Pharmaceutical compositions for the treatment of diseases involving fibrosis.

Abstract

To provide a compound that suppresses collagen production via a TGF-β signal, and is effective as a treatment drug for diseases that cause fibrillization of cells or tissue, and a medical composition containing the compound.SOLUTION: Provided are compounds represented by a general formula (1) [In formula (1), A1 and A2 are each independently a group represented by any one of general formulas (A-1) to (A-4) [In the formula, R11 is a C1-6 alkyl group or a C1-6 alkoxy group; n1 is an integer of 0 to 3; black circles are bonds}; X1 is an oxygen atom or -NH-; Z1 is a C1-6 alkylene group, a phenylene group, or a cycloalkylene group; R1 is a C1-6 alkoxy group or -N(R2)(R3); R2 and R3 are each independently a hydrogen atom or a C1-6 alkyl group].SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cells or tissues. line This relates to a pharmaceutical composition used in the treatment of diseases involving vascularization. [Background technology]

[0002] Epithelial-mesenchymal transition (EMT) is a transformation from an epithelial phenotype characterized by strong cell junctions to a mesenchymal phenotype characterized by weakened cell junctions and increased cell motility. Epithelial and mesenchymal cells differ not only in function but also in morphology. In epithelial cells, adjacent cells are adhered to each other by cell adhesion factors such as tight junctions, gap junctions, and adhesion junctions. They are also fixed at the base by the basement membrane. On the other hand, mesenchymal cells lack such polarity, have a spindle-shaped morphology, and interact with each other only at partial points. Epithelial cells express high levels of E-cadherin and cytokeratin, while mesenchymal cells express high levels of N-cadherin and vimentin; therefore, changes in the expression levels of these proteins are used as indicators of EMT.

[0003] EMTs are observed during the process of ontogeny, wound healing, tissue fibrosis, and the acquisition of metastatic properties in cancer cells. Furthermore, EMTs are involved in fibrotic diseases such as systemic scleroderma and idiopathic pulmonary fibrosis, as well as cancer metastasis.

[0004] Systemic sclerosis is an autoimmune disease in which collagen accumulates in the skin and tissues, causing hardening and fibrosis. Symptoms initially appear in the skin, including Raynaud's phenomenon (purple discoloration of the fingers), motor impairment due to contractures of the extremities, and peripheral necrosis due to ulceration, leading to a decline in the patient's quality of life. The rate of progression varies from patient to patient, but as it progresses throughout the body, various tissues such as the lungs, kidneys, and digestive organs become fibrotic, leading to serious complications that affect the prognosis, such as pulmonary fibrosis, scleroderma renal crisis, and reflux esophagitis, and in some cases, death. Systemic sclerosis is designated as an intractable disease, and there are currently around 20,000 patients in Japan. Current treatments mainly involve immunosuppressants, steroids, and herbal medicines that suppress inflammation, but these are all symptomatic treatments. In September 2021, rituximab, an antibody drug used as an anticancer drug and immunosuppressant, was approved as a curative drug for systemic sclerosis, but its effectiveness as a curative drug must be evaluated in the future. The biggest challenge with systemic sclerosis is that its pathogenesis remains unclear, which is hindering the development of an effective cure.

[0005] Systemic sclerosis is, line This disease is thought to be caused by the promotion of extracellular matrix production, such as collagen, in fibroblasts. This is a phenomenon closely related to EMT. Several signaling pathways are known to induce EMT. In fibrosis, EMT mainly involves TGF-β, etc. line Because it occurs by acting on fibroblasts, it is believed that increased TGF-β signaling leads to systemic sclerosis.

[0006] Compounds that inhibit EMT are expected to lead to curative treatments for fibrotic diseases. A common characteristic of fibrotic diseases is the increased accumulation of collagen mediated by the TGF-β / Smad signaling pathway. For example, compounds that have the activity to suppress collagen production mediated by the TGF-β / Smad signaling pathway are expected to lead to a cure for fibrotic diseases. line Maintenance diseaseIt is considered effective in preventing and treating [the condition] (Patent Document 1). In addition, NSAIDs such as oxaprozin (3-(4,5-diphenyloxazole-2-yl)propanoic acid) and diclofenac have been reported to have anti-fibrotic activity and suppress EMT (Patent Document 2). Furthermore, compound SB-431542, a structurally similar compound to oxaprozin, also has anti-fibrotic activity and can suppress EMT (Non-Patent Document 1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2020 / 158890 [Patent Document 2] International Publication No. 2015 / 182565 [Non-patent literature]

