Application of TLR8 inhibitor in preparation of medicine for treating systemic sclerosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a TLR8 inhibitor in preparation of a medicine for treating systemic sclerosis. The invention has the following advantages: the compound shown in the formula (I) and / or the pharmaceutically acceptable salt, the deuterated substance, the solvate and the precursor of the compound have obvious improvement on skin lesion of patients with systemic sclerosis. The invention provides application of the compound shown in the formula (I) and / or the pharmaceutically acceptable salt, the deuterated compound, the solvate and the precursor of the compound in preparation of drugs for treating systemic sclerosis, and is expected to provide a new strategy for treatment of systemic sclerosis. # imgabs0 # (I)

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine technology, and particularly relates to the use of a TLR8 inhibitor in preparing a drug for treating systemic sclerosis. Background Art

[0002] Systemic sclerosis (SSc), also known as scleroderma, is a connective tissue disease caused by a combination of genetic factors and recurrent, persistent acute and chronic infections. It is characterized by localized or diffuse skin thickening and fibrosis. It is more common in women than in men and is relatively rare in children. The severity and progression of the disease vary widely, affecting not only the skin but also internal organs (such as the heart, lungs, brain, and digestive tract).

[0003] Toll-like receptors (TLRs) are a key class of protein molecules involved in nonspecific immunity (innate immunity), serving as a bridge between nonspecific and specific immunity. TLR8, a member of the TLR family, has been implicated in the etiology of certain autoimmune diseases targeting self-RNA and DNA / protein complexes. Therefore, inhibitors targeting TLR8 hold great promise, but currently, relatively few are available.

[0004] The use of TLR8 inhibitors in the preparation of drugs for treating systemic sclerosis has not been reported in the prior art.

[0005] The present disclosure aims to develop the use of TLR8 inhibitors in the preparation of drugs for treating systemic sclerosis through efficacy experiments in animal models of systemic sclerosis (scleroderma). Summary of the Invention

[0006] The technical problem to be solved by the present disclosure is to develop the use of TLR8 inhibitors in the preparation of drugs for treating systemic sclerosis. The TLR8 inhibitors disclosed herein have significant improvements in skin lesions in patients with systemic sclerosis. The present disclosure provides the use of TLR8 inhibitors in the preparation of drugs for treating systemic sclerosis, which is expected to provide a new strategy for the treatment of systemic sclerosis.

[0007] To achieve the above technical objectives, the technical solutions adopted by this disclosure are: In one aspect, the present disclosure provides the use of a compound represented by formula (I) and / or a pharmaceutically acceptable salt, deuterated substance, solvate, or precursor thereof in the preparation of a medicament for treating systemic sclerosis. (I) Wherein, R1 is selected from H, -OH, -OC 1-6Alkyl or C 1-6 alkyl; R2 is selected from -OH, -NH2, -OC 1-8 Alkylene -OH or -OC 1-8 Alkylene -NH2; R3 is selected from H, -C 1-6 Alkyl or -C 1-6 alkyl halide; R4 is selected from H, -C 1-6 Alkyl or -C 1-6 Halogenated alkyl.

[0008] In some embodiments of the present disclosure, R1 is selected from -OH, -OC 1-6 alkyl; R2 is selected from -OH, -NH2, -OC 1-8 Alkylene-OH; R3 is selected from H, -C 1-6 Alkyl or -C 1-6 alkyl halide; R4 is selected from H, -C 1-6 alkyl.

[0009] In some embodiments of the present disclosure, the pharmaceutically acceptable salt of the compound represented by formula (I) is selected from one or more of inorganic acid salts or organic acid salts.

[0010] In some embodiments of the present disclosure, the inorganic acid salt includes, but is not limited to, one or more of hydrochloride, phosphate, hydrobromide, hydroiodide, hydrofluoride, nitrate, sulfate, and carbonate.

[0011] In some embodiments of the present disclosure, the organic acid salt includes, but is not limited to, one or more of malonate, succinate, formate, propionate, butyrate, and valerate.

[0012] In some preferred embodiments of the present disclosure, the pharmaceutically acceptable salt of the compound represented by formula (I) is the monohydrochloride salt of the compound represented by formula (I).

[0013] In some embodiments of the present disclosure, R1 is selected from H, -OH, -OC 1-5 Alkyl, C 1-5 alkyl.

[0014] In some embodiments of the present disclosure, R1 is selected from H, -OH, -OC 1-5 alkyl.

[0015] In some embodiments of the present disclosure, R1 is selected from H, -OH, -OC 1-3 alkyl.

[0016] In some embodiments of the present disclosure, R1 is selected from H, -OH, -OCH3, -OCH2CH3.

[0017] In some embodiments of the present disclosure, R1 is selected from -OH, -OC 1-5 alkyl.

[0018] In some embodiments of the present disclosure, R1 is selected from -OH, -OC 1-4 alkyl.

[0019] In some embodiments of the present disclosure, R1 is selected from -OH, -OC 1-3 alkyl.

[0020] In some embodiments of the present disclosure, R1 is selected from -OH, -OCH3, -OCH2CH3.

