Glycogen synthase kinase 3β inhibitor, composition and process for the preparation thereof

The present invention relates to a compound for inhibiting glycogen synthase kinase 3beta (GSK-3β) activity, a pharmaceutical composition containing the compound as an active ingredient and a process for the preparation thereof.

Glycogen synthase kinase 3 (GSK-3), the well-known target protein for the treatment of diabetes and dementia, is a serine/threonine protein kinase which inhibits the activity of glycogen synthase (GS) by way of phosphorylation.

In the fatty tissue of mice suffering from fatty diabetes, the GSK-3β activity has been observed to be 2 fold higher than that of a normal mouse (H. Eldar-Finkelman, Diabetes, 48:1662–1666 (1999)) and patients during the second type diabetes are characterized by a high expression level of GSK-3β than normal (S. E. Nikoulina et al., Diabetes, 49: 263–171 (2000)). Also, the GSK-3β activity in the brain of a dementia patient is high (Yamaguchi H. et al., Acta. Neuropathol., 92: 232–241 (1996)), and transgenic mice programmed to express GSK-3β in the brain have abnormal neurons caused by hyperphosphorylating tau of the neurofibrillary tangle which plays an important role in the dementia attack (Lucas J. J. et al., EMBO J. 20: 27–39 (2001)).

GSK-3β is further related to bipolar disorder which can be treated by lithium and valproic acid, well-known GSK-3β inhibitors (Elahi S. et al., J. Infect. Dis. 176: 217–226 (1997)).

Thus, there has existed a need to develop an effective inhibitor of GSK-3β for treating or preventing GSK-β-dependent diseases.

The present inventors have endeavored to develop an effective inhibitor of GSK-3β; and have unexpectedly found that a compound containing a hydroxybenzoimidazole carboxylic amide moiety can inhibit the activity of GSK-3β, and therefore, can be used for treating or preventing GSK-β-dependent diseases such as fatness, diabetes and dementia.

Accordingly, it is an object of the present invention to provide a GSK-3β inhibitor having high inhibitory activity against GSK-3β.

It is another object of the present invention to provide a process for preparing said inhibitor.

It is further object of the present invention to provide a pharmaceutical composition for inhibiting GSK-3β.

In accordance with one aspect of the present invention, there is provided a compound of formula (I), a pharmaceutically acceptable salt, hydrate, solvate or isomer thereof:

wherein:

n is 0, 1, 2 or 3;

R 1 , R 2 and R 3 are each independently hydrogen, hydroxy, halogen or morpholin-1-yl-ethylamino;

R 4 and R 5 are each independently hydrogen;

linear or cyclic C 1 –C 6 alkyl optionally having one or more substituents, the carbon of the alkyl being optionally replaced with nitrogen, sulfur or oxygen, wherein the substituent is: hydroxy; halogen; alkyloxy; alkyl; amino; alkylamino; carboxyl; nitro; sulfonylamido; alkanesulfonyl; amido; an aromatic group optionally having one or more substituents selected from the group consisting of hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxyl, nitro, amido, dioxoisoindole and sulfonylamino; an aromatic group having one or more substituents selected from the group consisting of hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxyl, nitro and amido, the aromatic ring having nitrogen, sulfur or oxygen; or cyclic C 3 –C 8 alkyl optionally having one or more substituents selected from the group consisting of hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxyl, nitro and amido;

an aromatic group optionally having one or more substituents, the aromatic ring having optional nitrogen, sulfur or oxygen, wherein the substituent is; hydroxy; halogen; alkyloxy; alkyl; amino; alkylamino; carboxyl; nitro; sulfonylamido, alkanesulfonyl; amido; or linear or cyclic C 1 –C 6 alkyl optionally having one or more substituents, the alkyl having an optional nitrogen, sulfur or oxygen linkage and the substituent of the alkyl being: hydroxy; halogen; alkyloxy; alkyl; amino; alkylamino; carboxyl; nitro; sulfonylamido, alkanesulfonyl; amido; an aromatic group optionally having one or more substituents selected from the group consisting of hydroxy; halogen; alkyloxy; allyl; amino; alkylamino; carboxyl; nitro; amido; dioxoisoindole; and a sulfonylamino having an aromatic group substituted with hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxyl, nitro, sulfonylamido, alkanesulfonyl or amido; an aromatic group optionally having one or more substituents selected from the group consisting of hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxyl, nitro, sulfonylamide, alkanesulfonyl and amido, the aromatic ring containing nitrogen, sulfur or oxygen; or a cyclic C 3 –C 8 alkyl optionally having one or more substituents selected from the group consisting of hydroxy, halogen, alkyloxy, alkyl, amino, alkylamino, carboxyl, nitro and amido; or

form, together with the —N—(CH 2 ) n — moiety to which they are attached, a nitrogen heterocycle optionally having one or more substituents selected from the group consisting of OH, NH 2 , NO 2 , the heterocycle containing optional nitrogen or oxygen.

