Patent Application
20080027092
a) α-(2-fluoro-5-methylphenyl)-4-quinolinemethanol
n-BuLi (2.5M in hexanes, 5.1 ml) was added dropwise to 3-bromo-4-fluorotoluene (2 g) in THF (100 ml) at −78° C. The reaction mixture was stirred for 10 min and then 4-quinolinecarboxaldehyde (1.7 g) in THF (10 ml) was added dropwise and stirred for 40 min. The reaction mixture was quenched (water) allowed to reach RT and then extracted (EtOAc), dried (MgSO4) and concentrated in vacuo. The residue was purified by chromatography (silica, eluting EtOAc:hexane; 6:4) to give the subtitle compound as a white solid (1.15 g).
MS ESI+ 267 [M+1]
b) (2-fluoro-5-methylphenyl)-4-quinolinyl-methanone
DMSO (1.26 ml) was added dropwise to a solution of oxalyl chloride (1.12 ml) in dichloromethane (40 ml) at −78° C. The solution was stirred for 30 min and then the product from step a (1.11 g) in dicloromethane (10 ml) was added dropwise. The reaction mixture was allowed to reach 0° C. over 2 h. Triethylamine (1.25 ml) was added keeping the temperature between (0-10° C.) and stirred for 5 min. The reaction mixture was quenched (water), separated the 2 layers. Then reextracted with dicloromethane. The combined organic extracts were dried (MgSO4) and concentrated in vacuo. The residue was purified by chromatography (silica, eluting EtOAc:hexane; 1:1) to give the subtitle compound as a yellow solid (1.2 g).
MS ESI+ 265 [M+1]
c) 4-(5-methyl-1H-indazol-3-yl)-quinoline
Hydazine monohydrate (0.48 ml) was added to the product of step b) (0.52 g) in toluene (6 ml), and heated for 3 days at 110° C. The reaction was concentrated in vacuo and the residue was purified by chomatography (silica, eluting EtOAc:hexame 4:6), to give the subtitle compound (0.25 g).
MS ESI+ 260 [M+1]
d) 5-methyl-3-(4-quinolinyl)-1H-indazole-1-acetic acid, ethyl ester
NaH (60% dispersion in mineral oil, 50 mg) was added to the product from step c (245 mg) in THF (8 ml) under nitrogen. The reaction mixture was stirred for 10 min and then ethyl bromoacetate (0.11 ml) was added dropwise and the mixture stirred for a further 1 h. The reaction mixture was quenched with water, extracted (EtOAc). The organic phase was dried (MgSO4) and concentrated in vacuo. The residue was purified by chromatography (silica, eluting EtOAc:hexame, 3:7), to give the subtitle compound (100 mg).
MS ESI+ 346 [M+1]
e) 5-methyl-3-(4-quinolinyl)-1H-indazole-1-acetic acid
The product from step b (70 mg) was treated with NaOH (2M, 0.2 ml), THF (1 ml) and methanol (1 ml). The resulting solution was stirred at room temperature for 3 h. The reaction mixture was concentrated in vacuo, then purified by reverse phase HPLC (eluting with ammonia and acetonitile), to give the title compound as a pale yellow solid (15 mg).
1H NMR (DMSO) δ 2.43 (s, 3H), 5.38 (s,2H), 7.35 (d,1H), 7.5-7.83 (m,5H), 8.12 (d,1H), 8.53 (d, 1H), 9.03 (d, 1H).
MS APCI+ 318 [M+1]
a) 4-fluoro-3-(hydroxy-4-quinolinylmethyl)-benzonitrile
The subtitle compound was prepared by the method of example 1 part a, using 2-fluro-4-cyanobromobenzene and quinoline-4-aldehyde.
1H NMR (CDCl3) δ 2.84 (d,1H), 6.85 (d,1H), 7.18-7.22 (m,1H), 7.52-7.89 (m,5H), 7.92 (d, 1H), 8.08 (d, 1H) and 8.97 (d,1H).
b) 4-fluoro-3-(4-quinolinylcarbonyl)-benzonitrile
The subtitle compound was prepared by the method of example 1 part b, using the product of step a).
