Co-crystal form of RNA m6a modulator and preparation method therefor and use thereof

Abstract

Provided are a co-crystal form of an RNA m6A modulator represented by formula (I) and a preparation method therefor and a use thereof. The crystal form of a co-crystal of the RNA m6A modulator has the beneficial effects of low hygroscopicity and good stability. The preparation method for the crystal form of the co-crystal of the RNA m6A modulator involves a simple process, the crystallization process is easy to control, and the reproducibility is good.

Description

A co-crystal form of an RNA m6A regulator and its preparation method and application

[0001] The present disclosure claims priority to Chinese patent application No. 2023116845011, filed with the Patent Office of China on December 8, 2023, entitled “A co-crystal form of an RNA m6A regulator, its preparation method and application”. The entire contents of the above application are incorporated into the present disclosure by reference. Technical Field

[0002] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a cocrystal form of an RNA m6A regulator, a preparation method thereof, and an application thereof. Background Art

[0003] Hand-foot syndrome (HFS) and hand-foot skin reaction (HFSR) are erythematous skin lesions on the palms and soles, primarily caused by cytotoxic chemotherapy and targeted tumor drugs. Severe cases can lead to loss of self-care ability. The main pathological features of HFS and HFSR are vacuolar degeneration of basal keratinocytes, perivascular lymphocytic infiltration of the skin, keratinocyte apoptosis, and skin edema. Microscopic examination also reveals inflammatory changes, vasodilation, edema, and leukocyte infiltration.

[0004] Drugs that can cause hand-foot syndrome (HFS) include chemotherapy drugs such as capecitabine, liposomal doxorubicin, cytarabine, docetaxel, vinorelbine, continuous infusion doxorubicin, and gemcitabine. Drugs that can cause HFS include targeted drugs such as sonitinib (Sutent), sorafenib (Nexavar), imatinib (Gleevec), and erlotinib (Tarceva). The World Health Organization (WHO) categorizes HFS into four grades: Grade 1: numbness, paresthesia, or tingling in the hands and feet; Grade 2: discomfort with holding objects and walking, painless swelling or erythema; Grade 3: painful erythema, edema of the palms and soles, periungual erythema and swelling; and Grade 4: peeling, ulceration, blistering, and severe pain. Currently, severe HFS and HFS are often treated with superficial skin care alone to alleviate symptoms, or even require discontinuation of medication. Existing treatments only address the symptoms, severely limiting the use of first-line cancer chemotherapy drugs. This represents a significant 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 medical needs worldwide.

[0005] m6A methylation is the most common RNA modification in mammals, regulating multiple signaling pathways and cellular processes (such as growth, development, and disease), thereby playing a key biological role (Frye M., et al. Science 2018, 361, 2073-2092; Yang C., et al. Cell Death & Disease 2020, 11, 960; Meyer KD & Jaffrey SR Nature Review Molecular Cell Biology 2014, 15, 313-326). m6A methylation of mRNA, miRNA, circRNA, and lncRNA is a dynamic and reversible process. Methyltransferases (such as METTL3, METTL14, and METTL16) attach methyl groups to RNA, while demethylases (FTO and ALKBH5) remove methyl groups from RNA, forming the regulatory basis of m6A. m6A affects RNA processing, translation, and degradation by recruiting specific binding proteins (such as YTHDF1, YTHDF2, YTHDC1, and IGF2BP), thereby leading to changes in downstream protein function and cell biological behavior (Hsu PJ, et al. Journal of Biological Chemistry 2019, 294, 19889-19895; Yao Y., et al. FASEB Journal 2019, 33, 7529-7544).

[0006] Currently, there are few known direct associations between RNA m6A and skin-related diseases. Most m6A-related methylases or demethylases play a role 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. Furthermore, the mRNA expression level of METTL3 is significantly higher in patients with advanced acral melanoma than in patients with early-stage acral melanoma (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 serve as novel biomarkers (Li TD, et al. Cancer Cell 2020, 20, 239). In keratinocytes, only long-term, low-level arsenic exposure has been reported to inhibit selective autophagy caused by m6A demethylase, thereby inducing the occurrence of skin tumors (Cui YH, et al. Nature Communications 2021, 12, 2183). In summary, there is currently no method to prevent and treat skin diseases by regulating RNA m6A methylation.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] Based on the unpredictable existence form and quantity of polymorphic compounds, the present invention provides a co-crystal form of an RNA m6A regulator as shown in formula (I), a preparation method and application thereof.

[0010] Solutions for solving problems

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

[0012] The drug co-crystal formed by the compound represented by formula (I) and isonicotinamide,

[0013] The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566; preferably, 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, 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; the most preferred X-ray powder diffraction pattern expressed in diffraction angle 2θ is shown in Figure 1.

[0014] The present invention provides a pharmaceutical co-crystal formed by a compound represented by formula (I) and glycolic acid.

[0015] The X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ has characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125; preferably, 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, 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, there are 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 shown in Figure 4.

[0016] The present invention provides a pharmaceutical co-crystal formed by a compound represented by formula (I) and L-proline.

[0017] The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably 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 have characteristic peaks; preferably, there are 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 shown in Figure 7.

[0018] The present invention provides a pharmaceutical co-crystal formed by a compound represented by formula (I) and nicotinamide.

[0019] The X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ has characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573; preferably, 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, 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, Characteristic peaks are present at 27.028, 30.766, 32.820 and 36.741; preferably, characteristic peaks are present 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 shown in Figure 10.

[0020] Effects of the Invention

[0021] The pharmaceutical cocrystals formed by the compound of formula (I) and isonicotinamide, the pharmaceutical cocrystals formed by the compound of formula (I) and glycolic acid, the pharmaceutical cocrystals formed by the compound of formula (I) and L-proline, and the pharmaceutical cocrystals formed by the compound of formula (I) and nicotinamide provided by the present invention have advantages in physical and chemical properties, formulation processing performance, and bioavailability, for example, advantages in at least one of melting point, solubility, hygroscopicity, purification effect, stability, adhesion, compressibility, flowability, in vitro and in vivo dissolution, and bioavailability. The pharmaceutical cocrystals formed by combining the compound of formula (I) and the cocrystal former according to the present invention have excellent physical and chemical stability, a high yield of crystalline forms prepared from the same starting materials, and significant advantages in solubility, hygroscopicity, stability, mechanical stability, flowability, compressibility, and adhesion, providing a new and better option for the development of RNA m6A modulator drugs, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and isonicotinamide.

[0023] FIG2 is a TGA spectrum of the drug co-crystal formed by the compound represented by formula (I) and isonicotinamide.

[0024] FIG3 is a DSC spectrum of the drug co-crystal formed by the compound represented by formula (I) and isonicotinamide.

[0025] FIG4 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and glycolic acid.

[0026] FIG5 is a TGA spectrum of the drug co-crystal formed by the compound represented by formula (I) and glycolic acid.

[0027] FIG6 is a DSC spectrum of the drug co-crystal formed by the compound represented by formula (I) and glycolic acid.

[0028] FIG7 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and L-proline.

[0029] FIG8 is a TGA spectrum of the drug co-crystal formed by the compound represented by formula (I) and L-proline.

[0030] FIG9 is a DSC spectrum of the drug co-crystal formed by the compound represented by formula (I) and L-proline.

[0031] FIG10 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and nicotinamide.

[0032] FIG11 is a TGA spectrum of the drug co-crystal formed by the compound represented by formula (I) and nicotinamide.

[0033] FIG12 is a DSC spectrum of the drug co-crystal formed by the compound represented by formula (I) and nicotinamide.

[0034] FIG13 depicts exemplary results of cellular m6A levels and NAP1L2 m6A levels measured by nanopore sequencing 24 hours after administration of sorafenib, capecitabine, docetaxel, and osimertinib to keratinocytes HaCat.

[0035] FIG14 depicts exemplary results of cellular m6A levels and NAP1L2 m6A levels in mouse hind limb skin tissue in Example 3.

[0036] FIG15 depicts exemplary results of NAP1L2 mRNA expression measured by RT-PCR 24 hours after administration of human recombinant METTL3 protein, human recombinant FTO protein, or transfection of METTL3 siRNA and FTO siRNA to keratinocytes HaCat.

[0037] 16 depicts exemplary results of mRNA and protein expression of matrix metalloproteinases measured by RT-PCR and Western blotting 24 hours after administration of human recombinant NAP1L2 protein, sorafenib, capecitabine, docetaxel, and osimertinib, or transfection of NAP1L2 siRNA to keratinocytes HaCat.

[0038] 17 depicts exemplary results of mRNA and protein expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin measured by RT-PCR and Western blotting 24 hours after administration of human recombinant HBEGF protein, HBEGF antibody, or transfection of siRNA of METTL3 and NAP1L2 to keratinocytes HaCat.

[0039] FIG18 depicts exemplary results of measuring the level of cellular m6A methylation inhibition by LC-MS 24 hours after administration of the compound of formula (I), nicotinamide, and UZH2 to THP-1 cells.

[0040] FIG19 depicts exemplary results of mRNA expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin measured by RT-PCR 24 hours after administration of human recombinant HBEGF protein and the compound of formula (I) to keratinocytes HaCaT.

