BR112016030018B1 - SALT COMPOSED OF HETEROCYCLIC SUBSTITUTED WITH HALOGEN, LPA RECEPTOR ANTAGONIST, PHARMACEUTICAL COMPOSITION AND USE OF SUCH SALT - Google Patents

Abstract

HETEROCYCLIC COMPOUND SALT SUBSTITUTED WITH HALOGEN. A novel a-halogen substituted thiophene compound salt is provided which has a potent LPA receptor antagonistic action and is useful as a pharmaceutical. A salt represented by the general formula (I): wherein R is a hydrogen atom or a methoxy group; X is a halogen atom; A is selected from the group consisting of the following groups: A; M is an alkali metal or an alkaline earth metal and n is 1 when M is an alkali metal and is 2 when M is an alkaline earth metal].

Description

TECHNICAL FIELD

[001] The present invention relates to a new salt composed of thiophene substituted with a-halogen useful as a medicine. The a-halogen substituted thiophene salt of the present invention has a lysophosphatidic acid (LPA) receptor antagonist action and is therefore useful for the prevention and/or treatment of diseases induced by LPA.

PRIOR TECHNIQUE

[002] Lysophosphatidic acid (LPA) is a physiologically active phospholipid that is present in a living body. By binding to specific G protein-coupled receptors (LPA1, LPA2, LPA3, LPA4, LPA5 and LPA6), LPA transduces signals in cells and modulates proliferation, differentiation, survival, migration, adhesion, infiltration and morphogenesis of the cells. Additionally, it is known that LPA is involved in diseases accompanied with fibrosis in various organs.

[003] It has been described that in the liver, LPA stimulates the proliferation or contraction of stellate cells that play an important role in the process of hepatic fibrosis and stimulates the migration of myofibroblasts (see Non-Patent Documents 1, 2 and 3).

[004] It has been reported that in the kidney either LPA production or LPA1 expression is enhanced in mice with unilateral urethral ligation as an animal model of renal fibrosis and that renal fibrosis is suppressed by LPA1 deficiency or treated with a receptor antagonist LPA (see Non-Patent Documents 4 and 5).

[005] With regard to the lung, it has been reported that the bronchoalveolar lavage fluids of patients with idiopathic pulmonary fibrosis have an increased concentration of LPA and that LPA1 is the receptor most expressed in fibroblasts that play an important role in the fibrosis process lung and LPA induces fibroblast migration. Additionally, LPA deficiency or administration of an LPA1 receptor antagonist has been reported to suppress fibrosis in mice treated intratracheally with bleomycin as an animal model of pulmonary fibrosis (see Non-Patent Documents 6 and 7).

[006] With regard to the skin, it has been reported that cutaneous fibrosis is suppressed by LPA1 deficiency or the administration of an LPA receptor antagonist in mice that are treated subcutaneously with bleomycin as models of scleroderma (see Paper of Non-Patent 8).

[007] It is also known that LPA is involved in immunological or inflammatory diseases. LPA has been reported to stimulate human monocyte migration and is involved in T cell proliferation or infiltration. Additionally, synovial cells from patients with rheumatoid arthritis have been reported to express LPA receptors and migrate or produce IL-6 and IL- 8 by LPA stimulation and that these actions are inhibited by an LPA receptor antagonist (see Non-Patent Documents 9, 10 and 11).

[008] Additionally, LPA and LPA1 have been described to be involved in the development of neuropathic pain (see Patent Document 12), that LPA causes contraction of urethral specimens and extracted prostatic specimens and increased intraurethral pressure, and is involved in diseases neurological disorders (see Patent Document 1) and that LPA is involved in cancer-related diseases by stimulating prostate cancer cell proliferation (see Non-Patent Documents 13, 14 and 15).

[009] Based on these reports, a drug that antagonizes LPA receptors (in particular, the LPA1 receptor) is considered to be useful for the prevention and/or treatment of diseases that are accompanied by fibrosis, immunological or inflammatory diseases, diseases of the central or peripheral nervous system, urological diseases and cancer-related diseases, etc.

[0010] On the other hand, Patent Documents 2 to 23 and Non-Patent Documents 5, 7, 8 and 16 describe ([1,1'-biphenyl]-4-yl) acetic acid derivatives, the Patent 17 describes (2'-methoxy-[1,1'-biphenyl]-4-yl)acetic acid derivatives and Patent Document 19 describes 3-chloroisothiazole derivatives as a compound having an antagonistic function on LPA receptors, not there is description of compounds according to the present invention.

PRIOR ART DOCUMENTS Patent Documents

[0011] Patent Document 1: WO 2002/062389

[0012] Patent Document 2: WO 2010/077882

[0013] Patent Document 3: WO 2010/077883

[0014] Patent Document 4: WO 2010/141761

[0015] Patent Document 5: WO 2010/141768

[0016] Patent Document 6: WO 2011/017350

[0017] Patent Document 7: WO 2011/041461

[0018] Patent Document 8: WO 2011/041462

[0019] Patent Document 9: WO 2011/041694

[0020] Patent Document 10: WO 2011/041729

[0021] Patent Document 11: WO 2011/091167

[0022] Patent Document 12: WO 2011/159632

[0023] Patent Document 13: WO 2011/159633

[0024] Patent Document 14: WO 2011/159635

[0025] Patent Document 15: WO 2012/078593

[0026] Patent Document 16: WO 2012/078805

[0027] Patent Document 17: WO 2012/138648

[0028] Patent Document 18: WO 2012/138797

[0029] Patent Document 19: WO 2013/025733

[0030] Patent Document 20: WO 2013/085824

[0031] Patent Document 21: WO 2013/189862

[0032] Patent Document 22: WO 2013/189864

[0033] Patent Document 23: WO 2013/189865

Non-patent Documents

[0034] Non-Patent Document 1: Biochemical and Biophysical Research Communications, 248(1998)436-440

[0035] Non-Patent Document 2: Biochemical and Biophysical Research Communications, 277 (2000) 72-78

[0036] Non-Patent Document 3: Journal of Biomedical Science, 10 (2003) 352-358

[0037] Non-Patent Document 4: Journal of the American Society of Nephrology, 18 (2007) 3110-3118

[0038] Non-Patent Document 5: The Journal of Pharmacology and Experimental Therapeutics, 336 (2011) 693-700

[0039] Non-Patent Document 6: Nature Medicine, 14 (2008) 45-54

[0040] Non-Patent Document 7: British Journal of Pharmacology, 160 (2010) 1699-1713

[0041] Non-Patent Document 8: Arthritis & Rheumatism, 63(2011) 1405-1415

[0042] Non-Patent Document 9: Journal of Biological Chemistry, 270 (1995) 25549-25556

[0043] Non-Patent Document 10: Biochimica et Biophysica Acta, 1582 (2002) 168-174

[0044] Non-Patent Document 11: Molecular Pharmacology, 73 (2008) 587-600

[0045] Non-Patent Document 12: Nature Medicine, 10 (2004) 712-718

[0046] Non-Patent Document 13: Biochemical and Biophysical Research Communications, 193 (1993) 497-503

[0047] Non-Patent Document 14: Biochemical Journal, 309 (1995) 933-940

[0048] Non-Patent Document 15: The Journal of Urology, 163 (2000) 1027-1032

[0049] Non-Patent Document 16: Journal of Medicinal Chemistry, 55 (2012) 7920-7939

DESCRIPTION OF THE INVENTION Problems to be Solved by the Invention

[0050] The present inventors have carried out research on various halogen-substituted heterocyclic compound salts in order to develop an excellent medicine for the treatment or prevention of diseases accompanied by fibrosis, inflammatory or immunological diseases, diseases of the central and peripheral nervous system, diseases urologic and cancer-related diseases. As a result, the present inventors have found that a novel salt composed of a-halogen substituted thiophene having a specific structure exhibits an excellent LPA receptor antagonistic action and is useful as a medicine (in particular, for the prevention and/or or treatment of diseases that are accompanied by fibrosis, inflammatory or immunological diseases, diseases of the central and peripheral nervous system, urological diseases and cancer-related diseases). The present invention was finalized based on this finding.

[0051] The present invention provides a novel salt composed of a-halogen substituted thiophene which has a potent LPA receptor antagonistic action and is useful as a drug for the treatment and/or prevention (preferably, a drug for the treatment) of, in particular, diseases that are accompanied by fibrosis, inflammatory or immunological diseases, diseases of the central and peripheral nervous system, urological diseases and cancer-related diseases.

Ways to Solve Problems

[0052] The present invention provides; (1) A salt represented by the general formula (I): Chemical Structure 1 where R is a hydrogen atom or a methoxy group, X is a halogen atom, A is selected from the group consisting of: Chemical Structure 2 M is an alkali metal or an alkaline earth metal and n is 1 when M is an alkali metal and is 2 when M is an alkaline earth metal. (2) The salt according to (1), wherein the alkali metal or alkaline earth metal is sodium, potassium or calcium. (3) (R)-1-[4'-(5-Chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy[1] acid salt ,1'-biphenyl]-4-yl]cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. (4) The salt according to (3), wherein the alkali metal or alkaline earth metal is sodium, potassium or calcium. (5) A (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorphenyl)ethoxy]carbonyl}amino)thiophen-2-yl] acid salt -2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. (6) The salt according to (5), wherein the alkali metal or alkaline earth metal is sodium, potassium or calcium. (7) A salt of (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2' acid -methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. (8) The salt according to (7), wherein the alkali metal or alkaline earth metal is sodium, potassium or calcium. (9) A salt of (R)-1-{4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. (10) The salt according to (9), wherein the alkali metal or alkaline earth metal is sodium, potassium or calcium. (11) A salt of (R)-1-{4'-[5-fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl] acid -[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. (12) The salt according to (11), wherein the alkali metal or alkaline earth metal is sodium, potassium or calcium. (13) An LPA receptor antagonist comprising the salt according to any one of (1) to (12) as an active ingredient. (14) Pharmaceutical composition comprising the salt according to any one of (1) to (12) as an active ingredient. (15) Pharmaceutical composition according to (14) for the treatment or prevention of a disease that is accompanied by fibrosis, inflammatory or immunological diseases, central and peripheral nervous system diseases, urological diseases and cancer-related diseases.

[0053] Specific examples of the compounds represented by the general formula (I) of the present invention include compounds described in Table 1 below. In Table 1, OMe represents a methoxy group and “Racemic” and “(R)-“ represent the configuration of the carbon atom marked “*” in the general formula (I) below. Chemical Structure 3 Table 1

Effects of the Invention

[0054] The a-halogen substituted thiophene compound salts of the present invention which are represented by the general formula (I) possess a potent LPA receptor antagonistic action and are therefore useful as medicaments for the prevention and/or treatment of diseases that are accompanied by fibrosis, inflammatory or immunological diseases, diseases of the central and peripheral nervous system, urological diseases and cancer-related diseases.

METHOD OF CARRYING OUT THE INVENTION

[0055] The preferred embodiments of each substituent group in the salts represented by the general formula (I) will be described below.

[0056] Examples of “halogen atoms” represented by X include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

[0057] Preferably, the "halogen atom" represented by X is a fluorine atom, a chlorine atom or a bromine atom, and is more preferably a fluorine atom or a chlorine atom.

[0058] Examples of the “alkali metals” represented by M include lithium, sodium, potassium, rubidium and cesium, with sodium and potassium being preferred.

[0059] Examples of the "alkaline earth metals" represented by M include magnesium, calcium, strontium, and barium, with calcium being preferred.

[0060] In the case where the salts of the present invention represented by the general formula (I) have optical isomers, geometric isomers and rotational isomers, these isomers are within the scope of the present invention. Furthermore, in a case where proton tautomerism is present, these tautomers are also within the scope of the present invention.

[0061] In the general formula (I), the group represented by: Chemical Structure 4 is preferably the following group: Chemical Structure 5

[0062] The salts of the invention represented by the general formula (I) can form hydrates or solvates, each of which and mixtures thereof are also within the scope of the invention.

[0063] One or more types of atoms constituting the salts of the present invention represented by formula (I) may have atomic isotopes in an unnatural ratio. Examples of atomic isotopes include deuterium (2H), tritium (3H), carbon-14 (14C), fluoro-18 (18F), sulfur-35 (35S), and iodine-125 (125I). Such compounds are useful as drugs for treatment or prevention, research reagents such as assay reagents, and diagnostic agents such as in vivo diagnostic imaging agents. All isotopic variants of the salts of the invention represented by formula (I) are within the scope of the present invention, irrespective of whether they are radioactive or not.

[0064] A general process for producing the compound of the present invention is shown below. Each specific process for producing the compound of the present invention will be individually described in detail in the Examples later on. Chemical Structure 6 Step 1 Chemical Structure 7 Step 2 Chemical Structure 8 Step 3

[0065] (In the formulas, R, X, A, M and n are the same as defined above, M' and n' have the same definitions as M and n, respectively, Z- is an anion such as a hydroxide ion , a halide ion or an acet ion and R1 is a carboxylic acid protecting group such as an alkyl group that is deprotected by hydrolysis).

[0066] The salts of the present invention represented by the general formula (I) can be synthesized by any of steps 1 to 3 described above.

[0067] In the reaction in each of steps 1 to 18 described below, any solvent can be used without limitation as long as the solvent does not inhibit the reaction and can dissolve part of the raw starting materials. Examples of the solvents include aliphatic hydrocarbons such as hexane, pentane, heptane, petroleum ethers and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene and ethylbenzene; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone; esters such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate and butyl acetate; nitriles such as acetonitrile, propionitrile, butyronitrile and isobutyronitrile; carboxylic acids such as acetic acid and propionic acid; alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol and 1,2-propanediol; amides such as formamide, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, dimethylimidazolone and hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide; sulfones such as sulfolane; water; and a mixture of these solvents.

[0068] In the reaction in each of steps 1 to 18 described below, the reaction temperature is variable depending on conditions such as solvents, raw starting materials and reagents and the reaction time is variable depending on conditions such as solvents, raw starting materials, reagents and reaction temperatures.

[0069] Step 1: Compound (1) can be reacted in a reaction solvent that uses an alkali metal or alkaline earth metal hydroxide to synthesize a salt of general formula (I).

[0070] The reaction solvent is preferably water or a water/organic solvent mixture and is more preferably water, acetonitrile/water mixture or acetonitrile/tetrahydrofuran/water mixture.

[0071] Step 2: Compound (I') and Compound (2) may be subjected to a base exchange in a reaction solvent to synthesize a salt of general formula (I).

[0072] The reaction solvent is preferably water or a water/organic solvent mixture and is more preferably water or an acetonitrile/water mixture.

[0073] Step 3: Compound (3) can be hydrolyzed in a reaction solvent using an alkali metal or alkaline earth metal hydroxide to synthesize a salt of general formula (I).

[0074] The reaction solvent is preferably water or a water/organic solvent mixture and is more preferably a 2-propanol/water mixture or a 2-propanol/tetrahydrofuran/water mixture.

[0075] A general process for producing a synthetic intermediate of the compound of the present invention will be described below. Each specific process for producing the compound of the present invention will be individually described in detail in the Examples later on.

[0076] In the synthetic routes illustrated below, R, X, A, M, M', n, n', Z, and R1 have the same meanings as described above. L, La and M are necessary substituents for the coupling reaction and, for example, in the case where L or La is a chlorine atom, a bromine atom, an iodine atom or a trifluoromethanesulfonyloxy group, etc. , M is boronic acid, a boronate ester or a trialkyltin, etc., and in each case where L or La is boronic acid, a boronate ester or a trialkyltin, etc., M is a chlorine atom , a bromine atom, an iodine atom or a trifluoromethanesulfonyloxy group, etc. Chemical Structure 9 Step 4

[0077] Step 4: According to a method described, for example, in Tetrahedron, 64 (2008), pp. 9733-9737 or Bioorganic and Medicinal Chemistry Letters, 21 (2011), pp. 528-530, Compound (4) can be halogenated with a halogenating agent in a reaction solvent to synthesize Compound (5).

[0078] Preferred examples of the reaction solvents include aliphatic hydrocarbons, halogenated hydrocarbons, ethers, nitriles, carboxylic acids, amides, sulfoxides, water and a mixture of these solvents. N,N-dimethylformamide is more preferable.

[0079] Examples of halogenating agents include iodine, N-iodosuccinimide, bromine, N-bromosuccinimide, 1,2-dibromoethane, 1,2-dibromo-1,1,2,2-tetrafluoroethane, chlorine, N-chlorosuccinimide , xenon difluoride, N-fluorobenzenesulfonimide and N-fluoro-N'-(chloromethyl)triethylenediamine bis(tetrafluoroborate).

