JP2023142282A - Medical composition for treating disease that generate filamentation - Google Patents

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 The present invention relates to a pharmaceutical composition used for the treatment of diseases in which cell or tissue fibrosis occurs.

Epithelial mesenchymal transition (EMT) is a transition from an epithelial phenotype in which cells have strong junctions to a mesenchymal phenotype in which cell-to-cell junctions are weakened and motility is increased. Epithelial cells and mesenchymal cells differ in morphology as well as function. In epithelial cells, adjacent cells adhere to each other by cell adhesion factors such as tight junctions, gap junctions, and adherens junctions. Also, at the bottom, it is fixed by a basement membrane. On the other hand, mesenchymal cells do not have such polarity, but instead take a spindle-like form and interact with each other only at partial points. Epithelial cells express high amounts of E-cadherin, cytokeratin, etc., and mesenchymal cells express high amounts of N-cadherin, vimentin, etc., so changes in the expression levels of these proteins are considered to be indicators of EMT. be done.

EMT is observed during the ontogenetic process, wound healing, tissue fibrosis, and metastatic acquisition of cancer cells. EMT is also involved in fibrotic diseases such as systemic sclerosis and idiopathic pulmonary fibrosis, and cancer metastasis.

Systemic scleroderma is an autoimmune disease in which collagen accumulates in the skin and tissues, causing them to harden and become fibrotic. Initially, symptoms begin to appear on the skin, including Raynaud's syndrome, in which the hands and fingers turn purple, movement disorders due to contracture of the extremities of the hands and feet, and peripheral necrosis due to ulceration, leading to a decline in the patient's QOL. The rate of progression varies from patient to patient, but when it progresses to the whole body, various tissues such as the lungs, kidneys, and digestive organs become fibrotic, leading to life-threatening conditions such as pulmonary fibrosis, scleroderma renal crisis, and reflux esophagitis. It can lead to serious illness and even death. Systemic scleroderma is designated as an incurable disease, and there are currently around 20,000 patients in Japan. Current treatments mainly involve drugs that suppress inflammation, such as immunosuppressants, steroids, and Chinese herbal medicine, but all of these are symptomatic treatments. In September 2021, the antibody drug rituximab, which is used as an anticancer drug and immunosuppressant, was approved as a radical cure for systemic scleroderma, but its evaluation as a radical cure will have to wait until the future. No. The biggest challenge with systemic scleroderma is that the pathogenic mechanism is still not clear, and this is a hindrance to the development of effective curative drugs.

Systemic scleroderma is a disease thought to be caused by accelerated production of extracellular matrices such as collagen in fibroblasts. This is a phenomenon closely related to EMT. Several signaling pathways are known to induce EMT. EMT in fibrosis is mainly caused by the action of TGF-β and the like on fibroblasts, and therefore, enhancement of TGF-β signals is said to lead to systemic scleroderma.

Compounds that inhibit EMT are expected to lead to radical treatments for fibrotic diseases. It is common to fibrotic diseases that collagen accumulation is accelerated due to TGF-β/Smad signals. For example, compounds having the activity of suppressing collagen production via TGF-β/Smad signaling are said to be effective in preventing and treating fibrotic lesions (Patent Document 1). Additionally, NSAIDs such as oxaprozin (3-(4,5-diphenyloxazol-2-yl)propanoic acid) and diclofenac have anti-fibrotic activity and have been reported to suppress EMT (Patent Document 2). Furthermore, compound SB-431542, which is a structurally similar compound to oxaprozin and the like, also has antifibrotic activity and can suppress EMT (Non-Patent Document 1).

International Publication No. 2020/158890 International Publication No. 2015/182565

Hasegawa et al, Journal of Dermatological Science, 2005, vol.39, p.33-38. Pickett, Tetrahedron Letters, 2015, vol.56, p.3023-3026, Supplementary Data. Ammirati et al, ACS Medicinal Chemistry Letters, 2015, vol.6, p.1128-1133, Supporting Information.