[0041] FIG. 20 depicts exemplary results of the measurement of stratum corneum thickness of rat paw plantar skin in Example 10.

[0042] FIG21 depicts exemplary histopathological results of epithelial blisters, inflammatory cell infiltration, and skin tissue hyperemia in rat paw plantar skin after tissue staining in Example 10.

[0043] FIG22 depicts exemplary results of histopathological scoring of epithelial blisters, inflammatory cell infiltration, and skin tissue hyperemia in rat paw plantar skin after tissue staining in Example 10.

[0044] FIG23 depicts exemplary results of mouse hind limb skin in Example 11.

[0045] FIG24 depicts exemplary results of the degree of swelling of the mouse hind limb toes in Example 11.

[0046] FIG. 25 depicts exemplary histopathological results of mouse hind limb skin tissue in Example 11.

[0047] FIG26 depicts exemplary results of immunohistochemistry for IL-8 in the skin tissues of the hind limbs and toes of mice in the capecitabine modeling series in Example 11.

[0048] FIG27 depicts exemplary results of immunohistochemistry for IL-6 in the skin tissues of the hind limbs and toes of mice in the docetaxel modeling series in Example 11.

[0049] FIG28 depicts exemplary results of mouse hind limb skin in Example 12.

[0050] FIG29 depicts exemplary results of the degree of swelling of the mouse hind limb toes in Example 12.

[0051] FIG30 depicts exemplary histopathological results of mouse hind limb skin tissue in Example 12.

[0052] FIG31 depicts exemplary results of immunohistochemistry for IL-1β in the skin tissues of the hind limbs and toes of mice in the Sorafenib model series in Example 12.

[0053] FIG32 depicts exemplary results of immunohistochemistry for IL-1β in the skin tissues of the hind limbs and toes of mice in the osimertinib modeling series in Example 12. DETAILED DESCRIPTION

[0054] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate 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 field to which this application belongs.

[0055] The present invention provides a pharmaceutical co-crystal formed by a compound represented by formula (I) and isonicotinamide.

[0056] The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566; preferably, 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, 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; the most preferred X-ray powder diffraction pattern expressed in diffraction angle 2θ is shown in Figure 1.

[0057] The present invention further provides a method for preparing a pharmaceutical cocrystal formed by the compound represented by formula (I) and isonicotinamide, comprising the steps of: mixing the compound represented by formula (I) and isonicotinamide, adding solvent 1, stirring, and filtering.

[0058] In certain embodiments, the solvent 1 is selected from alcohol solvents.

[0059] In certain embodiments, the solvent 1 is selected from C 1-4 alcohol.

[0060] In certain embodiments, the solvent 1 is selected from one or more of methanol, ethanol and isopropanol.

[0061] In certain embodiments, the solvent 1 is selected from ethanol.

[0062] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing or drying.

[0063] The present invention further provides a pharmaceutical co-crystal formed by the compound represented by formula (I) and glycolic acid,

[0064] The X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ has characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125; preferably, 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, 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, there are 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 shown in Figure 4.

[0065] The present invention further provides a method for preparing a pharmaceutical cocrystal formed by the compound represented by formula (I) and glycolic acid, comprising the steps of: mixing the compound represented by formula (I) and glycolic acid, adding solvent 2, stirring, and filtering.

[0066] In certain embodiments, the solvent 1 is selected from ester solvents.

[0067] 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.

[0068] In certain embodiments, the solvent 1 is selected from isopropyl acetate.

[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 co-crystal formed by the compound represented by formula (I) and L-proline,

[0071] The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably 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 have characteristic peaks; preferably, there are 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 shown in Figure 7.

[0072] The present invention further provides a method for preparing a pharmaceutical cocrystal formed by a compound represented by formula (I) and L-proline, comprising the steps of: mixing the compound represented by formula (I) and L-proline, adding solvent 3, stirring, and filtering.

[0073] In certain embodiments, the solvent 3 is selected from alcohol solvents.

[0074] In certain embodiments, the solvent 3 is selected from C 1-4 alcohol.

[0075] In certain embodiments, the solvent 3 is selected from one or more of methanol, ethanol and isopropanol.

[0076] In certain embodiments, the solvent 3 is selected from ethanol.

[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 co-crystal formed by the compound represented by formula (I) and nicotinamide,

[0079] The X-ray powder diffraction pattern expressed in terms of a diffraction angle 2θ has characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573; preferably, 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, 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, Characteristic peaks are present at 27.028, 30.766, 32.820 and 36.741; preferably, characteristic peaks are present 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 shown in Figure 10.

[0080] The present invention further provides a method for preparing a pharmaceutical co-crystal formed by the compound represented by formula (I) and nicotinamide, comprising the steps of: mixing the compound represented by formula (I) and nicotinamide, adding solvent 4, stirring, and filtering.

[0081] In certain embodiments, the solvent 1 is selected from nitrile solvents.

[0082] In certain embodiments, the solvent 1 is selected from one or more of acetonitrile, trimethylacetonitrile, propionitrile and valeronitrile.

[0083] In certain embodiments, the solvent 1 is selected from acetonitrile.

[0084] In certain embodiments, the preparation method of the present invention further comprises steps such as centrifugation, washing or drying.

[0085] The present invention further provides a pharmaceutical composition prepared by combining a pharmaceutical co-crystal formed by combining the compound of the aforementioned formula (I) and a co-crystal former, or a pharmaceutical co-crystal formed by combining the compound of the aforementioned formula (I) with isonicotinamide, or a pharmaceutical co-crystal formed by combining the compound of the aforementioned formula (I) with glycolic acid, or a pharmaceutical co-crystal formed by combining the compound of the aforementioned formula (I) with L-proline, or a pharmaceutical co-crystal formed by combining the compound of the aforementioned formula (I) with nicotinamide.

[0086] The present invention further provides a pharmaceutical composition comprising a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and a co-crystal former, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with isonicotinamide, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with glycolic acid, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with L-proline, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with nicotinamide, and optionally a pharmaceutically acceptable excipient.

[0087] "Pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, mixture, or vehicle that contributes to the consistency of the dosage form or 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.

[0088] 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, viscosity enhancers, antioxidants, preservatives, stabilizers, surfactants and buffers.

[0089] In certain embodiments, the pharmaceutical composition is a solid preparation.

[0090] In certain embodiments, the pharmaceutical composition is a film or coating.

[0091] In certain embodiments, the pharmaceutical composition is an ointment.

[0092] In certain embodiments, the pharmaceutical combination is a plaster.

[0093] In certain embodiments, the pharmaceutical combination is a cream.

[0094] In certain embodiments, the solid dosage form is a capsule.

[0095] In certain embodiments, the pharmaceutical combination is a tablet.

[0096] In certain embodiments, the ointment contains 0 to 50% by weight of the co-crystal of the compound represented by formula (I).

[0097] The present invention further provides a method for preparing a pharmaceutical composition, comprising the step of mixing a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and a co-crystal former, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with isonicotinamide, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with glycolic acid, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with L-proline, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) with nicotinamide, with a pharmaceutically acceptable excipient.

[0098] The present invention further provides a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and a co-crystal former, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and isonicotinamide, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and glycolic acid, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and L-proline, or a pharmaceutical co-crystal formed by combining the aforementioned compound of formula (I) and nicotinamide, or the aforementioned composition, or the use of the composition prepared by the aforementioned method in preparing a drug for treating and / or preventing diseases related to RNA m6A regulation.

[0099] In certain embodiments, the m6A regulation-related diseases are endocrine and metabolic diseases, nervous system diseases, tumors, cardiovascular diseases, infections, immune system diseases, genitourinary system diseases, skin and musculoskeletal diseases, respiratory system diseases, genetic diseases and deformities, digestive system diseases, oral and maxillofacial diseases, vascular and lymphatic system disease-related diseases or pain.

[0100] In certain embodiments, the m6A regulation-related disease is hand-foot syndrome, hand-foot skin reaction, cancer, or a dermatological disease.

[0101] The "2θ or 2θ angle" mentioned in the present invention refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2θ is ±0.2 (including the case where the number exceeding 1 decimal place is rounded off), which 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.

[0102] The precipitation methods of the present invention include but are not limited to stirring, cooling, volatilization, beating, and precipitation.

[0103] "Pulping" is a common term in the field of drug preparation, which usually refers to the mechanization or fluidization of solid drug raw materials so that the solid drug is dispersed or suspended in a solvent.

[0104] In some embodiments, the beating time is 5 hours to 30 hours.

[0105] According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "9103 Guiding Principles for Hygroscopicity of Drugs" in Part IV of the 2020 edition of the Chinese Pharmacopoeia,

[0106] Deliquescent: Absorbs sufficient water to form a liquid;

[0107] Highly hygroscopic: weight gain due to moisture absorption is not less than 15%;

[0108] Hygroscopic: weight gain due to moisture absorption is less than 15% but not less than 2%;

[0109] Slightly hygroscopic: weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0110] No or almost no hygroscopicity: weight gain due to moisture is less than 0.2%.

[0111] The "differential scanning calorimetry or DSC" mentioned in the present invention refers to measuring the temperature difference and heat flow difference between a sample and a reference object during the process of heating or maintaining the sample at a constant temperature, so as to characterize all physical and chemical changes related to thermal effects and obtain phase change information of the sample.