1H NMR (CDCl3) δ 7.23-7.31 (m,1H), 7.39 (d,1H), 7.62 (t, 1H), 7.82 (t,1H), 7.84-7.98 (m,1H), 8.13-8.18 (m,2H), 8.2-8.25 (d, 1H), 9.05 (d,1H).
c) 3-(4-quinolinyl)-1H-indazole-5-carbonitrile
The subtitle compound was prepared by the method of example 1 part c, using the product of step b).
MS APCI+ 279 [M+1]
d) 5-cyano-3-(4-quinolinyl)-1H-indazole-1-acetic acid, ethyl ester
The subtitle compound was prepared by the method of example 1 part d, using the product of step c).
1H NMR (CDCl3) δ 0.83 (t,3H), 4.31 (q,2H), 5.34 (s,2H), 7.54-7.76 (m,4H), 7.8-7.84 (m,1H), 8.14 (s,1H), 8.27-8.31 (m, 2H), 9.08 (d,1H).
e) 5-cyano-3-(4-quinolinyl)-1H-indazole-1-acetic acid
The title compound was prepared by the method of example 1 part e) using the product of step d).
1H NMR (DMSO) δ 5.16 (s,2H), 7.6-7.7 (m,1H), 7.78-7.96 (m,4H), 8.15-8.19 (d,1H), 8.42 (s, 1H), 8.78 (d,1H) and 9.05 (d,1H).
a) 7-fluoro-α-[2-fluoro-6-(trifluoromethyl)phenyl]-1-naphthalenemethanol
Prepared by the method of example 1 step a, using 2-bromo-1-fluoro-3-(trifluoromethyl)-benzene and 6-fluoroquinoline.
MS ESI+ 340 [M+1]
b) (6-fluoro-4-quinolinyl)[2-fluoro-6-(trifluoromethyl)phenyl]-methanone
Dess-martin periodinone (1.06 g) was added to the product of step a (0.85 g) in dichloromethane (25 ml). The solution was stirred for 2 h and then washed with sodium thiosulfate, sodium hydrogen carbonate and brine. The organic phase was dried (MgSO4) and then concentrated in vacuo. The residue was purified by chomatography (silica, eluting EtOAC:hexame 3:7), to give the subtitle compound (420 mg).
MS ESI+ 338 [M+1]
c) 6-fluoro-4-[4-(trifluoromethyl)-1H-indazol-3-yl]-quinoline
The subtitle compound was prepared by the method of example 1 step c) from the product of step c)
MS ESI+ 314 [M+1]
d) 3-(6-fluoro-4-quinolinyl)-4-(trifluoromethyl)-1H-indazole-1-acetic acid, ethyl ester
NaH (60% dispersion in mineral oil, 22 mg) was added to the product from step c (245 mg) in THF (8 ml) uder nitrogen. The reaction mixture was stirred for 10 min and then ethyl bromoacetate (0.1 ml) was added dropwise and the mixture stirred for a further 1 h. The reaction mixture was quenched with water, extracted (EtOAC). The organic phase was dried (MgSO4) and concentrated in vacuo. The residue was purified by chomatography (silica, eluting EtOAC:hexame 3:7), to give the subtitle compound (100 mg).
MS ESI+ 346 [M+1]
e) 3-(6-fluoro-4-quinolinyl)-4-(trifluoromethyl)-1H-indazole-1-acetic acid
The title compound was prepared by the method of example 1 part e) using the product of step d).
1H NMR (DMSO) δ 5.1 (s,2H), 7.14 (dd,1H), 7.57-7.62 (m,3H), 7.62-7.75 (m,1H), 8.051 (d, 1H), 8.18-8.22 (m,1H) and 9.01 (d,1H).
a) α-(2-fluoro-6-iodophenyl)-4-quinolinemethanol
n-BuLi (2.5M) was added dropwise to a stirred solution of diisopropylamine in THF (80 ml) at 0° C. under nitrogen. The reaction mixture was cooled to −78° C. and 1-iodo-3-fluorobenzene (10 g) was added dropwise. The reaction mixture was stirred at this temperature for 1.5 h and the treated with a solution of quinoline-4-aldehyde (7.1 g) in THF (30 ml) and stirred for 10 minutes before quenching with ammonium chloride solution and allowing to reach room temperature. The mixture was diluted with water and ethyl acetate. The organic phase was dried (MgSO4) and concentrated in vacuo. The sub-title compound was obtained as a white solid (6.65 g) after triuration with diethyl ether.