The present invention provides a compound that suppresses collagen production through TGF-β signals and is effective as a therapeutic agent for diseases that cause cell or tissue fibrosis, and a compound containing the compound as an active ingredient that causes cell or tissue fibrosis. The object of the present invention is to provide a pharmaceutical composition for use in treating diseases.

As a result of intensive research aimed at solving the above problems, the present inventors discovered that a new compound with a structure similar to that of oxaprozin and SB-431542 has the effect of suppressing collagen production via TGF-β signals. completed the invention.

[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 each independently represent 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 circles are bonds}
X ¹ is an oxygen atom or -NH-; Z ¹ is an alkylene group, phenylene group, or cycloalkylene group having 1 to 6 carbon atoms; R ¹ is , an alkoxy group having 1 to 6 carbon atoms, or -N(R ² )(R ³ ); 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

[2] In the compound of [1] above, it is preferable that A ¹ and A ² are both (A-1), X ¹ is an oxygen atom, and Z ¹ is a phenylene group.
[3] In the compound of [2] above, R ¹ is preferably a methoxy group or an amino group.
[4] The pharmaceutical composition according to the second aspect of the present invention contains the compound of any one of [1] to [3] above, or a pharmacologically acceptable salt thereof, or a solvate thereof. Use as an active ingredient.
[5] The pharmaceutical composition of [4] above is preferably used for the treatment of diseases in which cell or tissue fibrosis occurs.
[6] The pharmaceutical composition of [4] or [5] is preferably administered orally.

The compound according to the present invention has the activity of suppressing cell fibrosis caused by collagen production. Therefore, the pharmaceutical composition containing the compound according to the present invention as an active ingredient can suppress the symptoms of diseases characterized by cell fibrosis due to increased collagen production, which causes cell or tissue fibrosis. Suitable for use in treating diseases.

This is a microscopic image of the cell morphology after TGF-β stimulation of A549 cells treated with oxaprozin, compound (AF-1), compound (AF-2), or compound (AF-3) in Example 1. FIG. 2 is a diagram showing the results of measuring the expression level of type I collagen by Western blotting after TGF-β stimulation of adult skin fibroblasts treated with oxaprozin or compound (AF-3) in Example 1. . A diagram showing the results of measuring the expression level of type I collagen by the direct adsorption method of ELISA after TGF-β stimulation of adult skin fibroblasts treated with oxaprozin or compound (AF-3) in Example 1. It is.

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 cell fibrosis.

In the general formula (1), A ¹ and A ² are each independently a group represented by any one of the following general formulas (A-1) to (A-4). A ¹ and A ² may be the same group or different groups.

In general formulas (A-1) to (A-4), R ¹¹ is an alkyl group having 1 to 6 carbon atoms (C _1-6 alkyl group) or an alkoxy group having 1 to 6 carbon atoms (C _1-6 alkoxy basis). Also, the black circles are bonds.

When R ¹¹ is a C _1-6 alkyl group, the C _1-6 alkyl group may be linear or branched. Specifically, 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- Examples include pentyl group and n-hexyl group.

When R ¹¹ is a C _1-6 alkoxy group, the alkyl group portion of the C _1-6 alkoxy group may be linear or branched. Specifically, 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, n-hexyloxy group and the like.

In general formulas (A-1) to (A-4), n11 is an integer of 0 to 3. As the compound according to the present invention, a compound in which n11 is 0 is preferable.

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

In the general formula (1), Z ¹ is an alkylene group having 1 to 6 carbon atoms (C _1-6 alkylene group), a phenylene group, or a cycloalkylene group. When Z ¹ is a C _1-6 alkylene group, the C _1-6 alkylene group may be linear or branched. Specifically, 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-butylene group, -pentylene group, n-hexylene group, etc. As the compound according to the present invention, in the general formula (1), a compound in which Z ¹ is a C _1-6 alkylene group or a phenylene group is preferable, and a compound in which Z ¹ is a phenylene group is more preferable.