[0021] In some embodiments of the present disclosure, R1 is selected from -OH, -OCH3.

[0022] In some embodiments of the present disclosure, R2 is selected from -OH, -NH2, -OC 1-8 Alkylene-OH.

[0023] In some embodiments of the present disclosure, R2 is selected from -OH, -NH2, -OC 1-6 Alkylene-OH.

[0024] In some embodiments of the present disclosure, R2 is selected from -OH, -NH2, -OC 1-5 Alkylene-OH.

[0025] In some embodiments of the present disclosure, R2 is selected from -OH, -NH2, -OC 1-4 Alkylene-OH.

[0026] In some embodiments of the present disclosure, R2 is selected from -OH, -NH2, -OC 1-4 Alkylene-OH.

[0027] In some embodiments of the present disclosure, R2 is selected from -OH, -NH2, -O(CH2)4-OH.

[0028] In some embodiments of the present disclosure, R3 is selected from H, -C 1-5 Alkyl or -C 1-5 Halogenated alkyl.

[0029] In some embodiments of the present disclosure, R3 is selected from H, -C 1-3 Alkyl or -C 1-3 Halogenated alkyl.

[0030] In some embodiments of the present disclosure, R3 is selected from H, methyl, ethyl, -CF3, -CCl3, -CBr3, -CH2F, -CHF2, -CH2Cl, -CHCl2, -CH2Br, -CHBr2.

[0031] In some embodiments of the present disclosure, R3 is selected from H, methyl, ethyl, -CF3, -CH2F, -CHF2.

[0032] In some embodiments of the present disclosure, R3 is selected from H, methyl, -CF3.

[0033] In some embodiments of the present disclosure, R4 is selected from H, -C 1-5 alkyl.

[0034] In some embodiments of the present disclosure, R4 is selected from H, -C 1-3 alkyl.

[0035] In some embodiments of the present disclosure, R4 is selected from H, methyl, ethyl.

[0036] In some embodiments of the present disclosure, R4 is selected from H, methyl.

[0037] In some embodiments of the present disclosure, the compound represented by formula (I) is selected from the following compounds: 、 、 、 、 、 、 .

[0038] Hydroxyproline (HYP) is one of the imino acids, a non-essential amino acid, one of the main components of collagen tissue, and a unique amino acid in collagen.

[0039] In some embodiments of the present disclosure, the medicament is used for one or more uses selected from the following: Reduce the level of connective tissue growth factor (CTGF) in plasma; Used to reduce the hydroxyproline (HYP) content in the skin and / or reduce the degree of skin collagen fibrosis; Used to reduce epidermal thickness and / or reduce dermal thickness.

[0040] In some embodiments of the present disclosure, the medicament is used to reduce the hydroxyproline (HYP) content in the skin and / or reduce the degree of skin collagen fibrosis.

[0041] In some embodiments of the present disclosure, the medicament is used to reduce the thickness of the epidermis layer and / or reduce the thickness of the dermis layer.

[0042] In some embodiments of the present disclosure, the compound of formula (I) and / or its pharmaceutically acceptable salts, deuterated forms, solvates, or precursors thereof significantly reduce both epidermal and dermal thickness. Compared to penicillamine, penicillamine significantly reduced dermal thickness while maintaining no significant difference in epidermal thickness. The compound of formula (I) and / or its pharmaceutically acceptable salts, deuterated forms, solvates, or precursors significantly reduced both epidermal and dermal thickness.

[0043] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; the pharmaceutical preparation contains the compound represented by formula (I) and / or its pharmaceutically acceptable salt, deuterated substance, solvate or precursor as the sole active ingredient.

[0044] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; the pharmaceutical preparation comprises a compound represented by formula (I) and / or a pharmaceutically acceptable salt, deuterated substance, solvate, or precursor thereof, and further comprises a pharmaceutically acceptable excipient.

[0045] In some embodiments of the present disclosure, the pharmaceutically acceptable excipients include one or more of enteric coating materials, binders, fillers, disintegrants, glidants, lubricants, solvents, emulsifiers, flavoring agents, colorants, pH regulators, stabilizers, surfactants, and preservatives.

[0046] In some embodiments of the present disclosure, the pharmaceutically acceptable excipients include one or more of an enteric coating material, a binder, a filler, a disintegrant, a glidant, and a lubricant.

[0047] In some embodiments of the present disclosure, the pharmaceutically acceptable excipients include enteric coating materials, binders, fillers, disintegrants, glidants, and lubricants.

[0048] Fillers, also known as diluents, include, but are not limited to, wheat starch, tapioca starch, corn starch, potato starch, dextrin, microcrystalline cellulose, lactose, and the like. Examples of flavoring agents include, but are not limited to, steviol glycosides, glycyrrhizin, mogroside, acesulfame potassium, aspartame, sucralose, and isomaltulose. Examples of lubricants include, but are not limited to, magnesium stearate, talc, micronized silica gel, and magnesium lauryl sulfate.