1H NMR (CDCl3) δ 2.97-3 (m, 1H), 6.76 (d, 1H), 7-7.5 (m,1H), 7.42 (m, 1H), 7.52-7.58 (m,1H), 7.64-7.79 (m, 2H), 8.02 (d, 1H) and 8.18 (d, 1H).
b) 4-(4-iodo-1H-indazol-3-yl)-quinoline
Prepared using the product of step a) by the methods of example 3 parts b and c.
MS ESI+ 372 [M+1]
c) 4-iodo-3-(4-quinolinyl)-1H-indazole-1-acetic acid, 1,1-dimethylethyl ester
The sub-title compound was prepared by the method of example 1 part d) using the product of part b) (0.82 g) and tertiary-butyl bromoacetate (0.5 ml). The product was used in the next step without any further purification.
d) 4-iodo-3-(4-quinolinyl)-1H-indazole-1-acetic acid
The product of step c) (0.2 g) was dissolved in dichloromethane (4 ml) and treated with TFA (1 ml), stirred overnight at room temperature and concentrated in vacuo. The subtitle compound was further purified by reverse phase HPLC to give the sub-title compound as a yellow solid (93 mg).
1H NMR (DMSO) δ 5.4 (s,2H), 7.2-7.23 (m,1H), 7.42-7.9 (m,5H), 8.16 (d,1H) and 9.07 (d, 1H).
a) 3-[(4-chlorophenyl)thio]-5-iodo-1H-indazole
5-iodoindazole (0.3 g) inDMF (8 ml) was treated with potassium-tertiary-butoxide solution (1.5 ml, 1M in THF) and bis(4-chlorophenyl)disulfide and heated at 65 C for 4 days after which the reaction was quenched with water and extracted with ethyl acetate, dried the organics (MgSO4) and then concentrated in vacuo. Purified by silica chromatography to afford the product as a white solid.
MS ES+ 387 [M+1]
b) 3-[(4-chlorophenyl)thio]-5-iodo-1H-indazole-1-acetic acid
The sub-title compound was prepared by the methods of example 1 part d) and example 1 part e) using the product from step a).
1H NMR (CDCl3) δ 4.98 (s, 2H), 7.17 (dd, 2H), 7.36 (dd,2H), 7.51 (d, 1H), 7.63 (dd,1H) and 7.87 (s,1H)
a) 7-chloro-4-(2-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-quinoline
2-methyl-1H-pyrrolo[2,3-b]pyridine (0.4 g), 4-chloroquinoline (0.6 g) and N-methyl pyrrolidine (1 ml) were stirred at 100° C. over 2 days. The reaction mixture was triturated with diethyl ether and filtered to give a solid, which was further purified by silica chromatography eluting with ethyl acetate: isoheaxane (3:7) to give the sub-title compound (31 mg).
MS ES+ 293 [M+1]
b) 3-(7-chloro-4-quinolinyl)-1H-pyrrolo[2,3-b]pyridine-1-acetic acid, ethyl ester
The sub-title compound was prepared from the product of step a) by the method of example 1 part d).
MS ES+ 380 [M+1]
c) 3-(7-chloro-4-quinolinyl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-1-acetic acid, sodium salt
The product of part b) (30 mg, 0.09 mmol), sodium hydroxide (0.09 ml), methanol (0.2 ml) and THF (0.2 ml), were stirred at room temperature overnight. The solution was concentrated in vacuo and then triturated with diethyl ether to give the title compound as a whiter solid (20 mg).