[0008] [Non-Patent Document 1] Hasegawa et al, Journal of Dermatological Science, 2005, vol.39, p.33-38. [Non-Patent Document 2] Pickett, Tetrahedron Letters, 2015, vol.56, p.3023-3026, Supplementary Data. [Non-Patent Document 3] Ammirati et al, ACS Medicinal Chemistry Letters, 2015, vol.6, p.1128-1133, Supporting Information. [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention suppresses collagen production via TGF-β signaling and inhibits the production of cells or tissues. lineA compound effective as a therapeutic agent for a disease in which fibrosis occurs, and a cell or tissue containing the compound as an active ingredient line An object of the present invention is to provide a pharmaceutical composition used for treating a disease in which fibrosis occurs.

Means for Solving the Problems

[0010] As a result of intensive studies to solve the above problems, the present inventors have found that novel compounds structurally similar to oxaprozin or SB-431542 have an action of suppressing collagen production via TGF-β signal, and have completed the present invention.

[0011] [1] The compound according to the first aspect of the present invention has the following general formula (1) [In formula (1), A 1 and A 2 are each independently a group represented by any of the following general formulas (A-1) to (A-4) {In formulas (A-1) to (A-4), R 11 is an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms; n11 is an integer of 0 to 3; the black circle is a bond} ; X 1 is an oxygen atom or -NH-; Z 1 is an alkylene group having 1 to 6 carbon atoms, a phenylene group, or a cycloalkylene group; R 1 is an alkoxy group having 1 to 6 carbon atoms or -N(R 2 )(R 3 ); the R 2 and R 3 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms] A compound represented by

[0012]

Chemical formula

[0013]

Chemical formula

[0014] [2] The compound in [1] is the A 1 and A 2 All of these are (A-1) above, and X 1 is an oxygen atom, and the Z 1 It is preferable that the group is a phenylene group. [3] The compound in [2] is R 1 However, it is preferable that the group be a methoxy group or an amino group. [4] A pharmaceutical composition according to a second aspect of the present invention comprises any of the compounds described in [1] to [3] above, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient. [5] The pharmaceutical composition in [4] above may include cells or tissue line It is preferable to use it in the treatment of diseases in which vascularization occurs. [6] The pharmaceutical composition of [4] or [5] is preferably administered orally. [Effects of the Invention]

[0015] The compound according to the present invention has the activity to suppress cellular fibrosis caused by collagen production. For this reason, a pharmaceutical composition containing the compound according to the present invention as an active ingredient can suppress the symptoms of diseases characterized by cellular fibrosis due to increased collagen production, and can suppress cells or tissues. line It is suitable for use in the treatment of diseases that involve vascularization. [Brief explanation of the drawing]

[0016] [Figure 1] The images show the cell morphology of A549 cells treated with oxaprozin, compound (AF-1), compound (AF-2), or compound (AF-3) in Example 1 after TGF-β stimulation. [Figure 2] This figure shows the results of measuring the expression level of type I collagen in adult dermal fibroblasts treated with oxaprozin or compound (AF-3) after TGF-β stimulation, as measured by Western blotting in Example 1. [Figure 3]This figure shows the results of measuring the expression level of type I collagen in adult dermal fibroblasts treated with oxaprozin or compound (AF-3) after TGF-β stimulation, using the direct adsorption method of ELISA in Example 1. [Modes for carrying out the invention]

[0017] The compound according to the present invention is a compound represented by the following general formula (1). This compound is structurally similar to oxaprozin and SB-431542, and like these compounds, it has the effect of suppressing collagen production and inhibiting cellular fibrosis.

[0018] [ka]

[0019] In general formula (1), A 1 and A 2 Each of these is an independent group represented by one of the following general formulas (A-1) to (A-4). 1 and A 2 These may be groups of the same species or groups of different species.

[0020] [ka]

[0021] In general formulas (A-1) to (A-4), R 11 C1-C6 alkyl groups (C 1-6 Alkyl group) or alkoxy group (C) having 1 to 6 carbon atoms 1-6 It is an alkoxy group. The black circles represent bonding sites.

[0022] R 11 C 1-6 If it is an alkyl group, then C 1-6The alkyl group may be linear or branched. Specifically, examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, n-hexyl group, and the like.

[0023] R 11 C 1-6 In the case of an alkoxy group, the C 1-6 The alkyl group portion of the alkoxy group may be linear or branched. Specifically, examples include methoxy group, ethoxy group, n-propyloxy group, isopropyloxy group, n-butyloxy group, isobutyloxy group, sec-butyloxy group, tert-butyloxy group, n-pentyloxy group, isopentyloxy group, neopentyloxy group, tert-pentyloxy group, and n-hexyloxy group.

