Deuterated benzyl alcohol TRPV3 inhibitor and medical application thereof

Abstract

The invention discloses a deuterated benzyl alcohol TRPV3 inhibitor and medical application thereof, and belongs to the technical field of medicines. The structural formula of the deuterated benzyl alcohol compound is shown as a formula (I). The compound provided by the invention can be used as an inhibitor of a TRPV3 ion channel, and can be used for preparing medicines for improving or treating diseases related to excessive activation or expression increase of TRPV3. Compared with a TRPV3 inhibitor KM-001 which is clinically researched, the compound provided by the invention has better metabolic stability and oral availability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a class of deuterated benzyl alcohol TRPV3 inhibitors and their pharmaceutical uses. Background Technology

[0002] Transient receptor potential (TRP) ion channels are a class of six-transmembrane channel proteins widely distributed in the peripheral and central nervous systems. They are molecular receptors that sense changes in the cellular environment, transmit signals, and maintain cellular homeostasis. Based on sequence similarity, mammalian TRP ion channels can be divided into six subfamilies: TRPA, TRPC, TRPM, TRPML, TRPP, and TRPV. The transient receptor potential vanilloid (TRPV) subfamily is a class of non-selective cation channels located on the cell membrane and widely distributed in mammalian tissues and organs. The TRPV subfamily contains six members: TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6, with 30%–40% homology among the various TRPV channels.

[0003] TRPV3, in particular, can respond to non-noxious thermal stimuli (≥ 33 °C) and possesses unique sensitization properties. TRPV3 is highly expressed in the skin, testes, duodenum, colon, brain, spinal cord, and dorsal root ganglia, and expressed at low levels in the heart, lungs, esophagus, stomach, bladder, and ovaries. It can regulate skin barrier formation and hair growth, sensory transmission (temperature, itch, and pain, etc.), and vasodilation, and is closely related to a variety of diseases.

[0004] TRPV3 is primarily expressed in keratinocytes (KCs), the most abundant cells in the epidermis. In humans or rodents carrying the TRPV3 gain-of-function mutation (GOF), skin barrier function is lost, leading to the death or premature differentiation of hair follicle KCs, resulting in inhibited hair growth. Increased TRPV3 expression or activity may cause hair loss or alopecia areata, and TRPV3 expression increases with the severity of atopic dermatitis (AD). The TRPV3 gain-of-function mutation has been found to be the cause of Olmsted syndrome, a rare hereditary skin disease in humans. Kamari's TRPV3 inhibitor KM-001 is currently in clinical trials for the treatment of palmoplantar keratosis and neurodermatitis. The clinical anesthetic dacronin can also act as a TRPV3 inhibitor for local antipruritic purposes.

[0005] TRPV3 is considered a drug target for pain relief. The sensations of itching and pain have long been thought to be closely related; weak activation of pain receptors has been shown to mediate itching, while stronger activation leads to weak pain. Studies have shown that mice overexpressing TRPV3 in keratinocytes release prostaglandin E2, resulting in thermal pain and hyperalgesia. Glenmark's TRPV3 inhibitor, GRC15300, was developed for the treatment of neuropathic pain and osteoarthritis pain, but its clinical trials have been discontinued.

[0006] TRPV3 is also highly expressed in the intestinal mucosal epithelium. In patients with irritable bowel syndrome (IBS), TRPV3 expression in the duodenum is significantly elevated, which may mediate postprandial symptoms in IBS patients. However, the role of TRPV3 inhibitors in the treatment of intestinal diseases has not been reported.

[0007] TRPV3 has been confirmed to be expressed in small arteries of the brain parenchyma, radial arteries of the uterus, and pulmonary arteries. Quantitative PCR and immunohistochemical results further indicate that TRPV3 is expressed in both arterial smooth muscle cells and endothelial cells. In the cardiovascular system, TRPV3 is involved in the pathological process of myocardial hypertrophy. TRPV3 activation can induce arterial diastole through both endothelium-dependent and endothelium-independent pathways. Furthermore, TRPV3 is also expressed in cardiomyocytes and cardiac fibroblasts, and participates in hypoxia-induced cardiomyocyte apoptosis and inflammatory responses.

[0008] TRPV channels are associated with various human cancers, and alterations in their expression influence cancer progression by enhancing cell proliferation, altering cell differentiation, and impairing cell death. While changes in TRPV channel expression are generally thought to exert their effects only in late-stage cancer, studies have shown that TRPV3 can serve as a prognostic marker for clear cell renal cell carcinoma and breast cancer. In kidney cells (KCs), TRPV3 forms a signaling complex with EGFR, and activation of TRPV3 enhances EGFR signaling activity, thereby regulating epithelial cell proliferation. This suggests that TRPV3 inhibitors may play a synergistic role in EGFR-targeted therapy for cancers such as skin cancer, breast cancer, and non-small cell lung cancer.

[0009] Therefore, drugs that regulate TRPV3 function or expression may be used to treat a variety of diseases or symptoms associated with overactivation or elevated expression of TRPV3 channels, such as, but not limited to, atopic dermatitis, neurodermatitis, pruritus, Olmsted syndrome, palmoplantar keratosis, pain, hyperalgesia, inflammatory bowel disease, irritable bowel syndrome, hypertension, pulmonary hypertension, heart disease, myocardial injury, and cancer.

[0010] WO2021154966A1 discloses a compound represented by the general formula (XXXII), wherein the substituent R 2Defined as cyano, nitro, hydroxy, halogen, C1-C3 haloalkyl, C1-C3 haloalkoxy, C1-C6 alkyl, hydroxyalkyl, C3-C8 cycloalkyl, C1-C6 alkoxy, aryl, -N(R) a (R) b ), -C(O)R c -CH2R c -CO2R c , -C(O)N(R a (R) b ), -SO2N(R a (R) b ) or -SOR c ; Each R a and R b Independently H, hydroxyl, -OR c C1-C6 alkyl, -C(O)R c or -C(O)OR c ;R c H, C1~C6 alkyl, aryl, -OR a or -N(R) a (R) a ); p = 0, 1 or 2.

[0011] , Among them, compound KM-001, which is the focus of research in this application, has good TRPV3 inhibitory activity and also showed good efficacy in mouse models of itching and dermatitis. However, this compound has poor metabolic stability and is difficult to administer orally, so it can only be applied topically through the skin. Summary of the Invention

[0012] The purpose of this invention is to provide a class of deuterated benzyl alcohol compounds for use as TRPV3 inhibitors, their preparation methods, and therapeutic applications. Compared to the clinically investigated TRPV3 inhibitor KM-001, the compounds of this invention exhibit better metabolic stability and oral bioavailability.

