Patent Application
20070293561
The following are hypothetical examples of useful compounds:
A compound of the formula
or a pharmaceutically acceptable salt thereof, or a prodrug thereof;
wherein a dashed line represents the presence or absence of a bond;
A is —(CH2)6—, cis —CH2CH═CH—(CH2)3—, or —CH2C≡C—(CH2)3—, wherein 1 or 2 carbon atoms may be replaced by S or O; or A is —(CH2)m—Ar—(CH2)o— wherein Ar is interarylene or heterointerarylene, the sum of m and o is 1, 2, 3, or 4, and wherein one CH2 may be replaced by S or O;
alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms; or CF3; and
A compound which is a carboxylic acid or a bioisostere thereof, said carboxylic acid having a structure
or a pharmaceutically acceptable salt thereof, or a prodrug thereof;
wherein a dashed line represents the presence or absence of a bond;
A is —(CH2)6—, cis —CH2CH═CH—(CH2)3—, or —CH2C≡C—(CH2)3—, wherein 1 or 2 carbon atoms may be replaced by S or O; or A is —(CH2)m—Ar—(CH2)o— wherein Ar is interarylene or heterointerarylene, the sum of m and o is 1, 2, 3, or 4, and wherein one CH2 may be replaced by S or O;
alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms; or CF3; and
A compound according to claim 1 wherein Y is selected from CO2R2, CON(R2)2, CON(OR2)R2, CON(CH2CH2OH)2, CONH(CH2CH2OH), CH2OH, P(O)(OH)2, CONHSO2R2, SO2N(R2)2, SO2NHR2,
wherein R2 is independently H, C1-C6 alkyl, unsubstituted phenyl, or unsubstituted biphenyl.
A compound according to claim 1 or 3 of the formula
or a pharmaceutically acceptable salt thereof, or a prodrug thereof.
A compound according to claim 1 or 3 of the formula
or a pharmaceutically acceptable salt thereof, or a prodrug thereof.
A compound according to claim 1 or 3 of the formula
or a pharmaceutically acceptable salt thereof, or a prodrug thereof.
A compound according to any one of claims 1 to 6 wherein A is (3-methylphenoxy)methyl.
A compound according to any one of claims 1 to 6 wherein A is (4-but-2-ynyloxy)methyl.
A compound according to any one of claims 1 to 6 wherein A is 2-(2-ethylthio)thiazol-4-yl.
A compound according to any one of claims 1 to 6 wherein A is 2-(3-propyl)thiazol-5-yl.
A compound according to any one of claims 1 to 6 wherein A is 3-(methoxymethyl)phenyl.
A compound according to any one of claims 1 to 6 wherein A is 3-(3-propyl)phenyl.
A compound according to any one of claims 1 to 6 wherein A is 3-methylphenethyl.
A compound according to any one of claims 1 to 6 wherein A is 4-(2-ethyl)phenyl.
A compound according to any one of claims 1 to 6 wherein A is 4-phenethyl.
A compound according to any one of claims 1 to 6 wherein A is 4-methoxybutyl.
A compound according to any one of claims 1 to 6 wherein A is 5-(methoxymethyl)furan-2-yl.
A compound according to any one of claims 1 to 6 wherein A is 5-(methoxymethyl)thiophen-2-yl.
A compound according to any one of claims 1 to 6 wherein A is 5-(3-propyl)furan-2-yl.
A compound according to any one of claims 1 to 6 wherein A is 5-(3-propyl)thiophen-2-yl.
A compound according to any one of claims 1 to 6 wherein A is 6-hexyl.
A compound according to any one of claims 1 to 6 wherein A is (Z)-6-hex-4-enyl.
A compound according to any one of claims 1, 3, 4 and 7 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 7 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 6 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 6 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 6 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 6 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 6 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1, 3, and 6 to 22 having the formula
or a pharmaceutically acceptable salt thereof or a prodrug thereof.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is O.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is S.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is F.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is Cl.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is Br.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is 1.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is CN.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein U1 is O-alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is hydrogen.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is F.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is Cl.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is Br.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is 1.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is O.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is OH.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is CN.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is O-alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms.
