Patent Grant
7179832
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;
R1, R2 and R3 are each independently hydrogen, hydroxy, halogen or morpholin-1-yl-ethylamino;
R4 and R5 are each independently hydrogen;
linear or cyclic C1–C6 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 C3–C8 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 C1–C6 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 C3–C8 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—(CH2)n— moiety to which they are attached, a nitrogen heterocycle optionally having one or more substituents selected from the group consisting of OH, NH2, NO2, the heterocycle containing optional nitrogen or oxygen.
Among the compounds of formula (I) of the present invention, the preferred are:
those wherein n, R1, R2 and R3 have the same meanings as defined previously;
R4 and R5 are each independently hydrogen;
C1–C4 alkyl optionally having one or more substituents selected from the group consisting of OH, NH2, NO2, and an aromatic group, the aromatic group optionally having one or more substituents selected from the group consisting of OH, C1–C4 alkyloxy, NH2, NO2, methanesulfonylamino, ethanesulfonylamino, tolunesulfonylamino and dioxoisoindole; cyclic C3–C8 alkyl optionally having one or more substituents selected from the group consisting of OH, NH2 and NO2; C1–C4 alkyl carrying a morpholine or oxopyrrolidine group which is optionally substituted with OH, NH2, NO2 or —O—; C1–C4 alkyl or C1–C4 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, NH2, NO2, C1–C4 and phenyl;
cyclic C3–C8 alkyl optionally having one or more substituents selected from the group consisting of OH, NH2 and NO2;
an aromatic group optionally having one or more substituents selected from the group consisting of OH; NH2; hydroxyalkyl; aminoalkyl; NO2; and a C1–C4 alkyl group optionally having one or more substituents selected from the group consisting of OH, NH2, NO2, methanesulfonylamino, ethanesulfonylamino, tolunensulfonylamino, dioxoisoindole and thiophensulfonylamino; or
form, together with the —N—(CH2)n— moiety to which they are attached, a nitrogen heterocycle optionally having one or more substituents selected from the group consisting of OH, NH2 and NO2, 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, R1, R2 and R3 have the same meanings as defined previously; R4 and R5 are each independently hydrogen;
C1–C4 alkyl optionally having one or more substituents selected from the group consisting of OH, NH2, NO2, morpholine, nitropyridineamino, pyridine, oxopyrrolidine, imidazole optionally having a Cl, CH3 or phenyl substituent; and phenyl optionally having one or more substituents selected from the group consisting of OH, NH2, methoxy, NO2, methanesulfonylamino, ethanesulfonylamino, toluenesulfonylamino and dioxoisoindole;
cyclic C3–C8 alkyl optionally having one or more substituents selected from the group consisting of OH, NH2 and NO2;
phenyl optionally having one or more substituents selected from the group consisting of OH; NH2; NO2; and C1–C4 alkyl optionally having a OH, NH2, NO2, methanesulfonylamino, ethanesulfonylamino, tolunesulfonylamino, dioxoisoindole or thiophensulfonylamino substituent; or
form, together with —N—(CH2)n— moiety to which they are attached, a piperidine ring optionally having one or more substituents selected from the group consisting of OH, NH2 and NO2.
Important compounds of the present invention are listed in Table 1 below.
The inventive compound (except for the compound wherein R3 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, R1, R2, R3, R4 and R5 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 (Na2CO3, NaHCO3, K2CO2 or KHCO3 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., AlCl3 or BBr3) 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 (CsCO3, Na2CO3, NaHCO3, K2CO3 or KHCO3) 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 R4N(CH2)nR5 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 CH2Cl2, adding trifluoroacetic acid thereto and stirring the mixture at room temperature to obtain compound (Ia).
The inventive compound (wherein R3 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 IC50 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.
3-amino-4-methoxy benzoic acid (40 g, 0.239 mol) was dissolved in methanol, H2SO4 (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 NaHCO3, 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%.