[0112] The drying temperature in the present invention is generally 25° C. to 100° C., preferably 40° C. to 70° C., and the drying can be performed under normal pressure or reduced pressure.

[0113] The method of the present invention is described below by means of 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 to the scope of the following examples. The implementation conditions adopted in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are generally the conditions in routine experiments.

[0114] The abbreviations used in the present invention are explained as follows:

[0115] XRPD: X-ray powder diffraction

[0116] DSC: Differential Scanning Calorimetry

[0117] TGA: Thermogravimetric analysis

[0118] DVS: Dynamic Water Sorption

[0119] HPLC: High Performance Liquid Chromatography

[0120] Detection instruments and methods

[0121] X-ray powder diffraction (XRPD)

[0122] Crystalline analysis of the samples was performed using a Bruker D8 ADVANCE X-ray powder diffractometer. The 2θ scanning angle ranged from 3° to 40°, with a scan step size of 0.02° and a scan time of 0.12 s per step. The light tube voltage and current were 40 kV and 40 mA, respectively. During sample preparation, an appropriate amount of sample was placed on a sample tray and flattened with a glass slide or other tool to ensure a smooth surface.

[0123] Thermogravimetric analysis (TGA)

[0124] The samples were analyzed using a TA Instruments TGA Discovery 5500. The samples were placed in a tared aluminum pan, and the system automatically weighed the samples. The samples were then heated to the specified temperature at a rate of 10°C / min under nitrogen.

[0125] Differential Scanning Calorimetry (DSC)

[0126] The samples were analyzed using a TA Instruments Discovery 2500. 0.5-1.5 mg of sample was weighed and placed in a sample tray, and the sample was heated to the specified temperature at a rate of 10°C / min under the protection of nitrogen (50 ml / min).

[0127] Dynamic Water Sorption Analysis (DVS)

[0128] Samples were analyzed using the ProUmid SPSx-1μ Advance. Sample sizes ranged from 5 to 50 mg. The test chamber temperature was controlled at 25 ± 1°C, and the relative humidity was cycled from 40% to 95% and then from 0% to 95% to 40% in 10% steps. Each step lasted 240 minutes, and mass data was recorded every 20 seconds.

[0129] High-performance liquid chromatography (HPLC)

[0130] Solubility and stability tests were performed using an Agilent 1260 infinityII Binary Pump.

[0131] Example 1: Preparation of the compound represented by formula (I)

[0132] Ethyl 3-bromo-2-oxocyclohexane-1-carboxylate (20 mg, 0.08 mmol) and 4-chloroaniline (25 mg, 0.2 mmol) were mixed and heated to 150°C. After reacting for 3 hours, the reaction solution was cooled to room temperature, diluted with 100 mL of dichloromethane, washed three times with 100 mL of 1N HCl and once with 100 mL of saturated NaHCO3. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The mixture was separated and purified by silica gel column chromatography to obtain 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, and 2 mL of 2M LiOH solution was added. The mixture was stirred at room temperature for 1 hour. After rotary distillation, 20 mL of water was added to dilute the mixture and the pH was adjusted to 2. The mixture was then extracted three times with 50 mL of dichloromethane. The organic phases were combined, dried, concentrated, and then ammonia was added. The mixture was heated at 60°C for 24 hours. The mixture was purified by silica gel column chromatography to obtain 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (compound 1, 7.6 mg) as a white solid. (S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazolecarboxamide (compound represented by formula (I)) was obtained by separation using a Chiralpak AD chiral column. LCMS of compound represented by formula (I) [M+H] + :249. 1 H-NMR (400MHz, DMSO-d6): δ10.79 (s, 1H), 7.37-7.39 (m, 2H), 7.28 (d, J = 8.0Hz, 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).

[0133] Example 2: Drug-induced changes in keratinocyte mRNA methylation levels

[0134] 1*10 8HaCat cells were plated in culture dishes and treated with 50 nM of each drug (sorafenib, capecitabine, docetaxel, and osimertinib). The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was removed, the cells were washed with PBS, centrifuged, and the supernatant discarded. 400 μL of RLT cell lysis buffer (QIAGEN, Cat. #79216) was added to the cells. A portion of the sample was added with 400 μL of 70% ethanol, stirred thoroughly, and transferred to an RNeasy mini column. RNA was extracted using the RNeasy mini kit (QIAGEN, Cat. #74104). 2 μL of RNA sample was dissolved in 98 μL of 10 mM Tris-HCl buffer, and the absorbance at 260 nm was measured using a Nanodrop (Thermo Fisher Scientific) to determine RNA concentration.

[0135] 40 μg of RNA sample was taken and mRNA (PolyA+RNA) was enriched using the NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, Cat.#E7490). The 3' end of the enriched mRNA (PolyA+RNA) molecule was then connected to the reverse transcription adapter RTA using the Direct RNA Sequencing Kit (Oxfod Nanopore Technologies, Cat.#SQK-RNA002). The mRNA (PolyA+RNA) molecule was used as a template for reverse transcription reaction to synthesize its complementary chain. The reverse transcription adapter RTA end was connected to the sequencing adapter (RNA adapter) to form the final sequencing library, and the library was sequenced using Qubit TM dsDNA HS assay kit (Invitrogen, Cat.# LOT2133187) was used for quantitative detection.

[0136] After library quality control, the prepared sequencing library was loaded onto a PromethION Flow Cell chip (Oxford Nanopore Technologies, Cat.# FLO-MIN106D) and sequenced using a PromethION sequencer (Oxford Nanopore Technologies) in the matching sequencing mode. Sequencing data were base-called using guppy, and m6A methylation analysis was performed using the original Fast5 data.

[0137] The results are shown in Figure 13. Compared to the blank control group, the addition of sorafenib, capecitabine, docetaxel, and osimertinib increased the m6A methylation levels of HaCat cells by 5.05-fold, 6.01-fold, 3.97-fold, and 5.13-fold, respectively. This suggests that anti-cancer drugs (sorafenib, capecitabine, docetaxel, and osimertinib) can induce abnormal m6A methylation in human keratinocyte mRNA. Further bioinformatics analysis revealed significant differences in the m6A methylation levels of NAP1L2, primarily in the m6A methylation of the UGAGGACUCA fragment.

[0138] Example 3: Changes in mRNA methylation levels in a drug-induced mouse skin adverse reaction model

[0139] ICR male mice (5-6 weeks old, weighing approximately 35 grams) were acclimated for 7 days and then randomly divided into 6 groups: blank control, sorafenib, capecitabine, docetaxel, and osimertinib. Each group was gavaged with the corresponding modeling drug (sorafenib 100 mg / kg, capecitabine 200 mg / kg, docetaxel 25 mg / kg, osimertinib 10 mg / kg) once daily for 30 consecutive days before being sacrificed. Skin tissue from the hind limbs and toes of the mice was collected. A small amount of tissue sample was ground into a powder in a mortar filled with liquid nitrogen. The single-phase lysis buffer was added and the sample was allowed to stand at room temperature for 5 minutes before centrifugation (12,000 rpm) for 5 minutes. 1 mL of the supernatant was removed, 200 μL of chloroform was added, and the mixture was shaken and allowed to stand at room temperature for 15 minutes. The sample was then centrifuged (12,000 rpm, 4°C) for 15 minutes. The upper aqueous phase was aspirated and an equal volume of isopropanol was added. The sample was allowed to stand at -20°C for 1 hour and then centrifuged (12,000 rpm, 4°C). The supernatant was discarded. 400 μL of 70% ethanol was added to the pellet, mixed thoroughly, and transferred to an RNeasy mini column. RNA was extracted using the RNeasy mini kit (QIAGEN, Cat. #74104). 2 μL of RNA sample was dissolved in 98 μL of 10 mM Tris-HCl buffer, and the absorbance at 260 nm was measured using a Nanodrop (Thermo Fisher Scientific) to determine RNA concentration.

[0140] 40 μg of RNA sample was taken and mRNA (PolyA+RNA) was enriched using the NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, Cat.#E7490). The 3' end of the enriched mRNA (PolyA+RNA) molecule was then connected to the reverse transcription adapter RTA using the Direct RNA Sequencing Kit (Oxfod Nanopore Technologies, Cat.#SQK-RNA002). The mRNA (PolyA+RNA) molecule was used as a template for reverse transcription reaction to synthesize its complementary chain. The reverse transcription adapter RTA end was connected to the sequencing adapter (RNA adapter) to form the final sequencing library, and the library was sequenced using Qubit TM dsDNA HS assay kit (Invitrogen, Cat.# LOT2133187) was used for quantitative detection.

[0141] After library quality control, the prepared sequencing library was loaded onto a PromethION Flow Cell chip (Oxford Nanopore Technologies, Cat.# FLO-MIN106D) and sequenced using a PromethION sequencer (Oxford Nanopore Technologies) in the matching sequencing mode. Sequencing data were base-called using guppy, and m6A methylation analysis was performed using the original Fast5 data.

[0142] The results are shown in Figure 14. Compared with the positive control group, the m6A methylation level in the skin tissue of the hind limbs and toes of mice in the drug modeling group was significantly increased. The m6A methylation level of NAP1L2 in the drug-treated group was significantly different from that in the blank control group.

