining of rats are as follows: no blisters, score 0; the number of blisters is 1 - 3, score 1; the number of blisters is 4 - 6, score 2; the number of blisters is 7 - 9, score 3; the number of blisters is more than 10, score 4. The inflammatory area accounts for less than 10% of the total area of the section, score 0; the inflammatory area accounts for 10 - 25% of the total area of the section, score 1; the inflammatory area accounts for 25 - 50% of the total area of the section, score 2; the inflammatory area accounts for 50 - 75% of the total area of the section, score 3; the inflammatory area accounts for more than 75% of the total area of the section, score 4. The congestion area accounts for less than 10% of the total area of the section, score 0; the congestion area accounts for 10 - 25% of the total area of the section, score 1; the congestion area accounts for 25 - 50% of the total area of the section, score 2; the congestion area accounts for 50 - 75% of the total area of the section, score 3; the congestion area accounts for more than 75% of the total area of the section, score 4. The scoring adopts 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).

[0144] Table 3. Drug dosage and experimental results in the rat model

[0145]

[0146]

[0147] As shown in Table 3, sorafenib, erlotinib, afatinib, and osimertinib were 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 respectively, and the success rates of establishing the hand - foot skin reaction model were 75%, 83.3%, 66.7%, and 75% respectively. The compound shown in formula (I) can significantly reduce the incidence of hand - foot skin reactions at the application site at low doses (for example, the mass content of the compound in the ointment is 1%), and it can significantly reduce the incidence of hand - foot skin reactions 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%).

[0148] As Figure 14 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 paw plantar skin was significantly higher than that of normal rats. The compound shown in formula (I) can effectively reduce the stratum corneum thickness of the paw plantar skin of the modeled rats.

[0149] As Figure 15As 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 plantar, with inflammatory cell infiltration in the dermis layer 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 layer, and significantly improve the skin hyperemia phenomenon.

[0150] As Figure 16 As shown in the pathological scoring results, the modeling drug can cause blisters to form under the subcutaneous and epidermal layers of the rat paw plantar, with inflammatory cell infiltration in the dermis layer 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 layer, and significantly improve the skin hyperemia phenomenon.

[0151] The immunohistochemical staining results also show that in the 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 diseases related to hyperkeratosis.

[0152] Example 5: Preparation of a drug cocrystal formed by the compound shown in formula (I) and isoniazide

[0153] Weigh about 20 mg of the compound shown in formula (I) and 10.2 mg of isoniazide and place them in a 2 mL glass bottle. Add ethanol (0.2 mL) to obtain a suspension. The obtained sample is stirred under suspension at 5 °C for 3 days. The obtained suspension is centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid is vacuum dried at 25 °C for 4 hours to obtain the product. After X-ray powder diffraction detection, this product is a drug cocrystal formed by the compound shown in formula (I) and isoniazide, and the XRPD spectrum is as Figure 1 shown, and its characteristic peak positions are shown in Table 4. The DSC spectrum shows that the melting T onset is 160.45 °C.

[0154] Table 4: XRPD diffraction peak data of the drug cocrystal formed by the compound shown in formula (I) and isoniazide

[0155]

[0156] Example 6: Preparation of a drug cocrystal formed by the compound shown in formula (I) and glycolic acid

[0157] Weigh approximately 20 mg of the compound shown in formula (I), place it together with 6.3 mg of glycolic acid in a 2 mL glass bottle, add isopropyl acetate (0.2 mL) to obtain a suspension. The obtained sample is stirred while suspended at 5 °C for 3 days. The obtained suspension is centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid is dried in vacuo at 25 °C for 4 hours to obtain the product. After detection by X-ray powder diffraction, this product is a pharmaceutical cocrystal formed by the compound shown in formula (I) and glycolic acid, 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 T onset @135.68 °C.

[0158] Table 5: XRPD diffraction peak data of the pharmaceutical cocrystal formed by the compound shown in formula (I) and glycolic acid

[0159]

[0160]

[0161] Example 7: Preparation of a pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline

[0162] Weigh approximately 20 mg of the compound shown in formula (I), place it together with 9.7 mg of L-proline in a 2 mL glass bottle, add ethanol (0.25 mL) to obtain a suspension. The obtained sample is stirred while suspended at 5 °C for 3 days. The obtained suspension is centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid is dried in vacuo at 25 °C for 4 hours to obtain the product. After detection by X-ray powder diffraction, this product is a pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline, 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 melting T onset @183.55 °C.