[0024] In general formulas (A-1) to (A-4), n11 is an integer between 0 and 3. The compounds according to the present invention are preferably those in which n11 is 0.

[0025] The compound according to the present invention is one in which A in general formula (1) 1 and A 2 A compound in which at least one of the groups is represented by the general formula (A-1) is preferred, A 1 and A 2 Compounds in which both are groups represented by general formula (A-1) are more preferred, A 1 and A 2 Both are more preferably groups represented by the general formula (A-1) where n11 is 0 (phenyl group).

[0026] In general formula (1), Z 1 This is an alkylene group (C) having 1 to 6 carbon atoms. 1-6 It is an alkylene group, a phenylene group, or a cycloalkylene group. 1 C 1-6 If it is an alkylene group, then C 1-6The alkylene group may be linear or branched. Specifically, examples include methylene group, ethylene group, n-propylene group, isopropylene group, n-butylene group, isobutylene group, sec-butylene group, tert-butylene group, n-pentylene group, isopentylene group, neopentylene group, tert-pentylene group, n-hexylene group, etc. In the compound according to the present invention, Z in general formula (1) 1 C 1-6 Compounds with an alkylene group or a phenylene group are preferred, Z 1 Compounds in which the group is a phenylene group are more preferred.

[0027] In general formula (1), R 1 C 1-6 Alkoxy group or -N(R 2 )(R 3 ) is the above R 2 and R 3 Each of these is independently a hydrogen atom or C 1-6 It is an alkyl group. 1 C 1-6 In the case of an alkoxy group, the C 1-6 As for alkoxy groups, R 11 The same groups as those listed above can be cited. 2 and R 3 C 1-6 In the case of an alkyl group, the C 1-6 As an alkyl group, R 11 Similar to the bases listed above, these can be cited.

[0028] In general formula (1), X 1 is an oxygen atom or -NH-. In general formula (1), X 1 Compounds in which is an oxygen atom include those represented by the following general formulas (2) and (3), where in general formula (1), X 1 Compounds in which is -NH- include those represented by the following general formulas (4) and (5). In the following general formulas (2) to (5), A 1 , A 2 , Z 1 , R 2 , R 3 , and R 4is the same as the general formula (1). As the compound according to the present invention, a compound represented by the following general formula (2) or (3) is preferable.

[0029]

Chemical formula

[0030] As the compound according to the present invention, in the general formula (2), at least one of A 1 and A 2 is a group represented by the general formula (A-1), and Z 1 is a C 1-6 alkylene group or a phenylene group, or in the general formula (3), at least one of A 1 and A 2 is a group represented by the general formula (A-1), and Z 1 is a C 1-6 alkylene group or a phenylene group is preferable; in the general formula (2), both of A 1 and A 2 are groups represented by the general formula (A-1), and Z 1 is a methylene group, an ethylene group, an n-propylene group, an n-butylene group, or a phenylene group, or in the general formula (3), both of A 1 and A 2 are groups represented by the general formula (A-1), and Z 1 is a methylene group, an ethylene group, an n-propylene group, an n-butylene group, or a phenylene group is more preferable; in the general formula (2), both of A 1 and A 2 are phenyl groups, Z 1 is an ethylene group or a phenylene group, and R 2 is a methyl group, an ethyl group, an n-butyl group, or in the general formula (3), both of A 1 and A 2 are phenyl groups, Z 1 is an ethylene group or a phenylene group, and R 3 and R 4 are each independently a hydrogen atom or a methyl group is even more preferable; in the general formula (2), A 1 and A 2Both are phenyl groups, and Z 1 is an ethylene group or a phenylene group, and R 2 is a methyl group, or in general formula (3), A 1 and A 2 Both are phenyl groups, and Z 1 is an ethylene group or a phenylene group, and R 3 and R 4 are both hydrogen atoms, and the compound is more preferably; in general formula (2), A 1 and A 2 Both are phenyl groups, and Z 1 is a phenylene group, and R 2 is a methyl group, or in general formula (3), A 1 and A 2 Both are phenyl groups, and Z 1 is a phenylene group, and R 3 and R 4 are both hydrogen atoms, and the compound is particularly preferred.

