Patent Application
20080045534
The compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers.
As used herein, the term “pharmaceutically acceptable carrier” comprises such excipients, binders, lubricants, tabletting agents, and disintegrants as are typically used in the art of formulation of pharmaceuticals. Examples of such agents include—but are not limited to—microcrystalline cellulose, lactose, starch, and dicalcium phosphate, and Providone. However, in view of the incompatibility of primary amines with lactose, this invention does not contemplate compositions in which active ingredients with primary amine groups are combined with lactose. Additionally, disintegrants such as sodium starch glycolate; lubricants such as stearic acid and SiO2; and solubility enhancers such as cyclodextrins, among many other examples for each group, are contemplated. Such materials and the methods of using them are well known in the pharmaceutical art. Additional examples are provided in Kibbe, Handbook of Pharmaceutical Excipients, London, Pharmaceutical Press, 2000.
As used herein the term “potassium channel modulator” refers to a compound capable of causing an increase in potassium channel currents. It also refers to a compound capable of increasing the KCNQ2/3 channel open probability. For preliminary testing of compounds for potassium channel modulating ability, the inventors have employed the rubidium ion efflux test described below.
As contemplated by this invention, compounds of formula I are designed for oral dosing of up to approximately 2000 mg per day. Similarly, solutions and suspensions suitable for oral pediatric administration, comprising, in addition to compounds of formula I, a syrup such as sorbitol or propylene glycol, among many other examples, are also contemplated. Additionally, both chewable and non-chewable tablets comprising compounds of formula I, along with pharmaceutically acceptable tabletting agents and other pharmaceutically acceptable carriers and excipients, are also contemplated.
The invention also contemplates pharmaceutical formulations for oral or intravenous administration to animals, comprising a therapeutically effective amount of a compound of formula I and an acceptable carrier for use in veterinary medicine. Any animal that is susceptible to disorders that are amenable to treatment with potassium channel modulators is included within the scope of this invention.
Section I. The preparation of compounds of formula V is outlined in Scheme 1.
Section II. The preparation of compounds of formula VIII is outlined in Scheme 2.
Section III. The preparation of compounds of formula XI is outlined in Scheme 3.
Section IV. The preparation of compounds of formula XII is outlined in Scheme 4.
Section V. The preparation of compounds of formula VII is outlined in Scheme 5.
Section VI. An alternative preparation of compounds of formula VII is outlined in Scheme 6.
Section VII. The preparation of compounds of formula XIII is outlined in Scheme 7.
Section VIII. The preparation of compounds of formula XVI is outlined in Scheme 8.
Section IX. The preparation of compounds of formula XVII is outlined in Scheme 9.
Section X. The preparation of compounds of formula XVIII is outlined in Scheme 10.
Section XI. The preparation of compounds of formula VI is outlined in Scheme 11
Section XII. The preparation of compounds of formula IX is outlined in Scheme 12.
Section XIII. The preparation of compounds of formula XII is outlined in Scheme 13.
Section XIV. The preparation of compounds of formula XVII is outlined in Scheme 14.
Section XV. The preparation of compounds of formula XIV is outlined in Scheme 15.
3,3-Dimethylbutanoyl chloride (1.0 g, 5.4 mmol) was added to a solution of 5-bromo-2-methylaniline (0.796 g, 5.9 mmol) in acetonitrile (10 mL). The reaction mixture was stirred at room temperature overnight. Water was added to the mixture and the precipitate formed collected to yield the title compound (0.9 g, 60%) as a white powder.
Bis(dibenzylidineacetone)palladium (2 mg, 0.0035 mmol) and (2′-dicyclohexylphosphanyl-biphenyl-2-yl)-dimethylamine (3.3 mg, 0.0084 mmol) were added to dry toluene (10 mL purged with argon) and stirred for 15 minutes under argon. Potassium tert-butoxide (157 mg, 1.4 mmol), 1,2,3,4-tetrahydroisoquinoline (113 mg, 0.85 mmol) and N-(5-bromo-2-methylphenyl)-3,3-dimethylbutanamide (200 mg, 0.7 mmol) were then added and the reaction mixture was stirred at 90° C. overnight. The reaction mixture was then cooled to room temperature, concentrated and purified by thin layer chromatography (Dichloromethane:Methanol 10%) to afford compound the title compound as a solid. (160 mg, 68%). 1H NMR (DMSO-d6, 300 MHz) δ 1.02 (s, 9H), 2.07 (s, 3H), 2.19 (s, 2H), 2.88 (t, J=5.4 Hz, 2H), 3.43(t, J=5.7 Hz, 2H), 4.27 (s, 2H), 6.76 (dd, J=8.4, 2.7 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 7.19 (m, 4H), 9.06 (s, 1H).
The following compounds were prepared analogously:
See example 1. Yield: 70%. 1H NMR (DMSO-d6, 300 MHz) δ 0.99 (s, 9H), 2.22 (s, 2H), 3.00 (t, J=4.8 Hz, 4H), 3.72 (t, J=4.5 Hz, 4H), 6.7(m, 1H), 7.09 (t, J=9.0 Hz, 1H), 7.40 (m, 1H), 9.43 (s, 1H).
See example 1. Yield: 52%. 1H NMR (CDCl3, 300 MHz) δ 1.12 (s, 9H), 2.18 (s, 3H), 2.25 (s, 2H), 3.15 (t, J=4.8 Hz, 4H), 3.85 (t, J=4.8 Hz, 4H), 6.63 (dd, J=8.4, 2.4 Hz, 1H), 6.84 (s, 1H), 7.07 (d, J=8.4 Hz, 1H), 7.69 (d, J=2.1 Hz, 1H).