1H NMR (CDCl3): δ 7.87–7.78 (2H, m), 7.22 (1H, d), 3.93 (3H, s), 3.82 (3H, s)
MW: 181
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 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 MgSO4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent—MeOH/CDCl3=5:95, Merck, Silicagel 60) to obtain the title compound (31 g, 25.10 mmol) in a yield of 88%;
1H NMR (CDCl3): 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
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 NaHCO3 (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%.
1H NMR (CDCl3): 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
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%.
1H NMR (DMSO-d6): δ 8.29 (2H; d), 7.68 (1H, d), 7.56–7.49 (3H, m), 6.67 (1H, d)
MW: 254
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, H2SO4 (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 NaHCO3. Then, the neutralized residue was extracted with ethyl acetate and concentrated under a reduced pressure to obtain a residue which purified by recrystallization from CHCl3/MeOH/hexane to obtain the title compound (1.7 g, 5.89 mmol) in a yield of 83%.
1H NMR (CH3OH-d4): δ 7.82 (1H, d), 7.42–7.25 (5H, m), 6.64 (1H, d), 4.92 (3H, s)
MW: 268
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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O (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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 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 MgSO4 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%.
1H NMR (CH3OH-d4): δ 7.62 (2H, m), 7.22–7.15 (4H, m), 6.59 (1H, s), 4.00–3.80 (6H, d)
MW: 352
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 Na2CO3 (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%.
1H NMR (CDCl3): δ 8.15 (2H, d), 7.95 (1H, d), 7.51 (2H, m), 6.75 (1H, d), 4.06 (3H, s)
MW: 316
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%.
1H NMR (CH3OH-d4): δ 8.06 (3H, m), 7.50 (2H, m), 6.97 (1H, d)
MW: 288
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, H2SO4 (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 NaHCO3. 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/CDCl3=5/95, Merck, Silicagel 60) to obtain the title compound (166 mg, 0.55 mmol) in a yield of 80%.
1H NMR (CH3OH-d4): δ 10.75 (1H, Br), 7.89 (3H, m), 7.46 (2H, d), 6.82 (1H, d), 3.39 (3H, s)
MW: 302
(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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O (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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 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 MgSO4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent—MeOH:CDCl3=5:95, Merck, Silicagel 60) to obtain the title compound (18 g, 10.32 mmol) in a yield of 84%.
1H NMR (CDCl3): 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
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 NaHCO3 (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%.
1H NMR (CDCl3): 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
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%.
1H NMR (DMSO-d6): δ 8.19 (1H, d), 7.78 (1H, d), 7.62–7.55 (2H, m), 6.82 (1H, d)
MW: 323
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 H2SO4 (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 NaHCO3. 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%.
1H NMR (CDCl3): 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
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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O (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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent—MeOH:CDCl3=5:95, Merck, Silicagel 60) to obtain the title compound (22.67 g, 75.06 mmol) in a yield of 68%.
1H NMR (CDCl3): δ 7.92–7.75 (4H, m), 7.15–7.02 (3H, m), 3.87–3.81 (6H, d)
MW: 302
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, Na2CO3 (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%.
1H NMR (CDCl3): 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
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%.
1H NMR (MeOH-d4): δ 8.19–8.15 (2H, t), 8.06 (1H, d), 7.50–7.44 (2H, t), 7.00 (1H, d)
MW: 272
2-(4-fluoro-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (500 mg, 1.84 mmol) obtained in step 3 was dissolved in methanol, H2SO4 (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 NaHCO3. 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%.
1H NMR (CH3OH-d4): 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
(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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O (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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 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%.
1H NMR (CDCl3): 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
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, Na2l CO3 (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%.
1H NMR (CDCl3): 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
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%.
1H NMR (CH3OH-d4): δ 8.13–8.03 (2H, m), 7.47–7.33 (2H, m), 7.04 (1H, d)
MW: 290
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, SOCl2 (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 NaHCO3. 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%.
1H NMR (DMSO-d6): 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
(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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O 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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 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%.
1H NMR (CDCl3): δ 7.92–7.75 (4H, m), 7.15–7.02 (3H, m), 3.87–3.81 (6H, d)
MW: 336
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, Na2CO3 (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%.
1H NMR (CDCl3): 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
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%.
