WO2025119335A1 - Co-crystal form of rna m6a modulator and preparation method therefor and use thereof - Google Patents

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 RNA m6A regulator and its preparation method and application

The present disclosure claims the priority of a Chinese patent application filed with the Patent Office of China on December 8, 2023, with the invention name “A co-crystal form of an RNA m6A regulator, its preparation method and application” and application number 2023116845011. The entire contents of the above application are incorporated into the present disclosure by reference.

Technical Field

The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a co-crystal form of an RNA m6A regulator and a preparation method and application thereof.

Background Art

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

Drugs that can cause hand-foot syndrome include chemotherapy drugs such as capecitabine, liposomal doxorubicin, cytarabine, docetaxel, vinorelbine, continuous infusion doxorubicin, and gemcitabine; drugs that can cause hand-foot skin reactions include targeted drugs such as sonitinib (Sutent), sorafenib (Nexavar), imatinib (Gleevec), and erlotinib (Tarceva). The World Health Organization (WHO) divides HFS into 4 grades: Grade 1, numbness, paresthesia, or tingling in the hands and feet; Grade 2, discomfort when holding objects and walking, painless swelling or erythema; Grade 3, painful erythema, edema of the palms and soles, periungual erythema and swelling; Grade 4, peeling, ulceration, blistering, and severe pain. At present, severe hand-foot syndrome and hand-foot skin reactions in clinical practice are often relieved by superficial skin care only, and even need to be discontinued. Existing treatments only treat the symptoms and not the root cause, which seriously limits the use of first-line tumor chemotherapy drugs. Therefore, there is a huge unmet medical need. There is a need to develop a drug with good efficacy, few side effects, and low cost for the prevention and treatment of hand-foot syndrome and hand-foot skin reaction to meet the growing medical needs worldwide.

m6A methylation modification is the most common RNA modification in mammals. It can regulate a variety of signaling pathways and cellular processes (such as growth, development, and disease) to play a key biological role (Frye M., et al. Science 2018, 361, 2073-2092; Yang C., et al. Cell Death & Disease 2020, 11, 960; Meyer K.D. & Jaffrey S.R. Nature Review Molecular Cell Biology 2014, 15, 313-326). The m6A methylation modification of mRNA, miRNA, circRNA, and lncRNA is a dynamic and reversible process. Methyltransferases (such as METTL3, METTL14, and METTL16) bind methyl groups to RNA, while demethylases (FTO and ALKBH5) can erase the methyl groups on RNA, thus 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 P.J., et al. Journal of Biological Chemistry 2019, 294, 19889-19895; Yao Y., et al. FASEB Journal 2019, 33, 7529-7544).

Currently, there are few known direct associations between RNA m6A and skin-related diseases. Most of them are m6A-related methylases or demethylases that 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 acrofacial melanoma are significantly higher than those in adjacent tissues, and the mRNA expression level of METTL3 in patients with advanced acrofacial melanoma is significantly higher than that in patients with early-stage melanoma (Le Zhanghui, Preliminary study on the pathogenesis of LncRNA and RNA m6A methylation in acrofacial melanoma, dissertation, 2019). In the diagnosis of melanoma, m6A-specific binding proteins YTHDF1 and HNRNPA2B1 can be used as new biomarkers (Li T.D., et al. Cancer Cell 2020, 20, 239). In terms of keratinocytes, it has only been reported that long-term, low-level arsenic exposure can inhibit selective autophagy caused by m6A demethylase, thereby inducing the occurrence of skin tumors (Cui Y.H., et al. Nature Communications 2021, 12, 2183). In summary, there is currently no method to prevent and treat skin diseases by regulating RNA m6A methylation.

In view of this, the present invention is proposed.

Summary of the invention

Problem that the invention aims to solve

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

Solutions for solving problems

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,

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

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.

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

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.

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

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.

The present invention provides a drug co-crystal formed by a compound represented by formula (I) and nicotinamide.

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.

Effects of the Invention

The drug co-crystals formed by the compound of formula (I) and isonicotinamide, the drug co-crystals formed by the compound of formula (I) and glycolic acid, the drug co-crystals formed by the compound of formula (I) and L-proline, and the drug co-crystals formed by the compound of formula (I) and nicotinamide provided by the present invention have advantages in terms of physical and chemical properties, preparation processing performance and bioavailability, for example, at least one of the aspects of melting point, solubility, hygroscopicity, purification effect, stability, adhesion, compressibility, fluidity, in vivo and in vitro dissolution, and bioavailability has advantages. The drug co-crystals formed by combining the compound of formula (I) and the co-crystal former according to the present invention have good physical and chemical stability, a high yield of crystal forms prepared with the same starting materials, and obvious advantages in terms of solubility, hygroscopicity, stability, mechanical stability, fluidity, compressibility and adhesion, etc., which provide a new and better choice for the development of RNA m6A regulator drugs, and are of great significance.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

FIG. 7 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and L-proline.

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

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

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

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

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

FIG. 13 depicts exemplary results of cellular m6A levels and m6A levels of NAP1L2 measured by nanopore sequencing 24 hours after administration of sorafenib, capecitabine, docetaxel, and osimertinib to keratinocytes HaCat.

FIG. 14 depicts exemplary results of cellular m6A levels and m6A levels of NAP1L2 in mouse hind limb skin tissue in Example 3.

FIG. 15 depicts exemplary results of the expression level of NAP1L2 mRNA 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.

16 depicts exemplary results of mRNA and protein expression levels 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.

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.

FIG. 18 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.

FIG. 19 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.

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

FIG. 21 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.

FIG. 22 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.

FIG. 23 depicts exemplary results of mouse hind limb toe skin in Example 11.

FIG. 24 depicts exemplary results of the degree of swelling of the hind limb toes of mice in Example 11.

FIG. 25 depicts exemplary results of histopathology of mouse hind limb skin tissue in Example 11.

FIG. 26 depicts exemplary results of immunohistochemistry for IL-8 in the skin tissue of the hind limbs of mice in the capecitabine modeling series in Example 11.

FIG. 27 depicts exemplary results of immunohistochemistry for IL-6 in the skin tissue of the hind limbs of mice in the docetaxel modeling series in Example 11.

FIG. 28 depicts exemplary results of mouse hind limb skin in Example 12.

FIG. 29 depicts exemplary results of the degree of swelling of the mouse hind limb toes in Example 12.

FIG30 depicts exemplary results of histopathology of mouse hind limb skin tissue in Example 12.