Among the compounds of formula (I) of the present invention, the preferred are:

those wherein n, R 1 , R 2 and R 3 have the same meanings as defined previously;

R 4 and R 5 are each independently hydrogen;

C 1 –C 4 alkyl optionally having one or more substituents selected from the group consisting of OH, NH 2 , NO 2 , and an aromatic group, the aromatic group optionally having one or more substituents selected from the group consisting of OH, C 1 –C 4 alkyloxy, NH 2 , NO 2 , methanesulfonylamino, ethanesulfonylamino, tolunesulfonylamino and dioxoisoindole; cyclic C 3 –C 8 alkyl optionally having one or more substituents selected from the group consisting of OH, NH 2 and NO 2 ; C 1 –C 4 alkyl carrying a morpholine or oxopyrrolidine group which is optionally substituted with OH, NH 2 , NO 2 or —O—; C 1 –C 4 alkyl or C 1 –C 4 aminoalkyl carrying a pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, isoxazole, oxazole, isothiazole, thiazolidine, tiazole, 1,2,5-oxadiazole, 1,2,3-oxadiazole, 1,2,5-thiadiazole, 1,2,3-thiadiazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole, pyridine, pyrimidine or triazine group which is optionally having one or more substituents selected from the group consisting of Cl, OH, NH 2 , NO 2 , C 1 –C 4 and phenyl;

cyclic C 3 –C 8 alkyl optionally having one or more substituents selected from the group consisting of OH, NH 2 and NO 2 ;

an aromatic group optionally having one or more substituents selected from the group consisting of OH; NH 2 ; hydroxyalkyl; aminoalkyl; NO 2 ; and a C 1 –C 4 alkyl group optionally having one or more substituents selected from the group consisting of OH, NH 2 , NO 2 , methanesulfonylamino, ethanesulfonylamino, tolunensulfonylamino, dioxoisoindole and thiophensulfonylamino; or

form, together with the —N—(CH 2 ) n — moiety to which they are attached, a nitrogen heterocycle optionally having one or more substituents selected from the group consisting of OH, NH 2 and NO 2 , the heterocycle containing 1 to 3 nitrogen, sulfur or oxygen atom.

In the present invention, the compounds of formula (I) as the below are most preferred: those wherein n, R 1 , R 2 and R 3 have the same meanings as defined previously; R 4 and R 5 are each independently hydrogen;

C 1 –C 4 alkyl optionally having one or more substituents selected from the group consisting of OH, NH 2 , NO 2 , morpholine, nitropyridineamino, pyridine, oxopyrrolidine, imidazole optionally having a Cl, CH 3 or phenyl substituent; and phenyl optionally having one or more substituents selected from the group consisting of OH, NH 2 , methoxy, NO 2 , methanesulfonylamino, ethanesulfonylamino, toluenesulfonylamino and dioxoisoindole;

cyclic C 3 –C 8 alkyl optionally having one or more substituents selected from the group consisting of OH, NH 2 and NO 2 ;

phenyl optionally having one or more substituents selected from the group consisting of OH; NH 2 ; NO 2 ; and C 1 –C 4 alkyl optionally having a OH, NH 2 , NO 2 , methanesulfonylamino, ethanesulfonylamino, tolunesulfonylamino, dioxoisoindole or thiophensulfonylamino substituent; or

form, together with —N—(CH 2 ) n — moiety to which they are attached, a piperidine ring optionally having one or more substituents selected from the group consisting of OH, NH 2 and NO 2 .

Important compounds of the present invention are listed in Table 1 below.

The inventive compound (except for the compound wherein R 3 is morpholin-1-yl-ethylamino) of formula (Ia) may be prepared as in Scheme 1.

wherein, p-TSA is p-toluenesulfonic acid, DMF is dimethylformamide, THF is tetrahydrofuran, TFA is trifluoroacetic acid, EDCI is ethyl-dimethylaminopropyl-carbodiimide hyrochloride, DMAP is 4-dimethylaminoprydine, HOBt is N-hydroxybenzotriazole, n, R 1 , R 2 , R 3 , R 4 and R 5 have the same meanings as defined previously.