[0013] In a first aspect, the present invention provides compounds, stereoisomers, or pharmaceutically acceptable salts thereof of formula (I): ,

[0014] in, Ring A is phenyl or a 5-6 membered heteroaryl group; R 1 It is a halogen, a C1-C6 alkyl, a C1-C6 haloalkyl, a C1-C6 alkoxy, a C1-C6 haloalkoxy, or a cyano; Each R 2Each of them independently consists of a halogen, a C1-C6 alkyl, a C1-C6 haloalkyl, a C1-C6 alkoxy, a C1-C6 haloalkoxy, a C3-C8 cycloalkyl, a C3-C8 cycloalkoxy, or a deuterium atom; m and n are each independently 0, 1, 2, 3 or 4.

[0015] Furthermore, ring A is a 5-6 membered heteroaryl group, preferably pyridyl or piperazine.

[0016] Furthermore, R 1 It is a halogen, a C1-C3 haloalkyl, a C1-C3 alkoxy or cyano group, preferably a fluorine atom, a trifluoromethyl group, a methoxy group or a cyano group.

[0017] Furthermore, R 2 It can be a halogen, a C1-C3 haloalkyl, a C1-C3 haloalkoxy, a C3-C5 cycloalkyl, a C3-C5 cycloalkoxy, or a deuterium atom, preferably a fluorine atom, a trifluoromethyl, a trifluoromethoxy, a cyclopropyl, a cyclopropoxy, or a deuterium atom; Furthermore, m is 1 or 2.

[0018] Furthermore, n is either 1 or 2.

[0019] In another preferred embodiment, the compound of the present invention has a substructure as shown in formula (Ia): ,

[0020] in, Z1 and Z2 are each independently CH or N; The definitions of the other substituents are as described in equation (I).

[0021] In another preferred embodiment, the compound of the present invention has a substructure as shown in formula (Ib): ,

[0022] in, Z1 and Z2 are each independently CH or N; The definitions of the other substituents are as described in equation (I).

[0023] In another preferred embodiment, the compounds of the present invention are selected from the following: .

[0024] Secondly, the present invention provides a pharmaceutical composition comprising at least one effective therapeutic dose of a compound of a general formula, a stereoisomer, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. The pharmaceutical excipient may be a pharmaceutically acceptable carrier, excipient, sustained-release agent, odorant, flavoring agent, etc.

[0025] In the pharmaceutical composition, the compound of the present invention is the active ingredient, accounting for 0.1 to 99.9% of the total weight of the pharmaceutical composition, and the remainder is pharmaceutically acceptable excipients; the preferred ratio of the compound of the present invention to the excipients is: the compound of the present invention as the active ingredient accounts for more than 60% of the total weight, and the remaining part accounts for 0-40% of the total weight, preferably 1-20%, and most preferably 1-10%.

[0026] The compounds or pharmaceutical compositions described in this invention can be formulated into various dosage forms, such as tablets, capsules, powders, syrups, solutions, suspensions, sprays, creams, ointments, gels, and transdermal patches, based on conventional processes in the pharmaceutical formulation field, and can be contained in suitable solid or liquid carriers or diluents. The pharmaceutical compositions of this invention can also be stored in suitable sterile injection or infusion apparatus.

[0027] The compounds or pharmaceutical compositions described in this invention are suitable for use in mammals, including humans and animals. Routes of administration include oral, nasal inhalation, topical application, intravenous injection, intramuscular injection, and subcutaneous injection. In another preferred embodiment, the preferred routes of administration for the compounds or pharmaceutical compositions described in this invention are oral and topical application.

[0028] Solid dosage forms of the compounds or pharmaceutical compositions described in this invention for oral administration include capsules, tablets, pills, powders, and granules. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include sterile water, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), provided they are suitable for the characteristics of the active ingredient and the desired specific route of administration. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, colorings, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0029] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0030] When the compound or pharmaceutical composition thereof represented by formula (I) of this invention is used to prepare a topical skin medication, it can be formulated into a suitable dosage form, such as, but not limited to, creams, ointments, gels, transdermal patches, sprays, etc. The drug carrier can be selected from starch, oils, creams, foams, ointments, skin lotions, gels, etc., or it can be a drug delivery system, such as liposomes, liposomes, microsponges, microemulsions, microspheres, solid lipid nanoparticles, etc.

[0031] When used as a pharmaceutical preparation, the compound of formula (I) of this invention can be effectively dissolved, suspended, or dispersed in a liquid medium to form a solution, suspension, or dispersion, and can be applied topically to the skin by application or spraying, and can be further aided by sponges, patches, pads, or other cosmetic products for delivery. In some cases, a sustained-release delivery system for the compound may be crucial for achieving its therapeutic effect.

[0032] When used as a pharmaceutical preparation, the compounds shown in this invention are preferably administered in unit doses of 0.01 mg to 200 mg, preferably 0.5 mg to 50 mg, of the active ingredient, either once or in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Generally, a small dose is started, and the dose is gradually increased until the most suitable dose is found.

[0033] Thirdly, the present invention provides the use of compounds of formula (I), stereoisomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, said compounds being used as inhibitors of the TRPV3 ion channel, thereby enabling the preparation of pharmaceuticals for improving or treating diseases associated with overactivation or elevated expression of TRPV3.

[0034] In this invention, the diseases associated with the overactivation or elevated expression of TRPV3 include, but are not limited to, skin diseases (such as atopic dermatitis, neurodermatitis, pruritus, Olmsted syndrome, palmoplantar keratosis, etc.), pain, hyperalgesia, inflammatory bowel disease, irritable bowel syndrome, hypertension, pulmonary hypertension, heart disease, myocardial injury, cancer, etc.

[0035] In a preferred embodiment, the disease associated with the overactivation or elevated expression of TRPV3 is a skin disease. Furthermore, since TRPV3 is primarily expressed in keratinocytes, the skin disease is related to abnormal keratinocyte function, such as, but not limited to, psoriasis, atopic dermatitis, neurodermatitis, eczema, ichthyosis, keratosis, Olmsted's disease, toad skin disease or menopausal keratosis, tactile hypersensitivity, burns, pruritus, etc.

[0036] In another preferred embodiment, the skin disease associated with the overactivation or elevated expression of TRPV3 involves inappropriate skin differentiation. Skin diseases associated with inappropriate skin differentiation include keratosis, ichthyosis, or a combination thereof.

[0037] In another preferred embodiment, the skin condition associated with the overactivation or elevated expression of TRPV3 is keratosis. Specifically, the term "keratosis" is also known as palmar-plantar keratosis. Palmoplantar keratosis refers to a disease / condition characterized by thickening of the skin on the palms and soles. Keratosis is at least one of diffuse keratosis, focal keratosis, or punctate keratosis.