[0049] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; the pharmaceutical preparation comprises a compound represented by formula (I) and / or a pharmaceutically acceptable salt, a deuterated substance, a solvate, a precursor thereof, and one or more other drugs for treating systemic sclerosis.

[0050] In some embodiments of the present disclosure, the pharmaceutical preparation is a solid preparation, a semisolid preparation or a liquid preparation.

[0051] In some embodiments of the present disclosure, the solid preparation is a tablet, capsule, granule or pill.

[0052] In some embodiments of the present disclosure, the semisolid formulation is a gel, a suppository, or an ointment.

[0053] In some embodiments of the present disclosure, the liquid preparation is an emulsion, a mixture, a suspension or a solution.

[0054] In some embodiments of the present disclosure, the solid preparation is a tablet, capsule, granule or pill; the semisolid preparation is a gel, suppository or ointment; and the liquid preparation is an emulsion, mixture, suspension or solution.

[0055] In some embodiments of the present disclosure, the medicine is in the form of tablets, capsules, granules, pills, injections, syrups, powders, chewable tablets, gels, and suppositories.

[0056] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; a unit pharmaceutical preparation contains 10 to 800 mg of the compound represented by formula (I).

[0057] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; a unit pharmaceutical preparation contains 50 to 500 mg of the compound represented by formula (I).

[0058] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; a unit pharmaceutical preparation contains 100 to 400 mg of the compound represented by formula (I).

[0059] In some embodiments of the present disclosure, the drug is prepared as a pharmaceutical preparation for treating systemic sclerosis; a unit pharmaceutical preparation contains 200 to 300 mg of the compound represented by formula (I).

[0060] In some embodiments of the present disclosure, the drug is administered by one or more of oral administration, sublingual administration, intravenous administration, transdermal administration, rectal administration, inhalation administration, intraperitoneal administration or intramuscular administration.

[0061] In some embodiments of the present disclosure, the drug is administered by one or more of oral administration, sublingual administration, intravenous administration, transdermal administration, and rectal administration.

[0062] In some embodiments of the present disclosure, the drug is administered by one or more of oral administration, sublingual administration, intravenous administration, and transdermal administration.

[0063] In some preferred embodiments of the present disclosure, the drug is administered orally or intravenously.

[0064] In some preferred embodiments of the present disclosure, the drug is administered orally.

[0065] The present disclosure has the following advantages: (1) The compound represented by formula (I) disclosed herein has a significant effect on improving skin lesions in patients with systemic sclerosis.

[0066] (2) The compound represented by formula (I) disclosed herein can reduce the thickness of the epidermis and dermis.

[0067] (3) The present disclosure provides the use of the compound represented by formula (I) in the preparation of a drug for treating systemic sclerosis, which is expected to provide a new strategy for the treatment of systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings required for the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without inventive work. The drawings herein are incorporated into and constitute a part of the specification, illustrate embodiments consistent with the specification, and together with the specification, are used to explain the principles of the specification.

[0069] Figure 1 The relative content of α-SMA mRNA in human skin fibroblasts detected by q-PCR in Example 2 is shown.

[0070] Figure 2 The relative content of COL1A1 mRNA in human skin fibroblasts detected by q-PCR in Example 2 is shown.

[0071] Figure 3 The relative content of COL1A2 mRNA in human skin fibroblasts detected by q-PCR in Example 2 is shown.

[0072] Figure 4 The relative content of COL3A1 mRNA in human skin fibroblasts detected by q-PCR in Example 2 is shown.

[0073] Figure 5 The results of hydroxyproline (HYP) detection of each group of mice in Example 3 are shown.

[0074] Figure 6 The graph shows the results of epidermal skin thickness testing of each group of mice in Example 3.

[0075] Figure 7 The graph shows the dermal skin thickness test results of each group of mice in Example 3. DETAILED DESCRIPTION

[0076] Definition and Description In order to make it easier to understand the present disclosure, certain technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell and tissue culture, microbiology-related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in this application are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0077] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0078] As used herein, the terms "comprise," "comprises," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to such process, method, product, or device.

[0079] In the description herein, reference is made to “some embodiments,” “some implementation schemes,” or “some implementation plans,” which describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0080] The abbreviation PEG400 refers to polyethylene glycol 400.

[0081] The abbreviation PG refers to propylene glycol.

[0082] The abbreviation H2O refers to water.

[0083] DMSO (Dimethyl Sulfoxide) refers to dimethyl sulfoxide.

[0084] The abbreviation CAS NO. is a registration number assigned to chemical substances by the Chemical Abstracts Service (CAS). This number is an important tool for retrieving information about chemical substances with multiple names. It is a unique numerical identification number for a substance (compound, polymer, biological sequence, mixture, or alloy).

[0085] The abbreviation PBS refers to phosphate buffered saline.

[0086] The abbreviation ELISA refers to enzyme-linked immunosorbent assay. Its basic method is to adsorb known antigens or antibodies on the surface of a solid phase carrier (polystyrene microplate), allow the enzyme-labeled antigen-antibody reaction to proceed on the solid phase surface, and use a washing method to remove free components in the liquid phase.

