CN120118019A - A co-crystal form of RNA m6A regulator and its preparation method and application - Google Patents
A co-crystal form of RNA m6A regulator and its preparation method and application Download PDFInfo
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Abstract
本发明提供一种式(I)所示RNAm6A调控剂的共晶的结晶形式及其制备方法与应用。本发明的RNAm6A调控剂的共晶的晶型具有引湿性低、稳定性好的有益效果。本发明的RNA m6A调控剂的共晶的晶型的制备方法工艺简单，结晶过程易于控制，重现性好。
The present invention provides a crystalline form of a cocrystal of an RNAm6A regulator shown in formula (I), a preparation method and application thereof. The crystal form of the cocrystal of the RNAm6A regulator of the present invention has the beneficial effects of low hygroscopicity and good stability. The preparation method of the crystal form of the cocrystal of the RNAm6A regulator of the present invention is simple in process, the crystallization process is easy to control, and the reproducibility is good.
Description
Technical Field
The invention belongs to the technical field of medicinal chemistry, and particularly relates to a eutectic form of an RNAm6A modulator, and a preparation method and application thereof.
Background
Hand-foot syndrome (HFS) and hand-foot skin reaction (HFSR), which are a type of hand and foot erythema skin lesions mainly caused by cytotoxic chemotherapeutics and tumor targeting drugs, can cause patients to lose life self-care ability in severe cases. The main pathological characteristics of hand-foot syndrome and hand-foot skin reaction are basal keratinocyte vacuolation, perivascular lymphocyte infiltration, keratinocyte apoptosis and skin edema, and inflammatory changes, vasodilation, edema and leukocyte infiltration are visible under a microscope.
The medicine capable of causing hand and foot syndromes comprises capecitabine, liposomal doxorubicin, cytarabine, docetaxel, vinorelbine, continuous infusion of doxorubicin, gemcitabine and other chemotherapeutic medicines, and the medicine capable of causing hand and foot skin reactions comprises sonitinib (sotan), sorafenib (dogemex), imatinib (glifex), erlotinib (tarceva) and other targeted medicines. The World Health Organization (WHO) classifies HFS into grade 4, grade 1, hand and foot dysesthesia, paresthesia or tingling, grade 2, discomfort with holding and walking, no pain swelling or erythema, grade 3, painful erythema, palm and sole edema, periungual erythema and swelling, grade 4, desquamation, ulceration, blistering and severe pain. At present, serious hand-foot syndrome and hand-foot skin reaction occur clinically, symptoms are relieved only through surface skin care, even medicine is stopped, the existing treatment means can treat symptoms and root causes, and the use of first-line tumor chemotherapeutic medicines is severely limited, so that huge unmet medical needs exist. There is a need to develop a medicament for the prevention and treatment of hand-foot syndrome and hand-foot skin reactions with good effectiveness, few side effects and low cost to meet the growing medical demands worldwide.
M6A methylation modification is the most common RNA modification in mammals, and can regulate various signal paths and cellular processes (such as growth, development, diseases and the like) so as 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).mRNA、miRNA、circRNA and m6A methylation modification of lncRNA is a dynamic reversible process, methyltransferases (such as METTL, METTL, METTL and the like) bind methyl groups to RNA, and demethylases (FTO and ALKBH 5) erase methyl groups on RNA, so that a regulation basis of m6A is formed. m6A affects RNA processing, translation and degradation processes by recruiting specific binding proteins (e.g., YTHDF1, YTHDF, YTHDC1 and IGF2BP, etc.), resulting in changes in downstream protein function and cell biology (HsuP.J.,et al.Journal ofBiological Chemistry 2019,294,19889-19895;YaoY.,et al.FASEB Journal 2019,33,7529-7544).
The direct association of RNAm6A with skin related diseases is rarely known at present, most m6A related methylases or demethylases play a role in skin diseases, especially skin tumors. For example, mRNA expression levels of METTL and alk bh5 in tumor tissues of patients with acromegaly were significantly higher than those of paracancerous tissues, and mRNA expression levels of METTL in patients with advanced acromegaly were significantly higher than those of early patients (Le Zhanghui, lncRNA and preliminary study of RNAm6A methylation pathogenesis in acromegaly, academic paper, 2019), m 6A-specific binding proteins YTHDF1 and HNRNPA B1 could be used as novel biomarkers in melanoma diagnosis (lit.d., et al cancer Cell 2020,20,239). In the case of keratinocytes, it has been reported that only long-term, low-level arsenic exposure can inhibit selective autophagy by m6A demethylase, thereby inducing skin tumor development (Cui y.h., et al nature Communications 2021,12,2183). In view of the above, there is currently no method for preventing and treating skin diseases by modulating RNAm6A methylation.
In view of this, the present invention has been made.
Disclosure of Invention
Problems to be solved by the invention
Based on the unexpected existence form and quantity of the polymorphic compound, the invention provides a eutectic form of an RNAm6A modulator shown as a formula (I), and a preparation method and application thereof.