1H NMR (DMSO) δ 2.3 (s, 3H), 4.62 (d, 2H), 7.01-7.06 (M, 1H), 7.5-7.6 (m,3H), 7.8 (d, 1H), 8.15-8.22 (m,2H) and 8.98 (d,1H).
a) 3-[(4-Chlorophenyl)thio]-2,5-dimethyl-1H-pyrrolo[3,2-b]pyridine
A mixture of 2,5-dimethyl-1H-pyrrolo[3,2-b]pyridine (0.5 g) and potassium tert-butoxide (3.7 ml, 1M in tert-butanol) in tert-butanol (25 ml) was heated under reflux for 20 min then bis(4-chlorophenyl)disulphide (1.44 g) added. After heating for a further 1 h, the mixture was cooled and water (100 ml) added. The precipitate was filtered off, washed with water, diethylether and dried to give the title compound (930 mg).
MS ESI+ 289 [M+1]
b) 3-[(4-Chlorophenyl)thio]-2,5-dimethyl-1H-pyrrolo[3,2-b]pyridine-1-acetic acid, ethyl ester
The subtitle compound was prepared by the method of example 1 part d, using the product of step a).
MS ESI+ 375 [M+1]
c) 3-[(4-Chloro-2,4-cyclohexadien-1-yl)thio]-2,5-dimethyl-1H-pyrrolo[3,2-b] pyridine-1-acetic acid
The product from step b (355 mg) was treated with aqueous NaOH (1M, 0.95 ml), THF (15 ml) and water (2 ml). The resulting solution was stirred at room temperature for 5 h then evaporated under reduced pressure. The residue was triturated with ether, filtered and dried to give the title compound (0.302 g)
δH (DMSO) 2.39 (s,3H), 2.47 (s,3H), 4.46 (s,2H), 6.69-7.25 (m,5H), 7.65 (d,1H).
MS APCI− 345 [M−1]
a) 2,5-Dimethyl-3-[[4-(methylsulfonyl)phenyl]methyl]-1H-pyrrolo[3,2-b]pyridine
Ethylmagnesium bromide (1.2 ml, 3M in diethylether) was added to a stirred solution of 2,5-dimethyl-1H-pyrrolo[3,2-b]pyridine (0.44 g) in THF (20 ml) at RT. After 30 min p-methylsulfonylbenzyl bromide (0.7 g) was added and the mixture heated under reflux for 2 h. DMF (5 ml) was added, the mixture heated under reflux for 4 h, cooled and partitioned between ethylacetate and water. The organic layer was separated, washed with water, dried and evaporated under reduced pressure. The residue was purified by chromatography on silica eluting with 5% methanol/DCM to give the title compound (0.121 g)
MS ESI+ 315 [M+1]
b) 2,5-Dimethyl-3-[[4-(methylsulfonyl)phenyl]methyl]-1H-pyrrolo[3,2-b]pyridine-1-acetic acid, ethyl ester
The product from step a) (0.115 g), potassium carbonate (0.2 g) and ethylbromoacetate (0.05 ml) in DMF (5 ml) were heated at 50° C. for 5 h. Ethyl bromoacetate (0.1 ml) was added and the mixture heated for a further 4 h at 80° C., cooled then partitioned between diethylether and water. The organic layer was separated, washed with water, dried and evaporated under reduced pressure. The residue was purified by chromatography on silica eluting with 2-3% methanol/DCM to give the sub-title compound (73 mg).
MS ESI+ 401 [M+1]
c) 2,5-Dimethyl-3-[[4-(methylsulfonyl)-2,4-cyclohexadien-1-yl]methyl]-1H-pyrrolo[3,2-b]pyridine-1-acetic acid
The product from step b) (72 mg) was treated with aqueous NaOH (1M, 0.3 ml), THF (5 ml) and water (3 ml). The resulting solution was stirred at room temperature for 3 h then 2M HCl (3 ml) added. The aqueous layer was extracted with ethylacetate then evaporated under reduced pressure. The residue was recrystallised from water to give the sub-title compound (40 mg).
1H NMR (DMSO) δ 2.40 (s,3H), 2.81 (s,3H), 3.17 (s,3H), 4.52 (s,2H), 5.27 (s,2H), 7.45 (d,1H), 7.49 (d,2H), 7.82 (d,2H), 8.57 (d,1H), 15.74 (s,1H).