[0143] Example 4: m6A regulates NAP1L2 mRNA expression levels

[0144] 1*10 8HaCat cells were plated in culture dishes and treated with recombinant m6A methylase METTL3 protein (Abcam, Cat.#ab271611) or m6A demethylase FTO protein (Abcam, Cat.#ab271525), or transfected with siRNA for METTL3 (sh-METTL3, GeneChip) or siRNA for FTO (sh-FTO, GeneChip). The cells were cultured at 37°C in an incubator containing 5% carbon dioxide for 24 hours, washed with PBS, and collected by high-speed centrifugation. Total RNA was extracted using Trizol reagent (Sigma Aldrich). 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). RT-PCR was performed by adding 1.25 μL of primers (Beyotime, Cat. #QH18721S), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction mixture. After completion of the reaction, the reaction mixture was subjected to agarose gel electrophoresis to determine the expression level of NAP1L2 mRNA.

[0145] The results are shown in Figure 15. When the m6A methylase METTL3 or the m6A methylase FTO function was inhibited (sh-FTO) in keratinocytes HaCat, the mRNA expression of NAP1L2 increased significantly; when the m6A methylase FTO or the m6A methylase METTL3 function was inhibited (sh-METTL3) in keratinocytes HaCat, the mRNA expression of NAP1L2 decreased significantly. This indicates that m6A methylation can regulate the mRNA expression of NAP1L2.

[0146] Example 5: NAP1L2 regulates the expression of matrix metal proteins in human keratinocytes HaCat

[0147] 1*10 8 HaCat cells were plated in culture dishes and treated with recombinant human NAP1L2 protein (Abcam, Cat.#ab117213), sorafenib, capecitabine, docetaxel, and osimertinib, or transfected with NAP1L2 siRNA (sh-NAP1L2, GeneChip). The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After washing with PBS, the cells were harvested by high-speed centrifugation. RLT lysis buffer was added to the harvested cells, shaken at 4°C for half an hour, and the supernatant was collected after centrifugation for measurement of matrix metal protein expression by Western blotting.

[0148] Total RNA was extracted from the collected cells using Trizol reagent (Sigma Aldrich). 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). RT-PCR was performed by adding 1.25 μL of primers (SinoBiological, Cat. #HP100168 & HP100367), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction solution. Following completion of the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression level of the metal matrix protein.

[0149] The results are shown in Figure 16. When NAP1L2 recombinant protein was added to keratinocytes HaCat, the expression levels of the mRNA and protein of the metalloproteinases MMP2 and MMP9 increased significantly. When NAP1L2 siRNA was added to keratinocytes HaCat to inhibit the expression of NAP1L2, the expression levels of the mRNA and protein of the metalloproteinases MMP2 and MMP9 decreased significantly. Many studies have confirmed that abnormal expression of metalloproteinases is closely related to various skin-related diseases (Kumper M. et al., Am. J. Physiol. Cell. Physiol. 2022, 323(4): 1290-1303).

[0150] Example 6: m6A regulates the expression of human keratinocyte differentiation markers

[0151] 1*10 8 HaCat cells were plated in culture dishes and incubated with recombinant HBEGF protein (Thermo Fisher Scientific, Cat.#100-47-1mg) at 37°C in an incubator containing 5% CO2 for 24 hours. HBEGF antibody (Invitrogen, Cat.#406316) or transfection with siRNA for METTL3 (sh-METTL3, GeneChip) or NAP1L2 (sh-NAP1L2, GeneChip) were then added and incubated at 37°C in an incubator containing 5% CO2 for 24 hours. After washing with PBS, the cells were harvested by high-speed centrifugation. RLT lysis buffer was added to the harvested cells, shaken at 4°C for half an hour, and the supernatant was collected after centrifugation. Protein expression of the keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin was measured by Western blotting.

[0152] Total RNA was extracted using Trizol reagent (Sigma Aldrich), and 1 μg of RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). RT-PCR was performed by adding 1.25 μL of primers (primer sequences are shown in Table 2), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction solution. After completion of the reaction, the reaction solution was subjected to agarose gel electrophoresis to measure the mRNA expression levels of the keratinocyte differentiation markers KRT1, KRT10, loricrin, and involucrin.

[0153] The results are shown in Figure 17. When HBEGF recombinant protein was added to keratinocytes HaCat, the mRNA and protein expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin significantly increased, indicating that the keratinocytes HaCat were in a highly differentiated state. However, when HBEGF antibodies were added or METTL3 and NAP1L2 siRNAs were used to inhibit intracellular m6A methylation function, the mRNA and protein expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin significantly decreased. This indicates that m6A can regulate the expression of keratinocyte differentiation markers, thereby regulating keratinocyte differentiation.

[0154] Example 7: Effects of compounds on m6A methylation levels in THP-1 cells

[0155] 1*10 8THP-1 cells were plated in a culture dish and treated with 4 nM and 400 nM of the compound of formula (I), 4 nM and 4 μM nicotinamide, and 5 μM of the m6A methylase inhibitor UZH2. The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The medium was discarded, the cells were washed with PBS, and the supernatant was discarded by centrifugation. 400 μL of RLT cell lysis buffer was added and the cells were stored at -80°C. A portion of the sample was taken, 400 μL of 70% ethanol was added and mixed thoroughly. 700 μL of the sample was transferred to an RNeasy spin column and centrifuged for 15 seconds (8000 g, 25°C). 700 μL of RW1 buffer was added and centrifuged for 15 seconds (8000 g, 25°C). Then, 500 μL of RPE buffer was added and centrifuged for 16 seconds (8000 g, 25°C). This step was repeated twice, followed by centrifugation for 2 minutes to completely remove the eluate. Add 30 μL of nuclease-free water to the column, incubate for 5 minutes, and centrifuge for 2 minutes (12,000 g, 25°C). Repeat this step three times. Dissolve 2 μL of RNA sample in 98 μL of 10 mM Tris buffer and measure the absorbance at 260 nm using a Nanodrop to determine the RNA concentration.

[0156] Dissolve 50 μg of total RNA in 100 μL of nuclease-free water. Resuspend the Oligo(dT) magnetic beads, transfer 50 μL of beads to a 1.5 mL tube, add 500 μL of binding buffer, let stand, and remove the supernatant. Add 100 μL of RNA sample to 200 μL of magnetic bead suspension, mix, and incubate in a Theromixer at 25°C for 5 minutes. Discard the supernatant and add 200 μL of wash buffer. Repeat twice, then centrifuge for 10 seconds (2000 g, 25°C). Add 50 μL of elution buffer and mix thoroughly. Heat to 75°C, transfer the supernatant to a new 1.5 mL tube, and purify using the RNA Clean & Concentrator kit to determine the RNA concentration.

[0157] Add 20 μL (about 200 ng) of mRNA sample and 20 μL of Nuclease P1 digestion premix (0.5 μL of 2 unit / μL Nuclease P1, 0.4 μL of 5 M NaCl, 2 μL of 0.1 M ZnCl2 and 17.1 μL of PCR-grade water) to each test tube, stir and incubate at 37°C in a Theromixer for 2 hours, add 2 μL of 2M NH4HCO3 solution and 1 unit of alkaline phosphatase, stir and incubate at 37°C in a Theromixer for 2 hours, add 1 μL of 1.2 M HCl neutralization solution, centrifuge for 30 minutes (16000g, 4°C), take 20 μL of supernatant and analyze m6A methylation fragments by LC-MS, and calculate the m6A inhibition rate.

[0158] m6A inhibition rate (%) = (measured ion peak area - blank control) / (positive ion peak area - blank control) * 100

[0159] The results are shown in Figure 18. The compound of formula (I) can significantly inhibit the m6A methylation level of cellular mRNA in a concentration-dependent manner. At a concentration of 400 nM, the compound of formula (I) has an m6A inhibitory activity comparable to that of the m6A methylase inhibitor UZH2 at a concentration of 5 μM. However, nicotinamide had no inhibitory effect on the m6A methylation level of cellular mRNA at either low (4 nM) or high (4 μM) concentrations.

[0160] Example 8: Effects of compounds on m6A methylation levels in human keratinocytes

[0161] HaCat cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture dishes were placed in an incubator containing 5% CO2 and cultured at 37°C for 24 hours. An equal volume of DMSO was added to control group 1, and 50 nM sorafenib, capecitabine, docetaxel, and osimertinib were added to control groups A, B, C, and D, respectively. After adding 50 nM of the corresponding compound, 400 nM of the test compound or nicotinamide was added to sample groups A1, A2, B1, B2, C1, C2, D1, and D2. The cells were cultured for 24 hours, washed with PBS, and collected by high-speed centrifugation. Total RNA was extracted using Trizol reagent (Sigma Aldrich), and m6A methylation in the sample RNA was quantitatively detected using the EpiQuik m6A RNA Methylation Quantification Kit. The standard curve used positive control samples at concentrations of 0.01 ng / μl, 0.02 ng / μl, 0.05 ng / μl, 0.1 ng / μl, 0.2 ng / μl, and 0.5 ng / μl. The absorbance at 450 nm was read using a Tecan GENios microplate reader. The m6A quantitative calculation formula is as follows:

[0162] m6A (ng) = (absorbance of sample well – absorbance of background well) / slope of standard curve

[0163] m6A (%) = m6A (ng) / sample RNA amount (ng) * 100%

[0164] The results are shown in Table 1. Treatment with sorafenib, capecitabine, docetaxel, and osimertinib resulted in a significant increase in m6A methylation levels in human keratinocytes (HaCat). The compound of formula (I) effectively inhibited the abnormal increase in RNA m6A levels in human keratinocytes caused by chemotherapy drugs and multikinase inhibitors.