In general formula (1), R ¹ is a C _1-6 alkoxy group or -N(R ² )(R ³ ). The R ² and R ³ each independently represent a hydrogen atom or a C _1-6 alkyl group. When R ¹ is a C _1-6 alkoxy group, examples of the C _1-6 alkoxy group include the same groups as those listed for R ¹¹ . When R ² and R ³ are C _1-6 alkyl groups, examples of the C _1-6 alkyl groups include the same groups as those listed for R ¹¹ .

In general formula (1), X ¹ is an oxygen atom or -NH-. Examples of compounds in which X ¹ is an oxygen atom include compounds represented by the following general formulas (2) and (3), in which X ¹ is -NH- Examples of the compound include compounds 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 ⁴ are the same as in the above general formula (1). As the compound according to the present invention, a compound represented by the following general formula (2) or (3) is preferable.

In 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. group, or in general formula (3), at least one of A ¹ and A ² is a group represented by general formula (A-1), and Z ¹ is a C _1-6 alkylene group or a phenylene group. Preferably, a compound is represented by formula (2), where both A ¹ and A ² are groups represented by formula (A-1), and Z ¹ is a methylene group, ethylene group, n-propylene group, A compound that is an n-butylene group or a phenylene group, or in the general formula (3), both A ¹ and A ² are groups represented by the general formula (A-1), and Z ¹ is a methylene group, More preferred are compounds that are an ethylene group, n-propylene group, n-butylene group, or phenylene group; in general formula (2), both A ¹ and A ² are phenyl groups, and Z ¹ is an ethylene group or phenylene group. a compound in which R ² is a methyl group, ethyl group, or n-butyl group, or in general formula (3), both A ¹ and A ² are phenyl groups, and Z ¹ is an ethylene group or phenylene A compound in which R ³ and R ⁴ are each independently a hydrogen atom or a methyl group is more preferable; in general formula (2), both A ¹ and A ² 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), both A ¹ and A ² are phenyl groups, and Z ¹ is an ethylene group or a phenylene group. , R ³ and R ⁴ are all hydrogen atoms; in general formula (2), both A ¹ and A ² are phenyl groups, Z ¹ is a phenylene group, and R ² is A compound that is a methyl group, or a compound in which A ¹ and A ² are both phenyl groups, Z ¹ is a phenylene group, and R ³ and R ⁴ are both hydrogen atoms in the general formula (3) Particularly preferred.

The method for synthesizing the compound represented by general formula (1) is not particularly limited. For example, a compound in which R ¹ is a C _1-6 alkoxy group in general formula (1) can be synthesized by alkyl esterifying the carboxy group of oxaprozin. Similarly, a compound in which R ¹ is an amino group in general formula (1) can be synthesized by aminating the carboxy group of oxaprozin.

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 and Z ¹ are the same as in general formula (1). R ⁵ is a C _1-6 alkyl group. Thereafter, by ring-closing the obtained condensate in the presence of ammonium acetate, an alkyl ester having an oxazole ring or an imidazole ring (a compound in which R ¹ is a C _1-6 alkoxy group in general formula (1)) is obtained. Synthesize. By aminating the obtained alkyl ester, a compound in which R ¹ is an amino group in general formula (1) can be synthesized.

The compound represented by general formula (1) has the effect of suppressing collagen production. Therefore, the compound represented by general formula (1) is suitable as an active ingredient of a pharmaceutical composition used to treat diseases characterized by cell or tissue fibrosis caused by excessive collagen deposition. . The disease is not particularly limited as long as it causes cell or tissue fibrosis due to excessive collagen deposition, such as pulmonary fibrosis, liver fibrosis, renal fibrosis, gastrointestinal fibrosis, Examples include fibrosis of various organs including skin fibrosis, inflammatory bowel diseases including Crohn's disease and ulcerative colitis, and systemic scleroderma. Moreover, suppression of fibrosis also results in suppression of EMT. Therefore, a pharmaceutical composition containing the compound represented by the general formula (1) as an active ingredient can also be used to treat diseases, such as cancer, whose symptoms can be expected to be improved by suppressing EMT.

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 the inorganic salt 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 the organic salt include Tris salt, methylamine salt, ethylamine salt, ethanolamine salt, ammonium salt, and the like.