FIG31 depicts exemplary results of immunohistochemistry of cytokine IL-1β in the skin tissue of the hind limbs and toes of mice in the Sorafenib modeling series groups in Example 12.

FIG32 depicts exemplary results of immunohistochemistry of cytokine IL-1β in the skin tissue of the hind limbs and toes of mice in the osimertinib modeling series groups in Example 12.

DETAILED DESCRIPTION

In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

The present invention provides a drug co-crystal formed by a compound represented by formula (I) and isonicotinamide.

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.

The present invention further provides a method for preparing a drug co-crystal 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.

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

In certain embodiments, the solvent 1 is selected from C _1-4 alcohols.

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

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

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

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

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.

The present invention further provides a method for preparing a drug co-crystal formed by a 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.

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

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.

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

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

The present invention further provides a pharmaceutical co-crystal formed by a compound represented by formula (I) and L-proline,

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.

The present invention further provides a method for preparing a drug co-crystal formed by a compound of formula (I) and L-proline, comprising the steps of: mixing the compound of formula (I) and L-proline, adding solvent 3, stirring, and filtering.

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

In certain embodiments, the solvent 3 is selected from C _1-4 alcohols.

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

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

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

The present invention further provides a drug co-crystal formed by the compound represented by formula (I) and nicotinamide,

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.

The present invention further provides a method for preparing a drug 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.

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

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

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

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

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

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.

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

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

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

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

In certain embodiments, the pharmaceutical composition is an ointment.

In certain embodiments, the pharmaceutical combination is a plaster.

In certain embodiments, the pharmaceutical combination is a cream.

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

In certain embodiments, the pharmaceutical combination is a tablet.

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

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 the aforementioned compound of formula (I) and isonicotinamide, or a pharmaceutical co-crystal formed by the aforementioned compound of formula (I) and glycolic acid, or a pharmaceutical co-crystal formed by the aforementioned compound of formula (I) and L-proline, or a pharmaceutical co-crystal formed by the aforementioned compound of formula (I) and nicotinamide with a pharmaceutically acceptable excipient.

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

In some embodiments, the m6A regulation-related disease is an endocrine and metabolic disease, a nervous system disease, a tumor, a cardiovascular disease, an infection, an immune system disease, a genitourinary system disease, a skin and musculoskeletal disease, a respiratory system disease, a genetic disease and deformity, a digestive system disease, an oral and maxillofacial disease, a vascular and lymphatic system disease-related disease or pain.

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

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.

The precipitation methods described in the present invention include but are not limited to stirring, cooling, volatilization, pulping, and precipitation.

"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.

In some embodiments, the beating time is 5h-30h.

According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "9103 Drug Hygroscopicity Guidance Principles" in the fourth volume of the 2020 edition of the Chinese Pharmacopoeia,

Deliquesce: Absorbs enough water to form a liquid;

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

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

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

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

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 a constant temperature of the sample, so as to characterize all physical and chemical changes related to thermal effects and obtain the phase change information of the sample.

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.

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, and the implementation conditions not specified are usually the conditions in routine experiments.

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

XRPD: X-ray powder diffraction

DSC: Differential Scanning Calorimetry

TGA: Thermogravimetric analysis

DVS: Dynamic Water Sorption

HPLC: High Performance Liquid Chromatography

Testing instruments and methods

X-ray powder diffraction (XRPD)

The crystal form of the sample was analyzed using a Bruker D8 ADVANCE X-powder diffractometer. The sample 2θ scanning angle was 3° to 40°, the scanning step was 0.02°, and the scanning time per step was 0.12s/step. The light tube voltage and current were 40kV and 40mA, respectively. When preparing the sample, place an appropriate amount of sample on the sample tray and flatten it with a glass sheet or other tool to ensure that its surface is smooth and flat.

Thermogravimetric analysis (TGA)

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

Differential Scanning Calorimetry (DSC)

The samples were analyzed using 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).

Dynamic Water Sorption Analysis (DVS)

The samples were analyzed using ProUmid SPSx-1μAdvance. The test sample size was approximately 5-50 mg. The temperature of the test chamber was controlled between 25±1°C, the relative humidity cycle was 40-95-0-95-40%, the step was 10%, the balance was 240 minutes per step, and the mass data was recorded every 20 seconds.

High Performance Liquid Chromatography (HPLC)

Solubility and stability tests were performed using Agilent 1260 infinityII Binary Pump.

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

3-Bromo-2-oxocyclohexane-1-carboxylic acid ethyl ester (20 mg, 0.08 mmol) and 4-chloroaniline (25 mg, 0.2 mmol) were mixed and then heated to 150°C. After reacting for 3 hours, the reaction solution was cooled to room temperature, diluted with 100 mL of dichloromethane, washed 3 times with 100 mL of 1N HCl and once with saturated 100 mL of NaHCO _3. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The product was separated and purified by silica gel column chromatography to obtain 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid ethyl ester (16 mg). 10 mg of 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylic acid ethyl ester (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, and then 20 mL of water was added for dilution after rotary distillation to adjust the pH to 2. The mixture was then extracted 3 times with 50 mL of dichloromethane, and the organic phases were combined, dried and concentrated, and then ammonia water was added. The mixture was heated at 60° C. for 24 hours, and the mixture was separated and purified by silica gel column chromatography to obtain white solid 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (compound 1, 7.6 mg). The mixture was separated by Chiralpak AD chiral column to obtain (S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazolecarboxamide (compound shown in formula (I)). LCMS [M+H] ⁺ of the compound shown in formula (I): 249. ¹ H-NMR (400MHz, DMSO-d ₆ ): 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).

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

1*10 ⁸ HaCat cells were plated in a culture dish, and 50 nM drugs (sorafenib, capecitabine, docetaxel and osimertinib) were added respectively. The cells were cultured at 37°C in an incubator containing 5% CO ₂ for 24 hours. The culture medium was discarded and washed with PBS. After centrifugation and discarding the supernatant, 400 μL RLT cell lysis buffer (QIAGEN, Cat.#79216) was added. Some samples were taken, 400 μL 70% ethanol was added, stirred evenly, and transferred to RNeasy mini columns. RNA was extracted using RNeasy mini kit (QIAGEN, Cat.#74104). 2 μL RNA samples were dissolved in 98 μL 10 mM Tris-HCl buffer, and the absorbance at 260 nm was measured using Nanodrop (Thermo Fisher Scientific) to determine the RNA concentration.