[0087] The abbreviation mpk stands for milligrams per kilogram (milligrams of drug mass / kilogram of animal weight), which refers to the number of milligrams of drug administered per kilogram of animal body weight.

[0088] The abbreviation D42 refers to day 42.

[0089] The abbreviation D0 refers to day 0.

[0090] The abbreviation D1 refers to day 1.

[0091] Solvent, also known as solvent, is a liquid that can dissolve gases, solids, and liquids to form a uniform mixture.

[0092] HEK-Blue-hTLR8 is a genetically engineered cell line primarily used to study the human Toll-like receptor 8 (TLR8)-dependent NF-κB signaling pathway.

[0093] DMEM (Dulbecco's Modified Eagle Medium) is a basal culture medium widely used in cell culture.

[0094] The activator R848 (Resiquimod) is an immune response regulator and a small molecule Toll-like receptor (TLR) agonist that mainly acts on Toll-like receptors 7 and 8 (TLR7 / TLR8).

[0095] Quanti-Blue is a colorimetric enzyme assay reagent used to detect alkaline phosphatase (AP) activity in biological samples such as cell culture supernatants.

[0096] SkanIt is a microplate reader data analysis software developed by Thermo Fisher Scientific. It is a software that integrates data acquisition and result analysis, and can realize the entire microplate workflow from experimental setup to data analysis.

[0097] CCK-8 (Cell Counting Kit-8) is a WST-8-based kit that is widely used for rapid and highly sensitive detection of cell proliferation and cytotoxicity.

[0098] GraphPad Prism 7 is a powerful scientific graphing and data analysis software widely used in biomedicine, pharmacology and other fields.

[0099] Compound 1-Compound 7 refers to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7.

[0100] IC 50 IC50 refers to the concentration of an inhibitor that reduces a biological activity (such as cell proliferation) to 50% of its original value under specific experimental conditions. 50 It is an important parameter to measure the efficacy of drugs, IC 50 The smaller the value, the stronger the inhibitory effect of the drug can be exerted at a lower concentration. 50 It is calculated using conventional calculation methods in this field.

[0101] HSF cells usually refer to human skin fibroblasts.

[0102] TGF-β refers to transforming growth factor β, an important cytokine.

[0103] q-PCR (Quantitative Polymerase Chain Reaction) is a technology developed based on PCR that monitors nucleic acid amplification products in real time and performs quantitative analysis. q-PCR incorporates fluorescent chemicals into the PCR reaction system, utilizing the accumulated fluorescent signal to monitor PCR amplification products in real time and perform quantitative analysis.

[0104] cDNA (complementary DNA) is a type of DNA that is reverse transcribed from RNA (such as messenger RNA or microRNA) using reverse transcriptase.

[0105] α-SMA (α-smooth muscle actin) is an actin isoform that is mainly expressed in smooth muscle cells, but is also expressed in a variety of non-smooth muscle cells, such as fibroblasts and mesenchymal stem cells.

[0106] The COL1A1 gene encodes the α1 chain of type I collagen, one of the most abundant types of collagen in the human body and widely present in connective tissues.

[0107] COL1A2 is the gene that encodes type I collagen alpha2 chain, and its full name is collagen type I alpha2 chain.

[0108] COL3A1 is the gene encoding the α1 chain of type III collagen.

[0109] H&E staining, also known as HE staining, is hematoxylin-eosin staining. It is a commonly used histological staining technique, mainly used to observe the structure and cell morphology of tissues.

[0110] Hydroxyproline (HYP) is one of the imino acids, a non-essential amino acid, one of the main components of collagen tissue, and a unique amino acid in collagen.

[0111] Systemic sclerosis (SSc), also known as scleroderma, is an autoimmune disease characterized by localized or diffuse skin thickening and fibrosis. Common clinical manifestations of SSc include sclerosis and fibrosis of the skin, as well as sclerosis, fibrosis, and vascular lesions that affect organs throughout the body.

[0112] Improving skin thickness in patients with scleroderma has multiple benefits, particularly significant impacts on patients' quality of life and health. Specific benefits include: 1. Relieve symptoms: Scleroderma causes the skin to thicken and harden. Improving skin thickness can alleviate these symptoms and reduce pain and discomfort.

[0113] 2. Improve mobility: Thinning and softening the skin can increase joint flexibility, relieve limited mobility caused by tight skin, and make patients more comfortable in their daily activities.

[0114] 3. Preventing complications: Scleroderma not only affects the skin but can also affect internal organs. By improving skin thickness, it may be possible to slow the progression of the disease and prevent damage to other organs.

[0115] 4. Aesthetics and Psychological Well-being: Skin hardening and thickening often affect appearance, especially in exposed areas. Improving skin thickness can help restore appearance, reduce psychological stress, and improve self-esteem and life satisfaction.

[0116] 5. Reduce the risk of infection: Scleroderma skin may be more prone to cracking and ulceration. Restoring thickness and elasticity helps maintain skin integrity and reduce the risk of infection.

[0117] Improving skin thickness is a multidimensional treatment goal that not only alleviates symptoms but also improves the patient's overall quality of life.