[0031] The synthesis method of the compound represented by general formula (1) is not particularly limited. For example, by alkyl esterifying the carboxy group of oxaprozin, a compound in which R 1 is a C 1-6 alkoxy group in general formula (1) can be synthesized. Similarly, by aminating the carboxy group of oxaprozin, a compound in which R 1 is an amino group in general formula (1) can be synthesized.

[0032] For example, a compound in which R 1 is a C 1-6 alkoxy group in general formula (1) can be synthesized by the following method. First, the following compound (C1) and compound (C2) are condensed. In general formulas (C1) and (C2), A 1 , A 2 , X 1 , Z 1 are the same as in general formula (1). R 5 is C 1-6It is an alkyl group. Subsequently, the resulting condensate is cyclized in the presence of ammonium acetate to form an alkyl ester having an oxazole ring or an imidazole ring (in general formula (1), R 1 C 1-6 A compound containing an alkoxy group is synthesized. By amination of the resulting alkyl ester, in general formula (1), R 1 This allows for the synthesis of compounds in which the amino group is located.

[0033] [ka]

[0034] The compound represented by general formula (1) has the effect of suppressing collagen production. For this reason, the compound represented by general formula (1) is suitable as an active ingredient in pharmaceutical compositions used for the treatment of diseases characterized by fibrosis of cells or tissues due to excessive collagen deposition. The disease in question is characterized by fibrosis of cells or tissues due to excessive collagen deposition. line Diseases involving fibrosis are not particularly limited, and examples include fibrosis of various organs, such as pulmonary fibrosis, hepatic fibrosis, renal fibrosis, gastrointestinal fibrosis, and cutaneous fibrosis; inflammatory bowel diseases, including Crohn's disease and ulcerative colitis; and systemic scleroderma. line Inhibition of fibrosis also leads to inhibition of EMT. For this reason, pharmaceutical compositions containing the compound represented by general formula (1) as an active ingredient can be used for the treatment of diseases in which symptom improvement can be expected by inhibiting EMT, such as cancer.

[0035] The active ingredient of the pharmaceutical composition according to the present invention may be a pharmacologically acceptable salt of the compound represented by general formula (1). The pharmacologically acceptable salt of the compound represented by general formula (1) may be an inorganic salt or an organic salt. Examples of inorganic salts include alkali salts such as sodium salts and potassium salts, alkali metal salts such as calcium salts and magnesium salts, and aluminum salts. Examples of organic salts include Tris salts, methylamine salts, ethylamine salts, ethanolamine salts, and ammonium salts.

[0036] The active ingredient of the pharmaceutical composition according to the present invention may be a solvate of a compound represented by general formula (1), or a solvate of a pharmacokinetically acceptable salt of a compound represented by general formula (1). Examples of solvents that form the solvate include water and ethanol.

[0037] In this specification, “pharmacologically acceptable” is used to describe any compound, material, composition, and / or dosage form that is free from excessive toxicity, irritation, allergic reactions, or other problems or complications, is in proportion to a reasonable benefit-to-risk ratio, and is within the range of correct medical judgment to be suitable for use in contact with human and animal tissues.

[0038] The pharmaceutical compositions according to the present invention may consist solely of the compound represented by general formula (1), or they may contain other components. The pharmaceutical compositions according to the present invention can be formulated into various dosage forms by conventional formulation methods by combining the compound represented by general formula (1) with a pharmacologically acceptable carrier. A "pharmacologically acceptable carrier" means a component that is compatible with other components of the formulation and is not harmful to the subject. Such carriers include any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic, absorption retarder, salt, preservative, drug, drug stabilizer, gel, binder, additive, disintegrant, lubricant, sweetener, flavoring, dye, and / or materials and combinations thereof, as known to those skilled in the art. Formulating pharmaceutical compositions containing the compound represented by general formula (1) based on the disclosures of this specification is within the scope of the art. For example, it is possible to formulate pharmaceutical compositions containing the compound represented by general formula (1) according to standard techniques in the field of pharmaceuticals. For example, see Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Ed., (1990), Mack Publishing Co., Easton, Pa.

[0039] The pharmaceutical composition according to the present invention may optionally further contain one or more other active ingredients. The "other active ingredients" may have an inhibitory effect on collagen production or an anti- line Components having fibrosis-forming and anti-inflammatory effects are preferred. The pharmaceutical composition according to the present invention may also contain components having other active properties.

[0040] The route of administration of the pharmaceutical composition according to the present invention is not particularly limited. For example, it may be prescribed as tablets, capsules, granules, lozenges, drinks, etc. and administered orally. Alternatively, parenteral administration such as transdermal administration (e.g., patches), intraperitoneal administration, intravenous administration by intravenous drip or injection may be used. In addition, administration by intramuscular injection, enteral administration, local administration, etc. is also possible. In one embodiment, the pharmaceutical composition according to the present invention is administered orally, transdermally, intraperitoneally, or intravenously. Specific examples of formulations (formulations) of the pharmaceutical composition according to the present invention are shown below.