40 μg RNA sample was taken and enriched with NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, Cat.#E7490), and then the reverse transcription adapter RTA was connected to the 3' end of the enriched mRNA (PolyA+RNA) molecule 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, and the reverse transcription adapter RTA end was connected to the sequencing adapter (RNA adapter) to form the final sequencing library, and the Qubit ^TM dsDNA HS assay kit (Invitrogen, Cat.#LOT2133187) was used for quantitative detection.

After library quality inspection, the prepared sequencing library was loaded into the PromethION Flow Cell chip (Oxfod Nanopore Technologies, Cat. #FLO-MIN106D) and placed into the PromenthION sequencer (Oxfod Nanopore Technologies) to select the matching sequencing mode for sequencing. The sequencing data was base called by guppy, and the m6A methylation analysis was performed with the original fast5 data.

The results are shown in Figure 13. Compared with the blank control group, the m6A methylation levels of HaCat cells increased by 5.05 times, 6.01 times, 3.97 times and 5.13 times after adding sorafenib, capecitabine, docetaxel and osimertinib, respectively. This shows that anti-tumor drugs (sorafenib, capecitabine, docetaxel and osimertinib) can induce abnormal m6A methylation of human keratinocyte mRNA. Further bioinformatics analysis found that there was a significant difference in the m6A methylation level of NAP1L2, mainly reflected in the m6A methylation of the UGAGGACUCA fragment.

Example 3: Changes in mRNA methylation levels in drug-induced adverse skin reaction models in mice

ICR male mice (5-6 weeks old, weighing about 35 grams) were randomly divided into groups after 7 days of adaptive feeding, with 6 mice in each group: blank control group, sorafenib group, capecitabine group, docetaxel group and osimertinib group. Each group was given the corresponding modeling drugs (sorafenib 100 mg/kg, capecitabine 200 mg/kg, docetaxel 25 mg/kg, osimertinib 10 mg/kg) by gavage once a day, and the mice were killed after 30 consecutive days of administration, and the skin tissue of the hind limbs and toes of the mice was obtained. Take a small amount of tissue sample, put it into a mortar filled with liquid nitrogen and grind it into powder, add single-phase lysis solution, leave it at room temperature for 5 minutes, centrifuge (12000rpm) for 5 minutes, take 1mL supernatant, add 200μL chloroform, shake and mix, leave it at room temperature for 15 minutes, centrifuge (12000rpm, 4℃) for 15 minutes, aspirate the upper aqueous phase and add an equal volume of isopropanol, leave it at -20℃ for 1 hour, centrifuge (12000rpm, 4℃), and discard the supernatant. Add 400μL 70% ethanol to the precipitate, stir well and transfer it to the RNeasy mini column, and use the RNeasy mini kit (QIAGEN, Cat.#74104) to extract RNA. Take 2μL RNA sample and dissolve it in 98μL 10mM Tris-HCl buffer, and measure the absorbance at 260nm with Nanodrop (Thermo Fisher Scientific) to determine the RNA concentration.

40 μg RNA sample was taken and enriched with NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, Cat.#E7490), and then the reverse transcription adapter RTA was connected to the 3' end of the enriched mRNA (PolyA+RNA) molecule 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, and the reverse transcription adapter RTA end was connected to the sequencing adapter (RNA adapter) to form the final sequencing library, and the Qubit ^TM dsDNA HS assay kit (Invitrogen, Cat.#LOT2133187) was used for quantitative detection.

After library quality inspection, the prepared sequencing library was loaded into the PromethION Flow Cell chip (Oxfod Nanopore Technologies, Cat. #FLO-MIN106D) and placed into the PromenthION sequencer (Oxfod Nanopore Technologies) to select the matching sequencing mode for sequencing. The sequencing data was base called by guppy, and the m6A methylation analysis was performed with the original fast5 data.

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.

Example 4: m6A regulates the mRNA expression level of NAP1L2

1*10 ⁸ HaCat cells were plated in a culture dish, and m6A methylase METTL3 recombinant protein (Abcam, Cat.#ab271611) and m6A demethylase FTO recombinant protein (Abcam, Cat.#ab271525) were added, or METTL3 siRNA (sh-METTL3, GeneCare) and FTO siRNA (sh-FTO, GeneCare) were transfected, and the cells were placed in an incubator containing 5% carbon dioxide at 37°C for 24 hours. After washing with PBS, the cells were collected by high-speed centrifugation. Total RNA was extracted with Trizol reagent (Sigma Aldrich), and 1 μg RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. # AT311-03). 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 were added to prepare a 20 μL reaction solution for RT-PCR reaction. After the reaction was completed, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression level of NAP1L2.

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

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

1*10 ⁸ HaCat cells were plated in a culture dish, and human recombinant NAP1L2 protein (Abcam, Cat.#ab117213), sorafenib, capecitabine, docetaxel and osimertinib were added, or NAP1L2 siRNA (sh-NAP1L2, GeneCare) was transfected, and the cells were placed in an incubator containing 5% carbon dioxide at 37°C for 24 hours, washed with PBS, and collected by high-speed centrifugation. RLT lysis buffer was added to the collected cells, shaken at 4°C for half an hour, and the supernatant was collected after centrifugation, and the expression of metal matrix protein was measured by Western blotting.

The total RNA of the cells collected above was extracted with Trizol reagent (Sigma Aldrich), and 1 μg RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). 1.25 μL primers (Sino Biological, Cat. #HP100168 & HP100367), 10 μL iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125) and an appropriate amount of DEPC ultrapure water were added to prepare a 20 μL reaction solution for RT-PCR reaction. After the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression of metal matrix protein.

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

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

1*10 ⁸ HaCat cells were plated in a culture dish, recombinant HBEGF protein (Thermo Fisher Scientific, Cat.#100-47-1mg) was added, and the cells were placed in an incubator containing 5% carbon dioxide at 37°C for 24 hours. HBEGF antibody (Invitrogen, Cat.#406316) or transfected with siRNA of METTL3 (sh-METTL3, Jikai Gene) and siRNA of NAP1L2 (sh-NAP1L2, Jikai Gene) were added, and the cells were placed in an incubator containing 5% carbon dioxide at 37°C for 24 hours. After washing with PBS, the cells were collected by high-speed centrifugation. RLT lysis buffer was added to the above collected cells, shaken at 4°C for half an hour, and the supernatant was taken after centrifugation. The protein expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin and Involucrin were measured by Western blotting.

Total RNA was extracted with Trizol reagent (Sigma Aldrich), and 1 μg RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. # AT311-03). 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 were added to prepare a 20 μL reaction solution for RT-PCR reaction. After the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin and Involucrin.