The active ingredient of the pharmaceutical composition according to the present invention is a solvate of the compound represented by general formula (1) or a pharmacologically acceptable salt of the compound represented by general formula (1). It may be a solvate of. Examples of the solvent that forms the solvate include water, ethanol, and the like.

As used herein, "pharmacologically acceptable" means free from undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio, and Used to describe such compounds, materials, compositions, and/or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with animal tissue.

The pharmaceutical composition according to the present invention may consist only of the compound represented by general formula (1), or may contain other components. The pharmaceutical composition according to the present invention can be formulated into various dosage forms by conventional pharmaceutical methods by combining the compound represented by general formula (1) with a pharmacologically acceptable carrier. can. A "pharmacologically acceptable carrier" refers to an ingredient that is compatible with the other ingredients of the formulation and is not harmful to the subject. Such carriers may include any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, etc. known to those skilled in the art. Included are salts, preservatives, drugs, drug stabilizers, gels, binders, additives, disintegrants, lubricants, sweeteners, flavors, dyes, and/or materials and combinations thereof. Based on the disclosure herein, it is within the skill of the art to formulate pharmaceutical compositions containing compounds of general formula (1). For example, it is possible to formulate a pharmaceutical composition containing a compound of general formula (1) according to standard techniques in the pharmaceutical field. See, for example, Alphonso Gennaro, ed. , Remington's Pharmaceutical Sciences, 18th Ed. , (1990) Mack Publishing Co. , Easton, Pa.

The pharmaceutical composition according to the present invention may further contain one or more other active ingredients, if desired. Like the compound represented by general formula (1), the "other active ingredients" are preferably components that have a collagen production suppressing effect, an antifibrotic effect, an anti-inflammatory effect, and the like. The pharmaceutical composition according to the present invention may contain components having activities other than those described above.

The route of administration of the pharmaceutical composition according to the present invention is not particularly limited. For example, it may be formulated into tablets, capsules, granules, troches, drinks, etc. and administered orally. Alternatively, parenteral administration such as transdermal administration such as a patch, intraperitoneal administration, intravenous administration by intravenous drip or injection, etc. may be used. In addition, intramuscular administration such as intramuscular injection, enteral administration, local administration, etc. are also possible. In one aspect, the pharmaceutical composition according to the present invention is administered orally, transdermally, intraperitoneally, or intravenously. Specific examples of formulations of the pharmaceutical composition according to the present invention are shown below.

For example, tablets, powders, granules, troches, capsules, etc. for oral administration may be prepared by adding an excipient, a disintegrant, a binder, or a lubricant to a pharmaceutical composition containing the compound represented by formula (1). They can be prepared by compression molding with the addition of one or more solid inert ingredients such as, and then optionally coating for taste masking, enteric or long-lasting purposes.

Injections include, for example, a pharmaceutical composition containing the compound represented by general formula (1), for example, as an aqueous injection together with a dispersant, a preservative, an isotonizing agent, etc., or an aqueous injection using olive oil, sesame oil, cottonseed oil, corn oil, etc. It can be produced by dissolving, suspending, or emulsifying the mixture in vegetable oils such as, propylene glycol, etc., and molding it into an oil-based injection.

The external preparation is produced, for example, by preparing a pharmaceutical composition containing the compound represented by the general formula (1) into a solid, semisolid, or liquid composition. For example, the above-mentioned solid composition can be produced by powdering the pharmaceutical composition as it is or by adding and mixing excipients, thickeners, etc. The above-mentioned liquid composition is produced in almost the same way as an injection by forming it into an oil-based or aqueous suspension. The semi-solid composition is preferably in the form of an aqueous or oily gel or an ointment. Furthermore, all of these compositions may contain buffering agents, preservatives, and the like. External preparations include, for example, creams, lotions, gels, ointments, etc. for topical administration.

Suppositories are produced, for example, by converting a pharmaceutical composition containing the compound represented by general formula (1) into an oil-based 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, Huitepsols, etc.), intermediate fatty acids (e.g., miglyols, etc.), or vegetable oils (e.g., sesame oil, soybean oil, etc.). cottonseed oil, etc.). Examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers.