[0041] For example, tablets, powders, granules, lozenges, capsules, etc. for oral administration can be manufactured by adding one or more solid inert components, such as excipients, disintegrants, binders, or lubricants, to a pharmaceutical composition containing a compound represented by general formula (1), compressing and molding the mixture, and then, if necessary, applying a coating for taste masking, enteric coating, or sustained release.

[0042] Injectable preparations can be manufactured, for example, by dissolving, suspending, or emulsifying a pharmaceutical composition containing a compound represented by general formula (1) as an aqueous injectable preparation together with, for example, a dispersant, a preservative, an isotonic agent, etc., or by dissolving, suspending, or emulsifying it in a vegetable oil such as olive oil, sesame oil, cottonseed oil, or corn oil, or propylene glycol, and then molding it as an oily injectable preparation.

[0043] Topical preparations are manufactured, for example, by preparing a pharmaceutical composition containing a compound represented by general formula (1) as a solid, semi-solid, or liquid composition. For example, the solid composition is manufactured by preparing the pharmaceutical composition as is, or by adding or mixing excipients, thickeners, etc., to make it a powder. The liquid composition is manufactured in much the same way as in the case of injectable preparations, by preparing an oily or aqueous suspension. Semi-solid compositions are preferably aqueous or oily gels or ointments. These compositions may also contain buffers, preservatives, etc. Topical preparations include, for example, creams, lotions, gels, ointments, etc., for local administration.

[0044] Suppositories are manufactured by forming a pharmaceutical composition containing, for example, a compound represented by general formula (1) into an oily or aqueous solid, semi-solid, or liquid composition. Examples of oily bases used in such compositions include glycerides of higher fatty acids (e.g., cocoa butter, whitepsols, etc.), intermediate fatty acids (e.g., migliols, etc.), or vegetable oils (e.g., sesame oil, soybean oil, cottonseed oil, etc.). Examples of aqueous gel bases include natural gums, cellulose derivatives, vinyl polymers, acrylic acid polymers, etc.

[0045] The dosage and duration of administration of the pharmaceutical compositions according to the present invention are determined by the specific circumstances of each individual patient, including the size, mass, age, and sex of the recipient, the nature and stage of the disease being treated, the aggressiveness of the disease, the route of administration, and the specific toxicity of radiation. The dosage and duration of administration may also be determined experimentally using known test protocols or by extrapolation from in vivo or in vitro test data. The concentration ranges described herein are for illustrative purposes only and do not limit the scope or implementation of the claimed compositions.

[0046] For example, the dosage in a pharmaceutical composition containing the compound represented by general formula (1) is 0.01 to 2000 mg / kg / day, more preferably 0.05 to 1000 mg / kg / day, and even more preferably 1.0 to 500 mg / kg / day, in terms of the amount of the compound represented by general formula (1) that is the active ingredient. The pharmaceutical composition according to the present invention may be administered once a day, or it may be divided into several low doses administered simultaneously or at time intervals.

[0047] The animals to which the pharmaceutical composition according to the present invention is administered are not particularly limited and may be humans or other animals. Examples of non-human animals include mammals such as cattle, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs. [Examples]

[0048] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0049] <chromatography> In subsequent experiments, thin-layer chromatography was performed using silica gel ("Silica gel 60 F254," manufactured by Merck). For column chromatography, silica gel ("Silica gel 60N (spherical, neutral)," manufactured by Kanto Chemical Co., Ltd.) was used.

[0050] <nmr> In subsequent experiments, NMR was performed as follows. first, 1 H-NMR and 13 For 13C-NMR spectroscopy, an NMR spectrometer ("BRUKER AVANCE 600 (600MHz)", Bruker BioSpin) was used. Chemical shifts were expressed as δ values ​​(ppm) with tetramethylsilane (TMS) as the internal standard, and J values ​​were expressed in Hz. Mass spectrometry (FAB) and high-resolution mass spectrometry (HRMS) were measured using a mass spectrometer ("JMS-DX303HF MASS spectrometer", JOEL). Infrared absorption spectra (IR) were measured using an IR spectrometer ("Frontier", perkin Elmer). Elemental analysis was performed using an organic elemental analyzer ("MICRO CORDER JM10", J-Science Group).