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

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

1*10 ⁸ THP-1 cells were plated in a culture dish, and 4 nM and 400 nM of the compound of formula (I), 4 nM and 4 μM nicotinamide, and 5 μM of m6A methylase inhibitor UZH2 were added, respectively, and cultured at 37°C in an incubator containing 5% CO ₂ for 24 hours. The culture medium was discarded and washed with PBS, centrifuged and the supernatant was discarded, 400 μL of RLT cell lysis buffer was added, and stored at -80°C. A portion of the sample was taken, 400 μL of 70% ethanol was added and stirred evenly, 700 μL of the sample was transferred to an RNeasy centrifugal column and centrifuged for 15 seconds (8000g, 25°C), 700 μL of RW1 buffer was added and centrifuged for 15 seconds (8000g, 25°C), and then 500 μL of RPE buffer was added and centrifuged for 16 seconds (8000g, 25°C), and the above steps were repeated twice and centrifuged for 2 minutes to completely remove the eluent. Add 30 μL of nuclease-free water to the column, incubate for 5 minutes, centrifuge for 2 minutes (12000 g, 25°C), and repeat this step 3 times. Take 2 μL of RNA sample and dissolve it in 98 μL of 10 mM Tris buffer, and measure the absorbance at 260 nm with Nanodrop to determine the RNA concentration.

Take 50μg of total RNA and dissolve it in 100μL of nuclease-free water. Resuspend Oligo(dT) magnetic beads, transfer 50μL of magnetic beads to a 1.5mL test tube, add 500μL of binding buffer, let stand and remove the supernatant. Add 100μL of RNA sample to 200μL of magnetic bead suspension and mix, stir and incubate at 25℃ in a Theromixer for 5 minutes, discard the supernatant and add 200μL of wash buffer, repeat twice and centrifuge for 10 seconds (2000g, 25℃). Add 50μL of elution buffer and mix well, heat to 75℃, transfer the supernatant to a new 1.5mL test tube, and use RNA Clean&Concentrator kit to purify and measure the RNA concentration.

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 ZnCl ₂ 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 NH ₄ HCO ₃ solution and 1 unit 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 to analyze mA methylation fragments by LC-MS, and calculate mA inhibition rate.

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

As shown in Figure 18, the compound of formula (I) can significantly inhibit the m6A methylation level of cellular mRNA, and it is concentration-dependent. At a concentration of 400nM, the compound of formula (I) has an m6A inhibitory activity comparable to that of the m6A methylase inhibitor UZH2 at a concentration of 5μM. However, nicotinamide has no inhibitory effect on the m6A methylation level of cellular mRNA at either low concentration (4nM) or high concentration (4μM).

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

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

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

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

The results are shown in Table 1. Treatment with sorafenib, capecitabine, docetaxel and osimertinib can lead to a significant increase in the m6A methylation level of human keratinocytes HaCat (as shown in Table 1). The compound of formula (I) can effectively inhibit the abnormal increase of RNA m6A level in human keratinocytes caused by chemotherapeutic drugs and multikinase inhibitors.

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 / measured m6A amount in control 1.

Example 9: Inhibitory effect of compounds on keratinocyte HaCaT differentiation

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

Table 2 Some primer sequence information

As shown in Figure 19, after adding human recombinant HBEGF protein for induction, the mRNA expression levels of HaCaT differentiation markers KRT1, KRT10, Loricrin and Involucrin increased significantly. However, after adding the compound of formula (I), the mRNA expression levels of HaCaT differentiation markers KRT1, KRT10, Loricrin and Involucrin were significantly inhibited.

Example 10: Inhibitory effect of compounds on keratinocyte differentiation in rat hand-foot skin reaction model

After SD rats (weighing about 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% H ₂ O, respectively, and the modeling drugs were fixed to the required concentrations and administered by gavage once a day according to the doses shown in Table 3. One hour after gavage, 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, and 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 a day. After continuous administration for 30 days, the rats were euthanized, and the paw plantar skin tissue was taken, fixed in 10% neutral formaldehyde and cut into 5 μm slices, and embedded in paraffin after dehydration. The ointment used for the test compound was prepared by mixing the compound, white beeswax, white vaseline and light liquid paraffin in a certain ratio (the weight ratio of the three ointments was: 1:18:58:23, 3:18:57:22, 10:20:40:30). The blank matrix ointment was prepared by mixing white beeswax, white vaseline and light liquid paraffin in a certain ratio (weight ratio: 18:60:22).

Tissue staining: After dewaxing and rehydration of the paw plantar skin tissue sections of the above rats, stain them in hematoxylin solution for several minutes, wash them and soak them in 1% acidic alcohol until the sections fade to light blue-red, wash them in running water for 5 minutes and then stain them with eosin for 2-3 minutes, wash and remove excess dye, add xylene for transparent for several minutes after dehydration, seal them with neutral resin, observe the stratum corneum under an optical microscope, and measure the thickness of the epidermal stratum corneum using Dmetrix software.

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, blocked with 10% goat serum for 30 minutes after antigen retrieval, KRT1 antibody (Abcam, ab93652) and Loricrin antibody (Abcam, ab183646) were added dropwise, incubated overnight at 4°C, HRP secondary antibody (ZSGB-BIO, PV-6001) and DAB kit (ZSG-BIO, ZLI9017) were added for color development, counterstained with hematoxylin, washed and sealed with central resin, and the expression levels of KRT1 and Loricrin were observed under an optical microscope.

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

As mentioned above, the scoring criteria for rat tissue staining pathology are: no blisters, 0 points; 1-3 blisters, 1 point; 4-6 blisters, 2 points; 7-9 blisters, 3 points; more than 10 blisters, 4 points. The inflammation area accounts for less than 10% of the total area of the slice, 0 points; the inflammation area accounts for 10-25% of the total area of the slice, 1 point; the inflammation area accounts for 25-50% of the total area of the slice, 2 points; the inflammation area accounts for 50-75% of the total area of the slice, 3 points; the inflammation area accounts for more than 75% of the total area of the slice, 4 points. If the congestion area accounts for less than 10% of the total area of the slice, 0 points are given; if the congestion area accounts for 10-25% of the total area of the slice, 1 point is given; if the congestion area accounts for 25-50% of the total area of the slice, 2 points are given; if the congestion area accounts for 50-75% of the total area of the slice, 3 points are given; if the congestion area accounts for more than 75% of the total area of the slice, 4 points are given. The scoring system was double-blind, and after the statistics of each group were completed, the data were presented in the form of Mean+SEM (N=12).