[0038] In another preferred embodiment, the keratosis associated with the overactivation or elevated expression of TRPV3 is a congenital (hereditary) keratosis. Hereditary keratosis may be caused by gene abnormalities leading to, for example, abnormalities in skin proteins (keratin). Hereditary palmoplantar keratosis (PPK) is at least one of diffuse hereditary palmoplantar keratosis, focal hereditary palmar spicule keratosis, and punctate palmar spicule keratosis.

[0039] The compounds or pharmaceutical compositions thereof described in this invention can be used alone or in combination with drugs of other mechanisms for the treatment of skin diseases. These drugs include, but are not limited to, glucocorticoids such as mometasone, fluocinolone acetonide, triamcinolone acetonide, hydrocortisone, clobetasol, etc.; Janus kinase inhibitors such as ruxolitinib, tofacitinib, utpatinib, etc.; calcineurin inhibitors such as tacrolimus, pimecrolimus; biological immunomodulators such as adalimumab, infliximab, etanercept, etc.; and antihistamines such as diphenhydramine, chlorpheniramine, promethazine, terfenadine, astemizole, loratadine, desloratadine, cetirizine, and azelastine.

[0040] In another preferred embodiment, the disease associated with the overactivation or elevated expression of TRPV3 is a pain-related disease. This includes, but is not limited to, nociceptive pain, mechanical pain, inflammatory pain, neuropathic pain, osteoarthritis pain, rheumatoid arthritis pain, postherpetic neuralgia, burn-related pain, migraine, irritable bowel syndrome-related pain, and cancer pain. The pain may be chronic or acute.

[0041] In another preferred embodiment, the disease associated with the overactivation or elevated expression of TRPV3 is an arthritis-related disease, including but not limited to osteoarthritis, rheumatoid arthritis, etc.

[0042] In another preferred embodiment, the disease associated with the overactivation or elevated expression of TRPV3 is a cardiovascular condition. This condition includes, but is not limited to, hypertension, atherosclerosis, coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, myocardial hypertrophy, stroke, and pulmonary hypertension.

[0043] In another preferred embodiment, the disease associated with the overactivation or elevated expression of TRPV3 is cancer. This disease includes, but is not limited to, lung cancer, renal cell carcinoma, breast cancer, colorectal cancer, brain tumors, etc.

[0044] In another preferred embodiment, the disease associated with the overactivation or elevated expression of TRPV3 is an intestinal disease. Such diseases include, but are not limited to, ulcerative colitis, short bowel syndrome, irritable bowel syndrome, intestinal spasm, diarrhea, abdominal pain, etc.

[0045] In another preferred embodiment, the intestinal disease is ulcerative colitis.

[0046] Fourthly, the present invention provides methods for preparing the compounds of formula (I), their stereoisomers, and pharmaceutically acceptable salts thereof. The compounds of the present invention can be prepared by a variety of synthetic methods. The method shown in route A below is a representative general approach, which, combined with the synthetic methods of specific compounds in the examples, constitutes the method for preparing the compounds of the present invention. It should be understood that the method for preparing the compounds of the present invention is not limited to that shown in route A; it is for illustrative purposes only and does not limit the present invention in any way.

[0047] .

[0048] In route A, R 3 It is a C1-C6 alkyl group, and other substituents are defined as described above. The reaction steps in Route A include: (a) Starting material A-1 was subjected to a Suzuki coupling reaction with 3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylic acid tert-butyl ester to generate intermediate A-2. The Suzuki coupling reaction is a conventional method in the art, carried out in the presence of a palladium catalyst, a ligand, and a base. The palladium catalyst is, for example but not limited to, palladium acetate, tris(dibenzylacetone)palladium, tetra(triphenylphosphine)palladium, Pd(dppf)Cl2, Pd(PPh3)2Cl2, etc.; the ligand is, for example but not limited to, 1,1'-bis(diphenylphosphine)ferrocene, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (XPhos), 2-di-tert-butylphosphine-2',4',6'-triisopropylbiphenyl (t-BuXPhos), 4,5-bisdiphenylphosphine-9,9-dimethyloxanthracene (XantPhos), and 2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl (Ru). Phos), tri-tert-butylphosphine, triphenylphosphine, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP), etc.; the base includes inorganic bases and organic bases, such as, but not limited to, cesium carbonate, potassium carbonate, sodium carbonate, potassium phosphate, sodium acetate, potassium acetate, potassium tert-butoxide, sodium tert-butoxide, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylamine, N,N-diisopropylethylamine (DIPEA), pyridine; the reaction is carried out in a suitable organic solvent, such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), tetrahydrofuran (THF), xylene, chlorobenzene, acetonitrile, tert-butanol; the reaction temperature is generally from room temperature to 200 °C.

[0049] (b) Intermediate A-2 undergoes a Miyaura borylation reaction with pinacol diboronate to generate intermediate A-3; the reaction conditions are similar to those in step a.

[0050] (c) Intermediate A-3 undergoes a hydrogenation reduction reaction to generate intermediate A-4; this reaction is usually carried out under the catalysis of metals such as palladium / carbon, palladium oxide, platinum oxide, and Raney nickel; hydrogen is usually used as the hydrogen source, but it can also be generated in situ by reagents such as ammonium formate and cyclohexene; the reaction temperature is generally from room temperature to 120°C; the reaction solvent is, for example but not limited to, DMF, methanol, ethanol, ethyl acetate, THF, and 1,4-dioxane; the reaction can be carried out under normal pressure or under pressure, with the pressure of the pressurized reaction generally ranging from 0.1 to 30 MPa.

[0051] (d) Intermediate A-4 undergoes a Suzuki coupling reaction with starting material A-5 to generate intermediate A-6; the reaction conditions are as described in step a.

[0052] (e) Intermediate A-6 is reduced by a deuterated reagent to generate intermediate A-7; the deuterated reducing reagent is, for example, but not limited to, lithium aluminum tetradeuterium, sodium borodeuteride, etc.; the reaction is carried out in an aprotic solvent or a deuterated protic solvent, for example, but not limited to, THF, 1,4-dioxane, deuterated methanol, deuterated ethanol, etc.; the reaction temperature is generally from -20 °C to room temperature.

[0053] (f) Intermediate A-7 undergoes the removal of the tert-butyloxycarbonyl (Boc) protecting group to generate A-8; the reaction is usually carried out in the presence of an acid, such as, but not limited to, trifluoroacetic acid, hydrochloric acid, acetic acid, methanesulfonic acid, and p-toluenesulfonic acid; the reaction temperature is generally -20 to 50°C, preferably room temperature; the reaction solvent is, for example, but not limited to, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane, methanol, ethanol, etc.