[0080] Alternatively, Compound (4) can be converted to an anion in a reaction solvent using a base and subsequently treated with a halogenating agent to synthesize Compound (5) according to a method described, for example, in Tetrahedron Letters, 51 (2010), pp. 4526-4529 or Journal of Medicinal Chemistry, 54 (2011), pp. 2687-2700.

[0081] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers and a mixture of these solvents. Ethers, aliphatic hydrocarbons and a mixture of these solvents are more preferred.

[0082] Examples of the bases include alkyl lithiums such as n-butyl lithium, sec-butyl lithium and tert-butyl lithium; lithium amides such as lithium diisopropylamide and lithium 2,2,6,6-tetramethipiperidine; Grignard reagents such as ethylmagnesium bromide, ethylmagnesium chloride, isopropylmagnesium chloride and phenylmagnesium chloride; magnesium amides such as magnesium chloride diisopropylamide and 2,2,6,6-tetramethylpiperidine magnesium chloride; and disilazane bases such as lithium 1,1,1,3,3,3-hexamethyldisilazane and potassium 1,1,1,3,3,3-hexamethyldisilazane.

[0083] Examples of halogenating agents include iodine, N-iodosuccinimide, bromine, N-bromosuccinimide, carbon tetrabromide, 1,2-dibromoethane, 1,2-dibromo-1,1,2,2-tetrafluoroethane, chlorine , N-chlorosuccinimide, carbon tetrachloride, xenon difluoride, N-fluorobenzenesulfonimide and N-fluoro-N'-(chloromethyl)triethylenediamine bis(tetrafluoroborate). Chemical Structure 10 Step 5

[0084] Step 5: According to a method described, for example, in Tetrahedron, 58 (2002), pp. 9633-9695, Compound (6) and Compound (7) can be reacted in a reaction solvent in the presence of a coupling catalyst, a linker and/or a base to synthesize Compound (8).

[0085] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, ethers, ketones, esters, nitriles, alcohols, amides, sulfoxides, sulfones, water and a mixture of these solvents. The 1,4-dioxane/water mixture is more preferable.

[0086] Examples of coupling catalysts include palladium catalysts such as tetracis(triphenylphosphino)palladium(0), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) chloride methylene dichloride adduct, bis(triphenylphosphino)palladium(II) dichloride, tris(dibenzylideneacetone)dipalladium(0) and palladium(II) acetate; and nickel catalysts such as bis(triphenylphosphino) nickel E(II) dichloride.

[0087] Examples of ligands, sometimes present in the coupling catalysts themselves, include triphenylphosphine, [1,1'-bis(diphenylphosphine)ferrocene], dibenzylideneacetone, triphenylarsine, tri(o-tolyl)phosphine, tri-tert- butylphosphine and tricyclohexylphosphine.

[0088] Examples of bases include fluorine salts such as potassium fluoride and cesium fluoride; carbonate salts such as sodium hydrogen carbonate, sodium carbonate, potassium carbonate, cesium carbonate and thallium carbonate; metal hydroxides such as sodium hydroxide, potassium hydroxide, barium hydroxide and thallium hydroxide; phosphate salts such as potassium phosphate; and organic bases such as triethylamine and diisopropylethylamine. Sodium carbonate is preferable. Chemical Structure 11 Step 6

[0089] Step 6: According to a method described, for example, in the Journal of the American Chemical Society, 129 (2007), pp. 45954605, Compound (8) can be reacted in a reaction solvent in the presence of a palladium catalyst, a linker, a boronic acid reagent, and/or a base to synthesize Compound (9). For example, L represents a chlorine atom, a bromine atom, an iodine atom or a trifluoromethanesulfonyloxy group and M represents boronic acid or a boronate ester.

[0090] Preferred examples of reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, ethers, ketones, esters, nitriles, alcohols, amides, sulfoxides, sulfones, water and a mixture of these solvents. 1,4-dioxane is more preferable.

[0091] Examples of palladium catalysts include tetracis(triphenylphosphino)palladium(0), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride adduct, methylene chloride, bis(dichloride) triphenylphosphine) palladium (II), tris(dibenzylideneacetone)dipalladium (0) and palladium (II) acetate. Palladium(II) methylene chloride [1,1'-bis(diphenylphosphino)ferrocene] dichloride adduct and palladium(II) acetate are preferable.

[0092] Examples of ligands, sometimes present in the coupling catalysts themselves, include triphenylphosphine, [1,1'-bis(diphenylphosphine)ferrocene], dibenzylideneacetone, triphenylarsine, tri(o-tolyl)phosphine, tri-tert- butylphosphine and tricyclohexylphosphine. Tricyclohexylphosphine is preferred.

[0093] Examples of boronic acid reagents include 4,4,4',4', 5,5,5',5'-octanetyl-2,2-bi(1,3,2-dioxaborolane) and 4,4 ,5,5-tetramethyl-1,3,2-dioxaborolane.

[0094] Examples of bases include potassium acetate and sodium acetate.

[0095] Alternatively, according to a method described, for example, in Angewandte Chemie - International Edition, 45 (2006), pp. 1404-1408, Compound (8) in which L is a chlorine atom, a bromine atom, or an iodine atom, can be treated in a reaction solvent so as to subject the halogen of the group L to a metal exchange halogen using a base and subsequently the product can be treated with a boronic acid reagent to synthesize Compound (9).

[0096] Preferred examples of reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, amides, sulfoxides, sulfones and a mixture of these solvents. Ethers, aliphatic hydrocarbons and a mixture of these solvents are more preferred.

[0097] Examples of the bases include alkyl lithiums such as n-butyl lithium, sec-butyl lithium and tert-butyl lithium; and Grignard reagents such as ethylmagnesium bromide, ethylmagnesium chloride, isopropylmagnesium chloride and phenylmagnesium chloride.

[0098] Examples of boronic acid reagents include trimethyl borate, triisopropyl borate, trihexadecyl borate and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane . Chemical Structure 12 Step 7 Chemical Structure 13 Step 8

[0099] Step 7 and Step 8: Compound (5) or Compound (11) and Compound (9) can be reacted in a similar manner to Step 5 to synthesize Compound (10) or Compound (12), respectively. Chemical Structure 14 Step 9

[00100] Step 9: Compound (12) can be treated similarly to Step 4 to synthesize Compound (13). Chemical Structure 15 Step 10

[00101] Step 10: In a reaction solvent or without solvent, an acid is allowed to act on Compound (13) to deprotect the compound, thereby synthesizing Compound (14).

[00102] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, esters, nitriles, carboxylic acids, alcohols, amides, sulfoxides, sulfones, water and a mixture of these solvents. Methylene chloride is more preferable.

[00103] Examples of acids include inorganic acids such as hydrochloric acid and sulfuric acid; organic acids such as acetic acid, trifluoroacetic acid and trichloroacetic acid; and sulfonic acids such as methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid, with trifluoroacetic acid being preferred. Chemical Structure 16 Step 11

[00104] Step 11: In a reaction solvent, Compound (10) is hydrolyzed in the presence of an acid or a base to synthesize Compound (15).

[00105] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, nitriles, carboxylic acids, alcohols, amides, sulfoxides, sulfones, water and a mixture of these solvents. An ethanol/tetrahydrofuran/water mixture and a 2-propanol/tetrahydrofuran/water mixture are more preferable.

[00106] Examples of acids and bases include inorganic acids such as hydrochloric acid and sulfuric acid; organic acids such as acetic acid, trifluoroacetic acid and trichloroacetic acid; and sulfonic acids such as methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid; alkali metal hydroxides such as sodium hydroxide, potassium hydroxide; and alkali metal carbonate salts such as sodium carbonate and potassium carbonate. The use of a base is preferable and the use of lithium hydroxide or sodium hydroxide is more preferable. Chemical Structure 17 Step 12

[00107] Step 12: In a reaction solvent, Compound (15) can be condensed with trimethylsilylethanol using a condensing agent in the presence or absence of a base and in the presence or absence of an additive, thus synthesizing the Compound ( 16).

[00108] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, esters, nitriles, amides, sulfoxides, sulfones and a mixture of these solvents. N,N-dimethylformamide is more preferable.

[00109] Examples of bases include carbonate salts such as sodium hydrogen carbonate, sodium carbonate, potassium carbonate, cesium carbonate and thallium carbonate; pyridines such as pyridine, 2,6-lutidine and 4-picoline; and organic bases such as triethylamine and diisopropylethylamine. Diisopropylethylamine is preferred.

[00110] Examples of condensing agents include carbodiimide condensing agents such as N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate salt; imidazole condensing agents such as N,N'-carbonyldiimidazole; Triazine condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride and (4,6-dimethoxy-1,3,5- triazin-2-yl)-(2-octoxy-2-oxoethyl)dimethylammonium; phosphonium condensing agents such as 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate salt, 1H-benzotriazol-1-yloxytripyrolidinophosphonium hexafluorophosphate salt, and chlorotripyrolidinophosphonium hexafluorophosphate salt; and uronium condensing agents such as ({[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene)dimethylammonium hexafluorophosphate salt, O-(benzotriazole-1- yl)-N,N,N',N'-tetramethyluronium, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate salt, O-tetrafluoroborate salt -(N-succinimidyl)-N,N,N',N'-tetramethyluronium and O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-) tetrafluoroborate salt yl)-N,N-N',N'-tetramethyluronium, with O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate salt being preferred.

[00111] Examples of the additives include benzotriazoles such as 1-hydroxybenzotriazole and 1-hydroxyazabenzotriazole; pyridines such as N,N-dimethylaminopyridine; and combinations thereof. N,N-dimethylaminopyridine is preferred. Chemical Structure 18 Step 13

[00112] Step 13: According to a method described, for example, in Tetrahedron Letters, 47 (2006), pp. 5261-5264, Compound (17) can be reduced with a reducing agent in a reaction solvent to synthesize Compound (18).

[00113] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, esters, nitriles, alcohols, amides, sulfoxides, sulfones, water and a mixture of these solvents.

[00114] Examples of reducing agents include borohydrides such as lithium borohydride, sodium borohydride, potassium borohydride and sodium trimethoxyborohydride; and aluminum hydrides such as lithium aluminum hydride, sodium aluminum hydride, diisobutylaluminum hydride and lithium trimethoxyaluminium hydride.

[00115] Optically active Compound (18) can be synthesized using (R)- or (S)-5,5-diphenyl-2-methyl-3,4-propane-1,3,2-oxaborolidino, etc. according to a method described, for example, in Journal of Organic Chemistry, 56 (1991), pp. 763-769.

[00116] The optical purity of Compound (18) obtained by the above method can be increased by a known method such as using an enzyme or a resolving agent or by a combination of such methods. Chemical Structure 19 Step 14 Chemical Structure 20 Step 15

[00117] Step 14 and Step 15: In a reaction solvent or without solvent, Compound (10) or Compound (16) is subjected to a Hofmann rearrangement in the presence or absence of a base using Compound ( 18) and an oxidizing agent according to a method - described, for example, in Organic Synthesis, 66 (1988), pp. 132-137, thereby synthesizing Compound (3) or Compound (19), respectively.

[00118] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, esters, nitriles, amides, sulfoxides, sulfones and a mixture of these solvents. Toluene is more preferable.

[00119] Examples of bases include organic amines such as triethylamine and diisopropylethylamine; and pyridines such as pyridine, 2,6-lutidine and 4-picoline. Pyridine is preferred.

[00120] Examples of oxidizing agents include high valence iodine compounds such as [bis(acetoxy)iodo]benzene, [bis(trifluoroacetoxy)iodo]benzene, and iodosylbenzene, with [bis(trifluoroacetoxy)iodo]benzene being preferred . Chemical Structure 21 Step 16

[00121] Step 16: In a reaction solvent or without solvent, Compound 14 can be subjected to the Curtius rearrangement using Compound (18), diphenylphosphoryl azide and a base according to a method described, for example, in the Journal of the American Chemical Society, 94 (1972), pp. 6203-6205, thereby synthesizing Compound (3).

[00122] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, esters, nitriles, amides, sulfoxides, sulfones, water and a mixture of these solvents. Toluene is more preferable.

[00123] Examples of bases include organic amines such as triethylamine and diisopropylethylamine, with triethylamine being preferred. Chemical Structure 22 Step 17

[00124] Step 17: Compound (3) can be treated similarly to Step 11 to synthesize Compound (1). Chemical Structure 23 Step 18

[00125] Step 18: In a reaction solvent, Compound (19) can be deprotected with a deprotection reagent to synthesize Compound (1).

[00126] Preferred examples of the reaction solvents include aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, ketones, esters, nitriles, carboxylic acids, alcohols, amides, sulfoxides, sulfones, water and a mixture of these solvents. N,N-dimethylformamide is more preferable.

[00127] Examples of deprotection reagents include hydrofluoric acid; inorganic fluoride salts such as potassium fluoride; organic hydrofluoride salts such as pyridine hydrofluoride salt, triethylamine hydrofluoride salt, 1-hexadecane hydrofluoride salt; ammonium fluorides such as tetraethylammonium fluoride and tetrabutylammonium fluoride; and difluorotrimethylsilicate salts such as tris(dimethylamino)sulfonic difluorotrimethylsilicate. Tetrabutylammonium fluoride is preferred.

[00128] The Compound of formula (I') can be synthesized by treating the Compound of formula (I) according to Step 1 using M'n'+(OH-)n'.

[00129] The target compound produced in each reaction can be recovered from the reaction mixture liquid by a common method. In the case where, for example, the target compound is completely or partially precipitated, deposited or crystallized in the liquid reaction mixture, the solid containing the target compound can be obtained by filtration of the liquid reaction mixture. When the target compound is completely or partially dissolved in the liquid reaction mixture, the target compound can be obtained by removing the solvent (eg, by lyophilization) directly or after insoluble materials are removed by filtration. Alternatively, the target compound can be obtained by properly neutralizing the liquid reaction mixture, removing any insoluble materials by filtration, adding a water-immiscible solvent such as ethyl acetate, washing the liquid mixture with water, separating the organic phase containing the target compound, drying the organic phase with a desiccant such as anhydrous magnesium sulfate or anhydrous sodium sulfate, and evaporating the solvent by distillation.

[00130] Where necessary, the obtained target compound can be separated and purified by an appropriate combination of common methods such as washing with water, an organic solvent or a mixture of such solvents; recrystallization; reprecipitation; and methods commonly used for the separation and purification of organic compounds (for example, adsorption column chromatography methods using a carrier such as silica gel or alumina; ion exchange chromatography methods; column chromatography methods on normal phase and reversed phase (preferably high performance liquid chromatography) using silica gel or alkylated silica gel; and normal phase and reversed phase (preferably high performance liquid chromatography) column chromatography methods using a filler on which optically active molecules are attached or on which the silica gel is coated with the optically active molecules).

[00131] When the salts of the present invention represented by the general formula (I) are used as medicines, the salts themselves (as an ingredient) can be administered as such or can be administered orally or parenterally (such as intravenous administration, administration intramuscular administration, intraperitoneal administration, percutaneous administration, intratracheal administration, intracutaneous administration or subcutaneous administration) in the form of tablets, capsules, powders, syrups, granules, fine granules, pills, suspensions, emulsions, preparations for percutaneous absorption, suppositories, ointments, lotions, inhalants and products for injection, which are manufactured by mixing salts with excipients, pharmacologically appropriate diluents.

[00132] These preparations are manufactured by known methods using additives such as excipients, lubricants, binders, disintegrating agents, emulsifiers, stabilizers, flavoring agents and diluents,

[00133] Examples of excipients include organic excipients and inorganic excipients. Examples of the organic excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, α-starch and dextrin; cellulose derivatives such as crystalline cellulose; Arabic gum; dextran; and pullulan. Examples of inorganic excipients include anhydrous light silicic acid; and sulfate salts such as calcium sulfate.

[00134] Examples of lubricants include stearic acid; a metal salt of stearic acid such as calcium stearate and magnesium stearate; baby powder; colloidal silica; waxes such as beeswax and spermaceti wax; Boric acid; adipic acid; sulfate salts such as sodium sulfate; glycol; fumaric acid; sodium benzoate; D,L-leucine; Sodium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; and starch derivatives listed as excipients above.

[00135] Examples of binders include hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, macrogol and the compounds listed as excipients above.

[00136] Examples of disintegrants include low-substituted hydroxypropylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose and internally cross-linked calcium carboxymethylcellulose; cross-linked polyvinyl pyrrolidone; and chemically modified starch or cellulose derivatives such as carboxymethyl starch and sodium carboxymethyl starch.