As shown in Scheme I, the compound of formula (Ia) can be prepared by reacting 3-amino-4-methoxy benzoic acid (compound II) and an alcohol (e.g., methanol or ethanol) to obtain compound (III); adding anhydrous p-toluenesulfonic acid and benzonitrile to the compound (III) thus obtained, refluxing the mixture at 80 to 200° C., adding NaOCl thereto at room temperature and purifying by silica gel column chromatography to obtain compound (IV); dissolving the compound (IV) thus obtained in an alcohol (e.g., methanol or ethanol), adding an aqueous alkali solution (Na 2 CO 3 , NaHCO 3 , K 2 CO 2 or KHCO 3 solution) thereto and refluxing the mixture to obtain compound (V); dissolving the compound (V) thus obtained in an organic solvent, e.g., toluene, adding a Lewis acid (e.g., AlCl 3 or BBr 3 ) thereto and refluxing the mixture to obtain compound (VI); dissolving the compound (V) thus obtained in an alcohol, adding a strong acid, nitric acid or sulfuric acid, thereto at room temperature and refluxing the mixture to obtain compound (VII); dissolving the compound (VII) thus obtained and (4-bromomethylphenoxy)-methyl polystyrene Wang resin in an organic solvent, e.g., DMF, THF or chloroform, adding a base (CsCO 3 , Na 2 CO 3 , NaHCO 3 , K 2 CO 3 or KHCO 3 ) and KI thereto (1:3:3:3) and stirring the mixture at 50 to 60° C. for 1 to 24 hours to obtain compound (VIII); dissolving the compound (VIII) thus obtained in an organic solvent, adding an alcohol solution of an alkali hydroxide (e.g., LiOH, NaOH or KOH) thereto and refluxing the mixture to obtain compound (IX); dissolving the compound (IX) thus obtained in an organic solvent, adding R 4 N(CH 2 ) n R 5 and a coupling agent (e.g., EDCI/DMAP/HOBt, DCC or pyBop) thereto and stirring the mixture at room temperature to obtain compound (X); and dissolving the compound (X) thus obtained in CH 2 Cl 2 , adding trifluoroacetic acid thereto and stirring the mixture at room temperature to obtain compound (Ia).

The inventive compound (wherein R 3 is morpholin-1-yl-ethylamino) represented to formula (Ib) may be prepared, as in Scheme II.

As shown in Scheme II, the compound of formula (Ib) can be prepared by reacting 3-amino-4-methoxy benzoic acid (compound II) and an alcohol (e.g., methanol or ethanol) to obtain compound (III), adding p-toluenesulfonic acid, benzene and 4-nitrobenzonitrile thereto, refluxing the mixture at 80 to 200° C., adding NaOCl thereto at room temperature and purifying by silica gel column chromatography to obtain compound (XI); dissolving the compound (XI) thus obtained in an organic solvent, adding an aqueous alkali solution (e.g., Na2CO3 solution) thereto, refluxing the mixture and purifying by silica gel column chromatography to obtain compound (XII); dissolving the compound (XII) thus obtained in an alcohol, adding Pd/C thereto and refluxing the mixture to obtain compound (XIII); dissolving the compound (XIII) thus obtained in an organic solvent, adding a base (e.g., CsCO3, Na2CO3, NaHCO3, K2CO3 or KHCO3), 2-chloroethylmorphine and potassium iodide thereto and stirring the mixture at room temperature to obtain compound (XIV); dissolving the compound (XIV) obtained thus in an organic solvent, adding an alkali hydrate, stirring the mixture at room temperature to obtain compound (XV); dissolving the compound (XV) thus obtained in an organic solvent, adding 4,5-dichloro-1-(3-aminoprophyl)imidazole and a coupling agent (e.g., EDCI, DMAP or HOBt), stirring the mixture at room temperature and purifying by silica gel column chromatography to obtain compound (XVI); and dissolving the compound (XVI) thus obtained in MC, adding a Lewis acid thereto, stirring the mixture, concentrating the resulting solution under a reduced pressure and purifying by silica gel column chromatography to obtain compound (Ib).

A salt, hydrate, solvate and isomer of the inventive compound of formula (I) may be prepared by employing any of the known methods. The inventive compound of formula (I), a salt, hydrate, solvate or isomer thereof may used for the treatment of GSK-3β-dependent diseases including fatness, diabetes and dementia, by way of inhibiting GSK-3β activity, the inventive compound having an IC 50 value in the range of 1 to 10,000 nM.

Accordingly, the present invention includes a pharmaceutical composition which comprises a therapeutically effective amount of the compound of formula (I), a salt, hydrate, solvate or isomer thereof as an active ingredient and a pharmaceutically acceptable carrier; therefore, the pharmaceutical composition of the present invention exerts superior preventive and treating effects on GSK-β-dependent diseases such as fatness, diabetes and dementia and the like.

A pharmaceutical formulation may be prepared in accordance with any of the conventional procedures. In preparing the formulation, the active ingredient is preferably admixed or diluted with a carrier, or enclosed within a carrier, sachet or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient. Thus, the formulations may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.

Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a mammal by employing any of the procedures well known in the art.