[0054] (g) Intermediates A-8 and A-9 undergo an amide condensation reaction to generate a compound of general formula (I); the reaction is typically carried out in the presence of a condensing agent well known to those skilled in the art, examples of which can be found in the literature or reference books. Representative examples include, but are not limited to, dicyclohexylcarbodiimide (DCC), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / 1-hydroxybenzotriazole (ECDI / HOBt), 2-(1H-benzotriazolyl-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), and N,N'-carbonyldiimidazole (C The reaction is typically carried out in the presence of a base, including inorganic and organic bases. Examples of organic bases include, but are not limited to, pyridine, DIPEA, 4-dimethylaminopyridine (DMAP), triethylamine, DUB, and trihydroxyethylamine. Examples of inorganic bases include, but are not limited to, lithium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, potassium phosphate, and sodium acetate. The solvent may be any solvent that does not adversely affect the reaction, such as, but not limited to, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane, DMF, DMA, NMP, and acetonitrile. The reaction temperature is typically from -20 to 120°C.

[0055] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Due to space limitations, they will not be described in detail here.

[0056] the term In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0057] In this invention, the term "C1-C6" refers to having 1, 2, 3, 4, 5, or 6 carbon atoms, and so on. "3-8" refers to having 3, 4, 5, 6, 7, or 8 cyclic atoms, and so on.

[0058] In this invention, "alkyl" refers to a branched or straight-chain hydrocarbon group having a specific number of carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, and isopropyl.

[0059] In this invention, "alkoxy" refers to -O-alkyl. For example, the term "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 6 carbon atoms, and representative examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and butoxy.

[0060] In this invention, "halogenated alkyl" and "halogenated alkoxy" refer to groups formed by the partial or complete substitution of hydrogen atoms in "alkyl" and "alkoxy" having a specific number of carbon atoms by "halogen atoms".

[0061] In this invention, "cycloalkyl" represents a non-aromatic cyclic aliphatic hydrocarbon group with a specific number of cyclic carbon atoms, and "C3-C8 cycloalkyl" represents a cyclic aliphatic hydrocarbon group composed of 3 to 8 cyclic carbon atoms. The "cycloalkyl" in this invention includes not only monocyclic aliphatic hydrocarbon groups, but also fused ring, spiro ring, and bridged ring systems composed of multiple cyclic aliphatic hydrocarbons. Examples of "cycloalkyl" in this invention include, but are not limited to: , , .

[0062] In this invention, "aryl" is defined as a monocyclic or bicyclic system composed of a specific number of carbon atoms and obeying Hückel's rule. When it is a bicyclic system, both rings may be aromatic, or only one ring may be aromatic while the other is hydrogenated. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, and dihydroindenyl.

[0063] In this invention, "heteroaryl" is defined as a cyclic system having a specific number of cyclic atoms and containing 1, 2, 3 or 4 heteroatoms (selected from N, O, S) while obeying Hückel's rule; examples of "heteroaryl" include, but are not limited to, pyridine, piperazine, pyrrole, imidazole, thiophene, oxazole, etc.

[0064] In this invention, the "halogen atom" includes fluorine, chlorine, bromine, and iodine.

[0065] In this invention, "substitution" means being replaced by one or more groups (such as 2, 3, 4, or 5). Unless otherwise specified, substitution can occur on any atom where the number of substituents has not yet reached saturation. When multiple substituents are selected from the same series, they can be identical or different.

[0066] In this invention, "optionally" means that the defined group can be selected from a series of candidate groups, or it can be left unselected.

[0067] The "pharmaceutically acceptable salt" described in this invention can be a salt formed by an anion and a positively charged group on a general formula compound. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronate, lactate, glutarate, or maleate. Similarly, salts can be formed by a cation and a negatively charged group on a general formula compound. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium ions, such as tetramethylammonium ions.

[0068] In another preferred embodiment, "pharmaceutically acceptable salt" refers to a salt formed by a compound of the general formula with an acid selected from the group consisting of: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid; or a sodium salt, potassium salt, calcium salt, aluminum salt, or ammonium salt formed by a compound of the general formula with an inorganic base; or a methylamine salt, ethylamine salt, or ethanolamine salt formed by a compound of the general formula with an organic base.

[0069] The compounds of the general formula of this invention, or pharmaceutically acceptable salts thereof, are obtained by distillation, crystallization, or recrystallization from water or organic solvents, and may contain solvent molecules used. Furthermore, different crystallization conditions may result in different crystal forms of the compounds. Therefore, compounds of the general formula, or pharmaceutically acceptable salts thereof, containing different chemical dosages of crystallization solvents and all crystal forms, are within the scope of this invention.

[0070] In this invention, the "effective therapeutic dose" means that, compared with subjects who do not receive this dose of treatment, the subjects who receive this dose of treatment experience a cure, improvement, effective prevention, or a significant reduction in the incidence of lesions or side effects; in addition, it also includes an effective dose that enhances normal physiological functions.

[0071] In this invention, the term "inhibitor" may also be expressed as "antagonist" or "blocker," which have the same meaning in this invention, referring to a compound or mixture that can be used to reduce or inhibit biological activity.

[0072] Some compounds represented by the general formula of this invention have a chiral center, a latent chiral center, or an unsaturated bond, and can form various forms of stereoisomers, such as racemates, enantiomers, diastereomers, E / Z isomers, cis-trans isomers, tautomers, etc. Unless otherwise stated, the chemical formulas or names given in the specification and appended claims are intended to cover all forms of stereoisomers, mixtures of individual isomers in different proportions, and pharmaceutically acceptable salts thereof. Those skilled in the art can use commonly used laboratory separation methods to isolate compounds containing asymmetric centers in this invention to obtain single isomers, but this does not preclude the novelty of the compounds of this invention. Attached Figure Description

[0073] Figure 1 To improve DSS-induced ulcerative colitis in mice with compound 1 (Cmpd1). (A) Changes in body weight of mice in each group after free access to 3% DSS for 6 consecutive days, and after gavage administration of different doses (2, 5, and 10 mg / kg) of compound 1 twice daily. (B) Disease activity index scores of mice in each group. P <0.01, compared with the control group (Veh); # P <0.05, ## P <0.01, compared with the model group (3% DSS), two-way ANOVA combined with Dunnett's multiple comparison test was used for data statistics. (CD) Representative colon photographs of mice in each group (C) and quantitative results of colon length (D). (E) H&E staining of paraffin sections of colon tissue from mice in each group. Black arrows indicate the disappearance of crypt structures, and red boxes indicate edematous areas of the lamina propria containing inflammatory cell infiltration. Scale bar is 100 μm. (F) Pathological scoring results of colon tissue in each group. (G) Quantitative statistical graph of myeloperoxidase (MPO) activity in colon tissue in each group. (H) Inflammatory factors in colon tissue in each group ( Tnf , Il1b , Cxcl1 , Il6 mRNA expression levels of [missing data]. Data are expressed as mean ± standard error, * P <0.05,** P <0.01, compared with the control group (Veh); # P <0.05, ## P<0.01, compared with the model group (3% DSS), the data were statistically analyzed by one-way ANOVA combined with Bonferroni multiple comparison test.