[0118] Example To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of them. The following is merely a further description of the present disclosure, and the scope of protection of the present disclosure is not limited thereto.

[0119] The experimental conditions and methods involved in the following Examples 1 to 3 are as follows: 1. Experimental conditions and methods 1.1 Preparation of Compound 1-7 、 、 、 、 、 、 .

[0120] The preparation method of compound 1-7 is as follows: (1) The synthesis of compounds 1, 2, 3, 4, and 5 was carried out according to “Small-MoleculeTLR8 Antagonists via Structure-Based Rational Design, Z. Hu, H. Tanji, S.Jiang, S. Zhang, K. Koo, J. Chan, et al., Cell Chem Biol 2018 Vol. 25 Issue 10 Pages 1286-1291 e3”.

[0121] (2) Synthesis of compound 6 Step 1: Synthesis of intermediate 1, 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline To a 100 ml single-necked flask were added 2 g (10.75 mol, 1.0 eq) of 4-bromo-2-methylaniline, 3.28 g (12.9 mmol, 1.2 eq) of pinacol diboronate, 2.11 g (21.5 mmol, 2.0 eq) of potassium acetate, and 439 mg (0.54 mmol, 5% mmol) of PdCl2(dppf)CH2Cl2. Finally, 35 ml of 1,4-dioxane was added and the mixture was reacted at 95°C under nitrogen protection overnight. The reaction solution was spin-dried and dry-loaded with the sample, and column chromatography with PE:EA (5:1) was performed to obtain 2.45 g of intermediate 1 as a yellow oil in a yield of 97.5%.

[0122] Step 2: Synthesis of compound 6: To a 50 ml single-necked flask were added 300 mg (1.26 mmol, 1.0 eq) of 4-bromo-7-methoxyquinoline, 382 mg (1.64 mmol, 1.2 eq) of the intermediate 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, 4 ml of an aqueous solution of 267 mg (2.52 mmol, 2.0 eq), and 52 mg (0.06 mmol, 5% mmol) of PdCl2(dppf)CH2Cl2. Finally, 20 ml of 1,4-dioxane was added, and the mixture was reacted at 95°C under nitrogen overnight. The reaction solution was spin-dried, dry-loaded, and passed through a column chromatography column with PE:EA (1:1) to afford 214 mg of compound 6 as a yellow solid, in a yield of 64.3%.

[0123] 1H NMR (400 MHz, DMSO) of compound 6 δ 8.75 (d, J = 4.5 Hz, 1H), 7.92 (d,J = 9.3 Hz, 1H), 7.42 (d, J= 2.7 Hz, 1H), 7.25 – 7.16 (m, 2H), 7.16 – 7.07(m, 2H), 6.77 (d, J = 8.1 Hz, 1H), 5.22 (s, 2H), 3.93 (s, 3H), 2.15 (s, 3H).

[0124] (3) Synthesis of compound 7 Step 1: Synthesis of intermediate 2, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(trifluoromethyl)aniline: To a 100 ml single-necked flask were added 2 g (8.33 mol, 1.0 eq) of 4-bromo-2-(trifluoromethyl)aniline, 2.54 g (10 mmol, 1.2 eq) of pinacol diboron, 1.64 g (16.64 mmol, 2.0 eq) of potassium acetate, and 340 mg (0.42 mmol, 5% mmol) of PdCl2(dppf)CH2Cl2. Finally, 35 ml of 1,4-dioxane was added and the mixture was reacted at 95°C under nitrogen protection overnight. The reaction solution was spin-dried and dry-loaded with the sample, and column chromatography with PE:EA = 5:1 was performed to obtain 2.26 g of intermediate 2 as a yellow solid in a yield of 94.6%.

[0125] Step 2: Synthesis of compound 7: To a 50 ml single-necked flask were added 300 mg (1.26 mmol, 1.0 eq) of 4-bromo-7-methoxyquinoline, 434 mg (1.51 mmol, 1.2 eq) of the intermediate 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(trifluoromethyl)aniline, 4 ml of an aqueous solution of 267 mg (2.52 mmol, 2.0 eq) of sodium carbonate, and 52 mg (0.06 mmol, 5% mmol) of PdCl2(dppf)CH2Cl2. Finally, 20 ml of 1,4-dioxane was added. The mixture was reacted at 95°C under nitrogen protection overnight. The reaction solution was spin-dried and dry-loaded with the sample, and column chromatography using PE:EA (1:1) was performed to obtain 291 mg of compound 7 as a yellow solid, in a yield of 72.6%.

[0126] 1H NMR (400 MHz, DMSO) of compound 7 δ 8.80 (d, J = 4.5 Hz, 1H), 7.82 (d,J = 9.2 Hz, 1H), 7.53 – 7.43 (m, 3H), 7.26 (dd, J = 9.8, 3.4 Hz, 2H), 7.03(d, J = 8.4 Hz, 1H), 5.98 (s, 2H), 3.94 (s, 3H).