The dosage and administration period of the pharmaceutical composition according to the present invention are determined depending on the size, mass, age, and sex of the subject to be administered, the nature and stage of the disease to be treated, the aggressiveness of the disease, the route of administration, and the specific toxicity of radiation. determined by the specific circumstances of the individual patient, including: Dosages and durations of administration can also be determined experimentally using known test protocols or by extrapolation from in vivo or in vitro test data. The concentration ranges set forth herein are for exemplary purposes only and are not intended to limit the scope or implementation of the claimed compositions.

For example, the dosage in a pharmaceutical composition containing the compound represented by general formula (1) is 0.01 to 2000 mg/kg/day in the amount of the compound represented by general formula (1) as an active ingredient, More preferably 0.05 to 1000 mg/kg/day, still more preferably 1.0 to 500 mg/kg/day. The pharmaceutical composition according to the invention may be administered once a day or may be divided into a number of lower doses administered simultaneously or at intervals of time.

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

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

<Chromatography>
In the subsequent experiments, silica gel ("Silica gel 60 F254", manufactured by Merck & Co., Ltd.) was used for thin layer chromatography. For column chromatography, silica gel (Silica gel 60N (spherical, neutral), manufactured by Kanto Kagaku Co., Ltd.) was used.

<NMR>
In subsequent experiments, NMR was performed as follows.
First, an NMR apparatus ("BRUKER AVANCE 600 (600 MHz)", manufactured by Bruker Biospin) was used to measure ¹ H-NMR and ¹³ C-NMR spectra. The chemical shift is shown in δ value (ppm) using tetramethylsilane (TMS) as an internal standard, and the J value is shown in Hz. Mass spectrometry (FAB) and high-resolution mass spectrometry (HRMS) were measured using a mass spectrometer ("JMS-DX303HF MASS spectrometer", manufactured by JOEL). Infrared absorption spectra (IR) were measured using an IR device (“Frontier”, manufactured by Perkin Elmer). Elemental analysis was performed using an organic elemental analyzer ("MICRO CORDER JM10", manufactured by J Science Group).

[Example 1]
The present inventors focused on the structural characteristics of oxaprozin and SB-431542, and created new compounds (AF-1, AF- 2, AF-3) was synthesized and its activity was investigated.

<Synthesis of compounds>
According to the method of Pickett et al. (Non-Patent Document 2), a compound (C3-1) is prepared by condensing benzoin (compound (C1-1)) and methyl-4-(chloroformyl)benzoate (compound (C2-1)). , with a yield of 62%. Compound (C3-1) and ammonium acetate were mixed and refluxed under anhydrous conditions to obtain the target compound (AF-1) having an oxazole ring in a yield of 32%. Furthermore, according to the method of Ammirati et al. (Non-Patent Document 3), the ester moiety of compound (AF-1) was hydrolyzed to synthesize the target compound (AF-2) in a yield of 92%. The carboxy group of compound (AF-2) was amidated to obtain the target compound (AF-3) in a yield of 24%. The details are described below.

(1) Synthesis of compound (C3-1) (Methyl (2-oxo-1,2-diphenylethyl) terephthalate) Benzoin (compound (C1-1)) (1.06 g, 5 mmol) was added to 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 12 The mixture was refluxed for an hour to react. After the reaction was completed, the temperature was returned to room temperature, and the mixture was filtered with suction. The obtained filtrate was sequentially extracted with purified water, 5% hydrochloric acid, 5% sodium hydroxide, purified water, and saturated saline. Thereafter, the obtained organic layer was dried by adding sodium sulfate, and diethyl ether was distilled off under reduced pressure. Thereafter, it was purified by silica gel column chromatography (CH ₂ Cl ₂ ) to obtain compound (C3-1) (1.155 g, yield 62%).

MS (FAB) (M+H) ⁺ m/z 375.3.
¹ H NMR (600MHz, CDCl ₃ ) 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, CDCl ₃ ) 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.