MS APCI− 371 [M−1]
a) 2,5-Dimethyl-3-[[4-(methylsulfonyl)phenyl]thio]-1H-pyrrolo[3,2-b]pyridine
A mixture of 2,5-dimethyl-1H-pyrrolo[3,2-b]pyridine (0.4 g), potassium carbonate (0.7 g) and bis(4-methylsulphonylphenyl)disulphide (1.2 g) in DMF (15 ml) were stirred at RT for 4 days then heated at 50° C. for 6 h. The mixture was partitioned between ethylacetate and water, the organic layer was separated, washed with water, dried and evaporated under reduced pressure. The residue was triturated with ethylacetate and filtered to give the sub-title compound (0.3 g)
MS ESI+ 333 [M+1]
b) 2,5-Dimethyl-3-[[4-(methylsulfonyl)phenyl]thio]-1H-pyrrolo[3,2-b]pyridine-1-acetic acid, 1,1-dimethylethyl ester
The product from step a) (0.3 g), potassium carbonate (0.3 g) and tert-butylbromoacetate (0.13 ml) in DMF (8 ml) were stirred at RT for 18 h. The mixture was partitioned between ethylacetate and water, the organic layer separated, washed with water, dried and evaporated under reduced pressure. The residue was triturated with ethylacetate/isohexane to give the sub-title compound (0.175 g)
MS ESI+ 447 [M+1]
c) 2,5-Dimethyl-3-[[4-(methylsulfonyl)phenyl]thio]-1H-pyrrolo[3,2-b]pyridine-1-acetic acid
The product from step b) (0.175 g), trifluoracetic acid (5 ml) and DCM (10 ml) were stirred at RT for 16 h then evaporated under reduced pressure. The residue was triturated with diethylether and filtered to give the title compound (125 mg).
1H NMR (DMSO) δ 2.71 (s,3H), 3.16 (s,3H), 5.34 (s,2H), 7.21 (d,2H), 7.43 (brd,1H), 7.75 (d,2H), 8.46 (brs,1H).
MS APCI− 389 [M−1]
a) [2-Chloro-3-(1-propynyl)-4-pyridinyl]-carbamic acid, 1,1-dimethylethyl ester
A mixture of (2-chloro-3-iodo-pyridin-4-yl)-carbamic acid tert-butyl ester (3.4 g), copper(I) iodide (0.09 g), triethylamine (2.8 ml), propyne (approx. 1 g) and dichloro(bistriphenylphosphine)palladium (0.2 g) in DMF (30 ml) was heated at 50° C. for 5 h. The mixture was partitioned between diethylether and water, the organics separated, washed with water, dried and evaporated under reduced pressure. The residue was purified by chromatography on silica eluting with 10% ethylacetate/isohexane to give the sub-title compound (2.14 g).
MS ESI− 265/7 [M−1]
b) 4-Chloro-2-methyl-1H-pyrrolo[3,2-c]pyridine
The product from step a) (2.1 g) and copper(I) iodide (0.035 g) in DMF (50 ml) was heated at 90° C. for 6 h, cooled and partitioned between ethylacetate and brine. The organics were separated, washed with brine, dried and evaporated under reduced pressure. The residue was purified by chromatography on silica eluting with 40% ethylacetate/isohexane to give the sub-title compound (0.785 g).
1H NMR (DMSO) δ 2.42 (s,3H), 6.24 (s,1H), 7.29 (d,1H), 7.87 (d,1H), 11.76 (s,1H).
c) 4-Chloro-3-[(4-chlorophenyl)thio]-2-methyl-1H-pyrrolo[3,2-c]pyridine
The subtitle compound was prepared by the method of example 9 part a), using the product of step b).
MS ESI+ 309/11 [M+1]
d) 4-Chloro-3-[(4-chlorophenyl)thio]-2-methyl-1H-pyrrolo[3,2-c]pyridine-1-acetic acid, 1,1-dimethylethyl ester
The subtitle compound was prepared by the method of example 9 part b), using the product of step c).
MS ESI+ 423/5 [M+1]
e) 4-Chloro-3-[(4-chlorophenyl)thio]-2-methyl-1H-pyrrolo[3,2-c]pyridine-1-acetic acid
The title compound was prepared by the method of example 9 part c), using the product of step d).