[0051] [Example 1] The inventors focused on the structural characteristics of oxaprozin and SB-431542, and synthesized novel compounds (AF-1, AF-2, AF-3) by combining the 4,5-diphenyloxazole moiety of oxaprozin with the carbamoylphenyl moiety of SB-431542, and investigated their activity.

[0052] <Synthesis of Compounds> Following the method of Pickett et al. (Non-Patent Literature 2), compound (C3-1) was obtained in 62% yield by condensing benzoin (compound (C1-1)) and methyl-4-(chloroformyl)benzoate (compound (C2-1)). Compound (C3-1) was mixed with ammonium acetate and refluxed under anhydrous conditions to obtain the target compound (AF-1) having an oxazole ring in 32% yield. Furthermore, following the method of Ammirati et al. (Non-Patent Literature 3), the ester portion of compound (AF-1) was hydrolyzed to synthesize the target compound (AF-2) in 92% yield. The carboxyl group of compound (AF-2) was amidated to obtain the target compound (AF-3) in 24% yield. Details are described below.

[0053] [ka]

[0054] (1) Synthesis of compound (C3-1) (Methyl (2-oxo-1,2-diphenylethyl) terephthalate) Benzoin (compound (C1-1)) (1.06 g, 5 mmol) was dissolved in diethyl ether (125 mL) under anhydrous conditions. Methyl-4-(chloroformyl)benzoate (compound (C2-1)) (1.09 g, 5.5 mmol) and TEA (0.836 mL, 6 mmol) were added, and the mixture was reacted under reflux under anhydrous conditions for 12 hours. After the reaction was complete, the mixture was allowed to return to room temperature and then filtered by suction. The resulting filtrate was sequentially extracted with purified water, 5% hydrochloric acid, 5% sodium hydroxide, purified water, and saturated brine. Subsequently, sodium sulfate was added to the resulting organic layer and dried, and the diethyl ether was removed under reduced pressure. The mixture was then purified by silica gel column chromatography (CH2Cl2) to obtain compound (C3-1) (1.155 g, yield 62%).

[0055] MS (FAB) (M+H) + m / z 375.3. 1 H NMR (600MHz, CDCl3) δ; 3.93(s,3H), 7.10(s,1H), 7.37-7.43(m, 5H), 7.51-7.54(m, 1H), 7.56-7.58(m, 2H), 7.98-8.00(m, 2H), 8.09-8.11(m, 2H), 8.17-8.19(m, 2H) 13 C NMR (150MHz, CDCl3) δ; 52.44, 78.38, 128.73, 128.79, 178.87, 129.25, 129.51, 129.60, 129.97, 130.14, 133.25, 133.52, 133.61, 133.78, 134.31, 134.64, 165.27, 166.25, 193.36.

[0056] (2) Synthesis of compound (AF-1: Methyl 4-(4,5-diphenyloxazol-2-yl)benzoate) In a flask, compound (C3-1) (1.07 g, 2.86 mmol) was dissolved in glacial acetic acid (25 mL), then ammonium acetate (275 mL, 3.57 mmol) was mixed in, and the mixture was refluxed at 155°C under anhydrous conditions for 2 hours, followed by stirring at room temperature for 12 hours to allow the reaction to proceed. After the reaction was complete, purified water was added to remove the solvent. Ethanol was added to the viscous substance remaining in the flask, and the mixture was filtered by suction. Recrystallization was performed to obtain the target compound (AF-1) (330 mg, yield 32%).

[0057] MS (FAB)(M+H) + m / z 356.3. 1 H NMR (600MHz, CDCl3)δ: 3.95(s, 3H), 7.35-7.44(m, 6H), 7.68-7.69(m, 2H), 7.71-7.73(m, 2H), 8.14-8.16(m, 2H), 8.21-8.23(m, 2H). 13 C NMR (150MHz,CDCl3)δ: 52.30, 52.44, 78.37, 126.28, 126.71, 128.14, 128.43, 128.69, 128.71, 128.73, 128.77, 128.87, 128.89, 129.25, 129.51, 129.59, 129.59, 129.97, 130.08, 131,19, 131.46, 132.30, 133.60, 137.29, 146.38, 159.15, 166.52.

[0058] (3) Synthesis of compound (AF-2:4-(4,5-Diphenyloxazol-2-yl)benzoic acid) Compound (AF-1) (190 mg, 0.534 mmol) was mixed with acetone (10 mL) and then 1 M LiOH (3 mL). The mixture was stirred at 80 °C for 3 hours and then at room temperature for 18 hours. After the reaction was complete, 1 M HCl was added to adjust the pH to 2. The mixture was filtered by suction to obtain the target compound (AF-2) (170 mg, 92% yield).