[0165] Table 1 shows the fold change of m6A methylation level relative to control group 1. The specific calculation method is: fold change = measured m6A amount / control 1 measured m6A amount.

[0166] Example 9: Inhibitory effect of compounds on keratinocyte HaCaT differentiation

[0167] Human keratinocytes HaCaT were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. 6 Cells were plated in 96-well plates at a density of 10 cells per square centimeter and incubated at 37°C in a 5% CO2 incubator for 24 hours. An equal volume of DMSO was added to control well 1, and 2.5 ng / mL human recombinant HBEGF protein (Abcam, ab205523) was added to control well 2. Sample wells were then incubated for 24 hours. After washing with PBS, cells were harvested by high-speed centrifugation. Total RNA was extracted using Trizol reagent (Sigma Aldrich). 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, AT311-03). RT-PCR reactions were performed 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 to a 20 μL reaction mixture. After the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin.

[0168] Table 2 Some primer sequence information

[0169] As shown in Figure 19, after induction with human recombinant HBEGF protein, the mRNA expression levels of HaCaT differentiation markers KRT1, KRT10, Loricrin, and Involucrin significantly increased. However, after addition of the compound of formula (I), the mRNA expression levels of HaCaT differentiation markers KRT1, KRT10, Loricrin, and Involucrin were significantly suppressed.

[0170] Example 10: Inhibitory effect of the compound on keratinocyte differentiation in a rat hand-foot skin reaction model

[0171] After SD rats (weighing approximately 200 g) were bred and adapted for one week, they were divided into groups according to their weight, with 12 rats in each group. The modeling drugs (sorafenib, erlotinib, afatinib and osimertinib) were dissolved in solutions containing 5% DMSO, 45% PEG400 and 50% H2O, respectively, and the modeling drugs were diluted to the required concentrations and administered orally once a day according to the doses shown in Table 3. One hour after oral administration, 0.05 g of ointment containing different mass ratios of the test compound was evenly applied to the left paw of the rat, while the right paw was applied with a blank matrix ointment as a self-control. The rats in the blank control group were not applied with the drug. After applying the drug, the limbs were fixed for 2 hours, and then the residual drug was wiped off with clean water, the fixation was released and free movement was restored. The modeling drug, blank matrix ointment, and test compound ointment were all administered once daily for 30 consecutive days. The rats were euthanized, and paw plantar skin tissue was obtained, fixed in 10% neutral formaldehyde, cut into 5 μm sections, dehydrated, and embedded in paraffin. The test compound ointment was prepared by mixing the compound, white beeswax, white petrolatum, and light liquid paraffin in specific ratios (weight ratios of the three ointments were: 1:18:58:23, 3:18:57:22, and 10:20:40:30, respectively). The blank matrix ointment was prepared by mixing white beeswax, white petrolatum, and light liquid paraffin in specific ratios (weight ratio: 18:60:22).

[0172] Tissue staining: After dewaxing and rehydrating, the paw and plantar skin tissue sections of the above rats were stained in a hematoxylin solution for several minutes. After washing, the sections were immersed in 1% acidic alcohol until the sections faded to a light blue-red color. After washing with running water for 5 minutes, the sections were stained with eosin for 2-3 minutes. After washing to remove excess dye, the sections were dehydrated and transparentized in xylene for several minutes. The sections were then sealed with a neutral resin. The stratum corneum was observed under an optical microscope, and the thickness of the epidermal stratum corneum was measured using Dmetrix software.

[0173] Immunohistochemical staining: After dewaxing and hydration, the paw plantar skin tissue sections of the above rats were incubated with 3% H2O2 at room temperature for 30 minutes. After antigen retrieval, they were blocked with 10% goat serum for 30 minutes. KRT1 antibodies (Abcam, ab93652) and Loricrin antibodies (Abcam, ab183646) were added dropwise and incubated overnight at 4°C. HRP secondary antibodies (ZSGB-BIO, PV-6001) and DAB kits (ZSG-BIO, ZLI9017) were added for color development. The sections were counterstained with hematoxylin, washed, and mounted with central resin. The expression levels of KRT1 and Loricrin were observed under a light microscope.

[0174] The criteria for successful rat model establishment as described above are: (i) the appearance of symptoms such as erythema, swelling, desquamation, ulceration, or blisters on the paw; and / or (ii) a significantly higher stratum corneum thickness than normal rats in tissue staining; and / or (iii) a significant increase in markers such as KRT1, KRT5, and Loricrin. The incidence rate was calculated as the proportion of animals in each group meeting the above criteria for successful model establishment: incidence rate = (number of rats with successful model establishment / total number of rats in the group) * 100%.

[0175] The rat tissue staining pathology scoring criteria are as follows: no blisters, 0 points; 1-3 blisters, 1 point; 4-6 blisters, 2 points; 7-9 blisters, 3 points; and more than 10 blisters, 4 points. Inflammation occupying less than 10% of the total section area is scored as 0 points; inflammation occupying 10-25% of the total section area is scored as 1 point; inflammation occupying 25-50% of the total section area is scored as 2 points; inflammation occupying 50-75% of the total section area is scored as 3 points; and inflammation occupying more than 75% of the total section area is scored as 4 points. A score of 0 was assigned if the hyperemia area accounted for less than 10% of the total slice area; a score of 1 was assigned if the hyperemia area accounted for 10-25% of the total slice area; a score of 2 was assigned if the hyperemia area accounted for 25-50% of the total slice area; a score of 3 was assigned if the hyperemia area accounted for 50-75% of the total slice area; and a score of 4 was assigned if the hyperemia area accounted for more than 75% of the total slice area. Scoring was performed using a double-blind scoring system, and data from each group were presented as mean ± SEM (N = 12).

[0176] Table 3 Drug dosage and experimental results in rat model

[0177] As shown in Table 3, once-daily oral administration of sorafenib, erlotinib, afatinib, and osimertinib to SD rats at doses of 100 mg / kg, 70 mg / kg, 50 mg / kg, and 60 mg / kg, respectively, resulted in 75%, 83.3%, 66.7%, and 75% success rates for establishing hand-foot skin reaction models, respectively. The compound of formula (I) significantly reduced the incidence of hand-foot skin reaction at the application site at low doses (e.g., 1% by weight of the compound). The compound of formula (I) significantly reduced the incidence of hand-foot skin reaction at all paws of rats at high concentrations (e.g., 3% and 10% by weight of the compound).

[0178] As shown in Figure 20, the stratum corneum thickness measurements of rats successfully treated with sorafenib, erlotinib, afatinib, and osimertinib showed significantly higher stratum corneum thickness in the paw and plantar skin than in normal rats. The compound of formula (I) can effectively reduce the stratum corneum thickness in the paw and plantar skin of rats treated with the stratum corneum.

[0179] As shown in Figure 21, the tissue staining results show that the skin toxicity caused by the modeling drug can form blisters in the subcutaneous and epidermal regions of the rat paws and feet, inflammatory cell infiltration in the dermis, and congestion in the skin tissue. The compound of formula (I) can effectively ameliorate the formation of subcutaneous and epidermal blisters induced by the modeling drug, inhibit inflammatory cell infiltration in the dermis, and significantly improve skin congestion.

[0180] As shown in the pathological scoring results in Figure 22, the modeling drug can cause the formation of blister subcutaneously and intraepidermally in the plantar region of the rat paw, inflammatory cell infiltration in the dermis, and congestion in the skin tissue. The compound of formula (I) can effectively ameliorate the formation of blister subcutaneously and intraepidermally caused by the modeling drug, inhibit inflammatory cell infiltration in the dermis, and significantly improve skin congestion.

[0181] Immunohistochemical staining results also showed that KRT1 and Loricrin levels increased significantly in tissues of rats successfully treated with sorafenib, erlotinib, afatinib, and osimertinib. However, with the addition of the compound, KRT1 and Loricrin levels decreased significantly in tissues. In summary, the compound of formula (I) can effectively inhibit keratinocyte differentiation in vitro and in vivo, and has the potential to treat hyperkeratosis-related diseases.

[0182] Example 11: Effect of the compound of formula (I) in a mouse hand-foot syndrome model induced by chemotherapy drugs

[0183] After 7 days of adaptive feeding, SPF ICR mice (male, 5-6 weeks old, weighing approximately 35 grams) were randomly divided into the following groups: blank control group (6 mice), capecitabine model group (6 mice), docetaxel model group (6 mice), capecitabine + compound of formula (I) group (6 mice), docetaxel + compound of formula (I) group (6 mice), capecitabine + nicotinamide group (6 mice), and docetaxel + nicotinamide group (6 mice). Each group was orally administered with the corresponding modeling drug (capecitabine 200 mg / kg, docetaxel 25 mg / kg) by gavage once daily; the blank control group was given an equal volume of normal saline. After 2 weeks of chemotherapy drug administration, each group was topically applied with the corresponding ointment of the compound of formula (I) used in Example 10 (compound content 3%) or orally administered with nicotinamide (100 mg / kg) once daily for 16 consecutive days.