Solution for solving the problem
The invention provides a pharmaceutical co-crystal formed by combining a compound shown as a formula (I) and a co-crystal forming substance, wherein the co-crystal forming substance is selected from nicotinamide, isonicotinamide, L-proline and glycollic acid,
A pharmaceutical co-crystal formed by a compound shown in a formula (I) and isonicotinamide,
The X-ray powder diffraction patterns expressed in terms of diffraction angle 2 theta have characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566, preferably 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 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, and most preferably in terms of diffraction angle 2 theta are shown in figure 1.
The invention provides a pharmaceutical co-crystal formed by a compound shown in a formula (I) and glycollic acid,
The X-ray powder diffraction patterns expressed in terms of diffraction angles 2 theta are shown in FIG. 4, with characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125, preferably 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 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 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, and most preferably in terms of diffraction angles 2 theta.
The invention provides a pharmaceutical co-crystal formed by a compound shown in a formula (I) and L-proline,
The X-ray powder diffraction patterns expressed in terms of diffraction angles 2 theta have 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, preferably 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, and most preferably in terms of diffraction angles 2 theta are shown in FIG. 7.
The invention provides a pharmaceutical co-crystal formed by a compound shown in a formula (I) and nicotinamide,
The X-ray powder diffraction patterns expressed in terms of diffraction angle 2 theta have characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573, preferably at 9.860, 13.311, 17.017, 19.752, 20.856, 23.638, 24.147, 26.573, 27.028 and 32.820, preferably at 9.860, 13.311, 17.017, 19.752, 20.856, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.820 and 36.741, preferably 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, and most preferably in terms of diffraction angle 2 theta are shown in FIG. 10.
ADVANTAGEOUS EFFECTS OF INVENTION
The pharmaceutical co-crystal formed by the compound shown in the formula (I) and isonicotinamide, the pharmaceutical co-crystal formed by the compound shown in the formula (I) and glycollic acid, the pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline, and the pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide have advantages in terms of physical properties, preparation processing performance, bioavailability and the like, such as at least one of melting point, solubility, hygroscopicity, purification effect, stability, adhesiveness, compressibility, flowability, in-vivo and in-vitro dissolution, bioavailability and the like. The pharmaceutical eutectic formed by combining the compound shown in the formula (I) and the eutectic forming substance has good physical and chemical stability, the crystal form prepared by the same starting substance has high yield, and the invention has obvious advantages in the aspects of solubility, hygroscopicity, stability, mechanical stability, fluidity, compressibility, adhesiveness and the like, provides a new and better choice for the pharmaceutical development of the RNAm6A modulator, and has very important significance.
Drawings
FIG. 1 is an XRPD pattern for a pharmaceutical co-crystal of a compound of formula (I) with isonicotinamide.
FIG. 2 is a TGA spectrum of a pharmaceutical co-crystal formed by a compound of formula (I) and isonicotinamide.
FIG. 3 is a DSC of a pharmaceutical co-crystal formed from a compound of formula (I) and isonicotinamide.
Fig. 4 is an XRPD pattern of a pharmaceutical co-crystal formed by a compound of formula (I) and glycolic acid.
FIG. 5 is a TGA spectrum of a pharmaceutical co-crystal formed by a compound of formula (I) and glycolic acid.
FIG. 6 is a DSC of a pharmaceutical co-crystal of a compound of formula (I) with glycolic acid.
FIG. 7 is an XRPD pattern for a pharmaceutical co-crystal of a compound of formula (I) with L-proline.
FIG. 8 is a TGA spectrum of a pharmaceutical co-crystal formed by a compound of formula (I) and L-proline.
FIG. 9 is a DSC of a pharmaceutical co-crystal formed from a compound of formula (I) and L-proline.
FIG. 10 is an XRPD pattern for a pharmaceutical co-crystal of a compound of formula (I) with nicotinamide.
FIG. 11 is a TGA spectrum of a pharmaceutical co-crystal formed by a compound of formula (I) and nicotinamide.
FIG. 12 is a DSC of a pharmaceutical co-crystal formed from a compound of formula (I) and nicotinamide.
FIG. 13 is an exemplary result of mRNA expression levels of glial differentiation markers KRT1, KRT10, loricrin and Involurin determined by RT-PCR 24 hours after administration of human recombinant HBEGF protein and the compound of formula (I) to keratinocytes HaCaT.
Fig. 14 is an exemplary result of measurement of the thickness of the skin stratum corneum of the paw plantar of the rat in example 4.
FIG. 15 is a graph showing exemplary histopathological results of rat paw plantar skin epithelial bleeds, inflammatory cell infiltration, and skin tissue hyperemia following tissue staining in example 4.
FIG. 16 is an exemplary result of histopathological scoring of rat paw plantar skin epithelial bleeds, inflammatory cell infiltration, and skin tissue hyperemia following tissue staining in example 4.
Detailed Description
In order to make the technical scheme and the beneficial effects of the application more obvious and understandable, the following detailed description is given by way of example. Wherein the drawings are not necessarily to scale, and wherein local features may be exaggerated or minimized to more clearly show details of the local features, unless otherwise defined, technical and scientific terms used herein have the same meaning as those in the technical field to which the present application pertains.