[0074] Figure 2 Compound 1 (Cmpd1) inhibits IL-17-induced keratinocyte proliferation. (A) Representative BrdU immunofluorescence images of HaCaT cells treated with IL-17 (300 ng / mL) and different concentrations (0.1, 0.3, and 1.0 μM) of compound 1 for 12 hours. Scale bar: 75 μm. (B) Quantitative statistical graph of cell proliferation index for each group. P <0.01, compared with the control group (Veh); # P <0.05, compared with the IL-17 group, data were statistically analyzed using one-way ANOVA combined with Bonferroni multiple comparison test. Detailed Implementation

[0075] The present invention will be further illustrated below with specific embodiments. It should be particularly noted that these embodiments are for illustrative purposes only and are not intended to limit the invention in any way. All parameters and other descriptions in the examples, unless otherwise stated, are based on quality. Unless otherwise specified, the packing material used for column chromatography separation is silica gel. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention.

[0077] Abbreviations: DCM: dichloromethane; EA: ethyl acetate; PE: petroleum ether; DMAP: 4-dimethylaminopyridine; DIPEA: N,N-diisopropylethylamine; HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; TFA: trifluoroacetic acid; THF: tetrahydrofuran.

[0078] Preparation Examples

[0079] Reagents and conditions: (a) Pd(dppf)Cl2, Cs2CO3, H2O / 1,4-Dioxane (1:6), sealedtube, 100℃; (b) Pd(dppf)Cl2, AcOK, 1,4-Dioxane, 90℃; (c) Pd / C, H2, Ethylacetate / MeOH (1:1), rt. (d) Pd(dppf)Cl2, Cs2CO3, H2O / 1,4-Dioxane (1:6), sealedtube, 110℃; (e) LiAlD4, dry THF, 0℃; (f) Trifluoroacetic acid,Dichloromethane, rt; (g) 5-Fluoro-2-picolinic acid, HATU, DIPEA,Dichloromethane, rt; (h) Chiral separation. 3-(4-bromo-2-(methoxycarbonyl)phenyl)-2,5-dihydro-1H-pyrrole-1-carboxylic acid tert-butyl ester (Int-1)

[0080] Under nitrogen protection, methyl 5-bromo-2-iodobenzoate (1.3 g, 3.81 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate tert-butyl ester (938 mg, 3.18 mmol), and cesium carbonate (2.072 g, 6.36 mmol) were dissolved in 36 mL of 1,4-dioxane and 6 mL of water, and dichloro-1,1'-bis(diphenylphosphino)ferrocene-palladium (117 mg, 0.16 mmol) was added. The reaction mixture was then heated to 100°C and stirred for 3 hours. After the reaction was complete, insoluble residues were removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by rapid chromatography to give intermediate Int-1 (927 mg, 64%) as an oil. 1 H NMR (500 MHz, Chloroform- d ) δ 7.94 (dd, J = 3.8, 2.1 Hz, 1H), 7.60 (dd, J = 8.3, 2.1 Hz, 1H), 7.16 (dd, J= 8.3, 3.8 Hz, 1H), 5.74 – 5.65 (m, 1H), 4.39 – 4.23 (m, 4H), 3.88(s, 3H), 1.49 (s, 9H). MS (ESI) m / z 382.1 [M + H] + . 3-(2-(methoxycarbonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)-2,5-dihydro-1H-pyrrole-1-carboxylic acid tert-butyl ester (Int-2)

[0081] Under nitrogen protection, Int-1 (927 mg, 2.42 mmol), bis(pinacolyl)diboron (938 mg, 3.18 mmol), and potassium acetate (477 mg, 4.87 mmol) were dissolved in 40 mL of 1,4-dioxane, and dichloro-1,1'-bis(diphenylphosphino)ferrocene-palladium (179 mg, 0.24 mmol) was added. The reaction mixture was then heated to 90°C and stirred overnight. After the reaction was complete, insoluble residues were removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by rapid chromatography to give intermediate Int-2 (1 g, 96%) as an oil. 1 H NMR (500 MHz, Chloroform- d ) δ 8.26 – 8.20 (m, 1H), 7.95 – 7.86 (m, 1H), 7.39 (d, J = 7.9 Hz, 1H), 4.23 –4.15 (m, 1H), 4.11 (qt, J = 7.0, 3.2 Hz, 1H), 3.90 (t, J = 2.8 Hz, 3H), 3.80 –3.73 (m, 1H), 3.65 – 3.47 (m, 1H), 3.45 – 3.29 (m, 2H), 1.46 (d, J = 5.5 Hz,9H), 1.34 (m, 12H). MS (ESI) m / z 430.2 [M + H] + . 3-(2-(methoxycarbonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)pyrrolidine-1-carboxylic acid tert-butyl ester (Int-3)

[0082] Int-2 (1 g, 2.33 mmol) was dissolved in 20 mL of a methanol / ethyl acetate mixture (v / v = 1:1). Palladium on carbon (10%, 200 mg) was added to this solution, and the reaction mixture was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the catalyst was removed by filtration, and the filtrate was concentrated to give intermediate Int-3 (920 mg, 92%) as an oil. MS (ESI): m / z 432.3 [M + H]⁺. 3-(4'-fluoro-3-(methoxycarbonyl)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)pyrrolidine-1-carboxylic acid tert-butyl ester (Int-4)

[0083] Under nitrogen protection, a solution of Int-3 (920 mg, 2.13 mmol), 4-fluoro-1-iodo-2-(trifluoromethoxy)benzene (544 mg, 1.78 mmol), and cesium carbonate (1.16 g, 3.56 mmol) was dissolved in 20 mL of 1,4-dioxane and 3 mL of water, and then dichloro-1,1'-bis(diphenylphosphine)ferrocene-palladium (132 mg, 0.18 mmol) was added. The reaction mixture was then heated to 110°C and stirred for 3 hours. After the reaction was complete, insoluble residues were removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by rapid chromatography to give intermediate Int-4 (520 mg, 61%) as an oil. 3-(4'-fluoro-3-(hydroxymethyl-d2)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)pyrrolidine-1-carboxylic acid tert-butyl ester (Int-5)