[0127] 1.2 Preparation of test solution Solvent: 42.5% PEG400 + 42.5% PG + 15% H2O Preparation method: Accurately weigh the test sample (Compound 1-Compound 7), calculate the volume of the solvent according to the corresponding dose concentration, accurately measure the solvent, add it to the test sample and stir until a clear solution or suspension is obtained, and finally ultrasonicate for half an hour.

[0128] 1.3 Preparation of positive drug solution Solvent: 42.5% PEG400 + 42.5% PG + 15% H2O Preparation method: Accurately weigh the positive drug, calculate the volume of the solvent according to the corresponding dosage concentration, accurately measure the solvent, add it to the positive drug and stir until a clear solution or suspension is obtained, and finally ultrasonicate for half an hour.

[0129] 1.4 Other reagents and drugs 1.5 Main instruments Example 1: Activity determination of the compounds of the present invention (1) Aspirate the original culture medium in the HEK-blue-hTLR8 cell culture dish, pipette with DMEM, and count the cells. Evenly plate the cells in a 96-well plate, with 50,000 cells per well.

[0130] (2) Add the activator R848 to a final concentration of 2 μg / mL. Add different concentrations of the test molecule.

[0131] (3) After culturing in the incubator for 24 hours, take 50 μL of supernatant and transfer it to a new 96-well plate. Then add 50 μL of Quanti-Blue (developer). After the negative control well turns blue, test its absorbance at 620 nm (using skanIt software to measure the wavelength).

[0132] (4) Add 5 μL / well of CCK-8 to the remaining cell-containing plates. After the untreated cell wells turn yellow to a certain level, test their absorbance at 450 nm.

[0133] (5) Data analysis: Data were organized using Excel and visualized using Graphpad Prism7.

[0134] IC of compounds 1-7 50 See the table below. It can be seen that compounds 1 to 7 provided in the present application have a strong inhibitory effect on TLR8.

[0135] Table 1. TLR8 inhibitory activity of compounds 1-7 (IC 50 value) Example 2: Effects of the compounds of the present invention in the HSF cell fibrosis model (1) Aspirate the original culture medium in the HSF cell culture dish, blow with culture medium, and count the cells. Evenly spread the cells in a 12-well plate, with 200,000 cells per well.

[0136] (2) TGF-β, a factor that promotes HSF cell fibrosis, was added to a final concentration of 10 ng / mL. The molecule to be tested was added to a final concentration of 10 μM.

[0137] (3) After incubation for 24 h, all cells were collected, washed with PBS, RNA was extracted, and reverse transcribed into cDNA.

[0138] (4) q-PCR detection of the relative expression levels of α-SMA, COL1A1, COL1A2, and COL3A1 mRNA; the relevant test results are shown in the appendix of the instructions. Figure 1-4 .

[0139] Instructions attached Figure 1-4 DMSO was the solvent control group, TGF-β was the group with only TGF-β added, TGF-β+1 was the group with TGF-β and compound 1 added, TGF-β+2 was the group with TGF-β and compound 2 added, TGF-β+3 was the group with TGF-β and compound 3 added, TGF-β+4 was the group with TGF-β and compound 4 added, TGF-β+5 was the group with TGF-β and compound 5 added, TGF-β+6 was the group with TGF-β and compound 6 added, and TGF-β+7 was the group with TGF-β and compound 7 added.

[0140] TGF-β can induce fibrosis of skin fibroblasts. In this process, a large amount of collagen is expressed. αSMA, COL1A1, COL1A2 and COL3A1 are key genes for collagen synthesis. TGF-β can promote the expression of these key genes. Figure 1-4 It can be seen that the compounds of the present invention can inhibit the increased expression of αSMA, COL1A1, COL1A2 and COL3A1 genes induced by TGF-β, and inhibit the fibrosis process of skin fibroblasts.

[0141] Example 3: Pharmacological efficacy of the compounds of the present invention in SSc mouse models Experimental methods and results: 3.1 Experimental animals and experimental groups Female humanized TLR8 transgenic C57BL / 6 mice (Beijing Biocytogen Pharmaceuticals Co., Ltd.), 9–10 weeks old, were housed in an environment maintained at a temperature of 16–28°C and a relative humidity of 40–70%.

[0142] Since the TOLL-like receptor inhibitory compounds disclosed herein are mainly directed against the human TLR8 target, and the TLR8 gene of ordinary mice is different from that of humans, humanized TLR8 transgenic mice are used.

[0143] A mouse scleroderma model was established by subcutaneous injection of BLM in female mice. On the day of grouping (D0), the animals were randomly divided into five groups based on body weight: a healthy control group, a BLM modeling control group, a penicillamine-treated group, a compound 1-treated group, and a compound 6-treated group. Each group received 100 mpk of BLM solution. The model was induced on the day following grouping (D1) with a subcutaneous injection of 500 μg / mL of BLM solution via the back. The injection volume was 100 μL per mouse, and the induction dose was 50 μg per mouse. The induction frequency was every other day for 6 consecutive weeks. Treatment with the test article and positive control drug began on the same day of modeling. Compound 1 and compound 6 were both administered orally at a dose of 100 mg / kg once daily for 6 consecutive weeks. Penicillamine was selected as the positive control drug in this study, with a dose of 200 mg / kg, administered orally by gavage, once a day for 6 consecutive weeks.