The invention provides a pharmaceutical co-crystal formed by a compound shown in a formula (I) and isonicotinamide,
The X-ray powder diffraction patterns expressed in terms of diffraction angle 2 theta have characteristic peaks at 13.641, 14.019, 18.822, 21.196 and 26.566, preferably 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 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, and most preferably in terms of diffraction angle 2 theta are shown in figure 1.
The invention further provides a preparation method of a pharmaceutical co-crystal formed by the compound shown in the formula (I) and isonicotinamide, which comprises the steps of mixing the compound shown in the formula (I) and isonicotinamide, adding the solvent 1, stirring and filtering.
In certain embodiments, the solvent 1 is selected from an alcohol solvent.
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 methods of making described herein further comprise centrifugation, washing, or drying.
The invention further provides a pharmaceutical co-crystal formed by the compound shown in the formula (I) and glycollic acid,
The X-ray powder diffraction patterns expressed in terms of diffraction angles 2 theta are shown in FIG. 4, with characteristic peaks at 10.588, 17.645, 21.232, 21.527 and 23.125, preferably 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 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 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, and most preferably in terms of diffraction angles 2 theta.
The invention further provides a preparation method of a pharmaceutical co-crystal formed by the compound shown in the formula (I) and the glycollic acid, which comprises the steps of mixing the compound shown in the formula (I) and the glycollic acid, adding the solvent 2, stirring and filtering.
In certain embodiments, the solvent 1 is selected from the group consisting of 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 methods of making described herein further comprise centrifugation, washing, or drying.
The invention further provides a pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline,
The X-ray powder diffraction patterns expressed in terms of diffraction angles 2 theta have 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, preferably 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, and most preferably in terms of diffraction angles 2 theta are shown in FIG. 7.
The invention further provides a preparation method of a pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline, which comprises the steps of mixing the compound shown in the formula (I) and L-proline, adding a 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 methods of making described herein further comprise centrifugation, washing, or drying.
The invention further provides a pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide,
The X-ray powder diffraction patterns expressed in terms of diffraction angle 2 theta have characteristic peaks at 13.311, 17.017, 19.752, 23.638 and 26.573, preferably at 9.860, 13.311, 17.017, 19.752, 20.856, 23.638, 24.147, 26.573, 27.028 and 32.820, preferably at 9.860, 13.311, 17.017, 19.752, 20.856, 23.161, 23.638, 24.147, 24.780, 26.165, 26.573, 27.028, 30.766, 32.820 and 36.741, preferably 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, and most preferably in terms of diffraction angle 2 theta are shown in FIG. 10.
The invention further provides a preparation method of a pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide, which comprises the steps of mixing the compound shown in the formula (I) and nicotinamide, adding a 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 methods of making described herein further comprise centrifugation, washing, or drying.
The invention further provides a pharmaceutical composition prepared from a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and a co-crystal forming substance, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and isonicotinamide, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and glycollic acid, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline, or a pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide.
The invention further provides a pharmaceutical composition comprising a pharmaceutical co-crystal formed by combining a compound shown in the formula (I) and a co-crystal forming substance, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and isonicotinamide, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and glycolic acid, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline, a pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide, and optionally pharmaceutically acceptable excipients.
By "pharmaceutically acceptable excipient" is meant a pharmaceutically acceptable material, mixture or vehicle that is relevant to the consistency of the dosage form or pharmaceutical composition being administered. Suitable pharmaceutically acceptable excipients will vary depending upon the dosage form selected. Furthermore, pharmaceutically acceptable excipients may be selected according to their particular function in the composition.
In certain embodiments, the pharmaceutically acceptable excipients include excipients of the type diluents, fillers, permeation 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 formulation.
In certain embodiments, the pharmaceutical composition is a film and a film 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 formulation is a capsule.
In certain embodiments, the pharmaceutical combination is a tablet.
In some embodiments, the ointment comprises 0-50% by weight of the co-crystal of the compound of formula (I).
The invention further provides a preparation method of the pharmaceutical composition, which comprises the step of mixing a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and a co-crystal formed by combining the compound shown in the formula (I) and isonicotinamide, a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and glycolic acid, a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and L-proline, or a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and nicotinamide with a pharmaceutically acceptable excipient.
The invention further provides a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and a co-crystal forming substance, a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and isonicotinamide, a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and glycolic acid, a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and L-proline, a pharmaceutical co-crystal formed by combining the compound shown in the formula (I) and nicotinamide, or a composition, or application of the composition prepared by the method in preparing a medicament for treating and/or preventing RNAm6A regulation related diseases
In certain embodiments, the m6A modulation-associated disease is an endocrine and metabolic disease, a neurological disease, a tumor, a cardiovascular disease, an infection, an immune system disease, a genitourinary system disease, a skin and musculoskeletal disease, a respiratory disease, a genetic disease and deformity, a digestive system disease, an oromandibular disease, a vascular and lymphatic system disease-associated disease, or pain.
In certain embodiments, the m6A modulation-associated disease is hand-foot syndrome, hand-foot skin reaction, cancer, dermatological disease.