[00137] Examples of emulsifiers include colloidal clays such as bentonite and bee gum; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; and anionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and sucrose fatty acid ester.

[00138] Examples of the stabilizers include p-hydroxybenzoate esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; acetic anhydride; and sorbic acid.

[00139] Examples of flavoring agents include sweeteners such as sodium saccharin and aspartame; acidulants such as citric acid, malic acid and tartaric acid; and flavors such as menthol, lemon extract and orange extract.

[00140] Diluents are compounds generally used for dilution. Examples thereof include lactose, mannitol, glucose, sucrose, calcium sulfate, hydroxypropylcellulose, microcrystalline cellulose, water, ethanol, polyethylene glycol, propylene glycol, glycerol, starch, polyvinylpyrrolidone and mixtures thereof.

[00141] The dose of the salts of the present invention represented by the general formula (I) may vary depending on conditions such as symptoms, age and body weight of patients. In the case of oral administration, the lower and upper limits of doses per administration may be 0.001 mg/kg (preferably 0.01 mg/kg) and 20 mg/kg (preferably 10 mg/kg), respectively. In the case of parenteral administration, the lower and upper limits of doses per administration may be 0.0001 mg/kg (preferably 0.0005 mg/kg) and 10 mg/kg (preferably 5 mg/kg), respectively. In both cases, the number of administrations for adults can be from 1 to 6 per day depending on the symptoms.

EXAMPLES

[00142] The present invention will be described in more detail below by presenting Examples (Examples 1 to 15), Reference Examples (Reference Examples 1 to 32), Test Examples (Test Examples 1 to 8) and Preparation Examples (1 to 3). These examples only serve to aid in understanding the present invention and are not intended to limit the scope of the present invention.

[00143] Of the properties in the Examples and Reference Examples, the Rf values are values measured with a thin layer chromatograph (Merck Co., silica gel plate TLC 60F254 (trade name)). Development solvents (in their proportion by volume) are described in parentheses.

[00144] The expression COOH column in silica gel column chromatography indicates the column prepared with silica gel Chromatorex (trademark) Q-PACK COOH by Fuji Silysia Chemical Ltda.

[00145] Ultrapure water from Wako Pure Chemical Industries, Toda was used. (214-01301).

[00146] In the case where a plurality of mass spectral values were obtained due to the presence of isotopes, only the smallest m/z value was described. DUIS in mass spectroscopy is the mixed ionization mode of ESI and APCI.

[00147] In chemical structures, Me indicates a methyl group unless otherwise specified. (Example 1) (R)-1-[4'-(5-chloro-3-{[(1 -phenylethoxy)carbonyl]amino}thiophen2-yl)-2'-methoxy-[1,1'-biphenyl] Sodium -4-yl]cyclopropanecarboxylate (Compound No. I-2) Chemical Structure 24

[00148] In an ice bath and while stirring is carried out, 2.00 ml (2.00 mmol) of a 1N aqueous solution of sodium hydroxide were added to a suspension of acetonitrile (80 ml) of 1.10 g (R)-1-[4'-(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy[1,00 mmol] acid (2.00 mmol) Synthesized 1'-biphenyl]-4-yl]cyclopropanecarboxylic acid in analogy to Reference Example 29. Then ultrapure water (6 ml) was added and the mixture was ultrasonicated to give a uniform solution which was then stirred at room temperature for 3 hours . A small amount of ultrapure water was subsequently added to the liquid reaction mixture. The solvent was removed by lyophilization and the residue was dried by heating under vacuum to give the title compound weighing 1.08 g (1.89 mmol, 95% yield) as a white solid. Mass spectrum (ESI+, m/z): 570 [M+1]+. 1H-NMR spectrum (400 MHz, DMSO-d6) δ: 9.44 (1H, brs), 7.43-7.22 (10H, m), 7.21-7.14 (2H, m), 7.08 (1H, dd, J = 7.8, 1.4 Hz), 5.75 (1H, q, J = 6.1 Hz), 3.73 (3H, s), 1.56-1 .38 (3H, m), 1.16 (2H, dd, J = 5.6, 2.6 Hz), 0.66 (2H, dd, J = 5.7, 2.7 Hz). (Example 2) (R)-1-[4'-(5-chloro-3-{[(1 -phenylethoxy)carbonyl]amino}thiophen2-yl)-2'-methoxy-[1,1'-biphenyl] Potassium -4-yl]cyclopropane carboxide (Compound No. I-4) Chemical Structure 25

[00149] While stirring was carried out, 0.500 ml (0.500 mmol) of a 1N aqueous potassium hydroxide solution was added to a suspension of acetonitrile (20 ml)-ultrapure water (1.5 ml) 275 mg (0.501 mmol) of (R)-1-[4'-(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy[1,1'-biphenyl] acid ]-4-yl]cyclopropanecarboxylic acid synthesized in analogy to Reference Example 29, thereby preparing a uniform solution. Then, ultrapure water (6 ml) was added and the mixture was ultrasonicated. The resulting liquid reaction mixture was left at room temperature for 30 minutes. A small amount of ultrapure water was added. The solvent was removed by lyophilization and the residue was dried by heating under vacuum to give the title compound weighing 235 mg (0.401 mmol, 80% yield) as a white solid. Mass spectrum (ESI+, m/z): 586 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.44 (1H, brs), 7.43-7.14 (12H, m), 7.08 (1H, dd, J = 7.8, 1.4 Hz), 5.75 (1H, q, J = 6.2 Hz), 3.73 (3H, s), 1.54-1.39 (3H, m), 1.11 (2H, s). dd, J = 5.8, 2.7 Hz), 0.60 (2H, dd, J = 5.8, 2.6 Hz). (Example 3) 1/2 Calcium (R)-1-[4'-(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy [1, 1'-biphenyl]-4-yl]cyclopropanecarboxylate (Compound No. I-6) Chemical Structure 26

[00150] 0.180 ml (0.090 mmol) of an aqueous solution of calcium acetate 0.5 M was added to a solution in ultrapure water (25 ml) of 101 mg (0.177 mmol) of (R)-1-[4' Sodium -(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy[1,1'-biphenyl]-4-yl]cyclopropanecarboxylate obtained from Example 1. The mixture was stirred at room temperature for 2 days. The resulting suspension was filtered through a membrane filter (Millipore). The residue was washed with ultrapure water and dried by heating under vacuum to give the title compound weighing 40.4 mg (0.071 mmol, 40% yield) as a white solid. Mass spectrum (ESI+, m/z): 1133 [2M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.42 (1H, brs), 7.41-7.25 (10H, m), 7.19-7.15 (2H, m), 7 .07 (1H, dd, J = 7.9, 1.3 Hz), 5.75 (1H, q, J = 6.2 Hz), 3.72 (3H, s), 1.54-1, 38 (3H, m), 1.38-1.22 (2H, m), 0.900.80 (2H, m). (Example 4) Sodium (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino)thiophen-2-yl]-2'- methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-8) Chemical Structure 27

[00151] In an ice bath and while stirring is carried out, 1.00 ml (1.00 mmol) of a 1N aqueous solution of sodium hydroxide were added to a suspension of acetonitrile (40 ml)-ultrapure water (3 mL) of 584 mg (1.00 mmol) of (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino) acid thiophen-2-yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 32, thereby preparing a uniform solution. The solution was stirred at room temperature for 30 minutes. A small amount of ultrapure water was subsequently added to the liquid reaction mixture. The solvent was removed by lyophilization and the residue was dried by heating under vacuum to give the title compound weighing 534 mg (0.882 mmol, 88% yield) as a white solid. Mass spectrum (ESI+, m/z): 606 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.56 (1H, brs), 7.36-7.14 (10H, m), 7.08 (1H, dd, J = 7.8, 1.4 Hz), 5.91 (1H, q, J = 6.6 Hz), 3.75 (3H, s), 1.61-1.36 (3H, m), 1.16 (2H, s). dd, J = 5.6, 2.8 Hz), 0.66 (2H, dd, J = 5.6, 2.6 Hz). (Example 5) Potassium (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino)thiophen-2-yl]-2'- methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-10) Chemical Structure 28

[00152] In an ice bath and while stirring, 0.500 ml (0.500 mmol) of a 1N aqueous potassium hydroxide solution were added to a uniform solution of acetonitrile (20 ml)-ultrapure water (1, 5 mL) of 292 mg (0.500 mmol) of (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino)thiophen-2 acid) -yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 32. The mixture was stirred at temperature for 1 hour. A small amount of ultrapure water was subsequently added to the liquid reaction mixture. The solvent was removed by lyophilization and the residue was dried by heating under vacuum to give the title compound weighing 276 mg (0.444 mmol, 89% yield) as a white solid. Mass spectrum (ESI+, m/z): 622 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.56 (1H, brs), 7.33-7.14 (10H, m), 7.08 (1H, dd, J = 7.8, 1.1 Hz), 5.91 (1H, q, J = 6.1 Hz), 3.75 (3H, s), 1.58-1.40 (3H, m), 1.12 (2H, s). dd, J = 5.7, 2.7 Hz), 0.61 (2H, dd, J = 5.6, 2.5 Hz). (Example 6) 1/2 Calcium (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino)thiophen-2-yl]- 2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-12) Chemical Structure 29

[00153] 0.170 ml (0.085 mmol) of a 0.5 M aqueous solution of calcium acetate was added to a uniform ultrapure water solution (25 ml) of 100 mg (0.165 mmol) of (R)-1-{4 '-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino)thiophen-2-yl]-2'-methoxy-[1,1'-biphenyl]- 4- Sodium yl}cyclopropanecarboxylate obtained in Example 4. The mixture was stirred at room temperature for 2 days. The resulting suspension was filtered through a membrane filter (Millipore). The residue was washed with ultrapure water and dried by heating under vacuum to give the title compound weighing 47.8 mg (0.079 mmol, 48% yield) as a pale yellow solid. Mass spectrum (ESI+, m/z): 1205 [2M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.55 (1H, brs), 7.38-7.14 (10H, m), 7.07 (1H, dd, J = 7.8, 1.6 Hz), 5.90 (1H, q, J = 6.4 Hz), 3.75 (3H, s), 1.60-1.40 (3H, m), 1.40-1, 20 (2H, m), 0.91-0.78 (2H, m). (Example 7) Sodium (R)-1-{4'-[3-({[ 1 -(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2'-methoxy-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-14) Chemical Structure 30

[00154] In an ice bath and while stirring is carried out, 2.00 ml (2.00 mmol) of a 1N aqueous solution of sodium hydroxide were added to a suspension of acetonitrile (80 ml)-ultrapure water (6 mL) of 1.13 g (2.00 mmol) of (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5 acid -fluorothiophen-2-yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 31. The mixture was ultrasonicated to give a uniform solution, which was left to stand at room temperature for 3 hours. A small amount of ultrapure water was subsequently added to the liquid reaction mixture. The solvent was removed by lyophilization and the residue was dried by heating under vacuum to give the title compound weighing 1.15 g (1.96 mmol, 98% yield) as a white solid. Mass spectrum (ESI+, m/z): 588 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.55 (1H, brs), 7.62-7.22 (9H, m), 7.14-7.20 (1H, m), 7 .07 (1H, dd, J = 7.8, 1.2 Hz), 6.84 (1H, d, J = 2.5 Hz), 6.00 (1H, q, J = 5.6 Hz) , 3.76 (3H, s), 1.59-1.35 (3H, m), 1.16 (2H, dd, J = 5.7, 2.7 Hz), 0.66 (2H, dd , J = 5.6, 2.6 Hz). (Example 8) Potassium (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2'-methoxy-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-16) Chemical Structure 31

[00155] At room temperature, 0.500 ml (0.500 mmol) of a 1N aqueous potassium hydroxide solution were added to a suspension of acetonitrile (20 ml)-ultrapure water (1.5 ml) with 284 mg (0.501 mmol) of (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2'-methoxy-[1, Synthesized 1'-biphenyl]-4-yl}cyclopropanecarboxylic acid in analogy to Reference Example 31. The mixture was ultrasonicated to give a uniform solution, which was then allowed to stand at room temperature for 1 hour. A small amount of ultrapure water was subsequently added to the liquid reaction mixture. The solvent was removed by lyophilization, and the residue was dried by heating under vacuum to give the title compound weighing 270 mg (0.447 mmol, 89% yield) as a white solid. Mass spectrum (ESI+, m/z): 604 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.56 (1H, brs), 7.60-7.21 (9H, m), 7.19-7.14 (1H, m), 7 .07 (1H, dd, J = 7.9, 1.4 Hz), 6.84 (1H, d, J = 2.4 Hz), 6.00 (1H, q, J = 6.4 Hz) , 3.76 (3H, s), 1.55-1.37 (3H, m), 1.12 (2H, dd, J = 5.8, 2.7 Hz), 0.61 (2H, dd , J = 5.8, 2.6 Hz). (Example 9) 1/2 Calcium (R)-1-{4'-[3-({[ 1 -(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2'- methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-18) Chemical Structure 32

[00156] At room temperature and while stirring was carried out, 0.500 ml (0.500 mmol) of a 1N aqueous solution of sodium hydroxide was added to a suspension of acetonitrile (20 ml)-ultrapure water (1.5 ml) with 282 mg (0.498 mmol) of (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2'-methoxy acid - [1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 31. The mixture was ultrasonicated to give a uniform solution. Then, 0.500 ml (0.085 mmol) of a 0.5M aqueous calcium acetate solution was added to the liquid reaction mixture and stirring was carried out at room temperature for 1 hour. Acetonitrile was distilled from the liquid reaction mixture and ultrapure water was added. The resulting mixture was stirred at room temperature for 18 hours. The resulting suspension was filtered and the residue was washed with ultrapure water and dried by heating under vacuum to give the title compound weighing 213 mg (0.365 mmol, 73% yield) as a white solid. Mass spectrum (ESI+, m/z): 1169 [2M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.54 (1H, brs), 7.58-7.25 (9H, m), 7.17 (1H, d, J = 1.3 Hz ), 7.07 (1H, dd, J = 7.8, 1.1 Hz), 6.84 (1H, d, J = 2.5 Hz), 6.00 (1H, q, J = 6, 5 Hz), 3.75 (3H, s), 1.57-1.38 (3H, m), 1.38-1.18 (2H, m), 0.90-0.79 (2H, m ). (Example 10) Sodium (R)-1-{4'-[5-chloro-3-({[ 1 -(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ 1, 1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-20) Chemical Structure 33

[00157] At room temperature and while stirring was carried out, 2.00 ml (2.00 mmol) of a 1N aqueous solution of sodium hydroxide were added to a uniform solution of acetonitrile (80 ml) with 1.05 g ( 2.00 mmol) of (R)-1-{4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ acid 2.00 mmol) Synthesized 1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid in analogy to Reference Example 28. The mixture was ultrasonicated and stirred at temperature for 4 hours. The resulting suspension was filtered and the residue was washed with the mother liquor and dried by heating under vacuum to give the title compound weighing 1.06 g (1.94 mmol, 97% yield) as a white solid. Mass spectrum (ESI+, m/z): 546 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.34 (1H, brs), 7.70-7.65 (2H, m), 7.57-7.40 (6H, m), 7 .35-7.30 (2H, m), 7.23-7.16 (1H, m), 7.167.08 (1H, m), 5.82 (1H, q, J = 6.4 Hz), 1.61-1.40 (3H, m), 1.18 (2H, dd, J = 5.8, 2.8 Hz), 0.68 (2H, dd, J = 5.7, 2.7 Hz). (Example 11) Potassium (R)-1-{4'-[5-chloro-3-({[ 1 -(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ 1, 1'-biphenyl]-4-yl}cyclopropanecarboxylic acid (Compound No. I-22) Chemical Structure 34

[00158] At room temperature and while stirring was carried out, 0.500 ml (0.500 mmol) of a 1N aqueous solution of potassium hydroxide were added to a uniform solution of acetonitrile (20 ml) with 262 mg (0.500 mmol) of acid ( R)-1-{4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[1,1'-biphenyl]- 4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 28. The mixture was ultrasonicated and stirred at room temperature for 3.5 hours. The resulting suspension was filtered through a membrane filter (Millipore). The residue was washed with the mother liquor and dried by heating under vacuum to give the title compound weighing 220 mg (0.392 mmol, 78% yield) as a white solid. Mass spectrum (ESI+, m/z): 562 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.34 (1H, brs), 7.71-7.64 (2H, m), 7.58-7.39 (6H, m), 7 .33-7.27 (2H, m), 7.24-7.16 (1H, m), 7.167.07 (1H, m), 5.82 (1H, q, J = 6.5 Hz), 1.61-1.41 (3H, m), 1.13 (2H, dd, J = 5.8, 2.7 Hz), 0.62 (2H, dd, J = 5.8, 2.7 Hz). Example 12) 1/2 Calcium (R)-1-{4'-[5-chloro-3-({[ 1 -(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-24) Chemical Structure 35