(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 added to glacial acetic acid ( 25 mL), mixed with ammonium acetate (275 mL, 3.57 mmol), refluxed at 155°C under anhydrous conditions for 2 hours, and then stirred at room temperature for 12 hours to react. After the reaction was completed, purified water was added to remove the solvent. Ethanol was added to the viscous material remaining in the flask and filtered with suction. Recrystallization was performed to obtain the target compound (AF-1) (330 mg, yield 32%).

MS (FAB)(M+H) ⁺ m/z 356.3.
¹ H NMR (600MHz, CDCl ₃ )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).
^13C NMR (150MHz, CDCl ₃ )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, 1 29.25, 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.

(3) Synthesis of compound (AF-2: 4-(4,5-Diphenyloxazol-2-yl) benzoic acid) Acetone (10 mL) was added to compound (AF-1) (190 mg, 0.534 mmol), and 1M- LiOH (3 mL) was added. The mixture was stirred at 80° C. for 3 hours and at room temperature for 18 hours. After the reaction was completed, 1M HCl was added to adjust the pH to 2. The target compound (AF-2) was obtained by suction filtration (170 mg, yield 92%).

MS (FAB) (M+H) ⁺ m/z 342.3.
¹ H NMR (600MHz, CDCl ₃ )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.

(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, It was refluxed at 96°C for 3 hours. After the obtained reaction product was returned to room temperature, it was azeotroped with thionyl chloride. Then, it was dissolved in 1,4-dioxane, cooled to 0°C, 33% ammonium hydroxide (0.4 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The precipitated crystals were filtered with suction to obtain the target compound (AF-3) (29 mg, yield 24%).

MS (FAB) (M+H) ⁺ m/z 341.1.
¹ H NMR (600MHz, CDCl ₃ )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).
^13C NMR (150MHz, CDCl ₃ ) 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, 168.4 7.
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.

<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, 0.5×10 ⁵ cells/mL (1 mL of culture solution) of A549 cells (human alveolar basal epithelial adenocarcinoma cell line) were seeded in each well of a 6-well plate in an amount of 1 mL, and cultured overnight. Then, 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 in which A549 cells were seeded. The test compound was added at a final concentration of 10 μM, incubated for 1 hour, and then TGF-β (10 ng/mL) was added and cultured for 3 days. As a control, an equal volume of 1% DMSO solution was similarly added to wells seeded with A549 cells and incubated, and then TGF-β was added and cultured. The morphology of the cells after 3 days of incubation was observed using a fluorescence microscope ("BZ-9000", manufactured by KEYENCE).

Figure 1 shows microscopic images of each cell. As a result of the investigation, in the presence of TGF-β, A549 cells changed into a spindle-shaped cell morphology with a pointed tip, which is a characteristic of mesenchymal cells. Compound (AF-1) partially inhibited this change in cell morphology, and compound (AF-3) inhibited it as a whole. Compound (AF-1) and compound (AF-3) were found to have EMT inhibitory activity, as they suppressed the changes in cell morphology observed during the progression of EMT.

<Evaluation of antifibrotic activity based on collagen expression level - Western blotting method>
The antifibrotic activity of compound (AF-3) was evaluated based on the expression level of type I collagen in adult dermal fibroblasts treated with oxaprozin or compound (AF-3) and then stimulated with TGF-β. The expression level of type I collagen was examined by Western blotting. First, 1.0×10 ⁵ cells/mL of adult skin fibroblasts (1 mL of culture solution) were seeded in each well of a 24-well plate, and cultured overnight. Thereafter, a test compound (oxaprozin or compound (AF-3)) dissolved in 1% DMSO solution was added to the wells seeded with adult skin fibroblasts so that the final concentration of the test compound was 10 μM or 25 μM. After incubating for 1 hour, TGF-β (10 ng/mL) was added and cultured for 2 days. As a control, an equal volume of 1% DMSO solution was similarly added to wells seeded with adult dermal fibroblasts and incubated, and then TGF-β was added and cultured.