The invention relates to a diffraction angle, namely a Bragg angle, wherein the angle is expressed as an angle, the unit is an angle or an angle, and the error range of each characteristic peak 2 theta is +/-0.2 (including the condition that numbers exceeding 1 decimal are rounded off) and 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 mode of the invention comprises but is not limited to stirring, cooling, volatilizing, pulping and precipitating.
"Beating" is a common term in the art of pharmaceutical preparation and generally refers to the mechanical or fluid treatment of a solid pharmaceutical material such that the solid pharmaceutical material is dispersed or suspended in a solvent.
In some embodiments, the beating time is 5h to 30h.
According to the description of the hygroscopicity characteristic and the definition of the hygroscopicity weight gain in the '9103 medicine hygroscopicity guiding principle' in the fourth edition of China pharmacopoeia 2020,
Deliquescence, which is the absorption of sufficient water to form a liquid;
The moisture absorption performance is very good, and the weight gain is not less than 15 percent;
the moisture absorption property is that the weight gain is less than 15 percent but not less than 2 percent;
slightly wet permeability, wherein the weight gain is less than 2 percent but not less than 0.2 percent;
no or almost no moisture permeability, and the weight gain caused by moisture is less than 0.2 percent.
The differential scanning calorimetric analysis or DSC refers to the measurement of the temperature difference and the heat flow difference between a sample and a reference object in the process of heating or constant temperature of the sample so as to represent all physical changes and chemical changes related to thermal effects and obtain the phase change information of the sample.
The drying temperature in the invention is generally 25-100 ℃, preferably 40-70 ℃, and can be normal-pressure drying or reduced-pressure drying.
The process of the present invention will now be described by way of specific examples, which are to be understood as illustrative of the basic principles, main features and advantages of the present invention, without limiting the scope of the invention by the examples below, which may be further modified according to specific requirements, with the non-specified implementation conditions typically being those of routine experimentation.
The abbreviations used in the present invention are explained as follows:
XRPD X-ray powder diffraction
DSC differential scanning calorimeter
TGA thermogravimetric analysis
DVS dynamic moisture adsorption
HPLC high performance liquid chromatography
Detection instrument and method
X-ray powder diffraction (XRPD)
The samples were subjected to a crystal form analysis using a Bruker D8 ADVANCE X-powder diffractometer. The scanning angle of the sample 2 theta is 3-40 degrees, the scanning step length is 0.02 degrees, and the scanning time of each step is 0.12 s/step. The light pipe voltage and current were 40kV and 40mA, respectively. When in sample preparation, a proper amount of samples are placed on a sample carrying tray, and are flattened by tools such as glass sheets and the like, so that the smooth and flat surface of the samples is ensured.
Thermogravimetric analysis (TGA)
Samples were analyzed using TAInstruments TGADiscovery 5500. The samples were placed in a tared aluminum pan, the system automatically weighed, and then the samples were raised to the indicated temperature at a rate of 10 ℃ per minute under nitrogen protection.
Differential scanning calorimetric analysis (DSC)
Samples were analyzed using TAInstruments Discovery f 2500 f. Weighing 0.5-1.5 mg of the sample, placing the sample in a sample loading tray, and raising the sample to a specified temperature at a rate of 10 ℃ per minute under the protection of nitrogen (50 ml/min).
Dynamic moisture adsorption analysis (DVS)
Samples were analyzed using ProUmid SPSx-1. Mu. Advance. The amount of the test sample is about 5-50 mg. The temperature of the test chamber is controlled to be 25+/-1 ℃, the relative humidity is cycled to be 40-95-0-95-40%, the steps are 10%, the balance is 240 minutes, and the mass data is recorded every 20 seconds.
High Performance Liquid Chromatography (HPLC)
Solubility and stability tests were performed using Agilent 1260infinityII Binary Pump.
EXAMPLE 1 preparation of Compounds of 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, then heated to 150 ℃ and after 3 hours of reaction the reaction solution was cooled to room temperature, diluted with 100mL of dichloromethane, washed 3 times with 100mL of 1n HCl and 1 time with saturation 100mLNaHCO 3, the organic layer was dried over anhydrous Na 2SO4 and concentrated in vacuo, and 6-chloro-2, 3,4, 9-tetrahydro-1H-carbazole-1-carboxylic acid ethyl ester (16 mg) was isolated and purified by silica gel column chromatography. 10mg of ethyl 6-chloro-2, 3,4, 9-tetrahydro-1H-carbazole-1-carboxylate (0.036 mmol) is dissolved in 10mL of ethanol, 2mL of 2M LiOH solution is added, the mixture is stirred at room temperature for reaction for 1 hour, 20mL of water is added for dilution after rotary distillation, the pH is adjusted to 2, then 50mL of dichloromethane is used for extraction for 3 times, the organic phases are combined, ammonia water is added after drying concentration, reaction is carried out for 24 hours at 60 ℃ by heating, silica gel column chromatography is used for separating and purifying to obtain white solid 6-chloro-2, 3,4, 9-tetrahydro-1H-carbazole-1-carboxamide (compound 1,7.6 mg), and the (S) -6-chloro-2, 3,4, 9-tetrahydro-1H-carbazole carboxamide (compound shown in formula (I)) is obtained by using CHIRALPAKAD chiral column separation. Compounds of formula (I) LCMS[M+H]+:249.1H-NMR(400MHz,DMSO-d6):δ10.79(s,1H),7.37-7.39(m,2H),7.28(d,J＝8.0Hz,1H),7.08(s,1H),6.98-7.01(m,1H),3.64-3.67(m,1H),2.58-2.61(m,2H),1.66-2.04(m,4H).