[0084] Int-4 (520 mg, 1.08 mmol) was dissolved in dry tetrahydrofuran and cooled to 0°C in an ice bath. Then, under nitrogen protection, lithium deuterated aluminum hydride (55 mg, 1.31 mmol) was added. The reaction mixture was maintained at 0°C and stirred for 30 minutes. After the reaction was complete, the reaction was carefully quenched with water, extracted with ethyl acetate, and washed with saturated brine. The organic layer was dried over anhydrous magnesium sulfate, and the crude product was purified by rapid chromatography to give intermediate Int-5 (420 mg, 85%) as an oil. MS (ESI) m / z 458.2 [M + H] + . (4'-Fluoro-4-(pyrrolidone-3-yl)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-3-yl)methanol- d 2(Int-6) Int-5 (420 mg, 0.92 mmol) was dissolved in dichloromethane (3 mL), and 1 mL of trifluoroacetic acid was added to the solution. The mixture was stirred at room temperature for 30 minutes. After the reaction was complete, the solvent was removed under reduced pressure to give crude product Int-6 (307 mg, 93%), which was used directly in the next step. MS (ESI) m / z 358.1 [M + H] + . (5-Fluoropyridin-2-yl)(3-(4'-Fluor-3-(hydroxymethyl-) d 2)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)pyrrolidine-1-yl)methyl ketone (compound 1) A solution of 5-fluoropyridinecarboxylic acid (146 mg, 1.03 mmol), HATU (490 mg, 1.3 mmol), and N,N-diisopropylethylamine (449 μL, 2.58 mmol) was prepared in 15 mL of dichloromethane, and the mixture was stirred at room temperature for 30 minutes. Subsequently, a dichloromethane solution of Int-6 (307 mg, 0.86 mmol) was added, and the reaction mixture was stirred for another 2 hours. After the reaction was complete, the mixture was concentrated under reduced pressure, and the crude product was purified by preparative high-performance liquid chromatography using an acetonitrile / water (containing 0.1% TFA) gradient elution to give the target compound "Compound 1" (45 mg, 11%) as a white solid. 1 H NMR (500 MHz, DMSO -d 6, major isomer of the 4:3 stereoisomers) δ 8.59 (d,1H, J = 2.5), 7.94 – 7.89 (m, 2H), 7.57 (dd, J = 8.5, 6.5 Hz, 1H), 7.51 – 7.45(m, 2H), 7.44 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.5, 2.0 Hz, 1H), 5.14 (s, 1H), 4.10 – 4.04 (m, 1H), 3.85 – 3.79 (m, 1H), 3.72 – 3.68 (m, 2H), 3.66 – 3.59(m, 1H), 2.30 – 2.22 (m, 1H), 2.15 – 2.07 (m, 1H). 13 C NMR (126 MHz, DMSO-d 6,major isomer of the 4:3 stereoisomers) δ 164.73 (C=O), 161.67 (d, J = 248.0Hz), 159.87 (d, J = 257.5 Hz), 150.91 (d, J = 3.9 Hz), 145.90 (d, J = 10.7 Hz),140.91, 139.01, 136.64 (d, J = 24.2 Hz), 133.77, 132.37 (d, J = 9.3 Hz), 131.52,128.93, 128.32, 126.43, 126.26 (d, J = 5.3 Hz), 124.59 (d, J = 18.6 Hz), 120.31(q, J = 258.4 Hz, -CF3), 115.58 (d, J = 20.8 Hz), 109.94 (d, J = 25.6 Hz), 60.78(p, J = 20.3 Hz), 55.76, 47.15, 37.29, 31.18. HRMS (ESI) m / z [M + H] + calcd forC 24 H 18 D2F5N2O3 + , 481.1514; found, 481.1510. HPLC purity: 99.53%.

[0085] Compound 1 was separated into compounds 1-a and 1-b by chiral separation under the following conditions: (1) WonCract ODS-2 liquid chromatography column (4.6 ID × 250 mm, Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd.); (2) Flow rate: 1 mL / min; (3) Detection wavelength: UV 210 nm; (4) Mobile phase: n-hexane: isopropanol = 9: 1.

[0086] Following the preparation method of compound 1, the compounds listed in the table below can be synthesized:

[0087]

[0088]

[0089] .

[0090] Pharmacological Examples 1. TRPV3 inhibitory activity test (1) Test method HEK-293 cells expressing hTRPV3 were seeded into poly-L-lysine (PDL)-coated black-walled permeabilized 96-well plates, 2 × 10⁶ cells per well. 4 After culturing the cells for 8 hours, the original culture medium was discarded, and 60 μL of Fluo-4 / AM dye was added to a final concentration of 4 μM. The cells were incubated at 37°C for 60 minutes, followed by rinsing five times with Locke's buffer. The cell plate was then placed in a preheated 30°C FLIPR container. ®TETRA (Molecular Devices, Sunnyvale, CA, USA) Excitation was performed at 488 nm, and fluorescence signals were continuously recorded at a sampling frequency of 1 s in the 515-535 nm range. After 60 seconds of recording, a solvent control, the test compound, and the positive inhibitor KM-001 (a compound in WO2021154966A1, with a final concentration of 100 nM) were added, and signal acquisition continued for 300 seconds. Then, the agonist 2-APB was added, and fluorescence signal acquisition continued for another 600 seconds. The fluorescence signal in the trajectory plot is expressed as F / F0, where F is the fluorescence signal at different time points, and F0 is the baseline fluorescence signal, i.e., the average of the fluorescence signals at the first 10 time points. The dose-response plot was first calculated with F / F0=1 as the baseline, calculating the area under the curve (AUC) of the fluorescence intensity change after adding 30 μM 2-APB. Combining the AUC with the log value of the compound concentration, the IC50 was calculated using log[Inhibitor] vs. response-Variable slope. 50 value.

[0091] (2) Test Results As shown in Table 1, the compounds of the present invention have TRPV3 inhibitory activity comparable to the positive control KM-001, and have potential application value in the treatment of diseases related to TRPV3 channel overactivation or TRPV3 protein overexpression.

[0092] Table 1: Inhibitory activity of compounds against TRPV3

[0093] 2. Liver microsomal stability test (1) Test method First, a 0.1 M pH 7.4 Tris buffer was prepared. Then, this buffer was used to prepare a 100 mM MgCl2 solution and a 10 mM NADPH solution. The analyte was first prepared as a stock solution in DMSO, and then diluted to the working concentration with 0.1% (w / v) BSA-PBS solution before use. For the assay, the liver microsomes TRIS buffer (final concentration 0.33 mg / mL microsomal protein), MgCl2 solution (final concentration 5 mM), analyte solution (final concentration 1 μM), and NADPH solution (final concentration 1 mM) were incubated at 37°C. The reaction was terminated by adding methanol at 0, 7, 17, 30, and 60 minutes, respectively. The residual concentration of the analyte was determined by LC / MS / MS.