1H NMR (CH3OH-d4): δ 8.19–8.15 (2H, t), 8.06 (1H, d), 7.50–7.44 (2H, t), 7.00 (1H, d)
MW: 306
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, H2SO4 (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 NaHCO3. 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%.
1H NMR (CH3OH-d4): 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
(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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O 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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
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 NaHCO3 thereto. The resulting mixture was extracted with ethyl acetate, the extract was dried over MgSO4 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%.
1H NMR (CDCl3): 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
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, Na2CO3 (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%.
1H NMR (CDCl3): 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
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%.
1H NMR (MeOH-d4): δ 8.34 (1H, dd), 8.22–8.08 (2H, m), 7.62 (1H, t), 7.03 (1H, d)
MW: 306
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, SOCl2 (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 NaHCO3. 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%.
1H NMR (DMSO-d6): 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
(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, Cs2CO3 (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 CH2Cl2 and dried to obtain the title compound (608 mg, 0.65 mmol) in a yield of 98%.
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.H2O (99 mg, 2.35 mmol) in MeOH—H2O 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 CH2Cl2, and dried to obtain the title compound (551 mg, 0.42 mmol) in a yield of 90%.
Wang resin-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid (36 mg, 0.03 mmol) obtained in Preparation Example 1 was dissolved in 3 ml of DMF and aluminum chloride (5 mg, 0.09 mmol), EDCI (18 mg, 0.09 mmol), DMAP (11 mg, 0.09 mmol) and HOBt (12 mg, 0.09 mmol) were added thereto and the resulting mixture was stirred at room temperature. The resulting solution was filtered, the filtrate was washed with DMF, MeOH and CH2Cl2 and dried to obtain Wang resin-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid amide.
Then, 30 mg of Wang resin-supported 7-hydroxy-2-phenyl-1H-benzoimidazole-4-carboxylic acid amide was dissolved in 0.2 ml of CH2Cl2, 0.2 ml of trifluoroacetic acid was added thereto and stirred for 30 min. The resulting solution was filtered, the filtrate was washed with MeOH and CH2Cl2 and dried to obtain the title compound in a yield of 90%.
1H NMR (CH3OH-d4): δ 8.15 (2H, d), 7.84 (1H, d), 7.78–7.56 (3H, m), 6.83 (1H, m)
MW: 253
The same procedure as described in Example 1 was repeated using R4R5NH2 listed in Table 2 to obtain the compounds 2 to 203, respectively.
1H NMR(CH3OH-d4)
Anhydrous p-toluenesulfonic acid (6.30 g, 33.1 mmol) was added to 50 ml of benzene and the resulting mixture was refluxed while removing water using a dean-stock trap. Added thereto were 3-amino-4-methoxy benzoic acid methyl ester (3 g, 16.6 mmol) obtained in step 1 of Preparation Example 1 and 4-nitrobenzonitrile (2.94 g, 19.9 mol), followed by stirring at 160° C. for 8 hours. The resulting solution was cooled to room temperature, the reaction was stopped by adding NaHCO3 thereto, extracted with ethyl acetate, the extract was dried over MgSO4 and concentrated under a reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain the title compound (2.83 g, 8.59 mmol) in a yield of 52%.
1H NMR (CDCl3): δ 8.12–8.09 (m, 2H), 7.82 (d,1H), 7.70–7.69 (m, 1H), 6.98 (d, 1H), 4.91 (bs, 2H), 3.89(s, 6H)
3-[(4-nitro-benzimidoyl)-amino]-4-methoxy-benzoic acid methyl ester (1.63 g, 4.95 mmol) was dissolved in 50% methanol, and 5% NaOCl was added dropwise thereto at room temperature. After checking the reaction by TLC, Na2CO3 (1.05 g, 9.38 mmol) seas added dropwise thereto and refluxed for 40 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 (0.75 g, 2.28 mmol) in a yield of 46%.