A compound according to any one of claims 1 to 3, and 7 to 22, and 31 to 38 wherein J1 is CF3.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is hydrogen.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is F.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is Cl.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is Br.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is I.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is O.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is OH.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is CN.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is O-alkyl having 1, 2, 3, 4, 5 or 6 carbon atoms.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms.
A compound according to any one of claims 1 to 3, 7 to 22, and 31 to 49 wherein J2 is CF3.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted phenyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted thienyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted naphthyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted furyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted pyridinyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted benzothienyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted indanyl.
A compound according to any one of claims 1 to 60 wherein B is substituted or unsubstituted tetralonyl.
A compound according to any one of claims 1 to 60 wherein B has 1, 2, 3, 4, or 5 substituents, wherein each substituent has one or more carbon, fluorine, chlorine, bromine, or oxygen atoms; and wherein all substituents taken together consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms; 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 fluorine atoms; 0, 1, 2 or 3 chlorine atoms, 0, 1, 2 or 3 bromine atoms, and 0, 1, 2 or 3 oxygen atoms.
A compound according to any one of claims 1 to 60 wherein B has a substituent of the formula CaHbOc; wherein a is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9, b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; and c is 0, 1, 2, or 3.
A compound according to any one of claims 1 to 60 wherein B has 1, 2, 3, or 4 alkyl substituents having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
A compound according to any one of claims 1 to 60 wherein B has a hydroxyalkyl substituent having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and 1 or 2 hydroxy moieties.
A compound according to any one of claims 1 to 60 wherein B has an alkyl substituent having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
A compound according to any one of claims 1 to 60 wherein B has 1, 2, 3, or 4 halogen substituents.
A compound according to any one of claims 1 to 60 wherein B has 1, 2, 3, or 4 chloro substituents.
A compound according to any one of claims 1 to 60 wherein B has 1 chloro substituent.
A compound according to any one of claims 1 to 60 wherein B has 2 chloro substituents.
A compound according to any one of claims 1 to 60 wherein B has 1, 2, 3, or 4 trifluoromethyl substituents.
A compound according to any one of claims 1 to 60 wherein B has 1, 2, or 3 trifluoromethyl substituents.
A compound according to any one of claims 1 to 60 wherein B has 1 trifluoromethyl substituent.
A compound according to any one of claims 1 to 60 wherein B has 2 trifluoromethyl substituents.
A compound according to any one of claims 1 to 60 wherein B has a hydroxyl substituent.
A compound according to any one of claims 1 to 61 wherein B is unsubstituted phenyl.
A compound according to any one of claims 1 to 61 wherein B is 3,5-dichlorophenyl.
A compound according to any one of claims 1 to 61 wherein B is 3,5-di(trifluoromethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 2-chlorophenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-chlorophenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-chlorophenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(trifluoromethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-isopropylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-tert-butylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-hydroxyphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-methoxyphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(benzoyloxy)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 2,3-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3,4-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 2,4-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 2,5-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3,5-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 2,6-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(hydroxymethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(1-hydroxyethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(1-hydroxy-2-methylpropyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 2-(hydroxymethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(hydroxymethyl)-3,5-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(methoxymethyl)-3,5-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(1-hydroxybutyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(1-methoxybutyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(1-hydroxybutyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(2-hydroxyethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(2-hydroxyethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 2-(2-hydroxyethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(2-hydroxyethyl)-3,5-dimethylphenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(1-hydroxyhexyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 3-(acetoxymethyl)-5-chlorophenyl.
A compound according to any one of claims 1 to 61 wherein B is 1-oxo-2,3-dihydro-1H-inden-4-yl.
A compound according to any one of claims 1 to 61 wherein B is 1-hydroxy-2,3-dihydro-1H-inden-4-yl.
A compound according to any one of claims 1 to 61 wherein B is 5-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl.
A compound according to any one of claims 1 to 61 wherein B is 3-(1-hydroxy-2-phenylethyl)phenyl.
A compound according to any one of claims 1 to 61 wherein B is 4-(2-phenylpropan-2-yl)phenyl.
A compound according to any one of claims 1 to 60 wherein B is naphthalen-2-yl.
A compound according to any one of claims 1 to 60 wherein B is naphthalen-1-yl.
A compound according to any one of claims 1 to 60 wherein B is 4-chloronaphthalen-1-yl.