[00159] 0.190 ml (0.095 mmol) of a 0.5M aqueous solution of calcium acetate was added to a uniform solution of ultrapure water (20 ml)-acetonitrile (5 ml) of 104 mg (0.190 mmol) of (R) -1- {4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[1,1'-biphenyl]-4- Sodium yl}cyclopropanecarboxylate obtained in Example 10. The mixture was stirred at room temperature for 2 days. The resulting suspension was filtered through a membrane filter (Millipore). The residue was washed with small amounts of acetonitrile and ultrapure water and dried by heating under vacuum to give the title compound weighing 47.8 mg (0.079 mmol, 48% yield) as a white solid. Mass spectrum (ESI+, m/z): 1085 [2M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.32 (1H, brs), 7.74-7.64 (2H, m), 7.59-7.49 (5H, m), 7 .47-7.40 (1H, m), 7.40-7.34 (2H, m), 7.227.16 (1H, m), 7.16-7.08 (1H, m), 5.82 (1H, q, J = 6.4 Hz), 1.62-1.40 (3H, m), 1.40-1.20 (2H, m), 0.90-0.78 (2H, m ). (Example 13) Sodium (R)-1-{4'-[5-fluoro-3-({[ 1 -(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-26) Chemical Structure 36

[00160] In an ice bath and while stirring is carried out, 2.00 ml (2.00 mmol) of a 1N aqueous solution of sodium hydroxide were added to a suspension of acetonitrile (80 ml)-ultrapure water (6 ml) with 1.04 g (2.00 mmol) of (R)-1-{4'-[5-fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy] acid carbonyl}amino)thiophen-2-yl]-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 30. Acetonitrile (80 ml) and tetrahydrofuran (50 ml) were then added to the liquid reaction mixture and the resulting mixture was sonicated at room temperature for 30 minutes and stirred at room temperature for 25 hours. The resulting suspension was filtered and the residue dried by heating under vacuum to give the title compound weighing 760 mg (1.40 mmol, 70% yield) as a white solid. Mass spectrum (ESI+, m/z): 544 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.33 (1H, brs), 7.69-7.63 (2H, m), 7.54-7.39 (5H, m), 7 .35-7.29 (2H, m), 7.16 (1H, d, J = 1.9 Hz), 6.83 (1H, brs), 5.74 (1H, q, J = 6.5 Hz), 2.17 (3H, s), 1.59-1.43 (3H, m), 1.18 (2H, dd, J=5.8, 2.8 Hz), 0.68 (2H , dd, J = 5.8, 2.8 Hz). (Example 14) Potassium (R)-1-{4'-[5-fluoro-3-({[ 1 -(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-28) Chemical Structure 37

[00161] At room temperature and while stirring was carried out, 0.500 ml (0.500 mmol) of a 1N aqueous solution of potassium hydroxide were added to a uniform solution of acetonitrile (20 ml)-tetrahydrofuran (5 ml) of 260 mg (0.499 mmol) of (R)-1-{4'-[5-fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl] acid - [1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 30. The mixture was ultrasonicated and stirred at room temperature for 2 hours. The resulting suspension was filtered through a membrane filter (Millipore). The residue was washed with a small amount of acetonitrile and dried by heating under vacuum to give the title compound weighing 126 mg (0.226 mmol, 45% yield) as a white solid. Mass spectrum (ESI+, m/z): 560 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.34 (1H, brs), 7.69-7.63 (2H, m), 7.56-7.41 (5H, m), 7 .34-7.27 (2H, m), 7.16 (1H, d, J = 2.0 Hz), 6.83 (1H, brs), 5.74 (1H, q, J = 6.5 Hz), 2.17 (3H, brs), 1.57-1.45 (3H, m), 1.14 (2H, dd, J = 5.9, 2.8 Hz), 0.63 (2H , dd, J = 5.8, 2.6 Hz). (Example 15) 1/2 Calcium (R)-1-{4'-[5-fluoro-3-({[ 1 -(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl ]-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylate (Compound No. I-30) Chemical Structure 38

[00162] 0.190 ml (0.095 mmol) of an aqueous solution of calcium acetate 0.5M was added to a uniform solution of ultrapure water (25 ml) 103 mg (0.190 mmol) of (R)-1-{4'- Sodium [5-fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylate synthesized in analogy to Example 13. The mixture was stirred at room temperature for 2 days. The resulting suspension was filtered through a membrane filter (Millipore). The residue was washed with ultrapure water and dried by heating under vacuum to give the title compound weighing 27.7 mg (0.051 mmol, 27% yield) as a white solid. Mass spectrum (ESI+, m/z): 1081 [2M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.32 (1H, brs), 7.70-7.63 (2H, m), 7.57-7.31 (8H, m), 7 .14 (1H, d, J = 1.9 Hz), 6.82 (1H, brs), 5.74 (1H, q, J = 6.5 Hz), 2.17 (3H, s), 1 .61-1.41 (3H, m), 1.41-1.20 (2H, m), 0.91-0.78 (2H, m). [Reference Examples] (Reference Example 1) 2-bromothiophene-3-carboxylic acid tert-butyl ester Chemical Structure 39

[00163] In a nitrogen atmosphere, 7.6 ml (87 mmol) of oxalyl chloride were added dropwise to a solution in methylene chloride (70 ml) of 15 g (72 mmol) of 2-bromothiophene acid -3-carboxylic acid (Aldrich) and 0.60 ml (7.8 mmol) of N,N-dimethylformamide at room temperature while stirring. The mixture was stirred at temperature for 15 hours. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure. 2-Methyl-2-propanol (70 ml), 65 ml (372 mmol) of N,N-diisopropylethylamine and 0.90 g (7.4 mmol) of N,N-dimethylaminopyridine were added sequentially to the residue . In a nitrogen atmosphere, the resulting mixture was stirred for 2 hours while heating to 80°C. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure. Water was added to the residue and the mixture was extracted with toluene. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 100:0 to 90:10 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced to give the title compound weighing 12 g (32 mmol (71% by weight purity, 45% yield)) as a pale yellow oil. Mass spectrum (EI, m/z): 262 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.32 (1H, d, J = 5.8 Hz), 7.18 (1H, d, J = 5.8 Hz), 1.59 (9H, s).

[00164] The title compound was also synthesized as follows.

[00165] In an argon atmosphere, 1.80 g (9.70 mmol) of p-toluenesulfonyl chloride were added in small portions into a pyridine solution (9.6 ml) of 1.005 g (4.85 mmol) of 2-bromothiophene-3-carboxylic acid (Aldrich) in an ice bath while stirring. Then 0.46 ml (4.8 mmol) of 2-methyl-2-propanol was added. In an ice bath, the mixture was stirred for 2 hours. Stirring was then carried out at room temperature for 1 hour. Then 0.47 ml (5.0 mmol) of 2-methyl-2-propanol was added and the mixture was stirred at room temperature for 27 hours. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure. Ethyl acetate and a saturated aqueous sodium hydrogen carbonate solution were added to perform liquid separation. The organic phase was washed with saturated aqueous sodium hydrogencarbonate solution and subsequently with saturated brine. Subsequently, the organic phase was washed with a 5% by weight aqueous solution of potassium hydrogen sulfate and was washed with saturated brine again. The organic phase was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 99:1 to 94:6 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 1.22 g (4.64 mmol, 96% yield) as a pale yellow oil. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 7.63 (1H, d, J = 5.8 Hz), 7.28 (1H, d, J = 5.8 Hz), 1.53 ( 9H, s). (Reference Example 2) 2-Bromo-5-chlorothiophene-3-carboxamide Chemical Structure 40

[00166] In an argon atmosphere and while stirring is carried out, 8.70 g (65.2 mmol) of N-chlorosuccinimide were added in a solution of N,N-dimethylformamide (50 ml) of 4.48 g ( 21.7 mmol) of 2-bromothiophene-3-carboxamide (synthesized according to WO 10/036497). The mixture was stirred for 3 hours while heating was carried out at 60°C. After completion of the reaction, 50 ml of water and 100 ml of ethyl acetate were added to it in an ice bath. While stirring was carried out, 6.80 g (65.3 mmol) of sodium hydrogensulfite were added. The mixture was stirred at room temperature for 15 minutes. Afterwards, water was added to perform liquid separation. The organic phase was washed twice with 50 ml of a saturated aqueous sodium hydrogencarbonate solution. The organic phase was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was concentrated under reduced pressure to about half volume. Hexane was added to the resulting suspension and the mixture was sonicated. Subsequently the solid was collected by filtration, washed with hexane and dried under heating under vacuum to give the title compound weighing 3.64 g (15.1 mmol, 70% yield) as a white solid. Mass spectrum (DUIS+, m/z): 240 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 7.75 (1H, brs), 7.58 (1H, brs), 7.33 (1H, s).

[00167] The title compound was also synthesized as follows.

[00168] In a nitrogen atmosphere, 0.90 g (6.7 mmol) of N-chlorosuccinimide were added to a solution of N,N-dimethylformamide (16 ml) of 1.0 g (4.8 mmol ) of 2-bromothiophene-3-carboxylic acid (Aldrich) at room temperature while stirring is carried out. The mixture was stirred for 1 hour while heating at 80°C. After completion of the reaction, the liquid reaction mixture was allowed to cool. Water was added and the liquid was acidified by the addition of 2N hydrochloric acid. The mixture was extracted with ethyl acetate. The organic phase was washed sequentially with aqueous sodium hydrogensulfite solution and saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in methylene chloride (15 ml) and 0.80 ml (9.1 mmol) of oxalyl chloride was added dropwise to the solution, in a nitrogen atmosphere at 0°C while stirring was carried out. The temperature was raised to room temperature and the mixture stirred for 30 minutes. Then, 3.7 ml (48 mmol) of 28 wt% aqueous ammonia was added dropwise at room temperature while stirring was carried out and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, water was added to the liquid reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 69:31 to 48:52 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure and dried by heating under vacuum to give the title compound weighing 0.62 g (2.6 mmol, 53% yield) as a white solid. (Reference Example 3) 4-Bromo-1-iodo-2-methoxybenzene Chemical Structure 41

[00169] In an ice bath and while stirring was carried out, 0.75 g (11 mmol) of sodium nitrite were added to a solution of acetic acid (15 ml)-concentrated hydrochloric acid (1 ml) of 2, 0 g (9.0 mmol) of 4-bromo-2-methoxyaniline (Tokyo Chemical Industry Co., Ltd.) in such a way that the internal temperature does not exceed 10°C. The mixture was stirred at room temperature for 30 minutes. Then, the liquid reaction mixture was added dropwise to an aqueous solution of potassium iodide 1.0 g (30 mmol) in a hydrobromic acid 48 wt% aqueous solution (30 ml) at room temperature while stirring was carried out. . The resulting mixture was stirred at room temperature for 1 hour. After completion of the reaction, the liquid reaction mixture was added in small portions to an aqueous solution of sodium carbonate and methylene chloride. After the alkalinization of the aqueous phase was confirmed, the liquid was separated. The organic phase was washed sequentially with 10% by weight aqueous sodium hydrogensulfite solution, saturated aqueous sodium hydrogencarbonate solution and saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 100:0 to 91:9 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure. give the title compound weighing 2.2 g (7.2 mmol, 73% yield) as an orange solid. Mass spectrum (EI, m/z): 312 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.61 (1H, d, J = 8.3 Hz), 6.94 (1H, d, J = 2.1 Hz), 6.87 (1H, dd, J = 8.2, 2.1 Hz), 3.88 (3H, s). (Reference Example 4) 1-(4'-Bromo-2'-methoxy-[1,1'-biphenyl]-4-yl)cyclopropanecarboxylic acid ethyl ester Chemical Structure 42

[00170] A solution of 1,4-dioxane (15 ml)-water (10 ml) with 1.2 g (3.8 mmol) of 4-bromo-1-iodo-2-methoxybenzene synthesized analogously to the Reference Example 3.1.1 g (3.5 mmol) 1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]cyclopropanecarboxylic acid ethyl ester (synthesized according to the procedure described in WO 12/078593) and 1.1 g (10 mmol) of sodium carbonate was degassed and purged with nitrogen. Then, 0.10 g (0.12 mmol) of methylene chloride [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride adduct was added. The mixture was stirred under a nitrogen atmosphere for 1.5 hours while heating to 80°C. After completion of the reaction, water was added to the liquid reaction mixture and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 94:6 to 75:25 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure and dried by heating under vacuum to give the title compound weighing 0.72 g (1.9 mmol, 55% yield) as a white solid. Mass spectrum (EI, m/z): 374 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.45-7.41 (2H, m), 7.39-7.35 (2H, m), 7.19 (1H, d, J = 8, 0 Hz), 7.15 (1H, dd, J = 8.0, 1.8 Hz), 7.10 (1H, d, J = 1.8 Hz), 4.12 (2H, q, J = 7.1 Hz), 3.81 (3H, s), 1.61 (2H, dd, J = 7.0, 4.0 Hz), 1.22 (2H, dd, J = 7.0, 4 .0 Hz), 1.19 (3H, t, J = 7.1 Hz). (Reference Example 5) 1-(4'-chloro-2'-methoxy-[1,1'-biphenyl]-4-yl)cyclopropanecarboxylic acid ethyl ester Chemical Structure 43

[00171] A solution of 1,4-dioxane (20 ml)-water (20 ml) of 2.0 g (9.0 mmol) of 1-bromo-4-chloro-2-methoxybenzene (Tokyo Chemical Industry Co. , Ltd.), 2.6 g (8.2 mmol) 1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl] acid ethyl ester carboxylic cyclopropane (synthesized according to the process described in WO 12/078593) and 2.7 g (25 mmol) of sodium carbonate was degassed and purged with nitrogen. Then, 0.21 g (0.25 mmol) of [1,1'-bis(diphenylphosphino)ferrocene] palladium(II) chloride methylene dichloride adduct was added. The mixture was stirred in a nitrogen atmosphere for 2 hours while heating at 80°C. After completion of the reaction, water was added to the liquid reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, and the fraction with Rf = 0.5 (development solvent: hexane:ethyl acetate = 90:10 (V/V)) was concentrated under reduced pressure. and dried by heating under vacuum to give the title compound weighing 2.46 g (7.4 mmol, 90% yield) as a white solid. Mass spectrum (EI, m/z): 330 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.45-7.41 (2H, m), 7.39-7.35 (2H, m), 7.25 (1H, d, J = 8, 2 Hz), 7.00 (1H, dd, J = 8.2, 2.0 Hz), 6.96 (1H, d, J = 2.0 Hz), 4.12 (2H, q, J = 7.1 Hz), 3.81 (3H, s), 1.61 (2H, dd, J = 6.9, 3.9 Hz), 1.22 (2H, dd, J = 7.0, 4 .0 Hz), 1.19 (3H, t, J = 7.1 Hz). (Reference Example 6) 1-[2'-Methoxy-4'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[ 1, 1'-biphenyl]-4yl]cyclopropanecarboxylic acid Chemical Structure 44

[00172] A solution of 1,4-dioxane (10 ml) 0.72 g (1.9 mmol) of 1-(4'-bromo-2'-methoxy-[1,1'-biphenyl) acid ethyl ester ]-4-yl)cyclopropanecarboxylic acid synthesized analogously to Reference Example 4, 0.60 g (2.4 mmol) of 4,4,4',4',5,5,5',5'-octamethyl- 2,2'-bi(1,3,2-dioxaborolane) and 0.30 g (3.1 mmol) of potassium acetate was degassed and purged with nitrogen. Then, 0.10 g (0.12 mmol) methylene chloride [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride adduct was added. In a nitrogen atmosphere, the mixture was stirred for 3 hours while heating was carried out under reflux conditions. After completion of the reaction, water was added to the liquid reaction mixture, and the mixture was extracted with toluene. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 92:8 to 79:21 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure and dried by heating under vacuum to give the title compound weighing 0.81 g (1.9 mmol, quantitative yield) as a pale yellow solid. Mass spectrum (EI, m/z): 422 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.52-7.46 (3H, m), 7.40-7.33 (4H, m), 4.12 (2H, q, J = 7, 1 Hz), 3.86 (3H, s), 1.60 (2H, dd, J = 6.9, 3.9 Hz), 1.36 (12H, s), 1.22 (2H, dd, J = 7.0, 4.0 Hz), 1.19 (3H, t, J = 7.1 Hz).