The pharmaceutical composition of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous and intramuscular introduction. In case of human, a typical daily dose of the compound of formula (I) may range from about 0.01 to 100 mg/kg body weight, preferably 0.1 to 50 mg/kg body weight, and can be administered in a single dose or in divided doses. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.

The following examples are intended to further illustrate the present invention without limiting its scope.

PREPARATION EXAMPLE 1

Preparation of Wang Resin (p-benzyloxybenzyl Alcohol Resin)-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid (R 1 ═H, R 2 ═H and R 3 ═H)

(1) Preparation of 3-amino-4-methoxy benzoic acid methyl ester

3-amino-4-methoxy benzoic acid (40 g, 0.239 mol) was dissolved in methanol, H 2 SO 4 (38.14 ml, 0.717 mol) was added dropwise thereto and refluxed for 12 hours. The resulting mixture was cooled to room temperature and concentrated under a reduced pressure to remove methanol, neutralized with NaHCO 3 , extracted with ethyl acetate, and the extract was concentrated under a reduced pressure. The resulting residue was purified by recrystallization from ethyl acetate/hexane to obtain the title compound (39 g, 0.215 mol) in a yield of 90%.

1 H NMR (CDCl 3 ): δ 7.87–7.78 (2H, m), 7.22 (1H, d), 3.93 (3H, s), 3.82 (3H, s)

MW: 181

(2) Preparation of 4-methoxy-3-[(N-chloro-benzimidoyl)-amino]-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (41.99 g, 220.8 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (20 g, 110.38 mmol) obtained in step 1 and benzonitrile (22.77 g, 220.8 mmol) were added thereto and stirred at 180° C. for 5 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The concentrate was dissolved in 50% methanol and 5% NaOCl (56 Ml, 37.65 mmol) was added dropwise thereto. After 5 min, the resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent—MeOH/CDCl 3 =5:95, Merck, Silicagel 60) to obtain the title compound (31 g, 25.10 mmol) in a yield of 88%;

1 H NMR (CDCl 3 ): δ 7.78 (1H, d), 7.48(1H, s), 7.37–7.24 (5H, m), 6.95 (1H, d), 3.78 (6H, s)

MW: 318

(3) Preparation of 7-methoxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester

4-methoxy-3-[(N-chloro-benzimidoyl)-amino]-benzoic acid methyl ester (8 g, 25.10 mmol) obtained in step 1 was dissolved in 50 ml of 50% methanol and NaHCO 3 (5.32 g, 50.20 mmol) was added dropwise thereto at room temperature and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, and the extract was concentrated under a reduced pressure. The resulting residue was purified by recrystallization from ethyl acetate/hexane to obtain the title compound (6 g, 15.94 mmol) in a yield of 86%.

1 H NMR (CDCl 3 ): δ 10.65 (1H, br), 8.23 (2H, d), 7.49 (3H, m), 6.75 (1H, d), 4.13 (3H, s), 3.99 (3H, s)

MW: 282

(4) Preparation of 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid

7-methoxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester (4.5 g, 15.94 mmol) obtained in step 3 was dissolved in 100 ml of toluene, aluminum chloride (9.56 g, 71.73 mmol) was added thereto and refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (3.5 g, 13.77 mmol) in a yield of 86%.

1 H NMR (DMSO-d 6 ): δ 8.29 (2H; d), 7.68 (1H, d), 7.56–7.49 (3H, m), 6.67 (1H, d)

MW: 254

(5) Preparation of 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester

7-methoxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid (2.00 g, 7.46 mmol) obtained in step 4 was dissolved in 30 ml of methanol, H 2 SO 4 (2.00 ml, 37.28 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which purified by recrystallization from CHCl 3 /MeOH/hexane to obtain the title compound (1.7 g, 5.89 mmol) in a yield of 83%.

1 H NMR (CH 3 OH-d 4 ): δ 7.82 (1H, d), 7.42–7.25 (5H, m), 6.64 (1H, d), 4.92 (3H, s)

MW: 268

(6) Preparation of Wang resin (p-benzyloxybenzyl alcohol resin)-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester

p-nitrophenyl carbonate Wang resin (476 mg, 0.67 mmol) was dissolved in DMF, and 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol) obtained in step 5, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(7) Preparation of Wang resin-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester

Wang resin-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 6 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O (2:1) was added thereto and refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.