Table 3 Drug dosage and experimental results in rat model

As shown in Table 3, sorafenib, erlotinib, afatinib and osimertinib were administered orally once a day in SD rats at a dose of 100 mg/kg, 70 mg/kg, 50 mg/kg and 60 mg/kg, respectively, and the success rates of hand-foot skin reaction modeling were 75%, 83.3%, 66.7% and 75%, respectively. The compound of formula (I) can significantly reduce the incidence of hand-foot skin reaction at the application site at a low dose (for example, the mass content of the compound is 1%). The compound of formula (I) can significantly reduce the incidence of hand-foot skin reaction in all paws of rats at a high mass content (for example, the mass content of the compound is 3% and 10%).

The results of stratum corneum thickness measurement shown in Figure 20 show that the stratum corneum thickness of the paw and plantar skin of rats successfully modeled with sorafenib, erlotinib, afatinib and osimertinib was significantly higher than that of normal rats. The compound of formula (I) can effectively reduce the stratum corneum thickness of the paw and plantar skin of modeled rats.

As shown in the tissue staining results in Figure 21, the skin toxicity caused by the modeling drugs can form blisters in the subcutaneous and epidermal areas of the rat paws and feet, inflammatory cell infiltration in the dermis, and congestion symptoms in the skin tissue. The compound of formula (I) can effectively improve the formation of subcutaneous and epidermal blisters caused by the above-mentioned modeling drugs, inhibit the infiltration of inflammatory cells in the dermis, and significantly improve the skin congestion phenomenon.

As shown in the pathological scoring results in Figure 22, the modeling drugs can cause the formation of blisters in the subcutaneous and epidermal areas of the rat paws, inflammatory cell infiltration in the dermis, and congestion symptoms in the skin tissue. The compound of formula (I) can effectively improve the formation of subcutaneous and epidermal blisters caused by the above-mentioned modeling drugs, inhibit the infiltration of inflammatory cells in the dermis, and significantly improve the skin congestion phenomenon.

The results of immunohistochemical staining also showed that in rats successfully modeled with sorafenib, erlotinib, afatinib and osimertinib, KRT1 and Loricrin were significantly increased in tissues. However, with the addition of the compound, KRT1 and Loricrin were significantly reduced in tissues. In summary, the compound of formula (I) can effectively inhibit the differentiation of keratinocytes in vitro and in vivo, and has the potential to treat diseases related to hyperkeratosis.

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

SPF ICR mice (male, 5-6 weeks old, weighing about 35 grams) were randomly divided into blank control group (6 mice), capecitabine modeling group (6 mice), docetaxel modeling 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 given the corresponding modeling drug (capecitabine 200 mg/kg, docetaxel 25 mg/kg) by oral gavage once a day; 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 a day for 16 consecutive days.

Measurement of toe swelling: A toe swelling tester (KW-7C, Nanjing Calvin Biotechnology Co., Ltd.) was used to measure the swelling of the toes of the hind limbs of mice before administration of modeling drugs, 2 weeks after administration of modeling drugs, and before sacrifice. The same position was marked on the joint of the hind foot of the mice before administration, 2 weeks after administration, and before sacrifice. During measurement, the water was just on the same horizontal line as the marked position, and the value was recorded after it stabilized.

Skin tissue pathological staining: Take skin tissue samples of the hind limbs of mice, fix them in 4% paraformaldehyde at room temperature for 4 hours, take out the tissue and rinse it with running water for several hours, dehydrate it with 70%, 80% and 90% ethanol solutions, and then treat it with a mixed solution of pure alcohol and xylene in equal amounts for 15 minutes, and permeabilize it with xylene solution twice, 15 minutes each time, until the sample is transparent. Soak it in a mixture of half xylene and half paraffin for 15 minutes, and then add paraffin I and paraffin II for permeabilization for one hour each. After paraffin embedding, the samples were sliced, baked, dewaxed and hydrated, and then the hydrated sections were placed in hematoxylin solution (Zhongshan Jinqiao, catalog number 23041001) for staining for 3 minutes, and then added with hydrochloric acid ethanol differentiation solution for differentiation for 15 seconds. After washing with water, Scott blueing solution (Servicebio, catalog number 20230801) was added to return to blue for 15 seconds. After rinsing with running water, the sections were stained with eosin staining solution (Solarbio, catalog number 33535) for 3 minutes. After rinsing with running water, the sections were dehydrated, transparent, sealed and examined under a microscope.

Immunohistochemical staining: After baking, dewaxing and hydration, paraffin sections of mouse hind limb skin tissue were used for antigen repair with 0.2M citric acid buffer (1.534g citric acid, 3.1g sodium citrate, 100mL distilled water, pH 4.7). Endogenous hydrogen peroxide was removed by 3% hydrogen peroxide (Shandong Anjie High-Tech Disinfection Technology Co., Ltd., item number 20290902), incubated at room temperature for 10 minutes, and then fully rinsed with PBS buffer (NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 1.44g, 1M HCl, pH 7.4). 0.5% Triron-X-100 (Aimejie Technology, item number A-CSH436-100mL) was used for permeabilization, mouse anti-IL-1β and rabbit IL-8 were permeabilized for 10 minutes each, and rabbit anti-IL-6 was not permeabilized. After 5% BSA antigen blocking, 1/150 diluted mouse anti-IL-1β (Affinity Biosciences, Catalog No. BF8021), 1/200 diluted rabbit anti-IL-8 (Affinity Biosciences, Catalog No. DF6998) or 1/150 diluted rabbit anti-IL-6 (Affinity Biosciences, Catalog No. DF6087) was added to each slide and incubated in a humidified box at 4°C overnight. Take out the wet box incubated overnight, let it stand at room temperature for 45 minutes, wash the slides with PBS buffer 3 times, 15 minutes each time, add 1/100 diluted horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao, catalog number 234750811) to rabbit anti-IL-8 and rabbit anti-IL-6 indicators, add 1/100 diluted horseradish enzyme-labeled goat anti-mouse IgG (H+L) (Zhongshan Jinqiao, catalog number 226700804) to mouse anti-IL-1β indicators, incubate at 37°C for 30 minutes and then rinse thoroughly with PBS buffer. Add DAB (CWBIO, catalog number 13723) for color development for 3-5 minutes, wash with PBS buffer for 1 minute, counterstain with hematoxylin (Zhongshan Jinqiao, catalog number 23041001) for 3 minutes, differentiate with hydrochloric acid alcohol to turn blue, and wash with water. The sections were placed in 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II in sequence for rapid dehydration. Xylene I and xylene II were transparent. The sections were sealed with neutral resin glue (Solarbio, product number 20230725) and examined under a microscope.