[00173] The title compound was also synthesized as follows.

[00174] A 1,4-dioxane solution (30 ml) of 2.46 g (7.43 mmol) of 1-(4'-chloro-2'-methoxy-[1,1'-) acid ethyl ester biphenyl]-4-yl)cyclopropanecarboxylic acid synthesized analogously to Reference Example 5, 2.43 g (9.57 mmol) of 4,4,4',4',5,5,5',5'-octamethyl -2,2'-bi(1,3,2-dioxaborolane) and 1.1 g (11 mmol) of potassium acetate was degassed and purged with nitrogen. Then, 0.30 g (0.37 mmol) of methylene chloride [1,1'-bis(diphenylphosphino)ferrocene]palladium (II) dichloride adduct and 0.30 g (1.1 mmol) of tricyclohexylphosphine were added. The mixture was stirred under a nitrogen atmosphere for 24 hours while heating under reflux conditions. Then 0.15 g (0.18 mmol) of methylene chloride [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride adduct and 0.15 g (0.54 mmol) of tricyclohexylphosphine were added to the liquid reaction mixture and the mixture was stirred under a nitrogen atmosphere for 5 hours while heating was carried out under reflux conditions. After completion of the reaction, the liquid reaction mixture was cooled to room temperature. Toluene was added and insoluble materials were filtered off. The filtrate was washed sequentially with a saturated aqueous sodium hydrogencarbonate solution and saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 92:8 to 79:21 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure. . Hexane was added to the residue. The solid was collected by filtration, washed with hexane and dried under heating under vacuum to give the title compound weighing 1.89 g (4.5 mmol, 60% yield) as a white solid. (Reference Example 7) 1-[4'-(3-Carbamoyl-5-chlorothiophen-2-yl)-[1,1'-biphenyl]-4-yl]cyclopropanecarboxylic acid ethyl ester Chemical Structure 45

[00175] A solution of 1,4-dioxane (15 ml)-water (5 ml) of 486 mg (2.02 mmol) of 2-bromo-5-chlorothiophene-3-carboxamide synthesized in analogy to Reference Example 2 , 877 mg (2.23 mmol) 1-[4'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1'- acid ethyl ester) carboxylic biphenyl]-4yl]cyclopropane (synthesized according to the procedure described in WO 12/078593) and 658 mg (6.21 mmol) of sodium carbonate was degassed frozen in a dry ice/acetone bath and purged with argon. Subsequently, 230 mg (0.199 mmol) of tetracis(triphenylphosphino)palladium(0) was added and the mixture was stirred for 3 hours while heating at 90°C. After completion of the reaction, the liquid reaction mixture was cooled. Ethyl acetate and water were added to perform liquid separation. The organic phase was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (eluting solvent: hexane: ethyl acetate = 64:36 to 43:57 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced. A solution of hexane-ethyl acetate (2:1 (V/V)) was added to the residue and the precipitated solid was collected by filtration and dried by heating under vacuum to give the title compound weighing 705 mg (1.66 mmol, 82% yield) as a white solid. Mass spectrum (EI, m/z): 425 [M]+. 1H-NMR Spectrum (400MHz, CDCl3) δ: 7.69-7.65 (2H, m), 7.58-7.53 (4H, m), 7.46-7.42 (2H, m) , 7.33 (1H, s), 5.44 (2H, brs), 4.12 (2H, q, J = 7.1 Hz), 1.65 (2H, dd, J = 7.0, 4 .0 Hz), 1.23 (2H, dd, J = 7.0, 4.0 Hz), 1.19 (3H, t, J = 7.1 Hz). (Reference Example 8) 1-[4'-(3-Carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'-biphenyl]-4yl]cyclopropanecarboxylic acid ethyl ester Chemical Structure 46

[00176] A solution of 1,4-dioxane (30 ml)-water (10 ml) of 2.0 g (4.7 mmol) of 1-[2'-methoxy-4'-(4 ,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1'-biphenyl]-4-yl]cyclopropanecarboxylic acid synthesized in analogy to Reference Example 6, 1.25 g ( 5.2 mmol) of 2-bromo-5-chlorothiophene-3-carboxamide synthesized in analogy to Reference Example 2 and 1.5 g (14 mmol) of sodium carbonate was degassed. Then, 0.30 g (0.26 mmol) of tetracis(triphenylphosphino)palladium(0) was added. The mixture was stirred under a nitrogen atmosphere for 4.5 hours while heating to 90°C. After completion of the reaction, the liquid reaction mixture was allowed to cool. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Ethyl acetate was added to the residue. The solid was collected by filtration, washed with a small amount of ethyl acetate and dried by heating under vacuum to give the title compound weighing 1.53 g (3.4 mmol, 71% yield) as a white solid. Mass spectrum (EI, m/z): 455 [M]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 7.73 (1H, brs), 7.50 (1H, brs), 7.48-7.43 (2H, m), 7.39-7 .33 (3H, m), 7.32 (1H, s), 7.25 (1H, d, J = 1.6 Hz), 7.13 (1H, dd, J = 7.8, 1.6 Hz), 4.05 (2H, q, J = 7.1 Hz), 3.79 (3H, s), 1.51 (2H, dd, J = 6.8, 4.0 Hz), 1, 23 (2H, dd, J = 7.0, 4.0 Hz), 1.12 (3H, t, J = 7.1 Hz). (Reference Example 9) 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-[1,1'-biphenyl]-4-yl}thiophene-3-carboxylic acid tert-butyl ester Chemical Structure 47

[00177] To a solution of 1,4-dioxane (15 ml)-water (5 ml) of 0.80 g (2.0 mmol) of acid ethyl ester 1-[4'-(4,4,5 carboxylic ,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1'-biphenyl]-4-yl]cyclopropane (synthesized according to the process described in WO 12/078593), 0.50 g (1.9 mmol) of 2-bromothiophene-3-carboxylic acid tert-butyl ester synthesized in analogy to Reference Example 1 and 0.61 g (5.8 mmol) of sodium carbonate was degassed. Then, 0.10 g (0.089 mmol) of tetracis(triphenylphosphino)palladium(0) was added. The mixture was stirred under a nitrogen atmosphere for 14.5 hours while heating at 90°C. After completion of the reaction, the liquid reaction mixture was allowed to cool. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and the fraction having Rf = 0.41 (developing solvent: hexane:ethyl acetate = 90:10 (V/V)) was concentrated under reduced pressure and dried by heating under vacuum to give the title compound weighing 0.58 g (1.3 mmol, 68% yield) as a white solid. Mass spectrum (EI, m/z): 448 [M]+. 1H-NMR Spectrum (400MHz, CDCl3) δ: 7.64-7.60 (2H, m), 7.59-7.55 (2H, m), 7.55-7.51 (2H, m) , 7.48 (1H, d, J = 5.4 Hz), 7.45-7.41 (2H, m), 7.23 (1H, d, J = 5.3 Hz), 4.13 ( 2H, q, J = 7.1 Hz), 1.64 (2H, dd, J = 6.9, 3.9 Hz), 1.38 (9H, s), 1.23 (2H, dd, J = 7.2, 4.1 Hz), 1.20 (3H, t, J = 7.2 Hz). (Reference Example 10) 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-2-methoxy-[1,1'-biphenyl]-4-yl}thiophene-3-carboxylic acid tert-butyl ester Structure Chemistry 48

[00178] 2.96 g (27.9 mmol) of sodium carbonate was added to a solution of 1,4-dioxane (23 ml)-water (23 ml) of 3.81 g (9.02 mmol) of 1-[2'-Methoxy-4'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-[1,1'-biphenyl]-4-acid ethyl ester yl]cyclopropanecarboxylic acid synthesized in analogy to Reference Example 6 and 2.7 g (7.3 mmol (purity 71% by weight)) of 2-bromothiophene-3-carboxylic acid tert-butyl ester synthesized in Reference Example 1 The mixture was degassed. Then, 540 mg (0.467 mmol) of tetracis(triphenylphosphino)palladium(0) was added. The resulting mixture was stirred under a nitrogen atmosphere for 7 hours while heating to 90°C. After completion of the reaction, the liquid reaction mixture was allowed to cool. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Ethyl acetate was added to the residue. The resulting insoluble materials were filtered off and washed with a solution of hexane-ethyl acetate mixture (1:2 (V/V)). Subsequently, the mother liquor and washings were combined and concentrated under reduced pressure. The concentrate was dissolved in ethyl acetate and hexane was added until the solution became cloudy. The precipitated solid was filtered, washed with hexane and dried by heating under vacuum to give the title compound weighing 3.09 g (5.55 mmol (86 wt% purity, 61% yield) as a white solid. Mass spectrum (EI, m/z): 478 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.53-7.48 (2H, m), 7.47 (1H, d, J = 5.4 Hz), 7.41-7.36 (2H , m), 7.34 (1H, d, J = 7.8 Hz), 7.23 (1H, d, J = 5.4 Hz), 7.12 (1H, dd, J = 7.7, 1.6 Hz), 7.07 (1H, d, J = 1.6 Hz), 4.13 (2H, q, J = 7.1 Hz), 3.83 (3H, s), 1.62 (2H, dd, J = 6.8, 4.0 Hz), 1.40 (9H, s), 1.23 (2H, dd, J = 6.8, 3.8 Hz), 1.20 ( 3H, t, J = 7.1 Hz). (Reference Example 11) 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid tert-butyl ester Chemical Structure 49

[00179] In an argon atmosphere, 11 ml (12.0 mmol) of a 1.09 M solution of tetrahydrofuranhexane, lithium diisopropylamide (Kanto Chemical Co., Inc.) was added dropwise over a period 5 minutes to a dehydrated tetrahydrofuran solution (60 ml) of 4.50 g (10.0 mmol) of 2-{4'-[1-(ethoxycarbonyl)cyclopropyl]-[1] acid tert-butyl ester Synthesized ,1'-biphenyl]-4-yl}thiophene-3-carboxylic acid in analogy to Reference Example 9 while the system was cooled to -70°C or below in a dry ice-acetone bath. The mixture was stirred at room temperature for 30 minutes. Then, while freezing to -65°C or below, a tetrahydrofuran solution (15 mL) of 4.75 g (15.1 mmol) of N-fluorobenzenesulfonimide was added dropwise over a period of time. 5 minutes and the mixture was stirred at room temperature for 30 minutes. Then, the temperature was gradually raised and the reaction was stopped at -45°C by adding 40 ml of a saturated aqueous ammonium chloride solution. The temperature was raised to room temperature and the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. Methylene chloride was added to the residue. Insoluble materials were filtered off and the filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (eluting solvent: hexane:ethyl acetate = 100:0 to 79:21 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure and dried by heating under vacuum to give the title compound weighing 2.23 g (4.78 mmol, 48% yield) as a white solid. Mass spectrum (CI, m/z): 467 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 7.78-7.72 (2H, m), 7.687.63 (2H, m), 7.56-7.50 (2H, m), 7 .46-7.41 (2H, m), 7.03 (1H, d, J = 2.4 Hz), 4.05 (2H, q, J = 7.1 Hz), 1.52 (2H, dd, J = 6.9, 3.9 Hz), 1.31 (9H, s), 1.24 (2H, dd, J = 7.1, 4.1 Hz), 1.12 (3H, t , J = 7.1 Hz). (Reference Example 12) 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-2-methoxy-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3- tert -butyl ester carboxylic Chemical Structure 50

[00180] In an argon atmosphere, 6.56 ml (7.22 mmol) in a 1.1 M solution of lithium diisopropylamide/tetrahydrofuran were added dropwise to a solution in tetrahydrofuran (37 ml ) of 2.88 g (5.17 mmol (purity 86% by weight)) of 2-{4'-[1-(ethoxycarbonyl)cyclopropyl]-2-methoxy-[1,1') tert-butyl ester -biphenyl]-4-yl}thiophene-3-carboxylic acid synthesized in Reference Example 10, at -78°C while stirring was carried out. The mixture was stirred at room temperature for 30 minutes. Then a tetrahydrofuran solution (9.5 ml) of 2.85 g (9.04 mmol) of N-fluorobenzenesulfonimide was added dropwise and the mixture was stirred at temperature for 30 minutes. After completion of the reaction, a saturated aqueous solution of ammonium chloride and ethyl acetate was added to the liquid reaction mixture. The organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:dichloroethane = 100:0 to 30:70 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure. Hexane was added to the residue and the mixture was heated to give a solution, which was sonicated. The precipitated solid was collected by filtration, washed with hexane and dried by heating under vacuum to give the title compound weighing 496 mg (1.00 mmol, 20% yield) as a white solid. Mass spectrum (DUIS+, m/z): 497 [M+1]+. 1H-NMR mass spectrum (400MHz, CDCl3) δ: 7.52-7.46 (2H, m), 7.41-7.36 (2H, m), 7.33 (1H, d, J = 7 .8 Hz), 7.08 (1H, dd, J = 7.8, 1.6 Hz), 7.03 (1H, d, J = 1.5 Hz), 6.84 (1H, d, J = 2.3 Hz), 4.13 (2H, q, J = 7.1 Hz), 3.82 (3H, s), 1.62 (2H, dd, J = 6.9, 3.9 Hz ), 1.37 (9H, s), 1.23 (2H, dd, J = 6.5, 3.5 Hz), 1.20 (3H, t, J = 7.2 Hz). (Reference Example 13) 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid Chemical Structure 51

[00181] In an argon atmosphere, 5.0 ml (65 mmol) of trifluoroacetic acid was added to a solution in methylene chloride (20 ml) of 2.18 g (4.67 mmol) of tert-butyl ester of 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid synthesized in analogy to Reference Example 11, in a water bath ice while stirring. The mixture was stirred at room temperature for 1 hour and then at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was washed sequentially with diethyl ether and hexane and dried by heating under vacuum to give the title compound weighing 1.87 g (4.56 mmol, 98% yield) as a white solid, Mass spectrum (CI, m/z): 411 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 12.92 (1H, s), 7.74-7.70 (2H, m), 7.68-7.64 (2H, m), 7 .60-7.55 (2H, m), 7.46-7.41 (2H, m), 7.05 (1H, d, J = 2.4 Hz), 4.05 (2H, q, J = 7.1 Hz), 1.52 (2H, dd, J = 6.9, 3.9 Hz), 1.24 (2H, dd, J = 7.0, 4.1 Hz), 1.11 (3H, t, J = 7.1 Hz). (Reference Example 14) 2-{4'-[1-(Ethoxycarbonyl)cyclopropyl]-2-methoxy-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid Structure Chemistry 52

[00182] In an atmosphere of argon at room temperature, 1.1 ml (14 mmol) of trifluoroacetic acid were added to the methylene chloride solution (4.4 ml) of 491 mg (0.989 mmol) of tert-butyl ester of 2-{4'-[1-(ethoxycarbonyl)cyclopropyl]-2-methoxy-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid synthesized in analogy to Example Reference 12. The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure. Methylene chloride was added and the mixture was concentrated under reduced pressure. Hexane was added to the residue and the mixture was concentrated under reduced pressure and dried by heating under vacuum to give the title compound weighing 436 mg (0.99 mmol, quantitative yield) as a white solid. Mass spectrum (EI, m/z): 440 [M]+. 1H-NM R spectrum (400MHz, CDCl3) δ: 7.53-7.48 (2H, m), 7.40-7.37 (2H, m), 7.34 (1H, d, J = 7 .8 Hz), 7.14 (1H, d, J = 1.6 Hz), 7.13-7.10 (1H, m), 6.94 (1H, d, J = 2.3 Hz), 4.12 (2H, q, J = 7.1 Hz), 3.82 (3H, s), 1.62 (2H, dd, J = 6.9, 3.9 Hz), 1.23 (2H , dd, J = 7.0, 4.0 Hz), 1.20 (3H, t, J = 7.1 Hz). (Reference Example 15) 1-[4'-(3-Carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'-biphenyl]-4yl]cyclopropanecarboxylic acid Chemical Structure 53