1H NMR (DMSO) δ 2.46 (s,3H), 5.23 (s,2H), 6.98 (d,2H), 7.29 (d,2H), 7.69 (d,1H), 8.05 (d,1H).
MS APCI− 365/7 [M−1]
The title compound was prepared by the method of example 10.
1H NMR (DMSO) δ 2.71 (s,3H), 3.16 (s,3H), 5.34 (s,2H), 7.21 (d,2H), 7.43 (brd,1H), 7.75 (d,2H), 8.46 (brs,1H).
MS APCI− 389 [M-−b 1]
a) 4-Chloro-2-methyl-1H-pyrrolo[3,2-c]pyridine-1-carboxylic acid, 1,1-dimethylethyl ester
The product from example 10 part b) (0.5 g), di-tert-butyl dicarbonate (0.655 g) and 4-dimethyl aminopyridine (0.05 g) in DCM (20 ml) was stirred at RT for 72 h then partitioned between diethylether and water. The organics were separated, washed with water, dried and evaporated under reduced pressure to give the sub-title compound (0.8 g).
MS ESI+ 267/9 [N+1]
b) 2-Methyl-4-phenyl-1H-pyrrolo[3,2-c]pyridine-1-carboxylic acid, 1,1-dimethylethyl ester
The product from step a) (0.6 g), cesium fluoride (0.87 g), phenylboronic acid (0.45 g) and tetrakis(triphenylphosphine)palladium(0) (0.1 g) in dioxane (20 ml) were heated at 100° C. for 5 h, cooled and partitioned between diethylether and water. The organics were separated, washed with water, dried and evaporated under reduced pressure. The residue was purified by chromatography on silica eluting with 20% ethylacetate/isohexane to give the sub-title compound (0.627 g).
MS ESI+ 309 [M+1]
c) 2-Methyl-4-phenyl-1H-pyrrolo[3,2-c]pyridine, trifluoroacetate salt
The product from step b) (0.62 g), trifluoroacetic acid (5 ml) and DCM (15 ml) were stirred at RT for 24 h then evaporated under reduced pressure. Used crude in next step.
d) 3-[(4-Chlorophenyl)thio]-2-methyl-4-phenyl-1H-pyrrolo[3,2-c]pyridine
The subtitle compound was prepared by the method of example 9 part a), using the product of step c).
MS ESI+ 351/3 [M+1]
e) 3-[(4-Chlorophenyl)thio]-2-methyl-4-phenyl-1H-pyrrolo[3,2-c]pyridine-1-acetic acid, 1,1-dimethylethyl ester
The subtitle compound was prepared by the method of example 9 part b), using the product of step d).
MS ESI+ 465/7 [M+1]
f) 3-[(4-Chlorophenyl)thio]-2-methyl-4-phenyl-1H-pyrrolo[3,2-c]pyridine-1-acetic acid
The title compound was prepared by the method of example 9 part c), using the product of step e).
1H NMR (DMSO) δ 2.53 (s,3H), 5.47 (s,2H), 6.58 (d,2H), 7.14 (d,2H), 7.35-7.56 (m,5H), 8.28 (d,1H), 8.55 (d,1H).
MS APCI− 407/9 [M−1]
The title compound was prepared by the method of example 11.
1H NMR (DMSO) δ 2.53 (s,3H), 3.15 (s,3H), 5.49 (s,2H), 6.80 (d,2H), 7.29-7.51 (m,5H), 7.57 (d,2H), 8.30 (d,1H), 8.57 (d,1H).
MS APCI− 451 [M−1]
a) 2-[(7-chloro-4-quinolinyl)amino]-4-methyl-benzoic acid
A solution of 2-amino-4-methylbenzoic acid (2 g) and 4,7-dichloroquinoline (2.62 g) in NMP was stirred at 140° C. for 4 hours. The reaction mixture was cooled to room temperature and added to brine to afford a precipitate which was filtered, washed with water and dried to afford the sub-title compound (4.04 g).