[0163] Table 6: XRPD diffraction peak data of the pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline

[0164]

[0165]

[0166] Example 8: Preparation of a pharmaceutical cocrystal formed by the compound shown in formula (I) and L-proline

[0167] Weigh approximately 300 mg of the compound shown in formula (I) and 145.8 mg of L-proline (1.0 equivalent) and place them in a 20 mL glass bottle. Add 3.75 mL of ethanol, and stir at 5 °C for 2 min to obtain a suspension. Add approximately 5 mg of the seed crystal of the drug co-crystal formed by the compound shown in formula (I) and L-proline to the above suspension. Then add 3 mL of ethanol to the above suspension and stir at 5 °C for 4 days. Collect the solid part by centrifugation, and dry the obtained solid part under vacuum at room temperature for approximately 2.5 hours. A total of 250 mg of white powder is obtained, and the yield is 56%. It is detected by X-ray powder diffraction that the product is the drug co-crystal formed by the compound shown in formula (I) and L-proline.

[0168] Example 9: Preparation of the drug co-crystal formed by the compound shown in formula (I) and nicotinamide

[0169] Weigh approximately 20 mg of the compound shown in formula (I) and 10.3 mg of nicotinamide and place them in a 2 mL glass bottle. Add acetonitrile (0.25 mL) to obtain a suspension. The obtained sample is suspended and stirred at 5 °C for 3 days. Centrifuge the obtained suspension through a 0.45 μm nylon filter membrane at 14,000 rpm, and dry the obtained solid under vacuum at 25 °C for 4 hours to obtain the product. It is detected by X-ray powder diffraction that the product is the drug co-crystal formed by the compound shown in formula (I) and nicotinamide, and the XRPD spectrum is as Figure 10 shown, and the characteristic peak positions are shown in Table 7. The DSC spectrum shows that the melting T onset @ 156.75 °C.

[0170] Table 7: XRPD diffraction peak data of the drug co-crystal formed by the compound shown in formula (I) and nicotinamide

[0171]

[0172]

[0173] Example 10: Preparation of the drug co-crystal formed by the compound shown in formula (I) and nicotinamide

[0174] Weigh approximately 300 mg of the compound shown in formula (I) and 154.6 mg of nicotinamide (1.0 equivalent) and place them in a 20 mL glass bottle. Add 3.75 mL of acetonitrile, and stir at 5 °C for 2 min to obtain a suspension. Add approximately 5 mg of the seed crystal of the drug co-crystal formed by the compound shown in formula (I) and nicotinamide to the above suspension. Then add 2 mL of acetonitrile to the above suspension and stir at 5 °C for 4 days. Collect the solid part by centrifugation, and dry the obtained solid part under vacuum at room temperature for approximately 2.5 hours. A total of 365 mg of white powder is obtained, and the yield is 80%. It is detected by X-ray powder diffraction that the product is the drug co-crystal formed by the compound shown in formula (I) and nicotinamide.

[0175] Example 11: Stability Tests of the Pharmaceutical Cocrystals Formed by the Compound Shown in Formula (I) with L - Proline and the Pharmaceutical Cocrystals Formed by the Compound Shown in Formula (I) with Nicotinamide

[0176] Open containers containing the pharmaceutical cocrystals formed by the compound shown in formula (I) with L - proline and the pharmaceutical cocrystals formed by the compound shown in formula (I) with nicotinamide were respectively placed at 25°C / 92.5% RH for one week. The containers containing the pharmaceutical cocrystals formed by the compound shown in formula (I) with L - proline and the pharmaceutical cocrystals formed by the compound shown in formula (I) with nicotinamide were respectively placed in a sealed container at 60°C for one week. After evaluating the solid stability by XRPD and HPLC, the samples were characterized, and the color change of the samples was observed. The conclusions are shown in Table 8.

[0177] Table 8: Solid Stability of the Pharmaceutical Cocrystals Formed by the Compound Shown in Formula (I)

[0178]

[0179] As can be seen from Table 8, after the pharmaceutical cocrystals formed by the compound shown in formula (I) with L - proline and the pharmaceutical cocrystals formed by the compound shown in formula (I) with nicotinamide were placed in an open container at 25°C / 92% RH for one week or in a sealed container at 60°C for one week, the purity did not decrease significantly. However, the pharmaceutical cocrystals formed by the compound shown in formula (I) with L - proline partially dissociated into the free crystal form and L - proline at 25°C / 92.5% RH. The pharmaceutical cocrystals formed by the compound shown in formula (I) with nicotinamide were relatively stable under both conditions.