[00183] At room temperature and while stirring was carried out, 6.6 ml (6.6 mmol) of a 1N aqueous solution of sodium hydroxide was added to a suspension of ethanol (10 ml)-tetrahydrofuran (10 ml ) of 1.00 g (2.19 mmol) of 1-[4'-(3-carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'-biphenyl] acid ethyl ester Synthesized -4yl]cyclopropanecarboxylic acid in analogy to Reference Example 8. The mixture was stirred at temperature for 4 days. After completion of the reaction, the liquid reaction mixture was neutralized by adding 6.6 ml (6.6 mmol) of 1N hydrochloric acid. Water was added and the precipitated solid was filtered through a membrane filter (Millipore), washed with water, and dried by heating under vacuum to give the title compound weighing 884 mg (2.07 mmol, 94% yield) as a white solid. Mass spectrum (DUIS+, m/z): 428 [M+1]+. 1H-NMR mass spectrum (400MHz, DMSO-d6) δ: 12.35 (1H, brs), 7.75 (1H, brs), 7.52 (1H, brs), 7.45-7.41 ( 2H, m), 7.38-7.35 (2H, m), 7.33 (1H, d, J = 7.9 Hz), 7.32 (1H, s), 7.24 (1H, d , J = 1.8 Hz), 7.13 (1H, dd, J = 7.8, 1.6 Hz), 3.79 (3H, s), 1.47 (2H, dd, J = 6, 7, 3.8 Hz), 1.18 (2H, dd, J = 6.9, 3.9 Hz). (Reference Example 16) 1-[4'-(3-Carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'-biphenyl]-4yl] acid 2-(trimethylsilyl)ethyl ester Cyclopropanecarboxylic Chemical Structure 54

[00184] Toluene was added to 0.80 g (1.9 mmol) of 1-[4'-(3-carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'- biphenyl]-4-yl]cyclopropanecarboxylic acid synthesized in Reference Example 15. After azeotropic dehydration was performed, the atmosphere was replaced with argon. Subsequently, N,N-dimethylformamide (10 ml), 23.0 mg (0.188 mmol) of N,N-dimethylaminopyridine, 0.42 ml (2.8 mmol) of trimethylsilylethanol and 0.98 ml (5 .6 mmol) of N,N-diisopropylethylamine. Then, in an ice bath, 1.06 g (2.81 mmol) of o-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate salt was added. The mixture was stirred at room temperature for 18 hours. Subsequently, 64.6 mg (0.529 mmol) of N,N-dimethylaminopyridine was added to the liquid reaction mixture and the mixture was stirred at temperature for 1 day. After completion of the reaction, water and ethyl acetate were added to the liquid reaction mixture. Extraction was performed twice with ethyl acetate. The organic phase was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (eluting solvent: hexane:ethyl acetate = 71:29 to 50:50 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced and dried by heating under vacuum to give the title compound weighing 889 mg (1.68 mmol, 90% yield) as a pale yellow solid. Mass spectrum (DUIS+, m/z): 528 [M+1]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 7.75 (1H, brs), 7.52 (1H, brs), 7.46-7.42 (2H, m), 7.38-7 .34 (2H, m), 7.33 (1H, d, J = 7.8 Hz), 7.32 (1H, s), 7.24 (1H, d, J = 1.6 Hz), 7 .13 (1H, dd, J = 7.8, 1.7 Hz), 4.14-4.07 (2H, m), 3.78 (3H, s), 1.49 (2H, dd, J = 6.8, 3.8 Hz), 1.22 (2H, dd, J = 7.0, 4.1 Hz), 0.91-0.84 (2H, m), -0.05 (9H , s). (Reference Example 17) 1-(4-Methylthiophen-3-yl)ethanone Chemical Structure 55

[00185] In an argon atmosphere at -78°C, 4.0 ml (6.4 mm mol) of a 1.6 M solution in hexane of n-butyl lithium were added dropwise to a solution of diethyl ether ( 23 ml) of 1.0 g (5.3 mmol) of 3-bromo-4-methylthiophene (Tokyo Chemical Industry Co., Ltd.). The mixture was stirred at room temperature for 15 minutes. Then, a diethyl ether solution (1 ml) of 0.70 ml (6.9 mmol) of N-methoxy-N-methylacetamide was added dropwise at -78°C and the mixture was stirred at room temperature for 15 minutes and at room temperature for 23 hours. After completion of the reaction, a saturated aqueous ammonium chloride solution was added to the liquid reaction mixture, and the resulting mixture was extracted with ethyl acetate. The organic phase was washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 80:20 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 552 mg (3.94 mmol, 75% yield) as a pale yellow oil. Mass spectrum (CI, m/z): 141 [M+1]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.99 (1H, d, J = 3.1 Hz), 6.91 (1H, dq, J = 3.1, 1.0 Hz), 2, 53 (3H, s), 2.46 (3H, d, J = 1.0 Hz). (Reference Example 18) (RS)-1-(2,5-difluorophenyl)ethanol Chemical Structure 56

[00186] While stirring was carried out, 10 g (260 mmol) of sodium borohydride was added to an ethanol solution (200 ml) of 39.0 g (250 mmol) of 1-(4-fluoro) -2-methylphenyl)ethanone (a combination of products from Wako Pure Chemical Industries, Ltd. and Tokio Chemical Industri Co., Ltd.). The mixture was stirred at room temperature for 30 minutes. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure. Water was added and the resulting mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 90:10 to 69:31 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 41.5 g (243 mmol, 97% yield) as a colorless oil 1H-NMR spectrum (400MHz, CDCl3) δ: 7.25-7.19 (1H, m) , 7.01-6.87 (2H, m), 5.22-5.14 (1H, m), 1.88 (1H, d, J = 4.3 Hz), 1.50 (3H, d , J = 6.4 Hz). (Reference Example 19) (R)-1-(thiophen-3-yl)ethanol Chemical Structure 57

[00187] According to the procedure described in the Journal of Organic Chemistry, 72 (2007) pp. 1639-1644, 0.446 g (1.61 mmol) of (S)-5,5-diphenyl-2-methyl-3,4-propane-1,3,2-oxazaborolidine (Aldrich) was added to the tetra- hydrofuran (100 mL) of 2.023 g (16.03 mmol) of 1-(thiophen-3-yl)ethanone (Aldrich) dried with Molecular Sieves 4A 1/16 (trademark, Wako Pure Chemical Industries, Ltd.) in a argon atmosphere at room temperature while stirring was carried out. Then, while the temperature was controlled at around -30°C in a dry ice-ethanol bath and while stirring was carried out, 19.0 ml (17.1 mmol) of borane-tetrahydrofuran complex 0.9 M (Tokio Chemical Industri Co., Ltd.) was added dropwise over a period of 1 hour. The mixture was stirred at about -30°C for 1 hour. After completion of the reaction, 50 ml of water were added and subsequently 100 ml of ethyl acetate and 5 ml of 1N hydrochloric acid were added to perform liquid separation. The organic phase was washed with 50 ml of saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 95:5 to 74:26 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 1.81 g (14.1 mmol, 88% yield, optical purity 82.9% ee) as a colorless oil. Analysis of Optical Purity Conditions Column: CHIRALCEL OJ-RH (trade name, Daicel Corporation) Size: 0.46 cm I.D. x 25 cm L. Mobile phase: trifluoroacetic acid/acetonitrile aqueous solution 0.03% by volume = 75/25 (V/V) Flow rate: 1.0 ml/min. Temperature: 40°C Wavelength: 254 nm 1H-NMR Spectrum (400MHz, DMSO-d6) δ: 7.44 (1H, dd, J = 5.0, 3.0 Hz), 7.25 (1H , ddd, J = 3.0, 1.2, 0.9 Hz), 7.07 (1H, dd, J = 5.0, 1.2 Hz), 5.11 (1H, d, J = 4 .8 Hz), 4.80-4.71 (1H, m), 1.34 (3H, d, J = 6.4 Hz).

[00188] The title compound can be obtained with an increased optical purity in the following manner.

[00189] At room temperature and while stirring was carried out, 13 g of Lipase PS Amano SD (Wako Pure Chemical Industries, Ltd.) were added to a solution of diisopropyl ether (200 ml) of 13.1 g (102 mmol, increased optical purity 69% ee) of (R)-1-(thiophen-3-yl)ethanol synthesized in analogy to Reference Example 19 and 15.0 ml (163 mmol) of vinyl acetate. The liquid reaction mixture was stirred at 45°C for 6.5 hours and filtered. The filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 80:20 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure. to give (R)-1-(thiophen-3-yl)ethyl acetic acid ester weighing 11.8 g (67 mmol, 65% yield, increased optical purity >99% ee) as a pale yellow oil. Analysis of Optical Purity Conditions Column: CHIRALPAK IA (trade name, Daicel Corporation) Size: 0.46 cm I.D. x 25 cm L. Mobile phase: hexane:2-propanol = 99.5:0.5 (V/V) Flow rate: 1.0 ml/min. Temperature: 40°C Wavelength: 254 nm 1H-NMR Spectrum (400MHz, CDCl3) δ: 7.30 (1H, dd, J = 5.0, 2.9 Hz), 7.25-7.22 (1H, m), 7.09 (1H, dd, J = 5.0, 1.3 Hz), 6.00 (1H, q, J = 6.6 Hz), 2.07 (3H, s) , 1.56 (3H, d, J = 6.5 Hz).

[00190] Under a stream of nitrogen, 2.50 g (104 mmol) of lithium hydroxide was added to an ethanol (100 ml)-water (10 ml) solution of 11.8 g (R)-acid ester 1-(thiophen-3-yl)ethyl acetic acid obtained above (67.1 mmol, increased optical purity > 99% ee) at room temperature while stirring was carried out. The mixture was stirred at temperature for 1.5 hours. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure to remove ethanol. Water was added to the residue and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 95:5 to 74:26 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure. to give the title compound weighing 8.1 g (63 mmol, 95% yield, increased optical purity > 99.0% ee) as a pale yellow oil. (Reference Example 20) (R)-1-(4-methylthiophen-3-yl)ethanol Chemical Structure 58

[00191] In an argon atmosphere at -30°C to -27°C, 3.4 ml (3.1 mmol) of 0.9 M borane-tetrahydrofuran complex was added dropwise to a solution of tetra -hydrofuran (1.0 ml) from 78 mg (0.28 mmol) of (S)-5,5-diphenyl-2-methyl-3,4-propane-1,3,2-oxazaborolidine (Aldrich) . The mixture was stirred at room temperature for 30 minutes. Then a solution in tetrahydrofuran (20 ml) of 406 mg (2.90 mmol) of 1-(4-methylthiophen-3-yl)ethanone synthesized in analogy to Reference Example 17 was added dropwise at -30° C to -27°C and the mixture was stirred at room temperature for 1 h. After completion of the reaction, water and 1N hydrochloric acid were added to the liquid reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (eluting solvent: hexane:ethyl acetate = 95:5 to 70:30 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 387 mg (2.72 mmol, 94% yield) as a colorless oil. Mass spectrum (EI, m/z): 142 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 7.23-7.21 (1H, m), 6.92 (1H, dq, J = 3.1, 0.9 Hz), 4.92 (1H, qdd, J = 6.4, 4.8, 0.8 Hz) , 2.27 (3H, d, J = 0.9 Hz), 1.63 (1H, d, J = 4.6 Hz), 1.53 (3H, d, J = 6.4 Hz).

[00192] The title compound can be obtained with an increased optical purity in the following manner.

[00193] At room temperature and while stirring was carried out, 6.7 g of Lipase PS Amano SD (Wako Pure Chemical Industries, Ltd.) was added to a diisopropyl ether solution (67 ml) of 13.3 g (94 mmol, increased optical purity 90% ee) of (R)-1-(4-methylthiophen-3-yl)ethanol synthesized in analogy to Reference Example 20 and 16.3 ml (163 mmol) of vinyl acetate . The mixture was stirred at 45°C for 25 hours. The obtained liquid reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 90:10 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure to give the (R)-1-(4-methylthiophen-3-yl)ethyl acetic acid ester weighing 10.3 g (56.0 mmol, 60% yield, increased optical purity >99% ee) as a light yellow oil. Analysis of Optical Purity Conditions Column: CHIRALPAK IA (trade name, Daicel Corporation) Size: 0.46 cm I.D. x 25 cm L. Mobile phase: hexane:2-propanol = 99.5:0.5 (V/V) Flow rate: 1.0 ml/min. Temperature: 40°C Wavelength: 254 nm 1H-NMR spectrum (400MHz, CDCl3) δ: 7.23 (1H, d, J = 3.3 Hz), 6.94-6.90 (1H, m ), 5.94 (1H, qd, J = 6.5, 0.9 Hz), 2.23 (3H, d, J = 1.0 Hz), 2.07 (3H, s), 1.55 (3H, d, J = 6.5 Hz).

[00194] Under a stream of nitrogen, 2.0 g (84 mmol) of lithium hydroxide was added to an ethanol (50 ml)-water (5 ml) solution of 10.3 g (56.0 mmol) of lithium ester (R)-1-(4-methylthiophen-3-yl)ethyl acetic acid obtained above at room temperature while stirring was carried out. The mixture was stirred at temperature for 1.5 hours. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure to remove ethanol. Water was added to the residue and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 95:5 to 70:30 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced to give the title compound weighing 7.8 g (55 mmol, 98% yield, increased optical purity 99.5% ee) as a colorless oil. Analysis of Optical Purity Conditions Column: CHIRALPAK IA (trade name, Daicel Corporation) Size: 0.46 cm ID x 25 cm W. Mobile phase: Liquid A:Liquid B = 95:1 (V/V) Liquid A: Hexane :2-propanol = 99.5:0.5 (V/V) Liquid B: 2-propanol Flow rate: 1.0 ml/min. Temperature: 40°C Wavelength: 254 nm (Reference Example 21) (R)-1-(2,5-difluorophenyl)ethyl acetic acid ester Chemical Structure 59

[00195] At room temperature and while stirring was carried out, 33 g of Lipase PS Amano SD (Wako Pure Chemical Industries, Ltd.) were added to a solution of diisopropyl ether (200 ml) of 39.3 g (249 mmol) of (RS)-1-(2,5-difluorophenyl)ethanol synthesized in analogy to Reference Example 18 and 45.0 ml (450 mmol) of vinyl acetate. The mixture was stirred at 45°C for 46.5 hours. The obtained liquid reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (eluting solvent: hexane:ethyl acetate = 90:10 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure to give the compound of the title weighing 20.1 g (100 mmol, 40% yield, increased optical purity 97% ee) as a pale yellow oil. Analysis of Optical Purity Conditions Column: CHIRALPAK IA (trade name, Daicel Corporation) Size: 0.46 cm I.D. x 25 cm L. Mobile phase: hexane:2-propanol = 99.5:0.5 (V/V) Flow rate: 1.0 ml/min. Temperature: 40°C Wavelength: 254 nm 1H-NMR Spectrum (400MHz, CDCl3) δ: 7.11-7.05 (1H, m), 7.03-6.90 (2H, m), 6 .09 (1H, q, J = 6.6 Hz), 2.11 (3H, s), 1.52 (3H, d, J = 6.5 Hz).

[00196] (The compound obtained in this reaction (optical purity increased 97% ee) was hydrolyzed in the same manner as Reference Example 22 (optical purity increased 92.2% ee) and the product was treated under the same conditions as in the reaction above, thereby enhancing the increased optical purity).