MS ES+ 313 [M+1]
b) 1-(7-chloro-4-quinolinyl)-1,3-dihydro-6-methyl-2H-benzimidazol-2-one
A suspension of the product of part a) (2 g) in dry DMF was treated with triethylamine (0.9 ml) and stirred for 30 min. Diphenylphosphoryl azide (1.38 ml) was added and the reaction stirred for a further 2 hours, then at 60° C. for 4 hours. The reaction was cooled to room temperature and added to brine and the suspension extracted with ethyl acetate. The combined organic extracts were dried (MgSO4) and concentrated in vacuo. The resulting solid was triturated and dried to give the sub-title compound (1.06 g).
3-(7-chloro-4-quinolinyl)-2,3-dihydro-5-methyl-2-oxo-1H-benzimidazole-1-acetic acid
The sub-title compound was prepared by the methods of example 1 parts d) and e).
1H NMR (DMSO) δ 2.23 (s,3H), 4.74 (d,2H), 6.54 (s,1H), 7.07 (d, 1H), 7.25 (d,1H), 7.64 (m,2H), 7.77 (d,1H), 8.28 (s,1H), 9.15 (d, 1H) and 13.22 (s, 1H).
MS APCI+ 369 [M+1]
[3H]PGD2 was purchased from Perkin Elmer Life Sciences with a specific activity of 100-210 Ci/mmol. All other chemicals were of analytical grade.
HEK cells expressing rhCRTh2/Gα16 were routinely maintained in DMEM containing 10% Foetal Bovine Serum (HyClone), 1 mg/ml geneticin, 2 mM L-glutamine and 1% non-essential amino acids. For the preparation of membranes, the adherent transfected HEKcells were grown to confluence in two layer tissue culture factories (Fisher, catalogue number TKT-170-070E). Maximal levels of receptor expression were induced by addition of 500 mM sodium butyrate for the last 18 hours of culture. The adherent cells were washed once with phosphate buffered saline (PBS, 50 ml per cell factory) and detached by the addition of 50 ml per cell factory of ice-cold membrane homogenisation buffer [20 mM HEPES (pH 7.4), 0.1 mM dithiothreitol, 1 mM EDTA, 0.1 mM phenyl methyl sulphonyl fluoride and 100 μg/ml bacitracin]. Cells were pelleted by centrifugation at 220×g for 10 minutes at 4° C., re-suspended in half the original volume of fresh membrane homogenisation buffer and disrupted using a Polytron homogeniser for 2×20 second bursts keeping the tube in ice at all times. Unbroken cells were removed by centrifugation at 220×g for 10 minutes at 4° C. and the membrane fraction pelleted by centrifugation at 90000×g for 30 minutes at 4° C. The final pellet was re-suspended in 4 ml of membrane homogenisation buffer per cell factory used and the protein content determined. Membranes were stored at −80° C. in suitable aliquots.
All assays were performed in Corning clear bottomed, white 96-well NBS plates (Fisher). Prior to assay, the HEK cells membranes containing CRTh2 were coated onto SPA PVT WGA beads (Amersham). For coating membranes were incubated with beads at typically 25 μg membrane protein per mg beads at 4° C. with constant agitation overnight. (The optimum coating concentrations were determined for each batch of membranes) The beads were pelleted by centrifugation (800×g for 7 minutes at 4° C.), washed once with assay buffer (50 mM HEPES pH 7.4 containing 5 mM magnesium chloride) and finally re-suspended in assay buffer at a bead concentration of 10 mg/ml.
Each assay contained 20 μl of 6.25 nM [3H]PGD2, 20 μl membrane saturated SPA beads both in assay buffer and 10 μl of compound solution or 13,14-dihydro-15-keto prostaglandin D2 (DK-PGD2, for determination of non-specific binding, Cayman chemical company).
Compounds and DK-PGD2 were dissolved in DMSO and diluted in the same solvent to 100× the required final concentration. Assay buffer was added to give a final concentration of 10% DMSO (compounds were now at 10× the required final concentration) and this was the solution added to the assay plate. The assay plate was incubated at room temperature for 2 hours and counted on a Wallac Microbeta liquid scintillation counter (1 minute per well).
Compounds of formula (I) have an IC50 value of less than (<) 10 μM.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0303180-4 | Nov 2003 | SE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB04/04937 | 11/24/2004 | WO | 00 | 5/25/2007 |