[0180] Example 12: Solubility Tests of the Pharmaceutical Cocrystals Formed by the Compound Shown in Formula (I) with L - Proline and the Pharmaceutical Cocrystals Formed by the Compound Shown in Formula (I) with Nicotinamide

[0181] Precisely weigh 15.1 mg of the pharmaceutical cocrystal formed by the compound shown in formula (I) with L - proline and 14.9 mg of the pharmaceutical cocrystal formed by the compound shown in formula (I) with nicotinamide and place them respectively in 20 - mL glass bottles. Add 5 mL of solvent to each. The mass of the compound shown in formula (I) in the weighed cocrystals is equivalent to 10 mg of the free anhydrous crystal form. Stir the obtained suspension at 37°C at a speed of 400 rpm, and sample at 0.5 hour and 2 hours respectively. Centrifuge the samples at 14,000 rpm at 37°C for 5 min. Determine the supernatant concentration by HPLC and measure the pH value of the supernatant by a pH meter. Detect whether there is a crystal form change in the residual solid (wet product) after 2 hours by XRPD. The experimental results are shown in Table 9.

[0182] Table 9: Solid Solubility of the Pharmaceutical Cocrystals Formed by the Compound Shown in Formula (I)

[0183]

[0184] As can be seen from Table 9, the drug cocrystals formed by the compound shown in formula (I) with L-proline and the drug cocrystals formed with nicotinamide are transformed into the free crystal form of the compound shown in formula (I) in four different solvents. In addition, the drug cocrystals formed with L-proline and the drug cocrystals formed with nicotinamide have relatively high solubility in the remaining solvents, and the solubility of the drug cocrystals formed by the compound shown in formula (I) with nicotinamide after dissolving in the four solvents for 2 h is higher than that of the drug cocrystals formed with L-proline.

[0185] Example 13: Hygroscopicity test of the drug cocrystal formed by the compound shown in formula (I) with L-proline and the drug cocrystal formed by the compound shown in formula (I) with nicotinamide

[0186] The water absorption and dehydration behaviors of the drug cocrystal formed by the compound shown in formula (I) with L-proline and the drug cocrystal formed by the compound shown in formula (I) with nicotinamide were studied by DVS test at 25°C. The DVS cycle was 40 - 0 - 95 - 0 - 40% RH. The samples after DVS test were detected by XRPD to judge whether crystal form transformation occurred, and the results are shown in Table 10.

[0187] Table 10: Hygroscopicity test of the drug cocrystal formed by the compound shown in formula (I)

[0188]

[0189] As can be seen from the results in Table 10, the drug cocrystal formed by the compound shown in formula (I) with L-proline has slight hygroscopicity between 40% RH and 80% RH, and the weight gain due to moisture absorption is about 0.4%; it has extremely high hygroscopicity between 80% RH and 95% RH, and the weight gain due to moisture absorption is about 84.0%. After DVS test, this cocrystal is partially dissociated into the free crystal form and L-proline. The drug cocrystal formed by the compound shown in formula (I) with nicotinamide has slight hygroscopicity, and its weight gain due to moisture absorption is about 0.2% between 40% RH and 95% RH at 25°C. After DVS test, no crystal form transformation occurred.

[0190] 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 combined arbitrarily 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. A pharmaceutical cocrystal formed by the combination of the compound represented by formula (I) and a cocrystal former, wherein the cocrystal former is selected from nicotinamide, isonicotinamide, L-proline, and glycolic acid, 2. The pharmaceutical cocrystal according to claim 1, characterized in that the chemical ratio of the compound represented by formula (I) to the cocrystal former is 1:0.5 to 1:3, preferably 1:0.5, 1:1, 1:2 or 1:3, and most preferably 1:1 or 1:

2.

3. The pharmaceutical cocrystal according to claim 1, wherein the cocrystal former is nicotinamide.

4. A pharmaceutical cocrystal formed by the compound represented by formula (I) and isonicotinamide, characterized in that the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566; preferably has characteristic peaks at 9.386, 11.847, 13.641, 14.019, 15.097, 18.822, 21.196, 23.762, 24.300, 26.566 and 35.936; preferably has characteristic peaks at 6.139, 9.386, 11.847, 13.641, 14.019, 15.097, 16.746, 17.556, 17.893, 18.822, 21.196, 23.762, 24.300, 26.354, 26.566, 33.296 and 35.936; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 1.

5. A pharmaceutical cocrystal formed by the compound represented by formula (I) and glycolic acid, characterized in that the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125; preferably has characteristic peaks at 10.588, 12.575, 17.645, 18.231, 19.706, 21.232, 21.527, 23.125, 24.776, 25.300 and 27.608; preferably has characteristic peaks at 10.588, 12.575, 14.928, 17.645, 18.231, 18.444, 19.706, 21.232, 21.527, 23.125, 24.776, 25.300, 27.608, 28.937, 30.376 and 33.925; preferably has characteristic peaks at 10.588, 12.575, 14.928, 17.645, 18.231, 18.444, 19.706, 20.417, 21.232, 21.527, 23.125, 24.776, 25.300, 26.840, 27.608, 28.937, 30.116, 30.376, 32.070, 33.925, 35.159, 35.368 and 36.010; most preferably, the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 4.