[00197] At room temperature and while stirring was carried out, 16 g of Lipase PS Amano SD (Wako Pure Chemical Industries, Ltd.) were added to a solution of diisopropyl ether (70 ml) of 15.8 g (100 mmol) of (R)-1-(2,5-difluorophenyl)ethanol (optical purity increased 92.2% ee) and 20.0 ml (200 mmol) of vinyl acetate. The mixture was stirred at 45°C for 64.5 hours. The obtained liquid reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 90:10 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 19.2 g (96 mmol, 96% yield, increased optical purity 99.7% ee) as a pale yellow oil. (Reference Example 22) (R)-1-(2,5-difluorophenyl)ethanol Chemical Structure 60

[00198] Under a nitrogen flow, 3.5 g (150 mmol) of lithium hydroxide was added and an ethanol (100 ml)-water (10 ml) solution of 19.2 g (96 mmol, increased optical purity 99.7% ee) of (R)-1-(2,5-difluorophenyl)ethyl acetic acid ester synthesized in Reference Example 21 at room temperature while stirring was carried out. The mixture was stirred at temperature for 1.5 hours. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure to remove ethanol. Water was added to the residue and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 90:10 to 69:31 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced to give the title compound weighing 13.5 g (85.6 mmol, 89% yield, increased optical purity 99.0% ee) as a pale yellow oil. Analysis of Optical Purity Conditions Column: CHIRALPAK IA (trade name, Daicel Corporation) Size: 0.46 cm ID x 25 cm W. Mobile phase: Liquid A:Liquid B = 99:1 (V/V) Liquid A: Hexane :2-propanol = 99.5:0.5 (V/V) Liquid B: 2-propanol Flow rate: 1.0 ml/min. Temperature: 40°C Wavelength: 254 nm 1H-NMR Spectrum (400MHz, CDCl3) δ: 7.25-7.19 (1H, m), 7.01-6.87 (2H, m), 5 .22-5.14 (1H, m), 1.88 (1H, d, J = 4.3 Hz), 1.50 (3H, d, J = 6.4 Hz). (Reference Example 23) (R)-1-{4'-[5-Chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl) ethyl ester ]-[ 1, 1'-biphenyl]-4-yl}cyclopropanecarboxylic acid Chemical Structure 61

[00199] In an argon atmosphere, 340 mg (0.791 mmol) of [bis(trifluoroacetoxy)iodo]benzene were added to a solution in toluene (6 ml) of 300 mg (0.704 mmol) of 1-[ acid ethyl ester 4'-(3-carbamoyl-5-chlorothiophen-2-yl)-[1,1'-biphenyl]-4-yl]cycloropanecarboxylic acid synthesized in analogy to Reference Example 7 and 0.53 ml (6.6 mmol) of pyridine. The mixture was stirred at room temperature for 30 minutes. Then, 105 mg (0.819 mmol) of (R)-1-(thiophen-3-yl)ethanol synthesized in analogy to Reference Example 19 was added in an argon atmosphere at room temperature and the mixture was stirred for 2 hours while heating was carried out at 70°C. After completion of the reaction, ethyl acetate and water were added to the liquid reaction mixture and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 80:20 to 30:70 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 306 mg (0.55 mmol, 79% yield) as a brown oil. Mass spectrum (DUIS-, m/z): 550 [M-1]-. 1H-NMR Spectrum (400MHz, CDCl3) δ: 7.69-7.63 (2H, m), 7.63-7.51 (3H, m), 7.48-7.40 (4H, m) , 7.31 (1H, dd, J = 5.0, 2.9 Hz), 7.28-7.26 (1H, m), 7.11 (1H, dd, J = 5.0, 1, 3 Hz), 6.72 (1H, s), 5.99 (1H, q, J = 6.6 Hz), 4.13 (2H, q, J = 7.1 Hz), 1.64 (2H , dd, J = 7.0, 4.0 Hz), 1.62 (3H, d, J = 6.5 Hz), 1.23 (2H, dd, J = 7.0, 4.0 Hz) , 1.19 (3H, t, J = 7.1 Hz). (Reference Example 24) (R)-1-[4'-(5-Chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy[ 1,1'-biphenyl]-4-yl]cycloranecarboxylic acid Chemical Structure 62

[00200] In a nitrogen atmosphere at room temperature, 2.4 g (5.6 mmol) of [bis(trifluoroacetoxy)iodo]benzene were added to a solution in toluene (20 ml) of 2.0 g (4, 4 mmol) of 1-[4'-(3-carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'-biphenyl]-4-yl]cycloropanecarboxylic acid ethyl ester synthesized in analogy to Reference Example 8, 0.80 g (6.6 mmol) of (R)-1-phenylethanol (Tokio Chemical Industrial Co., Ltd.) and 1.2 ml (15 mmol) of pyridine. The mixture was stirred for 1.5 hours while heating at 60°C. After completion of the reaction, the liquid reaction mixture was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 99:1 to 80:20 (V/V)), and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 1.46 g (4.0 mmol (purity 72 wt%, yield 42%) as an orange oil. Mass spectrum (CI, m/z): 575 [M]+. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 9.39 (1H, brs), 7.40-7.26 (10H, m), 7.23-7.16 (2H, m), 7 .10 (1H, dd, J = 7.8, 1.6 Hz), 5.75 (1H, q, J = 6.4 Hz), 4.06 (2H, q, J = 7.1 Hz) , 3.73 (3H, s), 1.56-1.41 (5H, m), 1.23 (2H, dd, J = 7.0, 4.0 Hz), 1.13 (3H, t , J = 7.0 Hz). (Reference Example 25) (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorphenyl)ethoxy]carbonyl} acid 2-(trimethylsilyl)ethyl ester (Reference Example 25) amino)thiophen-2-yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cycloropanecarboxylic Chemical Structure 63

[00201] In an argon atmosphere at room temperature, 874 mg (2.03 mmol) of [bis(trifluoroacetoxy)iodo]benzene were added to a solution in toluene (10 ml) of 886 mg (1.68 mmol) of 1-[4'-(3-Carbamoyl-5-chlorothiophen-2-yl)-2'-methoxy-[1,1'-biphenyl]-4-yl]cycloropanecarboxylic acid 2-(trimethylsilyl)ethyl ester synthesized in analogy to Reference Example 16 and 0.65 ml (8.0 mmol) of pyridine. The mixture was stirred for 5 minutes. Then, 413 mg (2.61 mmol) of (R)-1-(2,5-difluorophenyl)ethanol (Enamine) was added. The mixture was stirred for 1 hour while heating was carried out at a bath temperature of 70°C. After completion of the reaction, water and ethyl acetate were added to the liquid reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 99:1 to 94:6 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced to give the title compound weighing 1.16 g (1.46 mmol (86 wt% purity, 87% yield) as a brown oil (semi-solid). Mass spectrum (DUIS-, m/z): 682 [M-1]-. 1H-NMR mass spectrum (400MHz, DMSO-d6) δ: 9.55 (1H, brs), 7.45-7.40 (2H, m), 7.39-7.17 (8H, m), 7.10 (1H, dd, J = 7.9, 1.5 Hz), 5.91 (1H, q, J = 6.3 Hz), 4.14-4.07 (2H, m), 3 .76 (3H, s), 1.55-1.42 (3H, m), 1.49 (2H, dd, J = 6.8, 3.9 Hz), 1.23 (2H, dd, J = 7.0, 4.0 Hz), 0.910.84 (2H, m), -0.05 (9H, s). (Reference Example 26) (R)-1-{4'-[5-Fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2 acid ethyl ester) -yl]-[1,1'-biphenyl]-4-yl}cycloropanecarboxylic acid ethyl ester Chemical Structure 64

[00202] In an argon atmosphere, 0.24 ml (1.7 mmol) of triethylamine and 0.29 ml (1.4 mmol) of diphenylphosphoryl azide were added to a toluene solution (10 ml) of 456 mg (1.11 mmol) of 2-{4'-[1-(ethoxycarbonyl)cyclopropyl]-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid synthesized in analogy to Reference Example 13. The mixture was stirred at room temperature for 30 minutes. Then, a toluene solution (1 ml) of 190 mg (1.34 mmol) of (R)-1-(4-methylthiophen-3-yl)ethanol which was synthesized in analogy to Reference Example 20 was added. dried to Molecular Sieves 4A (powder) (trade name, NACALAI TESQUE, INC.) (0.3 g). The resulting mixture was stirred for 2 hours while heating at 70°C. After completion of the reaction, ethyl acetate and a saturated aqueous ammonium chloride solution were added to the liquid reaction mixture and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 80:20 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 570 mg (1.04 mmol, 93% yield) as a colorless oil. Mass spectrum (DUIS-, m/z): 548 [M-1]-. 1H-NMR mass spectrum (400MHz, DMSO-d6) δ: 9.31 (1H, brs), 7.74-7.68 (2H, m), 7.66-7.61 (2H, m), 7.57-7.40 (5H, m), 7.17-7.13 (1H, m), 6.83 (1H, brs), 5.74 (1H, q, J = 6.5 Hz) , 4.05 (2H, q, J = 7.2 Hz), 2.17 (3H, brs), 1.60-1.43 (3H, m), 1.51 (2H, dd, J = 6 .8, 4.0 Hz), 1.23 (2H, dd, J = 7.1, 4.1 Hz), 1.11 (3H, t, J = 7.1 Hz). (Reference Example 27) (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl] acid ethyl ester -2'-methoxy-[1,1'-biphenyl]-4-yl}cycloropanecarboxylic Chemical Structure 65

[00203] In an argon atmosphere, 0.040 ml (0.29 mmol) of triethylamine and 0.045 ml (0.21 mmol) of diphenylphosphoryl azide were added to a solution in toluene (4.0 ml) of 72 mg (0 .16 mmol) of 2-{4'-[1-(ethoxycarbonyl)cyclopropyl]-2-methoxy-[1,1'-biphenyl]-4-yl}-5-fluorothiophene-3-carboxylic acid synthesized in analogy to Reference Example 14. The mixture was stirred at room temperature for 30 minutes. Then, 35 mg (0.22 mmol) of (R)-1-(2-chlorophenyl)ethanol (Shanghai AoBo Bio-pharm) was added. The mixture was stirred for 2 hours while heating to 70°C. After completion of the reaction, ethyl acetate and water were added to the liquid reaction mixture and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography on silica gel (eluting solvent: hexane:ethyl acetate = 93:7 to 72:28 (V/V)) and the fraction containing the target compound was concentrated under pressure. reduced to give the title compound weighing 84 mg (0.061 mmol (purity 43% by weight), 37% yield) as a colorless oil. Mass spectrum (EI, m/z): 593 [M]+. 1H-NMR spectrum (400MHz, CDCl3) δ: 752-7.48 (2H, m), 7.43-7.17 (9H, m), 7.06 (1H, dd, J = 7.8, 1.6 Hz), 6.97 (1H, d, J = 1.6 Hz), 6.23 (1H, q, J = 6.7 Hz), 4.13 (2H, q, J = 7, 1 Hz), 3.82 (3H, s), 1.63 (2H, dd, J = 6.9, 3.9 Hz), 1.57 (3H, d, J = 6.7 Hz), 1 .24 (2H, dd, J = 7.0, 4.0 Hz), 1.20 (3H, t, J = 7.2 Hz). (Reference Example 28) (R)-1-{4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[ acid 1,1'-biphenyl]-4-yl}cycloranecarboxylic acid Chemical Structure 66

[00204] 2.0 ml (4.0 mmol) of a 2N aqueous sodium hydroxide solution were added to a solution in 2-propanol (4 ml) of 304 mg (0.551 mmol) of acid ethyl ester (R) -1-{4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[1,1'-biphenyl]-4- yl}cycloropanecarboxylic acid synthesized in analogy to Reference Example 23. The mixture was stirred at room temperature for 42.5 hours. After completion of the reaction, the liquid reaction mixture was acidified by adding 2N hydrochloric acid and extracted with ethyl acetate. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (COOH column, elution solvent: hexane: ethyl acetate = 70:30 to 10:90 (V/V)) and the fraction containing the compound target was concentrated under reduced pressure. Hexane (10 ml) and ethyl acetate (3 ml) were added to the residue. The precipitated white solid was filtered and washed with a mixed solution of hexane-ethyl acetate (3:1 (V/V)). The mother liquor and washings were concentrated under reduced pressure to give the title compound weighing 65 mg (0.55 mmol, 23% yield) as a white solid. Mass spectrum (DUIS-, m/z): 522 [M-1]-. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 12.37 (1H, brs), 9.33 (1H, brs), 7.74-7.68 (2H, m), 7.65-7 .60 (2H, m), 7.58-7.50 (3H, m), 7.487.37 (3H, m), 7.25-7.07 (2H, m), 5.82 (1H, q , J = 6.4 Hz), 1.56-1.44 (3H, m), 1.48 (2H, dd, J = 6.7, 3.8 Hz), 1.19-1.16 ( 2H, m). (Reference Example 29) (R)-1-[4'-(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy-[ 1, 1'-biphenyl]-4-yl]cycloranecarboxylic acid Chemical Structure 67

[00205] 8.0 ml (16.0 mmol) of a 2N aqueous sodium hydroxide solution were added to a solution in 2-propanol (30 ml) of 1.46 g (4.0 mmol (72% purity in weight)) of (R)-1-[4'-(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2'-methoxy[1, 1'-biphenyl]-4-yl]cycloropanecarboxylic acid synthesized in Reference Example 24. The mixture was stirred at room temperature for 110 hours. After completion of the reaction, the liquid reaction mixture was acidified by adding 2N hydrochloric acid and extracted with methylene chloride. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 80:20 to 0:100 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure. The residue was dissolved in a small amount of ethanol. Water was added to the solution to precipitate the solid. The solid was collected by filtration, washed with water and dried by heating under vacuum to give the title compound weighing 588 mg (1.07 mmol, 58% yield) as a light red solid. Mass spectrum (DUIS-, m/z): 546 [M-1]-. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 12.34 (1H, brs), 9.40 (1H, brs), 7.45-7.25 (10H, m), 7.21-7 .16 (2H, m), 7.09 (1H, dd, J = 7.9, 1.6 Hz), 5.75 (1H, q, J = 6.4 Hz), 3.73 (3H, s), 1.54-1.41 (5H, m), 1.18-1.12 (2H, m). Reference Example 30) (R)-1-{4'-[5-fluoro-3-({[1-(4-methylthiophen-3yl)ethoxy]carbonyl}amino)thiophen-2-yl]- acid [ 1, 1'-biphenyl]-4-yl}cycloranecarboxylic acid Chemical Structure 68

[00206] 4.0 ml (8.0 mmol) of a 2N aqueous sodium hydroxide solution were added to a solution in 2-propanol (12 ml) of 565 mg (1.03 mmol) of acid ethyl ester ( R)-1-{4'-[5-fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy]carbonyl}amino)thiophen-2-yl]-[1,1'-biphenyl ]-4-yl}cycloropanecarboxylic acid synthesized in analogy to Reference Example 26. The mixture was stirred at room temperature for 91 hours. After completion of the reaction, the liquid reaction mixture was acidified by adding 1N hydrochloric acid and extracted with methylene chloride. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 50:50 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure. Then, 6 ml of hexane and 12 ml of ethyl acetate were added, and the mixture was heated to 50°C and cooled. The precipitated solid was filtered and washed with a mixed solution of hexane-ethyl acetate (50:50 (V/V)). The mother liquor and washings were concentrated under reduced pressure. To the residue were added 8 ml of acetonitrile, 4 ml of water and 3 ml of tetrahydrofuran. Lyophilization of the mixture resulted in the title compound weighing 193 mg (0.37 mmol, 36% yield) as a white solid. Mass spectrum (DUIS-, m/z): 520 [M-1]-. 1H-NMR spectrum (400MHz, DMSO-d6) δ: 12.38 (1H, brs), 9.33 (1H, brs), 7.73-7.67 (2H, m), 7.65-7 .59 (2H, m), 7.57-7.50 (2H, m), 7.497.38 (3H, m), 7.19-7.12 (1H, m), 6.83 (1H, brs ), 5.74 (1H, q, J = 6.4 Hz), 2.17 (3H, brs), 1.59-1.44 (3H, m), 1.48 (2H, dd, J = 6.7, 3.8 Hz), 1.18 (2H, dd, J = 6.9, 3.9 Hz). (Reference Example 31) (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophenyl]-2'-methoxy-[ 1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid Chemical Structure 69

[00207] 1.5 ml (3.0 mmol) of a 2N aqueous sodium hydroxide solution were added to a solution in 2-propanol (3.0 ml) 80 mg (0.058 mmol (purity 43% by weight)) of (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2-yl]-2'-methoxy-[ 1,1'-biphenyl]-4-yl}cycloropanecarboxylic acid synthesized in Reference Example 27. The mixture was stirred at room temperature for 23 hours. After completion of the reaction, the liquid reaction mixture was acidified by adding 2N hydrochloric acid and extracted with methylene chloride. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (COOH column, elution solvent: hexane:ethyl acetate = 80:20 to 20:80 (V/V)) and the fraction containing the target compound was concentrated under reduced pressure to give the title compound weighing 13 mg (0.023 mmol, 40% yield) as a white solid. Mass spectrum (DUIS-, m/z): 564 [M-1]-. 1H-NMR mass spectrum (400MHz, DMSO-d6) δ: 12.35 (1H, brs), 9.55 (1H, brs), 7.60-7.28 (9H, m), 7.18 ( 1H, d, J = 1.5 Hz), 7.09 (1H, dd, J = 7.8, 1.4 Hz), 6.84 (1H, d, J = 2.5 Hz), 6, 00 (1H, q, J = 6.1 Hz), 3.77 (3H, s), 1.55-1.39 (5H, m), 1.21-1.10 (2H, m). (Reference Example 32) (R)-1-{4'-[5-chloro-3-({[ 1 -(2,5difluorophenyl)ethoxy]carbonyl}amino)thiophen-2-yl]-2 acid '-methoxy-[1,1'-biphenyl]-4yl}cycloropanecarboxylic acid Chemical Structure 70

[00208] In an argon atmosphere at room temperature, 9.0 ml (9.0 mmol) of a 1 M solution in tetrahydrofuran of tetrabutylammonium fluoride were added to a solution of dimethylformamide (90 ml) of 3 .00 g (4.38 mmol) of (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy] acid 2-(trimethylsilyl)ethyl ester (4.38 mmol) carbonyl}amino)thiophen-2-yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid synthesized in analogy to Reference Example 25. The mixture was stirred at temperature for 2 hours . After completion of the reaction, ethyl acetate and water were added to the liquid reaction mixture, and the pH was adjusted to approximately 3 with 0.5N hydrochloric acid. The liquid reaction mixture was separated into phases. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 2-Propanol (40 ml) and water (40 ml) were added to the residue and the mixture was sonicated. The precipitated solid was filtered and washed with water. Thus, the crude title compound was obtained weighing 2.86 g. The crude compound was subjected to silica gel column chromatography (eluting solvent: hexane:ethyl acetate = 70:30 to 10:90 (V/V)), and the fraction containing the target compound was concentrated under pressure reduced. Heptane was added to the residue and the precipitated solid was filtered. In this way, the title compound weighing 1.62 g (2.77 mmol, 63% yield) was obtained as a white solid. Mass spectrum (DUIS-, m/z): 582 [M-1]-. 1H-NMR mass spectrum (400MHz, DMSO-d6) δ: 12.35 (1H, brs), 9.54 (1H, brs), 7.44-7.39 (2H, m), 7.39- 7.19 (7H, m), 7.18 (1H, d, J = 1.6 Hz), 7.10 (1H, dd, J = 7.9, 1.6 Hz), 5.91 (1H , q, J = 6.5 Hz), 3.76 (3H, s), 1.56-1.43 (3H, m), 1.47 (2H, dd, J = 6.7, 3.7 Hz), 1.18 (2H, dd, J = 6.8, 4.0 Hz).