The results of mouse skin appearance are shown in Figure 23. After 14 days of modeling with capecitabine and docetaxel, obvious lesions appeared on the skin of the hind limbs and toes of mice. Compared with the normal control group, the skin of the hind limbs and toes of mice in the capecitabine group and docetaxel group showed obvious redness, swelling, cracking and blisters.

The degree of swelling of the hind limb toes of mice is shown in Figure 24. The modeling drugs capecitabine and docetaxel can cause swelling of the hind limb toes of mice after continuous oral administration for 14 days. Local application of the ointment of the compound of formula (I) on the hind limb toes of mice can significantly improve the swelling of the toes caused by the modeling drugs (p<0.05). However, no significant improvement was observed in the swelling of the hind limb toes of mice after oral administration of 100 mg/kg nicotinamide.

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 the epidermis and stratum corneum in the skin tissue of the hind limbs of mice to increase significantly after continuous oral administration for 14 days, increase the number of granular cells in the basal layer, and visible inflammatory cell infiltration. The ointment of the compound of formula (I) applied locally to the hind limbs 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 of mice, with slightly more granular cells in the basal layer, and inflammatory cell infiltration is still seen.

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 hind limbs and toes of mice after continuous oral administration for 14 days (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. Local application of the compound ointment of formula (I) on the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-8 in the skin tissue of the hind limbs and toes of mice (p<0.05). However, oral administration of 100 mg/kg nicotinamide has no significant effect on the expression level of the cytokine IL-8 in the skin tissue of the hind limbs and toes of mice.

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 hind limbs and toes of mice after continuous oral administration for 14 days (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. Local application of the compound ointment of formula (I) on the hind limbs and toes of mice can significantly reduce the expression level of cytokine IL-6 in the skin tissue of the hind limbs and toes of mice (p<0.05). However, oral administration of 100 mg/kg nicotinamide has no significant effect on the expression level of cytokine IL-6 in the skin tissue of the hind limbs and toes of mice.

Example 12: Effect of the compound of formula (I) in the kinase inhibitor-induced mouse hand-foot skin reaction model

SPF ICR mice (male, 5-6 weeks old, weighing about 35 grams) were randomly divided into blank control group (6 mice), sorafenib modeling group (6 mice), osimertinib modeling group (6 mice), sorafenib + formula (I) compound group (6 mice), osimertinib + formula (I) compound group (6 mice), sorafenib + nicotinamide group (6 mice) and osimertinib + nicotinamide group (6 mice). Each group was given the corresponding modeling drug (sorafenib 100 mg/kg, osimertinib 10 mg/kg) by oral gavage once a day; 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 a day for 16 consecutive days.

Measurement of toe swelling: A toe swelling tester (KW-7C, Nanjing Calvin Biotechnology Co., Ltd.) was used to measure the swelling of the toes of the hind limbs of mice before administration of modeling drugs, 2 weeks after administration of modeling drugs, and before sacrifice. The same position was marked on the joint of the hind foot of the mice before administration, 2 weeks after administration, and before sacrifice. During measurement, the water was just on the same horizontal line as the marked position, and the value was recorded after it stabilized.

Skin tissue pathological staining: Take skin tissue samples of the hind limbs of mice, fix them in 4% paraformaldehyde at room temperature for 4 hours, take out the tissue and rinse it with running water for several hours, dehydrate it with 70%, 80% and 90% ethanol solutions, and then treat it with a mixed solution of pure alcohol and xylene in equal amounts for 15 minutes, and permeabilize it with xylene solution twice, 15 minutes each time, until the sample is transparent. Soak it in a mixture of half xylene and half paraffin for 15 minutes, and then add paraffin I and paraffin II for permeabilization for one hour each. After paraffin embedding, the samples were sliced, baked, dewaxed and hydrated, and then the hydrated sections were placed in hematoxylin solution (Zhongshan Jinqiao, catalog number 23041001) for staining for 3 minutes, and then added with hydrochloric acid ethanol differentiation solution for differentiation for 15 seconds. After washing with water, Scott blueing solution (Servicebio, catalog number 20230801) was added to return to blue for 15 seconds. After rinsing with running water, the sections were stained with eosin staining solution (Solarbio, catalog number 33535) for 3 minutes. After rinsing with running water, the sections were dehydrated, transparent, sealed and examined under a microscope.

Immunohistochemical staining: After baking, dewaxing and hydration, paraffin sections of mouse hind limb skin tissue were used for antigen repair with 0.2M citric acid buffer (1.534g citric acid, 3.1g sodium citrate, 100mL distilled water, pH 4.7). Endogenous hydrogen peroxide was removed by 3% hydrogen peroxide (Shandong Anjie High-Tech Disinfection Technology Co., Ltd., item number 20290902), incubated at room temperature for 10 minutes, and then fully rinsed with PBS buffer (NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 1.44g, 1M HCl, pH 7.4). 0.5% Triron-X-100 (Aimejie Technology, item number A-CSH436-100mL) was used for permeabilization, mouse anti-IL-1β and rabbit IL-8 were permeabilized for 10 minutes each, and rabbit anti-IL-6 was not permeabilized. After 5% BSA antigen blocking, 1/150 diluted mouse anti-IL-1β (Affinity Biosciences, Catalog No. BF8021), 1/200 diluted rabbit anti-IL-8 (Affinity Biosciences, Catalog No. DF6998) or 1/150 diluted rabbit anti-IL-6 (Affinity Biosciences, Catalog No. DF6087) was added to each slide and incubated in a humidified box at 4°C overnight. Take out the wet box incubated overnight, let it stand at room temperature for 45 minutes, wash the slides with PBS buffer 3 times, 15 minutes each time, add 1/100 diluted horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao, catalog number 234750811) to rabbit anti-IL-8 and rabbit anti-IL-6 indicators, add 1/100 diluted horseradish enzyme-labeled goat anti-mouse IgG (H+L) (Zhongshan Jinqiao, catalog number 226700804) to mouse anti-IL-1β indicators, incubate at 37°C for 30 minutes and then rinse thoroughly with PBS buffer. Add DAB (CWBIO, catalog number 13723) for color development for 3-5 minutes, wash with PBS buffer for 1 minute, counterstain with hematoxylin (Zhongshan Jinqiao, catalog number 23041001) for 3 minutes, differentiate with hydrochloric acid alcohol to turn blue, and wash with water. The sections were placed in 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II in sequence for rapid dehydration. Xylene I and xylene II were transparent. The sections were sealed with neutral resin glue (Solarbio, product number 20230725) and examined under a microscope.