1H NMR (CDCl3): δ 10.90 (bs, 1H), 8.36–8.31 (m, 4H), 7.95 (d, 1H), 6.78 (d, 1H), 4.16 (s, 3H), 4.01 (s, 3H)
2-(4-nitro-phenyl)-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (0.63 g, 1.92 mmol) obtained in step 2 was dissolved in 15 ml of EtOH, 0.1 g of 10% Pd/C was added thereto and stirred for 24 hours while hydrogen was supplied thereto from a balloon fulfilled with H2 gas. The resulting solution was filtered and dried to obtain the title compound (0.57 g, 1.92 mmol) in a yield of 100%.
1H NMR (CH3OH-d4): δ 10.48 (bs, 1H), 7.93 (d, 2H), 7.82 (d, 1H), 6.77 (d, 2H), 6.71 (d, 1H), 4.11 (s,3H), 3.98 (s, 3H)
2-(4-amino-phenyl)-7-hydroxy-1H-benzoimidazole-4-carboxylic acid (160 mg, 0.54 mmol) obtained in step 3 was dissolved in DMF, cesium carbonate (0.53 g, 1.61 mmol) was added thereto and stirred for 5 min. Added thereto were 2-chloroethylmorpholine (0.12 g, 0.64 mmol) and potassium iodide (0.18 g, 1.08 mmol), followed by stirring for 24 hours. Then, the resulting solution was extracted with ethyl acetate, the extract was concentrated under a reduced pressure, and the residue was purified by silica gel chromatography to obtain the title compound (91 mg, 0.22 mmol) in a yield of 41%.
1H NMR (CH3OH-d4): δ 7.97 (d, 1H), 7.57 (d, 2H), 6.77–6.73 (m, 3H), 4.54 (t, 2H), 4.11 (s, 3H), 3.99 (s, 3H), 3.57–3.55(m, 4H), 2.64 (t, 2H), 2.31–2.28 (m, 4H)
2-[(2-morpholinoethyl)-4-amino-phenyl]-7-methoxy-1H-benzoimidazole-4-carboxylic acid methyl ester (22 mg, 0.05 mmol) was dissolved in THF/H2O, LiOHH2O (6.7 mg, 0.16 mmol) was added thereto and stirred at room temperature. The resulting solution was filtered to remove residual LiOHH2O, and the solvent was removed. The residue was dried and dissolved in DMF. Added thereto were 4,5-dichloro-1-(3-aminopropyl)imidazole (12.5 mg, 0.06 mmol), EDCI (30.9 mg, 0.16 mmol), DMAP (65.6 mg, 0.54 mmol) and HOBt (21.8 mg, 0.16 mmol), followed by stirring at room temperature. The resulting solution was extracted with ethyl acetate and concentrated under a reduced pressure. The resulting residue was purified by silica gel chromatography to obtain the title compound (19 mg, 0.03 mmol) in a yield of 63%.
1H NMR (CH3OH-d4): δ 7.93 (d, 1H), 7.77–7.75 (m, 3H), 7.52 (d, 2H), 6.92 (d, 1H), 4.17 (t, 2H), 4.06–4.02 (m, 5H), 3.58–3.56 (m, 4H), 3.50 (t, 2H), 2.66 (t, 2H), 2.31–2.29 (m, 4H), 2.16 (q, 2H)
2-[(2-morpholinoethyl)-4-amino-phenyl]-7-methoxy-1H-benzoimidazole-4-carboxylic acid-[3-(4,5-dichloro-imidazol-1-yl)-propyl]-amide (15 mg, 0.03 mmol) obtained in step 5 was dissolved in MC, BBr3 (1.0M solution in MC, 0.3 mL, 0.3 mmol) was added thereto and stirred at room temperature for 48 hours. The reaction was stopped by adding water thereto and the resulting solution was extracted with MC/MeOH (7:1). The extract was concentrated under a reduced pressure and purified by silica gel chromatography to obtain the title compound (5.9 mg, 0.01 mmol) in a yield of 40%.