Example 2 Effect of Compounds of formula (I) on the methylation level of mRNA of THP-1 cells
8X 10 6 THP-1 cells were placed in a petri dish and added with DMEM medium, control group was added with an equal volume of DMSO, positive reference group 2 was added with 2. Mu.M UZH a (Shanghai Haiyuan medicine Co., ltd.), sample groups A, B and C were respectively added with 5nM, 100nM and 2. Mu.M of the compound of formula (I), placed in an incubator containing 5% carbon dioxide for 24 hours at 37℃and after washing with PBS, centrifuged at high speed, the supernatant was discarded and RLT lysate (QIAGEN) was added. After diluting the sample with 400. Mu.L of 70% ethanol, 700. Mu.L of the sample was taken and total RNA was extracted with Trizol reagent (SigmaAldrich) and the RNA sample was purified with Oligo (dT) beads. 200ng of the purified mRNA sample was taken, 20. Mu.L of nuclease P1 mixture (1 unit nuclease P1, 0.4. Mu.L of 2M NaCl, 2. Mu.L of 0.1M ZnCl 2, 17.1. Mu.L of PCR-grade water) was added and reacted at 37℃for 2 hours, 2. Mu.L of NH 4HCO3 solution and 1unit alkaline phosphatase were added and reacted at 37℃for 2 hours, 1.2M HCl was added and then centrifuged, 20. Mu.L of supernatant was collected and the ion peak area was detected by LC-MS (150.000549/150.000549). The inhibition ratio of m6A is calculated as follows, and the results are shown in Table 1:
m6a inhibition= (peak area (control group) -peak area (sample group))/peak area (control group) ×100% of the total amount of the active substance
Table 1. M6A inhibition of control and sample groups A-C.
UZH1a is a nanomolar inhibitor of the selective m6A methylase METTL3 (Moroz-Omori EV et al chemMed chem 2021,2021,16 (19): 3035-3043), and the inhibition of RNAm6A by UZH a in THP-1 cells is about 41.5%. The compound shown in the formula (I) inhibits m6A in a dose-dependent manner, and the inhibition rate of RNAm6A is higher (56.8%) at the same concentration as UZH a.
Example 3 inhibition of keratinocyte HaCaT differentiation by Compounds of formula (I)
Human keratinocytes HaCaT were cultured in DMEM medium containing 10% fetal bovine serum (Gibco, 10099141), 100U/mL Penicillin (Penicillin) and 100 μg/mL streptomycin (Streptomycin), plated in 96-well plates at a density of 2 x 10 6 cells per square centimeter and placed in an incubator containing 5% carbon dioxide for 24 hours at 37 ℃, control well 1 was added with an equal volume of DMSO, control well 2 was added with 2.5ng/mL human recombinant HBEGF protein (Abcam, ab 205523), sample wells were added with 2.5ng/mL human recombinant HBEGF protein and corresponding concentrations of the compound of formula (I), cultured continuously for 24 hours, washed with PBS, and cells were harvested by high speed centrifugation. Total RNA was extracted with Trizol reagent (SigmaAldrich), 1. Mu.g of RNA was reverse transcribed into cDNA using cDNA reverse transcription kit (TRANSGENE BIOTECH, AT 311-03), and 1.25. Mu.L of primer (see Table 2 for primer sequence), 10. Mu. L iTag Universal SYBR Green of super mix (Bio-Rad, 172-5125) and an appropriate amount of DEPC ultra pure water were added to prepare 20. Mu.L of reaction solution, followed by RT-PCR reaction. After the completion of the reaction, the reaction solution was subjected to agarose gel electrophoresis to measure the mRNA expression amounts of keratinocyte differentiation markers KRT1, KRT10, loricrin and involin.
TABLE 2 partial primer sequence information
As shown in FIG. 13, the mRNA expression levels of the differentiation markers KRT1, KRT10, loricrin and Involurin of HaCaT increased significantly upon induction with the addition of human recombinant HBEGF protein. However, after the compound of formula (I) was added, mRNA expression amounts of the differentiation markers KRT1, KRT10, loricrin and Involurin of HaCaT were significantly inhibited.