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

The degree of swelling of the hind limb toes of mice is shown in Figure 29. The modeling drugs sorafenib and osimertinib can cause significant swelling of the hind limb toes of mice after continuous oral administration for 14 days. Local application of the ointment of the compound of formula (I) on the hind limb toes of mice can effectively improve the swelling of the toes caused by the modeling drugs. However, no significant improvement was observed in the swelling of the hind limb toes of mice after oral administration of 100 mg/kg nicotinamide.

The results of pathological staining of toe skin tissue are shown in Figure 30. Compared with the normal control group, the modeling drugs sorafenib and osimertinib can cause the epidermis and stratum corneum in the skin tissue of the hind limbs of mice to increase significantly after continuous oral administration for 14 days, increase the granular cells in the basal layer, and visible inflammatory cell infiltration. The ointment of the compound of formula (I) applied locally to the hind limbs 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 of mice, and the morphology of the basal layer cells is slightly restored, and inflammatory cell infiltration is still seen.

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. Local application of the compound ointment of formula (I) on 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.

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 hind limbs and toes of mice after continuous oral administration for 14 days (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. Local application of the compound ointment of formula (I) on 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.

Example 13: Preparation of drug co-crystals formed by the compound represented by formula (I) and isonicotinamide

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 detection showed that the product was a drug co-crystal formed by the compound represented by formula (I) and isonicotinamide, and the XRPD spectrum was shown in Figure 1, and the characteristic peak positions were shown in Table 4. The DSC spectrum showed that the melting _Tonset was 160.45°C. The chemical dosage ratio of the obtained drug co-crystal was 1:1.

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

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

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 detection 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 T _onset @135.68°C. The chemical dosage ratio of the obtained drug co-crystal is 1:1.

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

Example 15: Preparation of a pharmaceutical co-crystal formed by the compound represented by formula (I) and L-proline

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 detection showed that the product was a drug co-crystal formed by the compound represented by formula (I) and L-proline, and the XRPD spectrum was shown in Figure 7, and the characteristic peak positions were shown in Table 6. The DSC spectrum showed that the melting T _onset @183.55°C. The chemical dosage ratio of the obtained drug co-crystal was 1:1.

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

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

Weigh about 300 mg of the compound of 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 min to obtain a suspension. Add about 5 mg of the seed crystals of the drug co-crystal formed by the compound of 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 part by centrifugation, and dry the obtained solid part under vacuum conditions 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 detection shows that the product is a drug co-crystal formed by the compound of formula (I) and L-proline.

Example 17: Preparation of drug co-crystals formed by the compound represented by formula (I) and nicotinamide

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 detection showed that the product was a drug co-crystal formed by the compound represented by formula (I) and nicotinamide, and the XRPD spectrum was shown in Figure 10, and the characteristic peak positions were shown in Table 7. The DSC spectrum showed that the melting T _onset @156.75°C. The chemical dosage ratio of the obtained drug co-crystal was 1:1.

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

Example 18: Preparation of drug co-crystals formed by the compound represented by formula (I) and nicotinamide

About 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 bottle. 3.75 mL of acetonitrile was added and stirred at 5°C for 2 min to obtain a suspension. About 5 mg of the seed crystals of the drug co-crystal formed by the compound of formula (I) and nicotinamide were added to the above suspension. 2 mL of acetonitrile was added to the above suspension and stirred at 5°C for 4 days. The solid part was collected by centrifugation, and the obtained solid part was placed under vacuum at room temperature and dried for about 2.5 hours. A total of 365 mg of white powder was obtained with a yield of 80%. The product was detected by X-ray powder diffraction as a drug co-crystal formed by the compound of formula (I) and nicotinamide.

In summary, four drug cocrystals were obtained, among which glycolic acid cocrystals usually have hygroscopicity problems, isonicotinamide is similar to nicotinamide, but nicotinamide is a GRAS agent (Generally Recognized As Safe), and isonicotinamide is a non-GRAS agent. Finally, L-proline cocrystals and nicotinamide cocrystals were selected to continue studying drug properties such as stability and hygroscopicity.

Example 19: Stability test of drug co-crystals formed by the compound represented by formula (I) and L-proline and drug co-crystals formed by the compound represented by formula (I) and nicotinamide

The open containers containing the drug co-crystals formed by the compound of formula (I) and L-proline and the drug co-crystals formed by the compound of formula (I) and nicotinamide were placed at 25°C/92.5%RH for one week. The drug co-crystals formed by the compound of formula (I) and L-proline and the drug co-crystals formed by the compound of formula (I) and nicotinamide were placed in a sealed container at 60°C for one week. The samples after solid stability evaluation were characterized by XRPD and HPLC, and the color change of the samples was observed. The conclusions are shown in Table 8.

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

As shown in Table 8, the drug co-crystals formed by the compound represented by formula (I) and L-proline and the drug co-crystals formed by nicotinamide were placed in an open container for one week at 25°C/92%RH or in a closed container for one week at 60°C. However, the drug co-crystals formed by the compound represented by formula (I) and L-proline were partially dissociated into free crystals and L-proline at 25°C/92.5%RH. The drug co-crystals formed by the compound represented by formula (I) and nicotinamide were relatively stable under both conditions.

Example 20: Solubility test of drug co-crystals formed by the compound represented by formula (I) and L-proline and drug co-crystals formed by the compound represented by formula (I) and nicotinamide

15.1 mg of the drug co-crystal formed by the compound represented by formula (I) and L-proline and 14.9 mg of the drug co-crystal formed by the compound represented by formula (I) and nicotinamide were accurately weighed and placed in 20 mL glass bottles. 5 mL of solvent was added to each. The mass of the compound represented by formula (I) in the weighed co-crystal was equivalent to 10 mg of free anhydrous crystalline form. The resulting suspension was stirred at 400 rpm at 37°C, and samples were taken at 0.5 hours and 2 hours respectively. The sample was centrifuged at 14,000 rpm at 37°C for 5 minutes. The concentration of the supernatant was determined by HPLC and the pH value of the supernatant was determined by a pH meter. Whether the residual solid (wet product) after 2 hours has a change in crystal form was detected by XRPD. The experimental results are shown in Table 9.