1H NMR (CH3OH-d4): δ 7.95 (d, 1H), 7.81–7.79 (m, 4H), 7.55 (d, 1H), 6.94 (d, 1H), 4.15 (t, 2H), 3.94 (t, 2H), 3.59 (t, 2H), 3.58–3.56 (m, 4H), 2.64 (t, 2H), 2.32–2.30 (m, 4H), 2.18 (q, 2H)
Assay for GSK-3β inhibiting activity
The GSK-3β inhibiting activity was determined in accordance with the method of Shultz et al. described in U.S. Pat. No. 6,153,618, with minor modifications by using phospho-CREB peptide as a substrate.
First, PCR (polymerase chain reaction) was carried out using human DNA as a template as well as primers which were designed to correspond to the 3′- and 5′ ends of the polynucleotide coding human GSK-3β gene (Genbank Accession No.: L33801). The BamH1/XhoI fragment of the amplified PCR product thus obtained was inserted into the pGex vector between the BamH1 and XhoI sites, and the vector obtained was transformed into E. coli BL21(DE3). The transformed cells thus obtained was incubated in LB agar plates (1% Bacto-trypton, 0.5% yeast extract, 1% NaCl) containing ampicillin (100 μl/ml) until the optical density at 600 nm reached about 0.5. The cultured mixture was cooled to 18° C. and isopropyl β-D-thiogalacto-pyranoside (IPTG) was added thereto to a final concentration of 0.5 mM. After 16 hours, the resultant was centrifuged at 10,000×g for 10 min, the collected cells were suspended in a buffer solution (30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5% glycerol, 2 mM DTT) and the cells were disrupted using Sonic Dismembrator (Fisher, U.S.A.) in a ice bath. The resulting solution was centrifuged at 16,000 rpm for 30 minutes. The supernatant was connected to GST (Glutathione-S-transferase) column (Pharmacia Biotech, U.S.A.) equilibrated in the same buffer solution, purified by glutathione affinity chromatography (eluent: 5 mM glutathione), and then, digested with thrombin to cleave the connecting site between the GST moiety and GSK-3β protein. The purified GSK-3β protein was diluted in a buffer solution (20 mM HEPES (pH 7.5), 5% glycerol, 2 mM DTT) to a final concentration of 50 mM NaCl and the resulting solution was subjected to mono S column chromatography (eluent: linear gradient from 0M to 1M NaCl buffer) using mono S column (Pharmacia Biotech, U.S.A.) equilibrated in the same buffer solution to obtain GSK-3β protein.
100 nM GSK-3β protein, 12.5 mM each of the compounds prepared in Examples 1 to 204 dissolved in DMSO, an assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT), 100 μM phospho-CREB peptide (NEB, USA), 100 μM ATP, 32P-ATP and 1 μCi were reacted at 30° C. for 1 hour. The reaction was stopped by adding 5 μl of 5% phosphoric acid to 25 μl of the resulting solution. The resulting mixture was centrifuged at 15,000 rpm for 10 min, 20 μl of the supernatant was added dropwise to Whatman p81 filter paper, and then, the resulting filter paper was washed with 0.5% phosphate buffer for 10 min. The filter paper was further washed 3 times and the enzymatic activity was determined by examining the extent of phospho-CREB peptide phosphorylation which is represented by the unit of count per minute (CPM), measured with a β-counter (Packard, USA).
The GSK-3β inhibiting activity was then calculated in accordance with the following equation:
wherein the blank represents a value obtained without the use of the enzyme and the compound of the present invention, and the control, in the absence of the compound of the present invention.
The IC50 value of the inventive compound was determined from the degree of inhibition (%) and the result is shown in Table 3.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2003-0004607 | Jan 2003 | KR | national |
| 10-2003-0004608 | Jan 2003 | KR | national |
| 10-2003-0042442 | Jun 2003 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2004/000097 | 1/20/2004 | WO | 00 | 7/22/2005 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2004/065370 | 8/5/2004 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5821258 | Matsunaga et al. | Oct 1998 | A |
| 6310082 | Griffin et al. | Oct 2001 | B1 |
| 6509365 | Lubisch et al. | Jan 2003 | B1 |
| Number | Date | Country |
|---|---|---|
| WO 9507263 | Mar 1995 | WO |
| WO-0029384 | May 2000 | WO |
| WO 02102978 | Dec 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060079521 A1 | Apr 2006 | US |