Example 4 inhibition of keratinocyte differentiation by the compound of formula (I) in the rat hand-foot skin reaction model SD rats (weighing about 200 g) were fed and adapted for one week and then grouped according to weight average, 12 rats per group. The modeling drugs (sorafenib, erlotinib, afatinib, and oritinib) were dissolved in solutions containing 5% dmso, 45% peg400, and 50% h 2 O, respectively, and the modeling drugs were fixed to the desired concentrations and administered by intragastric administration once daily at the doses shown in table 3. After 1 hour of gastric administration, the left paw of the rat was uniformly coated with 0.05g of an ointment containing the compound of formula (I) in different mass ratios, while the right paw was coated with a blank matrix as a self-control, and rats of the blank control group were not coated with the ointment. After the application, the limbs are fixed for 2 hours, and then the residual medicine is wiped off by clean water, so that the fixation is released and the free movement is restored. The molding agent and the test compound ointment were administered once a day, after 30 days of continuous administration, the rats were euthanized, the paw plantar skin tissue was taken, fixed in 10% neutral formaldehyde and cut into 5 μm slices, dehydrated and embedded in paraffin.
Tissue staining, namely dewaxing and rehydrating the skin tissue sections of the paw plantar of the rat, staining in a Hematoxylin (Hematoxylin) solution for a plurality of minutes, washing, soaking in 1% acid alcohol until the sections fade to light blue, washing with running water for 5 minutes, staining with Eosin (Eosin) for 2-3 minutes, washing to remove superfluous dye, dewatering, adding xylene for a plurality of minutes, sealing with neutral resin, observing the stratum corneum under an optical microscope, and measuring the thickness of the epidermis stratum corneum by using Dmetrix software.
Immunohistochemical staining after deparaffinization and hydration of the paw plantar skin tissue sections of the above rats, incubation with 3%H 2O2 for 30 minutes at room temperature, blocking with 10% goat serum for 30 minutes after antigen retrieval, dropping KRT1 antibody (Abcam, ab 93652) and Loricrin antibody (Abcam, ab 183646), incubation overnight at 4 ℃, addition of HRP secondary antibody (ZSGB-BIO, PV-6001) and DAB kit (ZSG-BIO, ZLI 9017) development, counterstaining in hematoxylin, blocking with a center resin after washing, and observing the expression levels of KRT1 and Loricrin under an optical microscope.
The criteria for successful modeling of the rat model as described above are (i) the appearance of symptoms such as erythema, swelling, desquamation, ulceration or blisters at the paw sites, and/or (ii) a significantly higher thickness of stratum corneum in tissue staining than in normal rats, and/or (iii) a significantly increased marker such as KRT1, KRT5 and Loricrin. The morbidity is calculated as the proportion of animals in each group meeting the criterion of modeling success described above, i.e. morbidity= (number of rats modeling success/total number of rats in the group) ×100%.
As described above, the tissue staining pathology scoring standard of the rat comprises no water bubbles, 0 score, 1-3 water bubbles, 1 score, 4-6 water bubbles, 2 scores, 7-9 water bubbles, 3 scores, and more than 10 water bubbles, 4 scores. The total area of the slice is less than 10%, 0min, 10-25%, 1 min, 25-50%, 2 min, 50-75%, 3min, 75% or more and 4 min. The total area of the slice is less than 10%, 0min, 10-25% and 1 min, 25-50% and 2 min, 50-75% and 3min, and more than 75% and 4 min. Scoring was done using a double-blind scoring system and each group of data was presented as mean+sem after statistics were completed (n=12).
TABLE 3 drug dosage and experimental results in rat model
As shown in table 3, sorafenib, erlotinib, afatinib, and oritinib were administered at doses of 100mg/kg, 70mg/kg, 50mg/kg, and 60mg/kg, respectively, once a day in SD rats with 75%, 83.3%, 66.7%, and 75% hand-foot skin reaction modeling success rates, respectively. The compound shown in the formula (I) can obviously reduce the incidence rate of the skin reaction of hands and feet of a medicine application part at a low dosage (for example, the mass content of the compound in the ointment is 1 percent), and can obviously reduce the incidence rate of the skin reaction of hands and feet of all feet of a rat at a high mass content (for example, the mass content of the compound in the ointment is 3 percent and 10 percent).
As shown in fig. 14, the stratum corneum thickness was significantly higher for the paw plantar skin stratum corneum thickness than for normal rats in rats successfully cast with sorafenib, erlotinib, afatinib, and octreotide. The compound shown in the formula (I) can effectively reduce the thickness of the skin cuticle of the paw plantar of a molding rat.
As shown in the tissue staining results of FIG. 15, skin toxicity caused by the modeling drug can form blisters under the paw plantar skin and in the epidermis of the rat, inflammatory cell infiltration occurs in the dermis layer, and hyperemia symptoms occur in the skin tissue. The compound shown in the formula (I) can effectively improve the formation of subcutaneous and intraepidermal blisters caused by the molding medicaments, inhibit inflammatory cell infiltration of dermis and remarkably improve skin congestion.
As shown in fig. 16, the modeling drug resulted in the formation of blisters in the skin and subcutaneous areas of the paw and plantar region of the rat, with inflammatory cell infiltration in the dermis and hyperemia in the skin tissue. The compound shown in the formula (I) can effectively improve the formation of subcutaneous and intraepidermal blisters caused by the molding medicaments, inhibit inflammatory cell infiltration of dermis and remarkably improve skin congestion.