[0144] 3.2 Hydroxyproline (HYP) detection After 42 days of administration, the skin of the affected side of each experimental group of animals was collected at the end of the experiment, and the HYP content of the skin of the affected side of each experimental group of animals was detected using a tissue HYP kit. The following indicators were detected using an enzyme marker: HYP; the test results are shown in the appendix of the instructions. Figure 5 . According to the statistical analysis results of various pharmacodynamic test indicators, the therapeutic effects of the compound 1 treatment group and the compound 6 treatment group of the present invention on disease model animals under the set treatment regimen were evaluated. The data were analyzed using GraphPad Prism 9.0 software and presented as mean values ​​(± standard deviation). If the data conform to the normal distribution, the P value is calculated using the paired sample t-test; otherwise, the two-tailed Wilcoxon paired t-test is applied. For multiple group comparisons, ANOVA or Kruskal-Wallis test is used. The statistical significance level is set at P < 0.05. The HYP expression level of each group of mice is shown in the appendix of the instruction manual. Figure 5 .

[0145] HYP is a non-essential amino acid and a major component of collagen. HYP is found in the highest concentration in collagen, and measuring HYP in skin homogenates can reveal the extent of collagen breakdown in the body, serving as an indicator of connective tissue breakdown.

[0146] Depend on Figure 5As can be seen, after BLM modeling, the HYP content in the mouse skin increased significantly. Compared with the BLM group, the HYP content in the skin of the penicillamine group, the group treated with compound 1 of the present invention, and the group treated with compound 6 decreased significantly (p < 0.01), and the differences were significant. This indicates that the penicillamine group, the group treated with compound 1 of the present invention, and the group treated with compound 6 can reduce HYP expression, which is beneficial to reducing the degree of skin collagen fibrosis.

[0147] 3.3 Epidermis and dermis skin thickness measurement After 42 days of dosing, the skin of the affected side of each experimental group was collected at the end of the experiment. The back skin was cut and stored in 4% tissue fixative at room temperature. Then, hematoxylin and eosin staining was performed to observe changes in skin thickness. Based on the H&E staining results, the thickness of the dermis of each experimental group of animals was measured using pathological analysis software to analyze the progression of dermal lesions. Based on the statistical analysis results of various pharmacodynamic test indicators, the therapeutic effects of the compound 1 and compound 6 treatment groups on the disease model animals under the established treatment regimen were evaluated.

[0148] The changes in the thickness of the epidermis and dermis of the mice were measured by HE staining. The results of the epidermis thickness test of each group of mice are shown in the appendix of the instruction manual. Figure 6 The results of the dermal skin thickness test of each group of mice are shown in the appendix of the instruction manual. Figure 7 .

[0149] After BLM modeling, the thickness of both the epidermis and dermis of the mouse skin increased significantly. Compared to the BLM group, the dermis thickness in the penicillamine group decreased significantly, but there was no significant difference in epidermal thickness. Compared to the BLM group, the epidermal thickness in the Compound 1 and Compound 6 groups decreased significantly (p < 0.05), and the dermal thickness in the Compound 1 and Compound 6 groups decreased significantly (p < 0.05). Both epidermal and dermal thicknesses returned to levels close to those of the healthy control group. This suggests that Compound 1 and Compound 6 treatments significantly improved the skin lesions in systemic sclerosis mice, helping to restore skin damage caused by SSc to normal levels, maintain skin integrity, reduce the risk of infection, alleviate pain and discomfort, improve mobility, and contribute to the restoration of aesthetic appearance and psychological well-being.

[0150] Through the above examples, it was found that the treatment groups of Compound 1 and Compound 6 of the present invention had strong inhibitory activity on TLR8 activity, inhibited the expression of collagen in the skin fibroblast fibrosis model, and significantly improved the skin lesions of mice with systemic sclerosis. In summary, Compounds 1 to 7 of the present invention had strong inhibitory activity on TLR8 activity, inhibited the expression of collagen in the skin fibroblast fibrosis model, and significantly improved the skin lesions of mice with systemic sclerosis.

[0151] The foregoing descriptions of specific exemplary embodiments of the present disclosure are for purposes of illustration and description. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the present disclosure and various options and modifications. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. Use of a compound represented by formula (I) and / or a pharmaceutically acceptable salt, deuterated form thereof, solvate thereof, or precursor thereof in the preparation of a medicament for treating systemic sclerosis. （I） in, R1 is selected from H, -OH, -OC 1-6 Alkyl or C 1-6 alkyl; R2 is selected from -OH, -NH2, -OC 1-8 Alkylene -OH or -OC 1-8 Alkylene-NH2; R3 is selected from H, -C 1-6 Alkyl or -C 1-6 alkyl halide; R4 is selected from H, -C 1-6 Alkyl or -C 1-6 Halogenated alkyl.