PREPARATION EXAMPLE 2

Preparation of 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (R 1 =H, R 2 =H and R 3 =Cl)

(1) Preparation of 3-[(4-chloro-N-chloro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (41.99 g, 220.76 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (20 g, 110.38 mmol) obtained in step 1 of Preparation Example 1 and 4-chlorobenzonitrile (22.78 g, 165.57 mol) were added thereto and stirred at 160° C. for 8 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding 1M NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The concentrate was dissolved in 500 ml of 50% methanol and 5% NaOCl (197 Ml, 132.46 mmol) was added dropwise thereto. After 5 min, the resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chlomatography (eluent—MeOH:CDCl3=5:95, Merck, Silicagel 60) to obtain the title compound (19.43 g, 55.19 mmol) in a yield of 50%.

1 H NMR (CH 3 OH-d 4 ): δ 7.62 (2H, m), 7.22–7.15 (4H, m), 6.59 (1H, s), 4.00–3.80 (6H, d)

MW: 352

(2) Preparation of 2-(4-chloro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester

3-[(4-chloro-N-chloro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (5.5 g, 15.63 mmol) obtained in step 1 was dissolved in 40 ml of 50% methanol and Na 2 CO 3 (3.53 g, 33.26 mmol) was added dropwise thereto at room temperature and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, the extract was concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (2.57 g, 8.13 mmol) in a yield of 52%.

1 H NMR (CDCl 3 ): δ 8.15 (2H, d), 7.95 (1H, d), 7.51 (2H, m), 6.75 (1H, d), 4.06 (3H, s)

MW: 316

(3) Preparation of 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

2-(4-chloro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (1.0 g, 3.16 mmol) obtained in step 2 was dissolved in 10 ml of toluene, aluminum chloride (2.11 g, 15.8 mmol) was added thereto and refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (745 mg, 2.59 mmol) in a yield of 82%.

1 H NMR (CH 3 OH-d 4 ): δ 8.06 (3H, m), 7.50 (2H, m), 6.97 (1H, d)

MW: 288

(4) Preparation of 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (200 mg, 0.69 mmol) obtained in step 3 was dissolved in 5 ml of methanol, H 2 SO 4 (0.18 ml, 3.45 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with 1M NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which was purified by silica gel column chromatography (eluent—MeOH/CDCl 3 =5/95, Merck, Silicagel 60) to obtain the title compound (166 mg, 0.55 mmol) in a yield of 80%.

1 H NMR (CH 3 OH-d 4 ): δ 10.75 (1H, Br), 7.89 (3H, m), 7.46 (2H, d), 6.82 (1H, d), 3.39 (3H, s)

MW: 302

(5) Preparation of Wang Resin-Supported 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

(4-bromomethylphenoxy)-methyl polystyrene Wang resin (476 mg, 0.67 mmol) was dissolved in 5 ml of DMF, and 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol) obtained in step 4, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(6) Preparation of Wang resin-supported 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

Wang resin-supported 2-(4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 5 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O (1:1) was added thereto and the resulting mixture was refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.

PREPARATION EXAMPLE 3

Preparation of 2-(2,4-dichloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (R 1 ═Cl, R 2 ═H and R 3 ═Cl)

(1) Preparation of 3-[(2,4-dichloro-N-chloro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (20.99 g, 110.04 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (10 g, 55.20 mmol) obtained in step 1 of Preparation Example 1 and 2,4-dichlorobenzonitrile (18.99 g, 110.04 mol) were added thereto and stirred at 180° C. for 6 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The concentrate was dissolved in 50% methanol and 5% NaOCl (30 ml, 20.64 mmol) was added dropwise thereto. After 5 min, the resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent—MeOH:CDCl 3 =5:95, Merck, Silicagel 60) to obtain the title compound (18 g, 10.32 mmol) in a yield of 84%.

1 H NMR (CDCl 3 ): δ 8.23 (1H, br), 7.75 (1H, d), 7.44 (1H, d), 7.36–7.26 (2H, m), 7.03 (1H, s), 6.88 (1H, d), 3.96 (3H, s), 3.76 (3H, s)

MW: 318

(2) Preparation of 2-(2,4-dichloro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester

3-[(2,4-dichloro-N-chloro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (4 g, 10.32 mmol) obtained in step 1 was dissolved in 50 ml of 50% methanol and NaHCO 3 (2.19 g, 20.64 mmol) was added dropwise thereto at room temperature and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, and the extract was concentrated under a reduced pressure. The resulting residue was purified by recrystallization from ethyl acetate/hexane to obtain the title compound (3.2 g, 5.47 mmol) in a yield of 88%.

1 H NMR (CDCl 3 ): δ 8.54 (1H, d), 7.94 (1H, d), 7.48 (1H, s), 7.42 (1H, d), 6.76 (1H,d), 4.44 (3H, s), 3.99 (3H, s)

MW: 351

(3) Preparation of 2-(2,4-dichloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

2-(2,4-dichloro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (1.9 g, 5.47 mmol) obtained in step 2 was dissolved in 100 ml of toluene, aluminum chloride (3.61 g, 27.05 mmol) was added thereto and refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (1.63 g, 5.03 mmol) in a yield of 92%.