[0184] Measurement of toe swelling: The degree of toe swelling of the mouse hind limbs was measured using a toe swelling tester (KW-7C, Nanjing Calvin Biotechnology Co., Ltd.) before administration of the modeling drug, 2 weeks after administration of the modeling drug, and before sacrifice. The same position was marked on the joint of the mouse hind foot before administration, 2 weeks after administration, and before sacrifice. During measurement, the water was exactly on the same horizontal line as the marked position, and the value was recorded after it stabilized.

[0185] Skin histopathological staining: Skin tissue samples from the hind limbs of mice were fixed in 4% paraformaldehyde at room temperature for 4 hours. The tissues were then removed and rinsed with running water for several hours. After dehydration with 70%, 80%, and 90% ethanol solutions, they were treated with a mixture of equal parts pure alcohol and xylene for 15 minutes. Permeabilization was performed twice with xylene for 15 minutes each, until the sample became transparent. The samples were then immersed in a mixture of 50% xylene and 50% paraffin for 15 minutes, followed by permeabilization with Paraffin I and Paraffin II for one hour each. After paraffin embedding, the samples were sliced, baked, dewaxed, and hydrated. The hydrated sections were then stained in hematoxylin solution (Zhongshan Jinqiao, Catalog No. 23041001) for 3 minutes, differentiated in hydrochloric acid ethanol solution for 15 seconds, washed with water, and blued in Scott blue solution (Servicebio, Catalog No. 20230801) for 15 seconds. After rinsing with running water, the sections were stained with eosin stain (Solarbio, Catalog No. 33535) for 3 minutes. After rinsing with running water, the sections were dehydrated, transparentized, mounted, and examined under a microscope.

[0186] Immunohistochemical staining: Paraffin sections of mouse hind limb skin tissue were baked, dewaxed, and hydrated, followed by antigen retrieval using 0.2 M citric acid buffer (1.534 g citric acid, 3.1 g sodium citrate, 100 mL distilled water, pH 4.7). Endogenous hydrogen peroxide was removed with 3% hydrogen peroxide (Shandong Anjie High-Tech Disinfection Technology Co., Ltd., Catalog No. 20290902). After incubation at room temperature for 10 minutes, sections were rinsed thoroughly with PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 1 M HCl, pH 7.4). Sections were permeabilized with 0.5% Triron-X-100 (Aimejie Technology, Catalog No. A-CSH436-100 mL). Mouse anti-IL-1β and rabbit anti-IL-8 were permeabilized for 10 minutes each, while rabbit anti-IL-6 was not permeabilized. After blocking with 5% BSA antigen, a 1 / 150 dilution of mouse anti-IL-1β (Affinity Biosciences, Catalog No. BF8021), a 1 / 200 dilution of rabbit anti-IL-8 (Affinity Biosciences, Catalog No. DF6998), or a 1 / 150 dilution of rabbit anti-IL-6 (Affinity Biosciences, Catalog No. DF6087) was added to each slide and incubated in a humidified chamber at 4°C overnight. Remove the wet chamber after overnight incubation and let it stand at room temperature for 45 minutes. Wash the slides three times with PBS buffer for 15 minutes each. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao, Cat. No. 234750811) to rabbit anti-IL-8 and rabbit anti-IL-6 markers. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-mouse IgG (H+L) (Zhongshan Jinqiao, Cat. No. 226700804) to mouse anti-IL-1β markers. Incubate at 37°C for 30 minutes and then rinse thoroughly with PBS buffer. Add DAB (CWBIO, Cat. No. 13723) for color development for 3-5 minutes, wash with PBS buffer for 1 minute, counterstain with hematoxylin (Zhongshan Jinqiao, Cat. No. 23041001) for 3 minutes, differentiate with hydrochloric acid and alcohol to turn blue, and wash with water. The sections were sequentially placed in 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II for rapid dehydration, and then transparentized with xylene I and xylene II. The sections were then sealed with neutral resin glue (Solarbio, product number 20230725) and examined under a microscope.

[0187] Figure 23 shows the skin appearance of mice. Fourteen days after the capecitabine and docetaxel treatment, significant lesions appeared on the skin of the hind limbs and toes of the mice. Compared with the normal control group, the capecitabine and docetaxel groups showed significant redness, swelling, cracking, and blisters on the skin of the hind limbs and toes of the mice.

[0188] The degree of swelling in the mouse hind limb toes is shown in Figure 24. The modeling drugs capecitabine and docetaxel caused swelling in the mouse hind limb toes after 14 consecutive days of oral administration. Topical application of the compound of formula (I) ointment to the mouse hind limb toes significantly improved the swelling caused by the modeling drugs (p < 0.05). However, oral administration of 100 mg / kg nicotinamide did not significantly improve the swelling in the mouse hind limb toes.

[0189] The results of pathological staining of the toe skin tissue are shown in Figure 25. Compared with the normal control group, the modeling drugs capecitabine and docetaxel can cause significant thickening of the epidermis and stratum corneum in the skin tissue of the hind limbs and toes of mice after continuous oral administration for 14 days, an increase in basal layer granular cells, and visible inflammatory cell infiltration. Local application of the ointment of the compound of formula (I) on the hind limbs and toes of mice can effectively improve the thickening of the epidermis and stratum corneum of the skin tissue, and the morphology of the basal layer cells returns to normal, and no inflammatory cell infiltration is seen. However, oral administration of 100 mg / kg nicotinamide has a slight improvement in the thickening of the epidermis and stratum corneum of the skin tissue of the hind limbs and toes of mice, with a slightly increased number of basal layer granular cells and still visible inflammatory cell infiltration.

[0190] The immunohistochemical results of the capecitabine modeling series are shown in Figure 26. Compared with the normal control group, the modeling drug capecitabine caused a significant increase in the cytokine IL-8 in the skin tissue of the mouse hind limbs and toes after 14 consecutive days of oral administration (p < 0.05). This shows that oral administration of capecitabine can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-8 in the skin tissue of the mouse hind limbs and toes (p < 0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-8 in the skin tissue of the mouse hind limbs and toes.

[0191] The immunohistochemical results of the docetaxel modeling series are shown in Figure 27. Compared with the normal control group, the modeling drug docetaxel caused a significant increase in the cytokine IL-6 in the skin tissue of the mouse hind limbs and toes after 14 consecutive days of oral administration (p < 0.05). This shows that oral administration of docetaxel can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-6 in the skin tissue of the mouse hind limbs and toes (p < 0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-6 in the skin tissue of the mouse hind limbs and toes.

[0192] Example 12: Effects of the compound of formula (I) in a kinase inhibitor-induced mouse hand-foot skin reaction model

[0193] After 7 days of adaptive feeding, SPF ICR mice (male, 5-6 weeks old, weighing approximately 35 grams) were randomly divided into the following groups: blank control group (6 mice), sorafenib modeling group (6 mice), osimertinib modeling group (6 mice), sorafenib + compound of formula (I) group (6 mice), osimertinib + compound of formula (I) group (6 mice), sorafenib + nicotinamide group (6 mice), and osimertinib + nicotinamide group (6 mice). Each group was orally administered with the corresponding modeling drug (sorafenib 100 mg / kg, osimertinib 10 mg / kg) by gavage once daily; the blank control group was given an equal volume of normal saline. After 2 weeks of chemotherapy, each group was topically applied with the corresponding ointment of the compound of formula (I) used in Example 10 (compound content 3%) or orally administered with nicotinamide (100 mg / kg) once daily for 16 consecutive days.

[0194] Measurement of toe swelling: The degree of toe swelling of the mouse hind limbs was measured using a toe swelling tester (KW-7C, Nanjing Calvin Biotechnology Co., Ltd.) before administration of the modeling drug, 2 weeks after administration of the modeling drug, and before sacrifice. The same position was marked on the joint of the mouse hind foot before administration, 2 weeks after administration, and before sacrifice. During measurement, the water was exactly on the same horizontal line as the marked position, and the value was recorded after it stabilized.

[0195] Skin histopathological staining: Skin tissue samples from the hind limbs of mice were fixed in 4% paraformaldehyde at room temperature for 4 hours. The tissues were then removed and rinsed with running water for several hours. After dehydration with 70%, 80%, and 90% ethanol solutions, they were treated with a mixture of equal parts pure alcohol and xylene for 15 minutes. Permeabilization was performed twice with xylene for 15 minutes each, until the sample became transparent. The samples were then immersed in a mixture of 50% xylene and 50% paraffin for 15 minutes, followed by permeabilization with Paraffin I and Paraffin II for one hour each. After paraffin embedding, the samples were sliced, baked, dewaxed, and hydrated. The hydrated sections were then stained in hematoxylin solution (Zhongshan Jinqiao, Catalog No. 23041001) for 3 minutes, differentiated in hydrochloric acid ethanol solution for 15 seconds, washed with water, and blued in Scott blue solution (Servicebio, Catalog No. 20230801) for 15 seconds. After rinsing with running water, the sections were stained with eosin stain (Solarbio, Catalog No. 33535) for 3 minutes. After rinsing with running water, the sections were dehydrated, transparentized, mounted, and examined under a microscope.