2. The use according to claim 1, characterized in that R1 is selected from -OH, -OC 1-6 alkyl; R2 is selected from -OH, -NH2, -OC 1-8 Alkylene-OH; R3 is selected from H, -C 1-6 Alkyl or -C 1-6 alkyl halide; R4 is selected from H, -C 1-6 alkyl; Preferably, R1 is selected from H, -OH, -OC 1-5 Alkyl, C 1-5 alkyl; Preferably, R1 is selected from H, -OH, -OC 1-5 alkyl; Preferably, R1 is selected from H, -OH, -OC 1-3 alkyl; Preferably, R1 is selected from H, -OH, -OCH3, -OCH2CH3; Preferably, R1 is selected from -OH, -OC 1-5 alkyl; Preferably, R1 is selected from -OH, -OC 1-4 alkyl; Preferably, R1 is selected from -OH, -OC 1-3 alkyl; Preferably, R1 is selected from -OH, -OCH3, -OCH2CH3; Preferably, R1 is selected from -OH, -OCH3; Preferably, R2 is selected from -OH, -NH2, -OC 1-8 Alkylene-OH; Preferably, R2 is selected from -OH, -NH2, -OC 1-6 Alkylene-OH; Preferably, R2 is selected from -OH, -NH2, -OC 1-5 Alkylene-OH; Preferably, R2 is selected from -OH, -NH2, -OC 1-4 Alkylene-OH; Preferably, R2 is selected from -OH, -NH2, -OC 1-4 Alkylene-OH; Preferably, R2 is selected from -OH, -NH2, -O(CH2)4-OH; Preferably, R3 is selected from H, -C 1-5 Alkyl or -C 1-5 alkyl halide; Preferably, R3 is selected from H, -C 1-3 Alkyl or -C 1-3 alkyl halide; Preferably, R3 is selected from H, methyl, ethyl, -CF3, -CCl3, -CBr3, -CH2F, -CHF2, -CH2Cl, -CHCl2, -CH2Br, -CHBr2; Preferably, R3 is selected from H, methyl, ethyl, -CF3, -CH2F, -CHF2; Preferably, R3 is selected from H, methyl, -CF3; Preferably, R4 is selected from H, -C 1-5 alkyl; Preferably, R4 is selected from H, -C 1-3 alkyl; Preferably, R4 is selected from H, methyl, ethyl; Preferably, R4 is selected from H, methyl.

3. The use according to claim 1 or 2, characterized in that The pharmaceutically acceptable salt of the compound represented by formula (I) is selected from one or more inorganic acid salts or organic acid salts; Preferably, the inorganic acid salt is selected from one or more of hydrochloride, phosphate, hydrobromide, hydroiodide, hydrofluoride, nitrate, sulfate, and carbonate; Preferably, the organic acid salt is selected from one or more of malonate, succinate, formate, propionate, butyrate, and valerate.

4. The use according to claim 1, characterized in that The compound represented by formula (I) is selected from the following compounds: 、 、 、 、 、 、 ； Preferably, the compound represented by formula (I) is selected from the following compounds: 、 、 、 、 。 5. The use according to any one of claims 1 to 4, characterized in that The medicine is used for one or more purposes selected from the following: Reduce the level of connective tissue growth factor (CTGF) in plasma; Used to reduce the hydroxyproline (HYP) content in the skin and / or reduce the degree of skin collagen fibrosis; Used to reduce epidermal thickness and / or reduce dermal thickness.

6. The use according to any one of claims 1 to 4, characterized in that The drug is prepared into a pharmaceutical preparation for treating systemic sclerosis; the pharmaceutical preparation uses the compound represented by formula (I) and / or its pharmaceutically acceptable salt, deuterated product, solvate or precursor as the sole active ingredient.

7. The use according to any one of claims 1 to 4, characterized in that The drug is prepared into a pharmaceutical preparation for treating systemic sclerosis; the pharmaceutical preparation comprises the compound represented by formula (I) and / or its pharmaceutically acceptable salt, deuterated substance, solvate, or precursor, and further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipients include one or more of solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants, and preservatives.

8. The use according to claim 7, characterized in that The pharmaceutical preparation may further comprise one or more other drugs for treating systemic sclerosis.

9. The use according to any one of claims 6 to 8, characterized in that The pharmaceutical preparation is a solid preparation, a semisolid preparation or a liquid preparation; Preferably, the solid preparation is a tablet, capsule, granule or pill; the semisolid preparation is a gel, suppository or ointment; the liquid preparation is an emulsion, mixture, suspension or solution; Preferably, the medicine is in the form of tablets, capsules, granules, pills, injections, syrups, powders, chewable tablets, gels and suppositories; Preferably, the drug is administered by one or more of oral administration, sublingual administration, intravenous administration, transdermal administration, rectal administration, inhalation administration, intraperitoneal administration or intramuscular administration.

10. The use according to any one of claims 1 to 8, characterized in that The drug is prepared into a pharmaceutical preparation for treating systemic sclerosis, and the dosage calculated based on the compound represented by formula (I) in a unit pharmaceutical preparation is 10 to 800 mg, preferably 50 to 500 mg, and more preferably 100 to 400 mg.

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