Immunohistochemical staining results also showed a significant increase in KRT1 and Loricrin in tissues in rats successfully modelled with sorafenib, erlotinib, afatinib and oritinib. However, with the addition of the compound of formula (I), KRT1 and Loricrin were significantly reduced in the tissue. In conclusion, the compound shown in the formula (I) can effectively inhibit the differentiation of keratinocytes in vitro and in vivo, and has the potential of treating diseases related to hyperkeratosis.
EXAMPLE 5 preparation of pharmaceutical Co-crystals of the Compound of formula (I) with isonicotinamide
About 20mg of the compound represented by formula (I) was weighed, placed in a 2mL glass bottle together with 10.2mg of isonicotinamide, and ethanol (0.2 mL) was added to obtain a suspension. The resulting sample was stirred in suspension at 5 ℃ for 3 days. The resulting suspension was centrifuged through a 0.45 μm nylon filter at 14,000rpm, and the resulting solid was dried under vacuum at 25 ℃ for 4 hours to give the product. The product is a pharmaceutical co-crystal formed by the compound shown in the formula (I) and isonicotinamide through X-ray powder diffraction detection, the XRPD spectrum is shown in figure 1, and the characteristic peak positions are shown in table 4. DSC profile showed a melting T onset 160.45 ℃.
TABLE 4 XRPD diffraction peak data for pharmaceutical co-crystals of the compound of formula (I) with isonicotinamide
EXAMPLE 6 preparation of pharmaceutical Co-crystals of the Compound of formula (I) with glycolic acid
About 20mg of the compound represented by the formula (I) was weighed, placed in a 2mL glass bottle together with 6.3mg of glycolic acid, and isopropyl acetate (0.2 mL) was added to obtain a suspension. The resulting sample was stirred in suspension at 5 ℃ for 3 days. The resulting suspension was centrifuged through a 0.45 μm nylon filter at 14,000rpm, and the resulting solid was dried under vacuum at 25 ℃ for 4 hours to give the product. The product is a pharmaceutical co-crystal formed by the compound shown in the formula (I) and glycollic acid through X-ray powder diffraction detection, an XRPD spectrum is shown in figure 4, and characteristic peak positions are shown in table 5. The DSC profile shows a melt T onset @135.68 ℃.
TABLE 5 XRPD diffraction peak data for pharmaceutical co-crystals of the compound of formula (I) with glycolic acid
EXAMPLE 7 preparation of pharmaceutical Co-crystals of the Compound of formula (I) with L-proline
About 20mg of the compound represented by the formula (I) was weighed, placed in a 2mL glass bottle together with 9.7mg of L-proline, and ethanol (0.25 mL) was added to obtain a suspension. The resulting sample was stirred in suspension at 5 ℃ for 3 days. The resulting suspension was centrifuged through a 0.45 μm nylon filter at 14,000rpm, and the resulting solid was dried under vacuum at 25 ℃ for 4 hours to give the product. The product is a pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline through X-ray powder diffraction detection, an XRPD spectrum is shown in figure 7, and characteristic peak positions are shown in table 6. The DSC profile shows a melt T onset @183.55 ℃.
TABLE 6 XRPD diffraction peak data for pharmaceutical co-crystals of the compound of formula (I) with L-proline
EXAMPLE 8 preparation of pharmaceutical Co-crystals of the Compound of formula (I) with L-proline
About 300mg of the compound of formula (I) and 145.8mg of L-proline (1.0 equivalent) were weighed into a 20mL glass bottle. 3.75mL of ethanol was added and stirred at 5℃for 2min to give a suspension. To the above suspension is added about 5mg of seed crystals of the pharmaceutical co-crystal of the compound of formula (I) with L-proline. To the above suspension was added 3mL of ethanol and stirred at 5 ℃ for 4 days. The solid fraction was collected by centrifugation and the resulting solid fraction was dried under vacuum at room temperature for about 2.5 hours. 250mg of white powder was obtained in a yield of 56%. The product is a pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline through X-ray powder diffraction detection.
EXAMPLE 9 preparation of pharmaceutical Co-crystals of the Compound of formula (I) with nicotinamide
About 20mg of the compound represented by formula (I) was weighed, placed in a 2mL glass bottle together with 10.3mg of nicotinamide, and acetonitrile (0.25 mL) was added to obtain a suspension. The resulting sample was stirred in suspension at 5 ℃ for 3 days. The resulting suspension was centrifuged through a 0.45 μm nylon filter at 14,000rpm, and the resulting solid was dried under vacuum at 25 ℃ for 4 hours to give the product. The product is a pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide through X-ray powder diffraction detection, an XRPD spectrum is shown in figure 10, and characteristic peak positions are shown in table 7. The DSC profile shows a melting T onset @156.75 ℃.