[0094] Calculation of half-life: T 1 / 2 =0.693 / k e ; Intrinsic clearance rate calculation: ; Calculation of clearance rate in vivo: ; Calculation of liver clearance rate: ; Metabolic utilization rate calculation: .

[0095] k e The slope of the linear regression line (absolute value of SLPOE) in the semi-logarithmic plot is calculated by taking the semi-logarithm of the remaining percentage of the substrate and plotting it against the reaction time. P: Microsomal protein concentration (mg / mL); Houston: Houston factor (45 mg microsomal protein / g liver); LW: Liver weight (g); HBF: Liver blood flow (mL / min); fu: Unbound percentage (usually fu=1).

[0096] (2) Test results As shown in Table 2, compared to the positive control KM-001, the half-life of the compound of the present invention in human liver microsomes was significantly prolonged, indicating better metabolic stability. Structure-activity relationship analysis shows that the improved metabolic stability of the compound of the present invention is related to the introduction of deuterium and fluorine atoms into the molecule.

[0097] Table 2: Half-life of the test compounds in human liver microsomes (t) 1 / 2 ) .

[0098] 3. Oral pharmacokinetic evaluation (1) Test method The test drug was dissolved in a 2:2:96 (V / V / V) aqueous solution of DMSO / Tween 80 / 0.5% (m / V) methylcellulose (model: A4M) to prepare the target concentration according to the dosage and volume. The oral (PO) administration volume was 10 mL / kg. Three male ICR rats weighing 30-38 g were used in each group. They were fasted for 12 h before the experiment but had free access to water. They were fed uniformly 4 h after drug administration. In group IV, the drug was administered via the tail vein. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 h after administration. In group PO, the drug was administered by gavage. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 h after administration. Blood samples of 30-40 μL were collected from the jugular or mandibular veins at the above time points and placed in anticoagulant tubes containing EDTA-K2. After collection, the whole blood was temporarily stored in an ice-water bath and centrifuged at 11000 rpm for 5 min within 30 min to separate the plasma. The plasma was then frozen at -70°C for analysis. Drug concentration in the samples was quantitatively determined using LC-MS / MS. Relevant pharmacokinetic parameters Tp were calculated using WinNonlin software. max C max AUC 0-∞ t 1 / 2 wait.

[0099] (2) Test results As shown in Table 3, the compound showed good plasma exposure in mice after oral administration of 10 mg / kg, indicating that the compound of the present invention has significant oral availability.

[0100] Table 3: Plasma pharmacokinetic parameters of compound 1 in mice after oral administration of 10 mg / kg (n = 3)

[0101] 4. The therapeutic effect of the compound on ulcerative colitis (1) Test method I. Mouse model of ulcerative colitis Male C57BL / 6 mice (7-8 weeks old, weighing 22-24 g) were purchased from Yangzhou University. Mice were housed in an animal room at a controlled temperature of 23 ± 2℃, with a 12-hour light-dark cycle and free access to food and water. After one week of acclimatization, mice were randomly assigned to groups. An ulcerative colitis model was established using free access to 3% sodium dextran sulfate (DSS) for 6 consecutive days. During DSS modeling, different doses (2, 5, 10 mg / kg / time) of compound 1 were administered twice daily by gavage for 6 consecutive days. The positive control drug was 5-aminosalicylic acid (200 mg / kg, twice daily for 6 consecutive days). All compounds were dissolved in a carrier solution (2% DMSO + 2% Tween-80 + 0.5% CMC-Na). The control and DSS model groups were administered the carrier solution according to the same protocol. The Disease Activity Index (DAI) was scored daily using the Sann method to comprehensively assess weight loss, hematochezia, and fecal characteristics. Mice were sacrificed on the seventh day, colon length was measured, distal colon tissue was harvested and fixed in 4% paraformaldehyde or flash-frozen in liquid nitrogen.

[0102] II. Colonic histopathological examination Fixed colon tissue samples were embedded in paraffin and prepared into 5 µm sections, which were then stained with hematoxylin and eosin. Images were acquired using a Hamamatsu NanoZoomer 2.0 RS slide scanning imaging system, and histological scoring was performed based on a comprehensive assessment of the degree of inflammation, the level of crypt damage, and the extent of the lesion.

[0103] III. MPO Activity Detection The activity of myeloperoxidase in mouse colon tissue was determined using an MPO activity assay kit (Cat# A114-1-1, Nanjing Jiancheng Biotechnology Co., Ltd.), following the manufacturer's instructions. The simplified steps are as follows: The supernatant of the colon tissue homogenate was mixed with hydrogen peroxide and o-anisidine. Quantification was performed by detecting the absorbance of oxidized o-anisidine at 460 nm, which showed a linear correlation with MPO activity. The final result was normalized to the absorbance of the control group sample and expressed as relative MPO activity.

[0104] IV. Detection of inflammatory factor expression levels Total RNA was extracted from colon tissue using Trizol reagent (Cat# R401-01, Novizan Biotech, Nanjing). Reverse transcription was performed using the HiScript II Q RT SuperMix with oligo (dT) 23 VN anchored primer reverse transcription kit (Cat# R223-01, Novizan Biotech, Nanjing). Real-time quantitative PCR was then performed using SYBR Green dye (Cat# Q221-01, Novizan Biotech, Nanjing) on ​​a QuantStudio 3 real-time quantitative PCR system (Thermo Fisher Scientific, USA). The results were analyzed using GAPDH as an internal reference gene. -ΔΔCt The mRNA expression level of the target gene was quantified using this method. All primers used in this study (see Table 4) were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0105] Table 4: Primer sequences for inflammatory factors

[0106] (2) Test results During DSS-induced ulcerative colitis, twice-daily gavage administration of compound 1 (Cmpd1) dose-dependently improved DSS-induced ulcerative colitis symptoms. Specifically, Cmpd1 significantly alleviated DSS-induced weight loss in animals. Figure 1 (A) Reduce disease activity index score ( Figure 1 (B) Improves colonic shortening ( Figure 1 (C, D), and reduce histopathological damage to the colon (colonic mucosal epithelial destruction, lamina propria edema, and inflammatory infiltration), Figure 1 (E, F). At a dose of 10 mg / kg, Cmpd1 reduced DSS-induced weight loss by 49.6 ± 4.0%, decreased the disease activity index score by 56.5 ± 5.4%, improved colonic shortening by 41.5 ± 4.7%, and reduced histopathological score by 71.4 ± 3.7%, which were statistically significant compared with the DSS model group mice.