[0196] Immunohistochemical staining: Paraffin sections of mouse hind limb skin tissue were baked, dewaxed, and hydrated, followed by antigen retrieval using 0.2 M citric acid buffer (1.534 g citric acid, 3.1 g sodium citrate, 100 mL distilled water, pH 4.7). Endogenous hydrogen peroxide was removed with 3% hydrogen peroxide (Shandong Anjie High-Tech Disinfection Technology Co., Ltd., Catalog No. 20290902). After incubation at room temperature for 10 minutes, sections were rinsed thoroughly with PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 1 M HCl, pH 7.4). Sections were permeabilized with 0.5% Triron-X-100 (Aimejie Technology, Catalog No. A-CSH436-100 mL). Mouse anti-IL-1β and rabbit anti-IL-8 were permeabilized for 10 minutes each, while rabbit anti-IL-6 was not permeabilized. After blocking with 5% BSA antigen, a 1 / 150 dilution of mouse anti-IL-1β (Affinity Biosciences, Catalog No. BF8021), a 1 / 200 dilution of rabbit anti-IL-8 (Affinity Biosciences, Catalog No. DF6998), or a 1 / 150 dilution of rabbit anti-IL-6 (Affinity Biosciences, Catalog No. DF6087) was added to each slide and incubated in a humidified chamber at 4°C overnight. Remove the wet chamber after overnight incubation and let it stand at room temperature for 45 minutes. Wash the slides three times with PBS buffer for 15 minutes each. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao, Cat. No. 234750811) to rabbit anti-IL-8 and rabbit anti-IL-6 markers. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-mouse IgG (H+L) (Zhongshan Jinqiao, Cat. No. 226700804) to mouse anti-IL-1β markers. Incubate at 37°C for 30 minutes and then rinse thoroughly with PBS buffer. Add DAB (CWBIO, Cat. No. 13723) for color development for 3-5 minutes, wash with PBS buffer for 1 minute, counterstain with hematoxylin (Zhongshan Jinqiao, Cat. No. 23041001) for 3 minutes, differentiate with hydrochloric acid and alcohol to turn blue, and wash with water. The sections were sequentially placed in 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II for rapid dehydration, and then transparentized with xylene I and xylene II. The sections were then sealed with neutral resin glue (Solarbio, product number 20230725) and examined under a microscope.

[0197] The results are shown in Figure 28. After 14 days of modeling with sorafenib and osimertinib, significant lesions appeared on the skin of the hind limbs and toes of the mice. Compared with the normal control group, the skin of the hind limbs and toes of the mice in the sorafenib and osimertinib groups showed significant redness, swelling, and cracking.

[0198] The degree of swelling in the mouse hind limbs and toes is shown in Figure 29. The modeling drugs sorafenib and osimertinib caused significant swelling in the mouse hind limbs and toes after 14 consecutive days of oral administration. Topical application of the compound of formula (I) ointment to the mouse hind limbs and toes effectively improved the toe swelling caused by the modeling drugs. However, oral administration of 100 mg / kg nicotinamide did not significantly improve the swelling in the mouse hind limbs and toes.

[0199] The results of pathological staining of the toe skin tissue are shown in Figure 30. Compared with the normal control group, the modeling drugs sorafenib and osimertinib can cause significant thickening of the epidermis and stratum corneum in the skin tissue of the hind limbs and toes of mice after continuous oral administration for 14 days, an increase in basal layer granular cells, and visible inflammatory cell infiltration. Local application of the ointment of the compound of formula (I) on the hind limbs and toes of mice can effectively improve the thickening of the epidermis and stratum corneum of the skin tissue, and the morphology of the basal layer cells returns to normal, and no inflammatory cell infiltration is seen. However, oral administration of 100 mg / kg nicotinamide has no significant improvement in the thickening of the epidermis and stratum corneum of the skin tissue of the hind limbs and toes of mice, and the morphology of the basal layer cells recovers slightly, and inflammatory cell infiltration is still seen.

[0200] The immunohistochemical results of the Sorafenib modeling series are shown in Figure 31. Compared with the normal control group, the modeling drug Sorafenib caused a significant increase in the cytokine IL-1β in the skin tissue of the hind limbs and toes of mice after continuous oral administration for 14 days (p<0.05). This shows that oral administration of Sorafenib can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-1β in the skin tissue of the hind limbs and toes of mice (p<0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-1β in the skin tissue of the hind limbs and toes of mice.

[0201] The immunohistochemical results of the osimertinib modeling series are shown in Figure 32. Compared with the normal control group, the modeling drug osimertinib caused a significant increase in the cytokine IL-1β in the skin tissue of the mouse hind limbs and toes after 14 consecutive days of oral administration (p < 0.05). This shows that oral administration of osimertinib can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-1β in the skin tissue of the mouse hind limbs and toes (p < 0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-1β in the skin tissue of the mouse hind limbs and toes.

[0202] Example 13: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and isonicotinamide

[0203] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 10.2 mg of isonicotinamide, and ethanol (0.2 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and isonicotinamide. The XRPD spectrum is shown in Figure 1, and the characteristic peak positions are shown in Table 4. The DSC spectrum showed that the melting point T onset 160.45°C. The chemical dosage ratio of the obtained drug cocrystal is 1:1.

[0204] Table 4 XRPD diffraction peak data of drug co-crystal formed by the compound represented by formula (I) and isonicotinamide

[0205] Example 14: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and glycolic acid

[0206] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 6.3 mg of glycolic acid, and isopropyl acetate (0.2 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and glycolic acid. The XRPD spectrum is shown in Figure 4, and the characteristic peak positions are shown in Table 5. The DSC spectrum shows that the melting point T onset @135.68℃. The obtained drug cocrystal has a chemical dosage ratio of 1:1.

[0207] Table 5 XRPD diffraction peak data of drug co-crystal formed by the compound represented by formula (I) and glycolic acid

[0208] Example 15: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and L-proline

[0209] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 9.7 mg of L-proline, and ethanol (0.25 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and L-proline. The XRPD spectrum is shown in Figure 7, and the characteristic peak positions are shown in Table 6. The DSC spectrum shows that the melting point T onset@183.55℃. The obtained drug cocrystal has a chemical dosage ratio of 1:1.

[0210] Table 6: XRPD diffraction peak data of the drug co-crystal formed by the compound represented by formula (I) and L-proline

[0211] Example 16: Preparation of a pharmaceutical co-crystal of the compound represented by formula (I) and L-proline

[0212] Weigh approximately 300 mg of the compound represented by formula (I) and 145.8 mg of L-proline (1.0 equivalent) into a 20 mL glass bottle. Add 3.75 mL of ethanol and stir at 5°C for 2 minutes to obtain a suspension. Add approximately 5 mg of seed crystals of the pharmaceutical co-crystal formed by the compound represented by formula (I) and L-proline to the above suspension. Add 3 mL of ethanol to the above suspension and stir at 5°C for 4 days. Collect the solid portion by centrifugation and dry the obtained solid portion under vacuum at room temperature for about 2.5 hours. A total of 250 mg of white powder is obtained with a yield of 56%. X-ray powder diffraction analysis shows that the product is a pharmaceutical co-crystal formed by the compound represented by formula (I) and L-proline.

[0213] Example 17: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and nicotinamide

[0214] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 10.3 mg of nicotinamide, and acetonitrile (0.25 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a pharmaceutical co-crystal formed by the compound represented by formula (I) and nicotinamide. The XRPD spectrum is shown in Figure 10, and the characteristic peak positions are shown in Table 7. The DSC spectrum showed that the melting point T onset @156.75℃. The obtained drug cocrystal has a chemical dosage ratio of 1:1.

[0215] Table 7 XRPD diffraction peak data of drug co-crystal formed by the compound represented by formula (I) and nicotinamide

[0216] Example 18: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and nicotinamide

[0217] Approximately 300 mg of the compound of formula (I) and 154.6 mg of nicotinamide (1.0 equivalent) were weighed and placed in a 20 mL glass vial. 3.75 mL of acetonitrile was added, and the mixture was stirred at 5°C for 2 minutes to obtain a suspension. Approximately 5 mg of seed crystals of the pharmaceutical co-crystal formed by the compound of formula (I) and nicotinamide were added to the suspension. 2 mL of acetonitrile was then added to the suspension, and the mixture was stirred at 5°C for 4 days. The solid portion was collected by centrifugation and dried under vacuum at room temperature for approximately 2.5 hours. A total of 365 mg of white powder was obtained, with a yield of 80%. X-ray powder diffraction analysis confirmed that the product was a pharmaceutical co-crystal formed by the compound of formula (I) and nicotinamide.

[0218] In summary, four drug cocrystals were obtained. Glycolic acid cocrystals often have hygroscopicity issues. Isonicotinamide is similar to niacinamide, but niacinamide is a GRAS (Generally Recognized As Safe) agent, while isonicotinamide is a non-GRAS agent. Ultimately, L-proline cocrystals and niacinamide cocrystals were selected for further study of druggable properties such as stability and hygroscopicity.