Potassium carbonate (99.99%, 2.25 g, 16.3 mmol) was added to phosphonate 2 (see Collect. Czech. Chem. Commun. 1994, 58,138-148, 2.90 g, 6.40 mmol) in DMF (11 mL) at 0° C. After 30 min at 0° C., a solution of known lactol 1 (1.20 g, 3.22 mmol) in DMF (11 mL+10 mL) was added. The reaction mixture was allowed to rt and stirred overnight. The reaction mixture was then poured into water and extracted with EtOAc (3×). The combined extracts were washed with brine then dried (MgSO4), filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography on silica gel (hexane→30% EtOAc/hexane, gradient) afforded 1.50 g (91%) of alkene 3.
Palladium on carbon (10 wt. %, 321 mg) was added to a solution of alkene 3 (1.50 g, 2.94 mmol) in EtOAc (59 mL). A hydrogen atmosphere was established by evacuating and refilling with hydrogen (5×) and the reaction mixture was stirred under a balloon of hydrogen for 6 h. The reaction mixture was filtered through celite, washing with EtOAc, and the filtrate was concentrated in vacuo to afford 1.51 g (quant.) of saturated compound 4.
Triethylamine (316 μL, 2.27 mmol) and methanesulfonyl chloride (142 μL, 1.80 mmol) were added sequentially to a solution of 4 (769 mg, 1.50 mmol) in CH2Cl2 (11.3 mL) at 0° C. The reaction mixture was allowed to warm to rt and stirred at rt overnight. Saturated aqueous NaHCO3 (20 mL) was added, CH2Cl2 was removed in vacuo, and the remaining mixture was extracted with EtOAc (3×20 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated in vacuo to afford 880 mg (99%) of the desired mesylate 5, which was used without further purification.
Tetrabutylammonium chloride (2.0 g, 7.2 mmol) was added to a solution of 5 (880 mg, 1.5 mmol) in toluene (15 mL). The reaction mixture was heated at 40° C. for 18 h. The cooled mixture was diluted with brine (30 mL) and extracted with EtOAc (3×50 mL). The combined organic extracts were dried (MgSO4), filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography on silica gel (hexane→EtOAc, gradient) afforded 230 mg (29%) of chloride 6 and 170 mg (27%) of alcohol 7.
Tetrabutylammonium fluoride (0.345 mL of a 1.0 M THF solution, 0.345 mmol) was added to a solution of 6 (61 mg, 0.11 mmol) in THF (5.4 mL) at rt. After 18 h at rt, the reaction mixture was partitioned between EtOAc (15 mL) and H2O (10 mL). The phases were separated and the aqueous phase was extracted with EtOAc (2×10 mL). The combined organic phase washed with brine (50 mL) then dried (MgSO4), filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography on silica gel (hexane→EtOAc, gradient) afforded 10 mg (21%) of alcohol 7.
Triphenylphosphine (160 mg, 0.61 mmol) and diisopropyl azodicarboxylate (DIAD, 90 μL, 0.49 mmol) were added to a solution of alcohol 7 (170 mg, 0.41 mmol) and phenol 8 (see U.S. Provisional Application No. 60/757,696, filed on Jan. 10, 2006, incorporated by reference herein, 81 mg, 0.40 mmol) in CH2Cl2 (2.0 mL). After stirring 18 h at rt, the mixture was partitioned between CH2Cl2 (15 mL) and saturated aqueous NaHCO3 (20 mL). The phases were separated and the aqueous phase was extracted with CH2Cl2 (2×20 mL). The combined organic phase washed with brine (15 mL) then the organic phase was dried (MgSO4) filtered and concentrated in vacuo. Purification of the residue by flash column chromatography on silica gel (hexane→EtOAc, gradient) afforded 170 mg (70%) of 9.
Pyridinium p-toluenesulfonate (PPTs, 7 mg, 0.028 mmol) was added to a solution of 9 (170 mg, 0.28 mmol) in methanol (2.8 mL) at rt. The solution was heated at 40° C. overnight, then cooled and concentrated in vacuo. Purification of the crude residue by flash column chromatography on silica gel (hexane→EtOAc, gradient) afforded 90 mg (62%) of 10.