[0107] Furthermore, Cmpd1 also exhibited dose-dependent anti-inflammatory effects: at a dose of 10 mg / kg, it significantly inhibited 64.8 ± 0.26% of the DSS-induced increase in MPO activity in colonic tissue and reduced tumor necrosis factor (TNF) by 62.7 ± 0.4%. Tnf ), 89.1 ± 0.2% interleukin-1b ( Il1b ), 94.1 ± 0.9% chemokine 1 ( Cxcl1 ) and 88.7 ± 1.1% interleukin-6 ( Il6The mRNA expression level of ) was significantly different from that of the DSS model group mice. Figure 1 (Zhong GH).

[0108] 10 mg / kg Cmpd1 showed better or equivalent improvement in symptoms and indicators of ulcerative colitis than the positive control drug 5-ASA (200 mg / kg). Figure 1 Therefore, it can be seen that the compounds of the present invention can significantly relieve the symptoms of ulcerative colitis and have the potential to improve or treat inflammatory bowel diseases.

[0109] 5. Effects of the compound on keratinocyte proliferation (1) Test method HaCaT cells were cultured in an environment containing 10% FBS, 10 mM HEPES, and 100 U / mL penicillin / streptomycin at 37°C, 5% CO2, and 95% humidity. When the cells reached 70-80% confluence, they were digested with trypsin-EDTA for 5-7 minutes. The cells were then pipetted to collect the cell suspension, centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in DMEM containing 2% FBS. The cells were then cultured at 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of cells / well in black-walled permeable 96-well plates. After cell attachment, 300 ng / mL IL-17 was added to induce cell proliferation, and different concentrations (0.1, 0.3, and 1.0 μM) of compound 1 were added, followed by culturing for another 12 hours.

[0110] Add 10 μM BrdU (5-bromodeoxyuridine) to the cell culture environment, continue culturing at 37°C for 3 hours, discard the solution in the wells, and wash three times with PBS; fix cells with 4% paraformaldehyde for 30 minutes, and wash three more times with PBS; treat cells with 0.1% Triton X-100 for 15 minutes, and wash three times with PBS; then treat with 2M HCl for 30 minutes, add 0.1M boric acid to neutralize for 10 minutes, wash three times with PBS, and then add 5% BSA to block at room temperature for 30 minutes; discard the liquid, add 1:200 diluted BrdU primary antibody (Boster Biologics, Wuhan), and incubate overnight at 4°C. The following day, the primary antibody was discarded, and the cells were washed five times with PBS. Under light-protected conditions, a 1:1000 dilution of fluorescent secondary antibody was added, and the cells were incubated at room temperature for 1 hour, followed by five washes with PBS. Under light-protected conditions, a 1:1000 dilution of Hoechst dye was added, and the cells were incubated at room temperature for 15-20 minutes, followed by two washes with PBS. An anti-fluorescence quencher was then added. Five to six fields of view were randomly selected under an inverted fluorescence microscope for observation and photography. The cell proliferation index was expressed as the percentage of cells displaying BrdU-positive fluorescence signals out of the total number of cells.

[0111] (2) Test results Cell proliferation was detected using a BrdU incorporation assay, and the results are as follows: Figure 2 As shown, after 12 hours of IL-17 treatment of HaCaT cells, the percentage of BrdU-positive cells increased from 7.5 ± 1.7% to 18.8 ± 3.2%, indicating that IL-17 promotes keratinocyte proliferation. Compound 1 (Cmpd1) was able to inhibit IL-17-induced cell proliferation in a dose-dependent manner, with inhibition rates of 45.5 ± 14.5% at different concentrations (0.1, 0.3, and 1.0 μM). P >0.05), 76.4 ± 10.2% ( P <0.05) and 89.9 ± 8.5% ( P <0.05). Therefore, the compounds of this invention can effectively inhibit keratinocyte proliferation, and thus can be used to improve or treat skin diseases related to incomplete differentiation or dysfunction of keratinocytes.

[0112] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The compound, stereoisomer, or pharmaceutically acceptable salt thereof represented by formula (I): ， in, Ring A is phenyl or a 5-6 membered heteroaryl group; R 1 It is a halogen, a C1-C6 alkyl, a C1-C6 haloalkyl, a C1-C6 alkoxy, a C1-C6 haloalkoxy, or a cyano; Each R 2 Each of them independently consists of a halogen, a C1-C6 alkyl, a C1-C6 haloalkyl, a C1-C6 alkoxy, a C1-C6 haloalkoxy, a C3-C8 cycloalkyl, a C3-C8 cycloalkoxy, or a deuterium atom; m and n are each independently 0, 1, 2, 3 or 4.

2. The compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Ring A is a 5-6 membered heteroaryl group, preferably pyridyl or piperazine.

3. The compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R 1 It is a halogen, a C1-C3 haloalkyl, a C1-C3 alkoxy or cyano group, preferably a fluorine atom, a trifluoromethyl group, a methoxy group or a cyano group.

4. The compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R 2 It can be a halogen, a C1-C3 haloalkyl, a C1-C3 haloalkoxy, a C3-C5 cycloalkyl, a C3-C5 cycloalkoxy, or a deuterium atom, preferably a fluorine atom, a trifluoromethyl, a trifluoromethoxy, a cyclopropyl, a cyclopropoxy, or a deuterium atom.

5. The compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, m is 1 or 2.

6. The compound, stereoisomer, or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, n is 1 or 2.

7. The following compounds, stereoisomers, or pharmaceutically acceptable salts thereof: 。 8. A pharmaceutical composition, characterized in that, It includes an effective amount of the active ingredient and pharmaceutically acceptable excipients; the active ingredient includes one or more of the compounds, stereoisomers or pharmaceutically acceptable salts thereof as described in any one of claims 1 to 7.

9. Use of the compound, stereoisomer, or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, or the pharmaceutical composition of claim 8, in the preparation of a drug that inhibits TRPV3 ion channels.

10. Use of the compound, stereoisomer, or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, or the pharmaceutical composition of claim 8, in the preparation of a medicament for improving or treating diseases associated with overactivation or elevated expression of TRPV3; preferably, the diseases associated with overactivation or elevated expression of TRPV3 include skin diseases, pruritus, pain, hyperalgesia, inflammatory bowel disease, irritable bowel syndrome, hypertension, pulmonary hypertension, heart disease, myocardial injury, cancer, etc.

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