Test Example 1 GTPYS binding test to the LPA1 receptor

[00209] 5 μg of a membrane fraction from RH 7777 cells expressing the human LPA1 receptor (A324, ChanTest) are suspended in a reaction buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 10 μM GDP, saponin 5 µg, 0.2% BSA, 0.1 nM [35S]GTPYS (NEG030X, Perkin Elmer), pH 7.4). Test compounds dissolved in DMSO at various concentrations are added to the suspension. After preincubation at 30°C for 15 minutes, LPA (L7260, Sigma, final concentration 100 nM) is added and the suspensions are incubated at 30°C for 30 minutes. Membrane fractions are collected on a glass fiber filter (GF/B, Whatman) by using a cell harvester (N30, Brandel) and are washed with a 10 mM phosphate buffer (pH 7.4). Membrane fractions are measured with a liquid scintillation analyzer (2990TR, Packard) and the concentration (IC50) of test compound required for 50% inhibition of LPA1 and [35S]GTPYS receptor binding is determined by non-regression analysis. linear using EXSAS (version 7.6.0, Arm Systex).

Test Example 2 cell migration test

[00210] Cell migration testing was performed using Chemo-Tx (trademark) (116-8, Neuro Probe). Human melanoma A2058 cells (obtained from the European Collection of Cell Culture) were cultured in serum-free EMEM medium for 24 hours and resuspended in EMEM medium containing 0.1% BSA to give a cell suspension. Test compounds dissolved in DMSA at various concentrations were each added to the cell suspension and the suspensions were cultured at 37°C for 15 minutes (final DMSO concentration 0.5%). LPA dissolved in DMEM medium containing 0.1% BSA (100 nM final concentration) was added to a 96-well Chemo-Tx plate and a Chemo-Tx filter coated with 0.001% Fibronectin on both sides was placed over the sign. Cultured cell suspensions (25,000 cells) were added to the top surface of the filter and further cultured at 37°C for 3 hours. Then, cells from the upper surface of the filter were removed. After the filter was removed and dried, cells that migrated to the bottom surface of the filter were stained with Diff-Quik dye (16920, Sysmex). The absorbance of the filter (570 nm) was measured and the concentration (IC50) of the test compound required for 50% inhibition of LPA cell migration activity was determined by non-linear regression analysis using EXSAS (version 7.6.0, Arm Systex).

[00211] In this test, the compounds of the present invention show excellent activity. For example, the IC50 values of the compounds of Examples 1 to 15 were not more than 200 nM.

Test Example 3 LPA-induced histamine release in mice

[00212] The LPA-induced histamine release test in mice was performed according to the method by Swaney et al. (The Journal of Pharmacology and Experimental Therapeutics, 336 (2011), pp. 693-700). The test compound was suspended in a 0.5% methylcellulose solution (133-14255, Wako Pure Chemical Industries, Ltd.) and orally administered to male CDI mice (30 to 40 g body weight, supplied by Charles River Laboratories Japan) at a dose of 10 ml/kg. 4 hours after administration, LPA (857130P, Avanti) dissolved in PBS containing 0.1% BSA was administered via the tail vein (300 µg/mouse). Immediately afterwards, each of the mice was anesthetized with isofluorane and blood was collected from a vein 2 minutes after LPA administration. Blood was placed in a test tube containing EDTA and centrifuged at 4°C, 200 xg for 10 minutes to provide plasma.

[00213] Plasma histamine concentration was measured with an EIA kit (62HTMPEB, Cisbio Bioassays).

[00214] The inhibition rate (%) in the 0.5% methylcellulose solution administration group was calculated in each subject based on the plasma histamine concentration in the mouse to which the test compound was administered and the rate of subjects who showed the inhibition rate of 80% or more was expressed as efficacy rate (%).

[00215] In this test, the compounds of the present invention showed excellent activity. For example, the compounds of Examples 1 to 15 achieved a 50% or greater efficacy rate at the 10 mg/kg dose.

Test Example 4 Models of bleomycin-induced pulmonary fibrosis

[00216] Bleomycin hydrochloride (Nippon Kayaku Co., Ltd) was administered to mice to prepare lung fibrosis models. The test compound was administered orally on each day starting from the day on which the administration of bleomycin was started. From Day 3 to Day 42 after bleomycin treatment, bronchoalveolar lavage fluids (BALFs) or lung fluids were collected under isofluorane anesthesia. BALFs were centrifuged at 800xg for 10 minutes to give supernatants. Supernatants were analyzed with the DC Protein Assay Kit (500-0114, Biorad) to determine amounts of protein and were analyzed with the Sircol Soluble Collagen Assay Kit (S111, Biocolor) to determine amounts of collagen. Additionally, biomarkers for inflammation and fibrosis in the supernatants were measured by ELISA method. With regard to the lungs, after their net weights were measured, the amounts of hydroxyproline in the tissues were measured by a modification of Woessner's method (Archives of Biochemistry and Biophysics, 93 (1961), pp.440-447). Portions of the lungs were fixed in 10% neutral formalin buffer and were observed for histopathological changes. Results were statistically analyzed using EXSAS (version 7.6.0, Arm Systex).

Test Example 5 Models of renal fibrosis due to unilateral ureteral obstruction (UUO)

[00217] An incision was made in the abdomen of mice anesthetized with isoflurane. The left ureter is ligated with a silk suture to prepare the UUO models. The test compound is administered orally each day starting on the day on which the UUO templates are prepared. On Day 8, Day 14 or Day 21 after UUO model preparation, the kidneys are collected and their net weights are measured. RNA is extracted from portions of the kidneys and expression levels of fibrosis marker genes are measured by the quantitative PCR method. Afterwards, the amounts of hydroxyproline or collagen in the kidney tissues are measured. Results were statistically analyzed using EXSAS.

Test Example 6 Models of liver fibrosis induced with carbon tetrachloride (CCk)

[00218] Diluted CCl4 (035-01237, Wako Pure Chemical Industries, Ltd.) is administered to mice twice a week to prepare liver fibrosis models. The test compound is administered orally on each day starting from the day on which the CCl4 administration is started. From Day 3 to Day 28 after starting CCl4 administration, livers are collected under isoflurane anesthesia and their net weights are measured. RNA is extracted from portions of the livers and expression levels of fibrosis marker genes are measured by the quantitative PCR method. Subsequently, the amounts of hydroxyproline or collagen in the liver tissues are measured. Portions of the livers were fixed in 10% neutral formalin buffer and observed for histopathological changes. Results were statistically analyzed using EXSAS.

Test Example 7 Non-alcoholic steatohepatitis (NASH) models in rats

[00219] NASH models are prepared by feeding mice a methionine/choline deficient (MCD) diet. Rats are allowed to freely ingest a regular diet or the MCD diet for 20 weeks. The test compound is administered orally each day from the day on which the MCD diet feeding is started. After rearing for 20 weeks, the livers are collected under isoflurane anesthesia and their net weights are measured. RNA is extracted from portions of the livers and expression levels of fibrosis marker genes are measured by the quantitative PCR method. Subsequently, the amounts of hydroxyproline or collagen in the liver tissues are measured. Portions of the livers were fixed in 10% neutral formalin buffer and were observed for histopathological changes. Results were statistically analyzed using EXSAS.

Test Example 8 Non-alcoholic steatohepatitis (NASH) models in mice

[00220] STAM (trademark) mice (available from Stelic Institute & Co., Inc.) are used as models of NASH. STAM mice (trademark) are prepared by administering 200 µg of streptozocin (Sigma Aldrich) subcutaneously once to the back of 2-day-old male mice and feeding the mice a high-fat diet (High Fat Diet 32, CLEA Japan, Inc.) 4 weeks after birth ( Medical Molecular Morphology, 46 (2013) pp. 141-152 ).

[00221] The test compound is administered orally every day after 5 or 6 weeks after birth. At the age of 9 or 10 weeks, blood and livers are collected under anesthesia. The blood is subjected to biochemical tests. After liver net weights are measured, RNA is extracted from liver portions and expression levels of inflammation and fibrosis marker genes are measured by the quantitative PCR method. Then the amounts of hydroxyproline or collagen in the liver tissues are measured. Paraffin sections or frozen sections are prepared from portions of the livers and subjected to histopathological testing to determine NAFLD activity scores, areas of fibrosis, or areas of inflammation. Results are statistically analyzed using EXSUS (version 8.0, CAC EXICARE CORPORATION) or Prism 4 (GraphPad Software, Inc.).

Test example 9

[00222] Non-alcoholic steatohepatitis (NASH) models in mice

[00223] NASH models are prepared by breeding mice with a choline deficiency, a high-fat diet containing 0.1% methionine (A06071302, Research Diets, Inc.) (International Journal of Experimental Pathology, 94 (2013) pp. 93-103).

[00224] The compound is administered orally every day from the day on which the CDAHFD feeding is started. After 8 to 12 weeks, the livers are collected under isflurane anesthesia and their weights are measured. RNA is extracted from portions of the livers, and expression levels of inflammation and fibrosis marker genes are measured by the quantitative PCR method. In addition the amounts of hydroxyproline or collagen in the liver tissues are measured. Portions of the livers are fixed in 10% formalin neutral buffer solution and are observed for histopathological changes. Results are statistically analyzed using EXSUS (version 8.0, CAC EXICARE CORPORATION).

Test Example 10 Pharmacokinetic studies in monkeys

[00225] A disposable catheter was inserted through the nasal cavity into the stomach of a cynomolgus monkey deprived of food since the night of the day before the test. Via a syringe, a 0.5% methylcellulose suspension or solution containing the test compound at 10 mg/2 ml was orally administered once at a dose of 2 ml/kg. Through a syringe, blood was sampled through the femoral vein before administration, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after administration. EDTA-2K was added to blood samples and samples were centrifuged (4°C, 1710 xg, 3000 rpm, 15 minutes) to provide plasma. Plasmas were deproteinized by adding acetonitrile (mixture of 50 μL of plasma + 200 μL of acetonitrile) and the mixtures were filtered (PTFE, 0.2 μm). The filtrates were analyzed with LC-MS/MS (QTrap 3200, AB SCIEX; and LC-20A or LC-30A series, Shimadzu Corporation) to determine test compound concentrations in plasmas. AUC (area under the plasma concentration curve) was calculated with Phoenix WinNonlin (CERTARA) based on changes in plasma concentration.

[00226] From the results of Test Examples 2 and 3, the a-halogen substituted thiophene compound salts of the present invention possess an LPA receptor antagonistic action and are particularly useful as drugs for the treatment and/or prevention ( preferably drugs for the treatment) of diseases that are accompanied by fibrosis, inflammatory or immunological diseases, diseases of the central or peripheral nervous system, urological diseases and cancer-related diseases.

Preparation Example 1 hard capsules

[00227] Standardized two-piece hard gelatin capsules are loaded with a powder (100 mg) of the compound salt of the Example, lactose (150 mg), cellulose (50 mg) and magnesium stearate (6 mg) to give gelatin capsules hard, which are washed and then dried.

Preparation Example 2 soft capsules

[00228] A mixture of a digestible oil such as soybean oil or olive oil and the compound salt of the Example is injected into gelatin to provide soft capsules containing 100 mg of the active ingredient, and the soft capsules are washed and then dried.

Preparation Example 3 Pills

[00229] According to a method known in the pharmaceutical field, tablets are produced using the compound salt (100 mg) of Examples, colloidal silicon dioxide (0.2 mg), magnesium stearate (0.2 mg), microcrystalline cellulose (0.2 mg), starch (0.2 mg) and lactose (98.8 mg). Tablets can be coated as needed.

INDUSTRIAL APPLICABILITY

[00230] The a-halogen substituted thiophene compound salts of the invention, represented by the general formula (I), have a potent LPA receptor antagonistic action and excellent properties such as long-lasting medicinal effects and solubility and are useful as medicines (drugs for the treatment and/or prevention of diseases that are accompanied by fibrosis, inflammatory or immunological diseases, diseases of the central or peripheral nervous system, urological diseases and cancer-related diseases).

Claims (16)

1. Salt, characterized by the fact that it is represented by the general formula (I): Chemical Structure 71 where R is a hydrogen atom or a methoxy group, X is a halogen atom, A is selected from the group consisting of: Chemical Structure 72 M is an alkali metal or an alkaline earth metal, and n is 1 when M is an alkali metal and is 2 when M is an alkaline earth metal. 2. Salt according to claim 1, characterized in that the alkali metal or alkaline earth metal is sodium, potassium or calcium. 3. Salt, characterized in that it is an acid salt (R)-1-[4'-(5-chloro-3-{[(1-phenylethoxy)carbonyl]amino}thiophen-2-yl)-2 '-methoxy[1,1'-biphenyl]-4-yl]cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. 4. Salt according to claim 3, characterized in that the alkali metal or alkaline earth metal is sodium, potassium or calcium. 5. Salt, characterized in that it is an acid salt (R)-1-{4'-[5-chloro-3-({[1-(2,5-difluorophenyl)ethoxy]carbonyl}amino) thiophene -2-yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. 6. Salt according to claim 5, characterized in that the alkali metal or alkaline earth metal is sodium, potassium or calcium. 7. Salt, characterized in that it is an acid salt (R)-1-{4'-[3-({[1-(2-chlorophenyl)ethoxy]carbonyl}amino)-5-fluorothiophen-2- yl]-2'-methoxy-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. 8. Salt according to claim 7, characterized in that the alkali metal or alkaline earth metal is sodium, potassium or calcium. 9. Salt, characterized in that it is an acid salt (R)-1-{4'-[5-chloro-3-({[1-(thiophen-3-yl)ethoxy]carbonyl}amino)thiophene -2-yl]-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. 10. Salt according to claim 9, characterized in that the alkali metal or alkaline earth metal is sodium, potassium or calcium. 11. Salt, characterized in that it is an acid salt (R)-1-{4'-[5-fluoro-3-({[1-(4-methylthiophen-3-yl)ethoxy]carbonyl}amino ) thiophen-2-yl]-[1,1'-biphenyl]-4-yl}cyclopropanecarboxylic acid with an alkali metal or an alkaline earth metal. 12. Salt according to claim 11, characterized in that the alkali metal or alkaline earth metal is sodium, potassium or calcium. 13. LPA receptor antagonist, characterized in that it comprises the salt as defined in any one of claims 1 to 12, as an active ingredient. 14. Pharmaceutical composition, characterized in that it comprises the salt as defined in any one of claims 1 to 12, as an active ingredient. 15. Pharmaceutical composition according to claim 14, characterized in that it is for the treatment or prevention of a disease that is accompanied by fibrosis, an inflammatory or immunological disease, a disease of the central or peripheral nervous system, a urological disease or a cancer-related illness. 16. Use of salt as defined in any one of claims 1 to 12, characterized in that it is for the preparation of a medicine for the treatment or prevention of a disease that is accompanied by fibrosis, an inflammatory or immunological disease , a disease of the central or peripheral nervous system, a urological disease or a cancer-related disease.