Dess-Martin periodinane (35 mg, 0.083 mmol) was added to a solution of 10 (35 mg, 0.068 mmol) in CH2Cl2 (3.0 mL) at 0° C. and the mixture was allowed to warm to rt. After 2 h at rt, the mixture was partitioned between CH2Cl2 (5 mL) and H2O (5 mL). The phases were separated and the aqueous phase was extracted with CH2Cl2 (2×5 mL). The combined organic phase washed with brine (5 mL) then the organic phase was dried (MgSO4) filtered and concentrated in vacuo. Purification of the residue by flash column chromatography on silica gel (hexane→EtOAc, gradient) afforded 30 mg (˜87%) of a mixture of 11 and 12 (approximately 4:1 in favor of 11).
Rabbit liver esterase (134 units/mg, 6 mg) was added to a mixture of 11 and 12 from step 8 above (15 mg, 0.03 mmol) and pH 7.2 buffer (2.4 mL). After 10 min at rt, MeCN (0.16 mL) was added. After stirring at rt for 24 h, the reaction mixture was concentrated to dryness. Purification of the resulting crude residue by flash column chromatography on silica gel (10% MeOH/CH2Cl2) afforded 5 mg (˜40%) of title compound 13.
Rabbit liver esterase (134 units/mg, 6 mg) was added to a mixture of 11 and 12 from Example 1, step 8 above (15 mg, ˜0.03 mmol) and pH 7.2 buffer (2.4 mL). After 10 min at rt, MeCN (0.16 mL) was added. After stirring at rt for 24 h, the reaction mixture was concentrated to dryness to afford 10 mg (˜73%) of a mixture of 12 and 13 (approximately 3:1 in favor of 12), which was taken on without further purification.
Palladium on carbon (10 wt. %, 1 mg) was added to a mixture of 12 and 13 (10 mg, ˜0.022 mmol) in EtOAc (0.42 mL). A hydrogen atmosphere was established by evacuating and refilling with hydrogen (10×) and the reaction mixture was stirred under a balloon of hydrogen for 6 h. The reaction mixture was filtered through celite, washing with EtOAc, and the filtrate was concentrated in vacuo. Purification of the resulting crude residue by flash column chromatography on silica gel (10% MeOH/CH2Cl2) afforded 7 mg (˜68%) of ester 14 and 2.7 mg (30%) of title compound 15.
It is envisioned that intermediates such as 12 may react with nucleophiles such as alkyl copper reagents to afford compounds that have alkyl groups at C-9. It is further envisioned that intermediates such as 14 may be reacted with lithium diisopropylamide (LDA, or some other suitable base) followed by an electrophile such as an alkyl halide or dimethyl dioxirane to afford compounds that have an alkyl group or a hydroxyl group at C-10. It is also envisioned that intermediates such as 12 may react with appropriate reagents such that an epoxide or diol at C-9-C-10 may result. Furthermore, intermediates such as 12 and 14 may serve as precursor to compounds in which the ketone has been replaced by a chloro, fluoro, or cyano group. In these cases, the ketone is first reduced to the corresponding alcohol, which is then converted into the corresponding mesylate, which then is converted into the desired halo or cyano moiety. The intermediate alcohol and its corresponding ether and ester derivatives are also desired.
Competition binding experiments were performed in a medium containing Hank's balanced salt solution, Hepes 20 mM, pH 7.3, membranes (˜60 μg protein) or 2×105 cells from HEK 293 cells stably expressing human EP2 receptors, [3H]PGE2 (10 nM) and various concentrations of test compounds in a total volume of 300 μl. Reaction mixtures were incubated at 23° C. for 60 min, and were filtered over Whatman GF/B filters under vacuum. Filters were washed three times with 5 ml ice-cold buffer containing 50 mM Tris/HCl (pH 7.3). Non-specific binding was estimated in the presence of excess unlabeled PGE2 (10 μM). Binding data fitted to the binding model for a single class of binding sites, using nonlinear regression analysis. IC50 values thus obtained were converted to Ki using the equation of Ki=(IC50/(1+[L]/KD) where [L] represents PGE2 concentration (10 nM) and KD the dissociation constant for [3H]PGE2 at human EP2 receptors (40 nM).
HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10N HCl is added to achieve a pH of 7.4).
The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4° C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2□ (5 nM) were performed in a 100 μl volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell harvester. The filters were washed 3 times with ice-cold buffer and oven dried for one hour.