[0219] Example 19: Stability Test of Pharmaceutical Co-crystals Formed by the Compound of Formula (I) and L-Proline and Pharmaceutical Co-crystals Formed by the Compound of Formula (I) and Nicotinamide

[0220] Open containers containing the pharmaceutical co-crystals formed by the compound of formula (I) and L-proline and the pharmaceutical co-crystals formed by the compound of formula (I) and nicotinamide were placed at 25°C / 92.5% RH for one week. The pharmaceutical co-crystals formed by the compound of formula (I) and L-proline and the pharmaceutical co-crystals formed by the compound of formula (I) and nicotinamide were placed in sealed containers at 60°C for one week. After solid stability evaluation, the samples were characterized by XRPD and HPLC, and the color changes of the samples were observed. The results are shown in Table 8.

[0221] Table 8 Solid stability of drug co-crystals formed by compounds represented by formula (I)

[0222] As shown in Table 8, the pharmaceutical co-crystals formed by the compound of formula (I) with L-proline and nicotinamide exhibited no significant decrease in purity after being stored in an open container for one week at 25°C / 92% RH or in a sealed container for one week at 60°C. However, the pharmaceutical co-crystals formed by the compound of formula (I) with L-proline partially dissociated into the free crystalline form and L-proline at 25°C / 92.5% RH. The pharmaceutical co-crystals formed by the compound of formula (I) with nicotinamide were relatively stable under both conditions.

[0223] Example 20: Solubility Test of Pharmaceutical Co-crystals Formed by the Compound of Formula (I) and L-Proline and Pharmaceutical Co-crystals Formed by the Compound of Formula (I) and Nicotinamide

[0224] Accurately weigh 15.1 mg of the pharmaceutical cocrystal formed by the compound of formula (I) and L-proline and 14.9 mg of the pharmaceutical cocrystal formed by the compound of formula (I) and nicotinamide, and place them separately in 20 mL glass vials. Add 5 mL of solvent to each. The mass of the compound of formula (I) in the weighed cocrystals is equivalent to 10 mg of the free anhydrous crystalline form. The resulting suspension was stirred at 400 rpm at 37°C, and samples were taken after 0.5 and 2 hours. The samples were centrifuged at 14,000 rpm at 37°C for 5 minutes. The supernatant concentration was determined by HPLC, and the pH of the supernatant was measured using a pH meter. The residual solid (wet product) after 2 hours was examined by XRPD to determine if there was any change in the crystal form. The experimental results are shown in Table 9.

[0225] Table 9 Solid solubility of drug co-crystals formed by compounds represented by formula (I)

[0226] As shown in Table 9, the drug co-crystals formed with the compound of formula (I) and L-proline, as well as the drug co-crystals formed with nicotinamide, transformed into the free crystalline form of the compound of formula (I) in all four different solvents. Furthermore, the drug co-crystals formed with L-proline and nicotinamide exhibited high solubility in the remaining solvents. Furthermore, the solubility of the drug co-crystals formed with nicotinamide after 2 hours of dissolution in all four solvents was higher than that of the drug co-crystals formed with L-proline.

[0227] Example 21: Hygroscopicity Test of Pharmaceutical Co-crystals Formed by the Compound of Formula (I) and L-Proline and Pharmaceutical Co-crystals Formed by the Compound of Formula (I) and Nicotinamide

[0228] The water absorption and dehydration behaviors of pharmaceutical cocrystals formed by the compound of formula (I) with L-proline and pharmaceutical cocrystals formed by the compound of formula (I) with nicotinamide were investigated by DVS testing at 25°C. The DVS cycle was 40%-0%-95%-0%-40% RH. XRPD analysis was performed on samples after DVS testing to determine whether crystal transformation occurred. The results are shown in Table 10.

[0229] Table 10 Hygroscopicity test of drug co-crystals formed by compounds represented by formula (I)

[0230] The results in Table 10 indicate that the pharmaceutical cocrystal formed by the compound of formula (I) and L-proline is slightly hygroscopic between 40% and 80% RH, with a weight gain of approximately 0.4% upon absorption. It is extremely hygroscopic between 80% and 95% RH, with a weight gain of approximately 84.0%. After DVS testing, the cocrystal partially dissociates into the free crystalline form and L-proline. The pharmaceutical cocrystal formed by the compound of formula (I) and nicotinamide is slightly hygroscopic, with a weight gain of approximately 0.2% upon absorption between 40% and 95% RH at 25°C. DVS testing reveals no change in crystalline form.

[0231] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A pharmaceutical co-crystal formed by combining a compound represented by formula (I) and a co-crystal former, wherein the co-crystal former is selected from nicotinamide, isonicotinamide, L-proline, glycolic acid, 2. The pharmaceutical co-crystal according to claim 1, characterized in that: The chemical ratio of the compound represented by formula (I) to the co-crystal 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 co-crystal according to claim 1, wherein the co-crystal former is nicotinamide.

4. A drug co-crystal formed by the compound represented by formula (I) and isonicotinamide, It is characterized in that The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566; preferably, 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, 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; the most preferred X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is shown in Figure 1.

5. A drug co-crystal formed by the compound represented by formula (I) and glycolic acid, It is characterized in that The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125; preferably, characteristic peaks are 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, characteristic peaks are 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, there are 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 represented by the diffraction angle 2θ is shown in Figure 4.

6. A drug co-crystal formed by the compound represented by formula (I) and L-proline, It is characterized in that The X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably 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 have characteristic peaks; preferably, there are 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 represented by the diffraction angle 2θ is shown in Figure 7.

7. A drug co-crystal formed by the compound represented by formula (I) and nicotinamide, It is characterized in that The X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573; preferably, there are 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, there are 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, 14.747, 24.780, 26.165, 26.573, 27.028, 30.766, 32.820 and 36.741; preferably, there are 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 represented by the diffraction angle 2θ is shown in Figure 10.

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

20.

9. The method for preparing the pharmaceutical co-crystal formed by the compound represented by formula (I) and isonicotinamide according to claim 4, characterized in that: The method comprises the steps of: mixing the compound represented by formula (I) with isonicotinamide, adding solvent 1, stirring, and filtering; preferably, the solvent 1 is selected from one or more of alcohol, ester, nitrile and water, preferably an alcohol solvent, more preferably C 1-4 The alcohol is more preferably one or more of methanol, ethanol and isopropanol, and most preferably ethanol.

10. A method for preparing a pharmaceutical co-crystal formed by the compound represented by formula (I) and glycolic acid as claimed in claim 5, characterized in that: The method comprises the steps of: mixing the compound represented by formula (I) with glycolic acid, adding solvent 2, stirring, and filtering; preferably, the solvent 2 is selected from one or more of alcohol, ester, nitrile 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 co-crystal formed by the compound represented by formula (I) and L-proline as claimed in claim 6, characterized in that: The method comprises the steps of: mixing the compound represented by formula (I) with L-proline, adding solvent 3, stirring, and filtering; preferably, the solvent 3 is selected from one or more of alcohol, ester, nitrile and water, preferably an alcohol solvent, more preferably C 1-4 The alcohol is more preferably one or more of methanol, ethanol and isopropanol, and most preferably ethanol.

12. The method for preparing the pharmaceutical co-crystal formed by the compound represented by formula (I) and nicotinamide as claimed in claim 7, characterized in that: The method comprises the steps of: mixing the compound represented by formula (I) with nicotinamide, adding solvent 4, stirring, and filtering; preferably, the solvent 4 is selected from one or more of alcohol, ester, nitrile 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 by combining a pharmaceutical co-crystal formed by combining the compound of formula (I) according to any one of claims 1 to 3 and a co-crystal former, or a pharmaceutical co-crystal formed by the compound of formula (I) and isonicotinamide according to claim 4, or a pharmaceutical co-crystal formed by the compound of formula (I) and glycolic acid according to claim 5, or a pharmaceutical co-crystal formed by the compound of formula (I) and L-proline according to claim 6, or a pharmaceutical co-crystal formed by the compound of formula (I) and nicotinamide according to claim 7.

14. A pharmaceutical composition comprising a pharmaceutical co-crystal formed by combining the compound of formula (I) according to any one of claims 1 to 3 and a co-crystal former, or a pharmaceutical co-crystal formed by the compound of formula (I) and isonicotinamide according to claim 4, or a pharmaceutical co-crystal formed by the compound of formula (I) and glycolic acid according to claim 5, or a pharmaceutical co-crystal formed by the compound of formula (I) and L-proline according to claim 6, or a pharmaceutical co-crystal formed by the compound of formula (I) and nicotinamide according to claim 7, and optionally a pharmaceutically acceptable excipient.

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

16. Use of a pharmaceutical co-crystal formed by combining the compound of formula (I) according to any one of claims 1 to 3 and a co-crystal former, or a pharmaceutical co-crystal formed by the compound of formula (I) and isonicotinamide according to claim 4, or a pharmaceutical co-crystal formed by the compound of formula (I) and glycolic acid according to claim 5, or a pharmaceutical co-crystal formed by the compound of formula (I) and L-proline according to claim 6, or a pharmaceutical co-crystal formed by the compound of formula (I) and nicotinamide according to claim 7, or a composition according to claim 13 or 14, or a composition prepared by the method according to 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 malformations, digestive system diseases, oral and maxillofacial diseases, vascular and lymphatic system diseases or pain, preferably hand-foot syndrome, hand-foot skin reaction, cancer, and dermatological diseases.

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