TABLE 7 XRPD diffraction peak data for pharmaceutical co-crystals of the compound of formula (I) with nicotinamide
EXAMPLE 10 preparation of pharmaceutical Co-crystals of the Compound of formula (I) with nicotinamide
About 300mg of the compound of formula (I) and 154.6mg of nicotinamide (1.0 equivalent) were weighed into a 20mL glass bottle. 3.75mL of acetonitrile was added and stirred at 5℃for 2min to give a suspension. To the above suspension is added about 5mg of seed crystals of the pharmaceutical co-crystal of the compound of formula (I) with nicotinamide. To the above suspension was added 2mL of acetonitrile and stirred at 5 ℃ for 4 days. The solid fraction was collected by centrifugation and the resulting solid fraction was dried under vacuum at room temperature for about 2.5 hours. 365mg of white powder was obtained in 80% yield. The product is a pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide through X-ray powder diffraction detection.
Example 11 stability test of pharmaceutical Co-crystals of Compounds of formula (I) with L-proline and pharmaceutical Co-crystals of Compounds of formula (I) with nicotinamide
The open containers containing the pharmaceutical co-crystal of the compound of formula (I) and L-proline and the pharmaceutical co-crystal of the compound of formula (I) and nicotinamide were placed in 25℃/92.5% RH for one week, respectively. And (3) placing the pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline and the pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide in a 60 ℃ closed container for one week respectively. The samples after solid stability evaluation were characterized by XRPD and HPLC and observed for color change, and the conclusions are shown in table 8.
TABLE 8 pharmaceutical co-crystal solid stability formed by the Compounds of formula (I)
As can be seen from Table 8, the pharmaceutical co-crystals of the compound of formula (I) and L-proline and nicotinamide do not significantly lower the purity after being placed in an open container at 25/92% RH for one week or in a closed container at 60 ℃ for one week, but the pharmaceutical co-crystals of the compound of formula (I) and L-proline are dissociated into free crystalline form and L-proline at 25/92.5% RH. The pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide is stable under both conditions.
EXAMPLE 12 solubility test of pharmaceutical Co-crystals of Compounds of formula (I) with L-proline and pharmaceutical Co-crystals of Compounds of formula (I) with nicotinamide
15.1Mg of the pharmaceutical co-crystal formed by the compound shown in the formula (I) and L-proline and 14.9mg of the pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide are accurately weighed and placed in a 20mL glass bottle respectively. 5mL of vehicle was added to each. The mass of the compound shown in the formula (I) in the weighed eutectic is equivalent to 10mg of free anhydrous crystal form. The resulting suspension was stirred at 400rpm at 37 ℃ and sampled at 0.5 hours and 2 hours, respectively. The sample was centrifuged at 14,000rpm for 5min at 37 ℃. The supernatant concentration was determined by HPLC and the pH of the supernatant was determined by a pH meter. The remaining solid (wet) was checked by XRPD for changes in crystalline form after 2 hours. The experimental results are shown in table 9.
TABLE 9 solid solubility of pharmaceutical Co-crystals formed from the Compounds of formula (I)
As can be seen from Table 9, the pharmaceutical co-crystals of the compound of formula (I) and L-proline and nicotinamide are converted into the free crystalline form of the compound of formula (I) in four different vehicles. In addition, the solubility of the pharmaceutical co-crystal formed by the compound shown in the formula (I) and the pharmaceutical co-crystal formed by the nicotinamide is higher in the rest solvents, and the solubility of the pharmaceutical co-crystal formed by the compound shown in the formula (I) and the nicotinamide after being dissolved in the four solvents for 2 hours is higher than that of the pharmaceutical co-crystal formed by the compound shown in the formula (I) and the pharmaceutical co-crystal formed by the L-proline.
Example 13 hygroscopicity test of pharmaceutical Co-crystals of Compounds of formula (I) with L-proline and pharmaceutical Co-crystals of Compounds of formula (I) with nicotinamide
The water absorption and dehydration behavior of the pharmaceutical co-crystals of the compound of formula (I) with L-proline and of the compound of formula (I) with nicotinamide were investigated by DVS testing at 25 ℃. The DVS cycle is 40-0-95-0-40% RH. XRPD detection was performed on the DVS-tested samples to determine if a crystalline transition occurred, and the results are shown in table 10.
TABLE 10 hygroscopicity test of pharmaceutical Co-crystals formed from the Compounds of formula (I)
As can be seen from the results in Table 10, the pharmaceutical co-crystal formed by the compound of formula (I) and L-proline has slight hygroscopicity between 40% RH and 80% RH, and has excellent hygroscopicity between 80% RH and 95% RH, and the hygroscopic weight is about 84.0%. After DVS testing, the eutectic moiety dissociates into the free crystalline form and L-proline. The pharmaceutical co-crystal formed by the compound shown in the formula (I) and nicotinamide has slight hygroscopicity, and the moisture absorption weight gain between 40% RH and 95% RH is about 0.2% at 25 ℃. After DVS testing, no conversion of the crystalline form occurred.
It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the invention. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.
Claims (17)
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| CN202311684501.1A CN120118019A (en) | 2023-12-08 | 2023-12-08 | A co-crystal form of RNA m6A regulator and its preparation method and application |
| PCT/CN2024/137463 WO2025119335A1 (en) | 2023-12-08 | 2024-12-06 | Co-crystal form of rna m6a modulator and preparation method therefor and use thereof |
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