[0059] MS (FAB) (M+H) + m / z 342.3. 1 H NMR (600MHz, CDCl3)δ: 7.43-7.51(m, 6H), 7.67-7.69(m,4H), 8.12(d, J = 8.3Hz, 2H), 8.22(d, J=8.3Hz, 2H), 13.26(s,1H). Anal. Calcd for C22H15NO3 1 / 2H2O: C, 75.42; H, 4.60; N, 4.00. Found: C, 75.69; H, 4.58; N, 4.04. IR (ATR) 2851, 1689 cm⁻¹.

[0060] (4) Synthesis of compound (AF-3:4-(4,5-Diphenyloxazol-2-yl)benzamide) Compound (AF-2) (120 mg, 0.351 mmol) and thionyl chloride (2 mL) were mixed and refluxed at 96°C for 3 hours. The resulting reaction product was allowed to return to room temperature and then azeotropically reacted with thionyl chloride. Next, it was dissolved in 1,4-dioxane, cooled to 0°C, and 33% ammonium hydroxide (0.4 mL) was added. The mixture was stirred at room temperature for 30 minutes. The precipitated crystals were filtered by suction to obtain the target compound (AF-3) (29 mg, yield 24%).

[0061] MS (FAB) (M+H) + m / z 341.1. 1 H NMR (600MHz, CDCl3)δ: 5.79-6.17(d, 2H), 7.37-7.43(m,6H), 7.68-7.70(m, 2H), 7.7.2-7.74(m, 2H), 7.93(d, J=8.4Hz, 2H), 8.24(d, J=8.4Hz, 2H). 13 C NMR (150MHz, CDCl3) δ: 128.05, 128.44, 129.18, 129.40, 129.47, 130.51, 132.28, 133.94, 134.55, 137.22, 146.34, 159.07, 168.47. Anal. Calcd for C22H16N2O2 3 / 2H2O: C,71.92; H, 5.21; N, 7.62. Found: C, 71.21; H, 5.21; N, 7.70. IR(ATR) 3356, 3182, 1667 cm-1.

[0062] <Evaluation of EMT inhibitory activity based on cell morphology> The EMT inhibitory activity of oxaprozin, compound (AF-1), compound (AF-2), and compound (AF-3) was evaluated by observing changes in cell morphology. Specifically, 0.5 × 10⁶ A549 cells (human alveolar basal epithelial adenocarcinoma cell line) were placed in a 6-well plate. 5 Cells were seeded in 1 mL of culture medium (1 mL) in each well and incubated overnight. Subsequently, a solution of the test compound (oxaprozin, compound (AF-1), compound (AF-2), or compound (AF-3)) dissolved in 1% DMSO solution was added to the wells seeded with A549 cells to a final concentration of 10 μM. After incubation for 1 hour, TGF-β (10 ng / mL) was added and the cells were incubated for 3 days. As a control, an equal volume of 1% DMSO solution was similarly added to the wells seeded with A549 cells and incubated, followed by the addition of TGF-β and subsequent incubation. The morphology of the cells after 3 days of incubation was observed using a fluorescence microscope ("BZ-9000", KEYENCE).

[0063] Figure 1 shows microscopic images of each cell. The results showed that in the presence of TGF-β, A549 cells changed to a pointed, spindle-shaped cell morphology characteristic of mesenchymal cells. Compound (AF-1) partially suppressed this morphological change, while compound (AF-3) suppressed it entirely. Since both compounds (AF-1) and (AF-3) suppressed the morphological changes observed during EMT progression, it was revealed that they possess EMT inhibitory activity.

[0064] <Anti-collagen expression levels based on collagen expression levels> line Evaluation of vascular activity - Western blot method > Adult skin treated with oxaprozin or compound (AF-3) and then stimulated with TGF-β. line The expression level of type I collagen in fibroblasts determines the resistance of compound (AF-3). line Fibrosis activity was evaluated. Type I collagen expression levels were examined by Western blotting. First, adult skin cells were placed in a 24-well plate. line 1.0 × 10⁶ fibroblast cells 5 Cells / mL (1 mL of culture medium) were seeded in 1 mL of each well and cultured overnight. Subsequently, adult skin cells were cultured. line In wells seeded with fibroblasts, a solution of the test compound (oxaprozin or compound (AF-3)) dissolved in 1% DMSO solution was added to achieve a final concentration of 10 μM or 25 μM of the test compound. After incubation for 1 hour, TGF-β (10 ng / mL) was added and the cells were cultured for 2 days. As a control, an equal volume of 1% DMSO solution was used in the same manner on adult skin cells. line After adding fibroblast cells to seeded wells and incubating them, TGF-β was added and the cells were cultured.