[3H-] PGE2 (specific activity 180 Ci mmol) was used as the radioligand for EP receptors. [3H] 17-phenyl PGF2, was employed for FP receptor binding studies. Binding studies employing EP1, EP2, EP4 and FP receptors were performed in duplicate in at least three separate experiments. A 200 μl assay volume was used. Incubations were for 60 min at 25° C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl, followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel). Competition studies were performed using a final concentration of 5 nM [3H]-PGE2, or 5 nM [3H] 17-phenyl PGF2α and non-specific binding determined with 10−5M of unlabeled PGE2, or 17-phenyl PGF2α, according to receptor subtype studied.
(a) Cell Culture
HEK-293(EBNA) cells, stably expressing one type or subtype of recombinant human prostaglandin receptors (prostaglandin receptors expressed: hDP/Gqs5; hEP1; hEP2/Gqs5; hEP3A/Gqi5; hEP4/Gqs5; hFP; hIP; hTP), were cultured in 100 mm culture dishes in high-glucose DMEM medium containing 10% fetal bovine serum, 2 mM 1-glutamine, 250 μg/ml geneticin (G418) and 200 μg/ml hygromycin B as selection markers, and 100 units/ml penicillin G, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B.
(b) Calcium Signal Studies on the FLIPR™
Cells were seeded at a density of 5×104 cells per well in Biocoat® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes.
Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR™, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration. The peak increase in fluorescence intensity was recorded for each well. On each plate, four wells each served as negative (HBSS-HEPES buffer) and positive controls (standard agonists: BW245C (hDP); PGE2 (hEP1; hEP2/Gqs5; hEP3A/Gqi5; hEP4/Gqs5); PGF2, (hFP); carbacyclin (hIP); U-46619 (hTP), depending on receptor). The peak fluorescence change in each drug-containing well was then expressed relative to the controls.
Compounds were tested in a high-throughput (HTS) or concentration-response (CoRe) format. In the HTS format, forty-four compounds per plate were examined in duplicates at a concentration of 10−5 M. To generate concentration-response curves, four compounds per plate were tested in duplicates in a concentration range between 10−5 and 10−11 M. The duplicate values were averaged. In either, HTS or CoRe format each compound was tested on at least 3 separate plates using cells from different passages to give an n>3.
cAMP Assay
A 384-well drug plate was prepared to contain 6 test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strepavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
The results of the binding and activity studies, presented in Table 1 below, demonstrate that the compounds disclosed herein are selective prostaglandin EP2 agonists, and are thus useful for the treatment of glaucoma, ocular hypertension, and other diseases or conditions.
The following are hypothetical examples demonstrating how a person may be treated with the compounds disclosed herein.
An aqueous liquid containing 0.1% of H1 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H2 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H3 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H4 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H5 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H6 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H7 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H8 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H9 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H10 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H11 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H12 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H13 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H14 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H15 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H16 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H17 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H18 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H19 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H2O is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H21 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H22 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H23 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H24 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H25 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H26 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H27 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H28 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H29 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H30 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H31 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H32 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H33 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H34 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H35 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H36 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H37 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H38 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H39 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H40 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H41 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H42 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H43 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H44 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H45 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H46 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H47 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H48 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H49 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H50 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H51 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H52 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H53 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H54 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H55 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H56 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H57 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H58 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H59 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H60 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H61 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H62 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H63 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
An aqueous liquid containing 0.1% of H64 is given topically to the eye of a person suffering from elevated intraocular pressure. A few hours after administration, the person's intraocular pressure is reduced. The drop is administered twice a day, and pressure remains low for as long as the treatment is continued.
The foregoing description details specific methods and compositions that can be employed to practice the present invention, and represents the best mode contemplated. However, it is apparent for one of ordinary skill in the art that further compounds with the desired pharmacological properties can be prepared in an analogous manner, and that the disclosed compounds can also be obtained from different starting compounds via different chemical reactions. Similarly, different pharmaceutical compositions may be prepared and used with substantially the same result. Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the lawful construction of the claims.
This application is based, and claims priority under 35 U.S.C. § 120 to U.S. Provisional Application Ser. No. 60/805,285, filed Jun. 20, 2006, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60805285 | Jun 2006 | US |