6. A pharmaceutical cocrystal formed by the compound of formula (I) and L - proline, characterized in that, the X - ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably has characteristic peaks at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.113, 20.643, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 15.763, 16.443, 18.719, 19.762, 20.113, 20.643, 21.217, 21.383, 21.899, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; most preferably, the X - ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 7.

7. A pharmaceutical cocrystal formed by the compound of formula (I) and nicotinamide, characterized in that, the X - ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573; preferably has characteristic peaks at 9.860, 13.311, 17.017, 19.752, 20.856, 23.638, 24.147, 26.573, 27.028 and 32.820; preferably has characteristic peaks at 9.860, 13.311, 17.017, 19.752, 20.856, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.820 and 36.741; preferably has characteristic peaks at 9.860, 11.529, 13.311, 14.036, 16.007, 17.017, 17.503, 19.752, 20.856, 21.661, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.284, 32.820 and 36.741; most preferably, the X - ray powder diffraction pattern expressed in terms of diffraction angle 2θ is as shown in Figure 10.

8. The pharmaceutical cocrystal according to any one of claims 1 - 7, characterized in that, the error range of the 2θ angle is ±0.

20.

9. A method for preparing a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 4 and isoniazide, characterized in that, Comprising the steps of: mixing the compound shown in formula (I) with isoniazid, adding solvent 1, stirring, and filtering; preferably, the solvent 1 is selected from one or more of alcohols, esters, nitriles, and water, preferably an alcohol solvent, more preferably a C 1-4 alcohol, more preferably one or more of methanol, ethanol, and isopropanol, and most preferably ethanol.

10. A method for preparing a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 5 and glycolic acid, characterized in that, comprises the steps of: mixing the compound of formula (I) with glycolic acid, adding solvent 2, stirring, and filtering; preferably, the solvent 2 is selected from one or more of alcohols, esters, nitriles and water, preferably an ester solvent, more preferably one or more of ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate and isobutyl acetate, and most preferably isopropyl acetate.

11. A method for preparing a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 6 and L-proline, characterized in that, It includes the steps of: mixing the compound shown in formula (I) with L-proline, adding solvent 3, stirring, and filtering; preferably, the solvent 3 is selected from one or more of alcohols, esters, nitriles, and water, preferably an alcohol solvent, more preferably a C 1-4 alcohol, more preferably one or more of methanol, ethanol, and isopropanol, and most preferably ethanol.

12. A method for preparing a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 7 and nicotinamide, characterized in that, comprises the steps of: mixing the compound of formula (I) with nicotinamide, adding solvent 4, stirring, and filtering; preferably, the solvent 4 is selected from one or more of alcohols, esters, nitriles and water, preferably a nitrile solvent, more preferably one or more of acetonitrile, trimethylacetonitrile, propionitrile and valeronitrile, and most preferably acetonitrile.

13. A pharmaceutical composition prepared from a pharmaceutical cocrystal formed by combining the compound of formula (I) as claimed in any one of claims 1-3 and a cocrystal former, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 4 and isoniazide, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 5 and glycolic acid, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 6 and L-proline, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 7 and nicotinamide.

14. A pharmaceutical composition comprising a pharmaceutical cocrystal formed by combining the compound of formula (I) as claimed in any one of claims 1-3 and a cocrystal former, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 4 and isoniazide, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 5 and glycolic acid, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 6 and L-proline, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 7 and nicotinamide, and a pharmaceutically acceptable excipient selected at will.

15. A method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutical cocrystal formed by combining the compound of formula (I) as claimed in any one of claims 1-3 and a cocrystal former, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 4 and isoniazide, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 5 and glycolic acid, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 6 and L-proline, or a pharmaceutical cocrystal formed by the compound of formula (I) as claimed in claim 7 and nicotinamide with a pharmaceutically acceptable excipient.

16. The use of a pharmaceutical co-crystal formed by combining the compound represented by formula (I) as described in any one of claims 1 to 3 and a co-crystal former, or a pharmaceutical co-crystal formed by the compound represented by formula (I) as described in claim 4 and isoniazide, or a pharmaceutical co-crystal formed by the compound represented by formula (I) as described in claim 5 and glycolic acid, or a pharmaceutical co-crystal formed by the compound represented by formula (I) as described in claim 6 and L-proline, or a pharmaceutical co-crystal formed by the compound represented by formula (I) as described in claim 7 and nicotinamide, or the composition as described in claim 13 or 14, or the composition prepared by the method as described in claim 15 in the preparation of a drug for treating and / or preventing diseases related to RNA m6A regulation.

17. The use according to claim 16, wherein 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 system, or pain, preferably hand-foot syndrome, hand-foot skin reaction, cancer, dermatological diseases.

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