Table 9 Solid solubility of drug co-crystals formed by the compound represented by formula (I)

As shown in Table 9, the drug co-crystals formed by the compound represented by formula (I) and L-proline and the drug co-crystals formed by nicotinamide are transformed into the free crystal form of the compound represented by formula (I) in four different solvents. In addition, the drug co-crystals formed with L-proline and the drug co-crystals formed with nicotinamide have high solubility in the remaining solvents, and the solubility of the drug co-crystals formed by the compound represented by formula (I) and nicotinamide after being dissolved in the four solvents for 2 hours is higher than that of the drug co-crystals formed with L-proline.

Example 21: Hygroscopicity test of drug co-crystals formed by the compound represented by formula (I) and L-proline and drug co-crystals formed by the compound represented by formula (I) and nicotinamide

The water absorption and dehydration behaviors of the drug co-crystals formed by the compound represented by formula (I) and L-proline and the drug co-crystals formed by the compound represented by formula (I) and nicotinamide were studied by DVS test at 25°C. The DVS cycle was 40-0-95-0-40% RH. XRPD was performed on the samples after DVS test to determine whether crystal transformation occurred. The results are shown in Table 10.

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

From the results in Table 10, it can be seen that the drug co-crystal formed by the compound shown in formula (I) and L-proline is slightly hygroscopic between 40% RH and 80% RH, and the weight gain by moisture absorption is about 0.4%; it is extremely hygroscopic between 80% RH and 95% RH, and the weight gain by moisture absorption is about 84.0%. After DVS test, the co-crystal is partially dissociated into free crystal form and L-proline. The drug co-crystal formed by the compound shown in formula (I) and nicotinamide is slightly hygroscopic, and its weight gain by moisture absorption is about 0.2% between 40% RH and 95% RH at 25°C. After DVS test, the crystal form did not change.

It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims (17)

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,
The pharmaceutical cocrystal according to claim 1, characterized in that the chemical ratio of the compound represented by formula (I) to the cocrystal former is 1:0.5 to 1:3, preferably 1:0.5, 1:1, 1:2 or 1:3, and most preferably 1:1 or 1:2. The pharmaceutical co-crystal according to claim 1, wherein the co-crystal former is nicotinamide. The drug co-crystal formed by the compound represented by formula (I) and isonicotinamide,
Characterized in that the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566; preferably, characteristic peaks are present 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 are present at ... Characteristic peaks are selected 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 represented by the diffraction angle 2θ is shown in Figure 1. The drug co-crystal formed by the compound represented by formula (I) and glycolic acid,
The invention is characterized in that the X-ray powder diffraction pattern represented by the 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. 4. The powder diffraction pattern is most preferably shown in Figure 5. The drug co-crystal formed by the compound represented by formula (I) and L-proline,
The invention is characterized in that the X-ray powder diffraction pattern represented by the diffraction angle 2θ has characteristic peaks at 10.302, 14.229, 14.871, 19.762 and 20.643; preferably has characteristic peaks at 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.643, 23.211, 28.621 and 31.158; preferably has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 19.762, 20.113, 20.643, 22.829, 23.211, 23. 7 has characteristic peaks at 6.585, 7.120, 10.302, 13.153, 14.229, 14.871, 15.763, 16.443, 18.719, 19.762, 20.113, 20.643, 21.217, 21.383, 21.899, 22.829, 23.211, 23.611, 26.465, 28.621, 31.158 and 37.176; most preferably, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is shown in Figure 7. The drug co-crystal formed by the compound represented by formula (I) and nicotinamide,
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, characteristic peaks are 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 are at 9.860, 13.311, 17.017, 19.752, 20.856, 23.161, 23.638, 24.147, 24.780, 26.165, 26.5 73, 27.028, 30.766, 32.820 and 36.741 have characteristic peaks; preferably 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 have characteristic peaks; most preferably, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is shown in Figure 10. The pharmaceutical cocrystal according to any one of claims 1 to 7, wherein the 2θ angle error range is ±0.20. The method for preparing the pharmaceutical cocrystal formed by the compound of formula (I) and isonicotinamide according to claim 4, characterized in that it comprises the steps of: mixing the compound of formula (I) and 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 a C _1-4 alcohol, more preferably one or more of methanol, ethanol and isopropanol, and most preferably ethanol. The method for preparing the drug cocrystal formed by the compound represented by formula (I) and glycolic acid as described in claim 5 is characterized in that it 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. The method for preparing the pharmaceutical cocrystal formed by the compound represented by formula (I) and L-proline as described in claim 6 is characterized in that it comprises the steps of: mixing the compound represented by formula (I) and 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 a C _1-4 alcohol, more preferably one or more of methanol, ethanol and isopropanol, and most preferably ethanol. The method for preparing the pharmaceutical cocrystal formed by the compound represented by formula (I) and nicotinamide as described in claim 7 is characterized in that it comprises the steps of: mixing the compound represented by formula (I) and 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. A pharmaceutical composition prepared by combining a drug 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 drug co-crystal formed by a compound of formula (I) and isonicotinamide according to claim 4, or a drug co-crystal formed by a compound of formula (I) and glycolic acid according to claim 5, or a drug co-crystal formed by a compound of formula (I) and L-proline according to claim 6, or a drug co-crystal formed by a compound of formula (I) and nicotinamide according to claim 7. A pharmaceutical composition comprising 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, and optionally a pharmaceutically acceptable excipient. 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 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, with a pharmaceutically acceptable excipient. A pharmaceutical co-crystal formed by combining the compound of formula (I) as described in any one of claims 1 to 3 and a co-crystal former, or a pharmaceutical co-crystal formed by the compound of formula (I) as described in claim 4 and isonicotinamide, or a pharmaceutical co-crystal formed by the compound of formula (I) as described in claim 5 and glycolic acid, or a pharmaceutical co-crystal formed by the compound of formula (I) as described in claim 6 and L-proline, or a pharmaceutical co-crystal formed by the compound of formula (I) as described in claim 7 and nicotinamide, or a composition as described in claim 13 or 14, or a composition prepared by the method as described in claim 15 in the preparation of a drug for treating and/or preventing diseases related to RNA m6A regulation. According to the use of claim 16, the diseases related to RNA m6A regulation are endocrine and metabolic diseases, nervous system diseases, tumors, cardiovascular diseases, infections, immune system diseases, urogenital system diseases, skin and musculoskeletal diseases, respiratory system diseases, genetic diseases and deformities, digestive system diseases, oral and maxillofacial diseases, vascular and lymphatic system disease-related diseases or pain, preferably hand-foot syndrome, hand-foot skin reaction, cancer, and dermatological diseases.