1 H NMR (DMSO-d 6 ): δ 8.19 (1H, d), 7.78 (1H, d), 7.62–7.55 (2H, m), 6.82 (1H, d)

MW: 323

(4) Preparation of 2-(2,4-dichloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

2-(2,4-dichloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (1.63 g, 5.03 mmol) obtained in step 3 was dissolved in 30 ml of methanol, and H 2 SO 4 (1.08 ml, 20.12 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which was purified by recrystallization from ethyl acetate/hexane to obtain the title compound (1.5 g, 3.62 mmol) in a yield of 86%.

1 H NMR (CDCl 3 ): δ 11.42 (1H, br), 8.21 (1H, d), 7.89 (1H, d), 7.56 (1H, s), 7.38 (1H, d), 6.82 (1H, d), 3.99 (3H, s)

MW: 337

(5) Preparation of Wang resin-supported 2-(2,4-chloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

p-nitrophenyl carbonate Wang resin (476 mg, 0.67 mmol) was dissolved in DMF, and 2-(2,4-dichloro-phenyl)-7-hydroxy-2H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol), obtained in step 4, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(6) Preparation of Wang resin-supported 2-(2,4-dichloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

Wang resin-supported 2-(2,4-dichloro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 5 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O (2:1) was added thereto and the resulting mixture was refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.

PREPARATION EXAMPLE 4

Preparation of Wang resin-supported 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (R 1 ═H, R 2 ═H and R 3 ═F)

(1) Preparation of 3-[(4-fluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (41.99 g, 220.76 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (20 g, 110.38 mmol) obtained in step 1 of Preparation Example 1 and 4-fluorobenzonitrile (20.00 g, 165.57 mol) were added thereto and stirred at 160° C. for 8 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent—MeOH:CDCl 3 =5:95, Merck, Silicagel 60) to obtain the title compound (22.67 g, 75.06 mmol) in a yield of 68%.

1 H NMR (CDCl 3 ): δ 7.92–7.75 (4H, m), 7.15–7.02 (3H, m), 3.87–3.81 (6H, d)

MW: 302

(2) Preparation of 2-(4-fluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester

3-[(4-fluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (10 g, 34.48 mmol) obtained in step 1 was dissolved in 50% methanol and 5% NaOCl (61 ml, 41.38 mmol) was added dropwise thereto at room temperature. After 5 min, Na 2 CO 3 (7.31 g, 68.96 mmol) was added dropwise thereto and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, and the extract was concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (5.66 g, 19.65 mmol) in a yield of 57%.

1 H NMR (CDCl 3 ): δ 8.18 (2H, t), 7.91 (1H, d), 7.30–7.25 (2H, t), 6.65 (1H, d), 6.85 (1H, d), 4.08 (3H, s), 3.98 (3H, s)

MW: 300

(3) Preparation of 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

2-(4-fluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (3 g, 10.00 mmol) obtained in step 2 was dissolved in toluene, aluminum chloride (6.67 g, 30.00 mmol) was added thereto and refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (1.96 g, 7.20 mmol) in a yield of 72%.

1 H NMR (MeOH-d 4 ): δ 8.19–8.15 (2H, t), 8.06 (1H, d), 7.50–7.44 (2H, t), 7.00 (1H, d)

MW: 272

(4) Preparation of 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (500 mg, 1.84 mmol) obtained in step 3 was dissolved in methanol, H 2 SO 4 (0.49 ml, 9.20 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which was purified by silica gel chromatography to obtain the title compound (397 mg, 1.39 mmol) in a yield of 76%.

1 H NMR (CH 3 OH-d 4 ): δ 8.22–8.18 (2H, t), 7.80 (1H, d), 7.32–7.26 (2H, t), 6.70 (1H, d), 3.97 (3H, s)

MW: 286

(5) Preparation of Wang resin-supported 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

(4-bromomethylphenoxy)-methyl polystyrene Wang resin (476 mg, 0.67 mmol) was dissolved in DMF, and 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol) obtained in step 4, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(6) Preparation of Wang Resin-Supported 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

Wang resin-supported 2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 5 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O (2:1) was added thereto and the resulting mixture was refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.

PREPARATION EXAMPLE 5

Preparation of Wang resin-supported 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (R 1 ═F, R 2 ═H and R 3 ═F)

(1) Preparation of 3-[(2,4-difluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (25.0 g, 137.43 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (10 g, 55.25 mmol) obtained in step 1 of Preparation Example 1 and 2,4-difluorobenzonitrile (11.53 g, 82.87 mol) were added thereto and stirred at 160° C. for 8 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (10.0 g, 31.22 mmol) in a yield of 57%.