[0065] After culturing, cells were disrupted with RIPA buffer, and the lysate was boiled at 100°C for 10 minutes to lyse the cells. The resulting cell lysate was subjected to electrophoresis, transferred to a membrane (Millipore), and reacted with goat anti-type I collagen antibody (Southern Biotech) as the primary antibody, followed by Western blotting. Detection was performed using a chemiluminescent imager ("ImmunoStar LD," Fujifilm Wako Pure Chemical Industries). The expression level of type I collagen was corrected using the expression level of β-actin as an internal standard.

[0066] Figure 2 shows the results of measuring the expression level of type I collagen. Figure 2(A) is the result of the Western blot, and Figure 2(B) is the relative expression level of type I collagen in each cell, determined from the Western blot. Adult skin line When fibroblasts are stimulated with TGF-β, type I collagen increases. line Fibrosis cells were activated. Adding compound (AF-3) to these activated cells suppressed the increase of type I collagen in a dose-dependent manner. Since compound (AF-3) suppressed collagen production, it was found to have anti-fibrotic activity. Although the type I collagen inhibitory effect of oxaprozin was not observed in this experiment, this was likely due to the low amount of oxaprozin added. It is presumed that anti-fibrotic activity could be confirmed with higher concentrations of oxaprozin.

[0067] <Anti-collagen expression levels based on collagen expression levels> line Evaluation of vascular activity - ELISA method Extracellular type I collagen was measured using the direct adsorption method of ELISA instead of Western blotting. First, adult skin cells were placed in a 24-well plate. line 1.0 × 10⁶ fibroblast cells 5 Cells / mL (1 mL of culture medium) were seeded in 1 mL of each well and cultured overnight. Subsequently, adult skin cells were cultured. line In wells seeded with fibroblasts, a solution of the test compound (oxaprozin or compound (AF-3)) dissolved in 1% DMSO solution was added to achieve a final concentration of 10 μM of the test compound. After incubation for 1 hour, TGF-β (10 ng / mL) was added and the cells were cultured for 1 day. As a control (1), an equal volume of 1% DMSO solution was used in the same manner to culture adult skin cells. line After adding the solution to wells containing seeded fibroblasts and incubating, the cells were cultured without the addition of TGF-β. As a control (part 2), an equal volume of 1% DMSO solution was used in the same manner on adult skin cells. line After adding fibroblast cells to seeded wells and incubating them, TGF-β was added and the cells were cultured.

[0068] After culturing, cells were fixed by incubation in 3% paraformaldehyde solution (Tokyo Chemical Industries, Ltd.) for 20 minutes. After stopping the fixation reaction by adding 1% glycine solution (Merck), the cells were blocked for 15 minutes with PBS solution of 5% goat serum (Fujifilm Wako Pure Chemical Industries, Ltd.). After blocking, PBS solution of goat anti-type I collagen antibody (MBL Corporation) containing 1% BSA (bovine serum albumin) was added to the cells and reacted as a primary antibody for 1 hour. Subsequently, HRP-labeled anti-mouse IgG antibody was reacted as a secondary antibody for 30 minutes. Detection was performed using OPD (o-phenylenediamine dihydrochloride) reagent (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0069] Figure 3 shows the results of measuring the expression level of type I collagen (relative value with the amount of type I collagen in control cells (part 1) set to 1). Similar to the results of the Western blot, it was confirmed that the amount of collagen was reduced in cells treated with compound (AF-3). These results indicate that compound (AF-3) has a collagen production inhibitory effect, and consequently, an anti-fibrotic effect.< / nmr>

Claims

1. The following general formula (1) 【Chemistry 1】 [In formula (1), A １ and A ２ Both are phenyl groups; X １ is an oxygen atom or -NH-; Z １ R is a phenylene group; １ [This is a methoxy group or an amino group.] A pharmaceutical composition comprising a compound represented by [formula], a pharmacokinetically acceptable salt thereof, or a solvate thereof as an active ingredient, used for the treatment of diseases in which fibrosis of cells or tissues occurs.

2. The aforementioned X １ The pharmaceutical composition according to claim 1, wherein is an oxygen atom.

3. A pharmaceutical composition according to claim 1 or 2, which is administered orally.

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