The cultured cells were disrupted by adding RIPA buffer, and the disrupted solution was boiled at 100° C. for 10 minutes to lyse the cells. The obtained cell lysate was electrophoresed and then transferred to a membrane (manufactured by Millipore), reacted with a goat anti-type I collagen antibody (manufactured by Southern Biotech) as a primary antibody, and subjected to Western blotting. Detection was performed using a Chemilumi imager (“ImmunoStar LD”, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The expression level of type I collagen was corrected using the expression level of β-actin as an internal standard.

The measurement results of the expression level of type I collagen are shown in FIG. FIG. 2(A) shows the results of Western blotting, and FIG. 2(B) shows the relative expression level of type I collagen in each cell determined from the Western blotting. Stimulation of adult dermal fibroblasts with TGF-β increased type I collagen and activated the fibroblasts. By adding the compound (AF-3) to these activated cells, the increase in type I collagen was suppressed in a dose-dependent manner. Compound (AF-3) was found to have antifibrotic activity because it suppressed collagen production. In this experiment, the type I collagen inhibitory effect of oxaprozin was not observed, but this was because the amount of oxaprozin added was small, and when treated with a higher concentration of oxaprozin, the antifibrotic activity was not observed. It was assumed that this could be confirmed.

<Evaluation of antifibrotic activity based on collagen expression level - ELISA method>
The amount of extracellular type I collagen was measured using ELISA direct adsorption method instead of Western blotting. First, 1.0×10 ⁵ cells/mL of adult skin fibroblasts (1 mL of culture solution) were seeded in each well of a 24-well plate, and cultured overnight. Thereafter, a solution of the test compound (oxaprozin or compound (AF-3)) dissolved in 1% DMSO solution was added to the wells seeded with adult skin fibroblasts so that the final concentration of the test compound was 10 μM. After incubating for 1 hour, TGF-β (10 ng/mL) was added and cultured for 1 day. As a control (Part 1), an equal volume of 1% DMSO solution was similarly added to wells seeded with adult dermal fibroblasts, incubated, and then cultured without adding TGF-β. As a control (Part 2), an equal volume of 1% DMSO solution was similarly added to wells seeded with adult dermal fibroblasts and incubated, and then TGF-β was added and cultured.

The cultured cells were fixed by incubating them for 20 minutes in a 3% paraformaldehyde solution (manufactured by Tokyo Kasei Co., Ltd.). After the immobilization reaction was stopped by adding 1% glycine solution (manufactured by Merck & Co., Ltd.), blocking was performed for 15 minutes using a PBS solution of 5% goat serum (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). A PBS solution of goat anti-type I collagen antibody (manufactured by MBL) containing 1% BSA (bovine serum albumin) was added to the cells after blocking, and reacted as a primary antibody for 1 hour. Thereafter, a HRP-labeled anti-mouse IgG antibody was reacted for 30 minutes as a secondary antibody. Detection was performed using OPD (o-phenylenediamine dihydrochloride) reagent (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.).

The measurement results of the expression level of type I collagen (relative value with the type I collagen level of control (part 1) cells as 1) are shown in FIG. As a result, it was confirmed that the amount of collagen was decreased in cells treated with compound (AF-3), similar to the results of Western blotting. These results showed that the compound (AF-3) had a collagen production suppressing effect and, in turn, an antifibrotic effect.

Claims (6)

General formula (1) below

[In formula (1), A ¹ and A ² each independently represent 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 circles are bonds}
X ¹ is an oxygen atom or -NH-; Z ¹ is an alkylene group, phenylene group, or cycloalkylene group having 1 to 6 carbon atoms; R ¹ is , an alkoxy group having 1 to 6 carbon atoms, or -N(R ² )(R ³ ); 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 The compound according to claim 1, wherein both A ¹ and A ² are (A-1), X ¹ is an oxygen atom, and Z ¹ is a phenylene group. The compound according to claim 2, wherein the R ¹ is a methoxy group or an amino group. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3, or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient. 5. The pharmaceutical composition according to claim 4, which is used for treating a disease in which cell or tissue fibrosis occurs. The pharmaceutical composition according to claim 4 or 5, which is orally administered.

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