1 H NMR (CDCl 3 ): δ 8.31–8.22 (1H, m), 7.82–7.79 (1H, d), 7.65 (1H, s), 7.02–6.85 (3H, m), 3.88 (6H, s)

MW: 320

(2) Preparation of 2-(2,4-difluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester

3-[(2,4-difluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (9.5 g, 29.66 mmol) obtained in step 1 was dissolved in 50% methanol and 5% NaOCl (53 ml, 35.71 mmol) was added dropwise thereto at room temperature. After 5 min, Na 2l CO 3 (6.29 g, 59.34 mmol) was added dropwise thereto and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, and the extract was concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (3.50 g, 11.0 mmol) in a yield of 37%.

1 H NMR (CDCl 3 ): δ 10.99 (1H, bs). 8.65–8.57 (1H, m), 0.92 (1H, d), 7.10–6.97 (2H, m), 6.76 (1H, d), 4.13 (3H, s), 4.00 (3H, s)

MW: 318

(3) Preparation of 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

2-(2,4-difluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (2.24 g, 7.04 mmol) obtained in step 2 was dissolved in toluene, aluminum chloride (3.75 g, 28.12 mmol) was added thereto and refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (1.70 g, 5.86 mmol) in a yield of 83%.

1 H NMR (CH 3 OH-d 4 ): δ 8.13–8.03 (2H, m), 7.47–7.33 (2H, m), 7.04 (1H, d)

MW: 290

(4) Preparation of 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (1.70 mg, 5.86 mmol) obtained in step 3 was dissolved in methanol, SOCl 2 (8.2 ml, 112 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which was purified by silica gel chromatography to obtain the title compound (1.50 mg, 1.64 mmol) in a yield of 84%.

1 H NMR (DMSO-d 6 ): δ 12.04 (1H, bs), 0.30–8.04 (1H, m), 7.73 (1H, d), 7.55–7.48 (1H, m), 7.33–7.27 (1H, m), 6.70 (1H, d), 4.01 (3H, s)

MW: 304

(5) Preparation of Wang resin-supported 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

(4-bromomethylphenoxy)-methyl polystyrene Wang resin (476 mg, 0.67 mmol) was dissolved in DMF, and 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol) obtained in step 4, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(6) Preparation of Wang resin-supported 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

Wang resin-supported 2-(2,4-difluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 5 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O was added thereto and the resulting mixture was refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.

PREPARATION EXAMPLE 6

Preparation of Wang resin-supported 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (R 1 ═Cl, R 2 ═H and R 3 ═F)

(1) Preparation of 3-[(2-chloro-4-fluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (41.99 g, 220.76 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (20 g, 110.38 mmol) obtained in step 1 of Preparation Example 1 and 2-chloro-4-fluorobenzonitrile (25.76 g, 165.57 mol) were added thereto and stirred at 160° C. for 8 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (26.70 g, 79.47 mmol) in a yield of 72%.

1 H NMR (CDCl 3 ): δ 7.92–7.75 (4H, m), 7.15–7.02 (3H, m), 3.87–3.81 (6H, d)

MW: 336

(2) Preparation of 2-(2-chloro-4-fluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester

3-[(2-chloro-4-fluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (10 g, 29.76 mmol) obtained in step 1 was dissolved in 50% methanol and 5% NaOCl (53 ml, 35.71 mmol) was added dropwise thereto at room temperature. After 5 min, Na 2 CO 3 (6.31 g, 59.52 mmol) was added dropwise thereto and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, the extract was concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (5.17 g, 15.48 mmol) in a yield of 52%.

1 H NMR (CDCl 3 ): δ 8.18 (2H, t), 0.91 (1H, d), 7.30–7.25 (2H, t), 6.65 (1H, d), 6.85 (1H, d), 4.08 (3H, s), 3.98 (3H, s)

MW: 334

(3) Preparation of 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

2-(2-chloro-4-fluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (3 g, 8.98 mmol) obtained in step 2 was dissolved in toluene and aluminum chloride (5.99 g, 44.90 mmol) was added thereto, refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (1.87 g, 6.11 mmol) in a yield of 68%.

1 H NMR (CH 3 OH-d 4 ): δ 8.19–8.15 (2H, t), 8.06 (1H, d), 7.50–7.44 (2H, t), 7.00 (1H, d)

MW: 306

(4) Preparation of 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (500 mg, 1.63 mmol) obtained in step 3 was dissolved in methanol, H 2 SO 4 (0.43 ml, 8.15 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which was purified by silica gel chromatography to obtain the title compound (393 mg, 1.23 mmol) in a yield of 67%.

1 H NMR (CH 3 OH-d 4 ): δ 8.22–8.18 (2H, t), 7.80 (1H, d), 7.32–7.26 (2H, t), 6.70 (1H, d), 3.97 (3H, s)

MW: 320

(5) Preparation of Wang resin-supported 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

(4-bromomethylphenoxy)-methyl polystyrene Wang resin (476 mg, 0.67 mmol) was dissolved in DMF, and 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol) obtained in step 4, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(6) Preparation of Wang resin-supported 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

Wang resin-supported 2-(2-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 5 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O was added thereto and the resulting mixture was refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.

PREPARATION EXAMPLE 7

Preparation of Wang resin-supported 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (R 1 ═H, R 2 ═Cl and R 3 ═F)

(1) Preparation of 3-[(3-chloro-4-fluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester

Anhydrous p-toluene sulfonic acid (10 g, 52.57 mmol) was melted at 120° C. and 3-amino-4-methoxy benzoic acid methyl ester (3.88 g, 21.44 mmol) obtained in step 1 of Preparation Example 1 and 3-chloro-4-fluorobenzonitrile (5.0 g, 32.14 mol) were added thereto and stirred at 160° C. for 8 hours. The resulting solution was cooled to room temperature and the reaction was stopped by adding NaHCO 3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO 4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (3.24 g, 9.62 mmol) in a yield of 45%.

1 H NMR (CDCl 3 ): δ 7.96–7.95 (1H, m), 7.76–7.73 (2H, m), 7.60 (1H, bs), 7.17–7.11 (1H, m), 6.93(1H, d), 3.85(3H, s), 3.84 (3H, d)

MW: 336

(2) Preparation of 2-(3-chloro-4-fluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester

3-[(3-chloro-4-fluoro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (3.24 g, 9.62 mmol) was dissolved in 50% methanol and 5% NaOCl (18 ml, 11.90 mmol) was added dropwise thereto at room temperature. After 5 min, Na 2 CO 3 (2.04 g, 19.25 mmol) was added dropwise thereto and refluxed for 5 min. The resulting solution was cooled to room temperature, extracted with ethyl acetate, and the extract was concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (0.95 g, 2.83 mmol) in a yield of 30%.

1 H NMR (CDCl 3 ): δ 10.68 (1H, bs), 8.23–8.20 (1H, m), 7.96–7.91 (1H, m), 7.87 (1H, d), 7.27–7.20 (1H, m), 6.73 (1H, d), 4.10 (3H, s), 3.97 (3H, s)

MW: 334

(3) Preparation of 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

2-(3-chloro-4-fluoro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (0.95 g, 8.98 mmol) obtained in step 2 was dissolved in toluene, aluminum chloride (1.5 g, 11.25 mmol) was added thereto and refluxed for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding 3 N HCl thereto and stirred for 30 min. The precipitate formed was filtered, washed with benzene and dried to obtain the title compound (0.81 g, 2.64 mmol) in a yield of 80%.

1 H NMR (MeOH-d 4 ): δ 8.34 (1H, dd), 8.22–8.08 (2H, m), 7.62 (1H, t), 7.03 (1H, d)

MW: 306

(4) Preparation of 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (800 mg, 2.64 mmol) obtained in step 3 was dissolved in methanol, SOCl 2 (1.93 ml, 26.41 mmol) was added dropwise thereto and refluxed for 15 hours. The resulting solution was cooled to room temperature, concentrated under a reduced pressure to remove methanol, and the residue was neutralized with NaHCO 3 . Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which was purified by silica gel chromatography to obtain the title compound (690 mg, 2.15 mmol) in a yield of 81%.

1 H NMR (DMSO-d 6 ): δ 12.39 (1H, bs), 8.56 (1H, d), 8.30 (1H, bs), 7.72 (1H, d), 7.59 (1H, t), 6.69 (1H, d), 3.90 (3H, s)

MW: 320

(5) Preparation of Wang resin-supported 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester

(4-bromomethylphenoxy)-methyl polystyrene Wang resin (476 mg, 0.67 mmol) was dissolved in DMF, and 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (567 mg, 2.01 mmol) obtained in step 4, Cs 2 CO 3 (655 mg, 2.01 mmol) and KI (334 mg, 2.01 mmol) were added thereto to be stirred at 50 to 60° C. for 12 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with DMF, MeOH and CH 2 Cl 2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.

(6) Preparation of Wang resin-supported 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid

Wang resin-supported 2-(3-chloro-4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid methyl ester (570 mg, 0.47 mmol) obtained in step 5 was dissolved in THF, LiOH.H 2 O (99 mg, 2.35 mmol) in MeOH—H 2 O was added thereto and the resulting mixture was refluxed for 5 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was washed with MeOH and CH 2 Cl 2 , and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.