See example 1. Yield: 19%. 1H NMR (CDCl3, 300 MHz) δ 1.12 (s, 9H), 2.23 (s, 3H), 2.26 (s, 2H), 2.89 (t, J=4.5 Hz, 4H), 3.86 (t, J=4.5 Hz, 4H), 6.91 (d, J=8.1, Hz, 2H), 7.21 (t, J=8.1 Hz, 1H), 7.54 (d, J=8.1 Hz, 1H).
See example 1. Yield: 17%. 1H NMR (CDCl3, 300 MHz) δ 1.15 (s, 9H), 2.21 (s, 6H), 2.30 (s, 2H), 2.88 (t, J=4.8 Hz, 4H), 3.84 (t, J=4.8 Hz, 4H), 6.63 (s, 1H), 6.93 (d, J=8.4, Hz, 1H), 7.07 (d, J=8.1 Hz, 1H).
3-cyclopentylpropionyl chloride (2.166 g, 13 mmol) was added to a mixture of 2,6-dimethylaniline (1.211 g, 10 mmol) and triethylamine (1.515 g, 15 mmol) in THF at 0° C. The reaction mixture was progressively warmed to room temperature and stirred overnight. Ethyl acetate (200 ml) was added to the reaction mixture which was washed twice with HCl aq., 1N (100 ml). The organic layer was dried over MgSO4 and concentrated. The oily crude product was subjected to crystallization form hexanes obtain the title compound (2.11 g, 8.6 mmol, 86%) as white crystal.
Fuming nitric acid (1.5 ml) was added dropwise to a mixture of 3-cyclopentyl-N-(2,6-dimethylphenyl)propanamide (1.0 g, 4.07 mmol) in acetic acid (3 ml) at 0° C. The reaction mixture was progressively warmed to room temperature and stirred for 2 hours and then poured onto cold water (50 ml). The mixture was extracted with ethyl acetate (100 ml). The organic layer was washed twice with NaOH aq., 1N (30 ml) and then dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 2:3) to obtain the desired product (0.841 g, 2.896 mmol, 72%) as off-white powder.
A suspension of Raney-Nickel (2 g) in a solution of 3-cyclopentyl-N-(2,6-dimethyl-3-nitrophenyl)propanamide (0.8 g, 2.75 mmol) in methanol (15 ml) and THF (5 ml) was vigorously stirred under hydrogen atmosphere (1 atm) at room temperature for 6 hours. The reaction mixture was filtered and then the filtrate was concentrated and chromatographed (EtOAc:Hexanes, 1:1) to obtain the title product (0.627 g, 2.4 mmol, 87%) as white powder.
A mixture of N-(3-amino-2,6-dimethylphenyl)-3-cyclopentylpropanamide (0.226 g, 0.87 mmol) and 4-fluorobenzaldehyde (0.14 g, 1.13 mmol) in THF (3 ml) was stirred at room temperature for 3 hours and then NaBH4 (0.15 g, 3.97 mmol) and MeOH (1 ml) were added simultaneously. The resulting mixture was stirred for an additional 2 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated, and chromatographed (EtOAc:Hexanes 1:3) to yield the title product (0.304 g, 0.825 mmol, 94%) as white powder. 1H NMR (DMSO-d6, 300 MHz) δ 1.00-1.17 (m, 2H), 1.40-1.62 (m, 6H), 1.70-1.81 (m, 3H), 1.93 (s, 6H), 2.28 (t, J=7.5 Hz, 2H), 4.27 (d, J=5.7 Hz, 2H), 5.47 (t, J=5.7 Hz, 1H), 6.17 (d, J=8.4 Hz, 1H), 6.70 (d, J=8.4 Hz, 1H), 7.08 (dd, J=8.7, 8.7 Hz, 2H), 7.35 (dd, J=8.3, 5.9 Hz, 2H), 9.05 (s, 1H).
A solution of N-(3-amino-2,6-dimethylphenyl)-3-cyclopentylpropanamide (0.13 g, 0.5 mmol) and 4-(trifluoromethyl)benzaldehyde (0.122 g, 0.7 mmol) in THF (3 ml) was stirred at room temperature for 3 hours and then NaBH4 (0.106 g, 2.8 mmol) and MeOH (1 ml) were added simultaneously. The resulting mixture was stirred for an additional 12 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to obtain the title compound (0.097 g, 0.232 mmol, 46%) as white powder. 1H NMR (DMSO-d6, 500 MHz) δ 1.05-1.22 (m, 2H), 1.40-1.52 (m, 2H), 1.55-1.65 (m, 4H), 1.70-1.82 (m, 3H), 1.93 (s, 3H), 1.95 (s, 3H), 2.27 (t, J=7.5 Hz, 2H), 4.40 (d, J=5.7 Hz, 2H), 5.62 (t, J=5.7 Hz, 1H), 6.13 (d, J=8.2 Hz, 1H), 6.96 (d, J=8.2 Hz, 1H), 7.52 (d, J=7.8 Hz, 2H), 7.63 (d, J=7.8 Hz, 2H), 9.05 (s, 1H).
ter-Butylacetyl chloride (3.63 ml, 26 mmol) was added to a mixture of 6-ethyl-o-toluidine (2.712 g, 20 mmol) and triethylamine (4.17 ml, 30 mmol) in THF at 0° C. The reaction mixture was progressively warmed to room temperature and stirred overnight. Ethyl acetate (200 ml) was added to the reaction mixture which was washed twice with HCl aq., 1N (100 ml). The organic layer was dried over MgSO4 and concentrated. The oily crude product was subjected to crystallization form hexanes to yield the title compound (3.982 g, 17 mmol, 85%) as white needle crystal.
Fuming nitric acid (3 ml) was added dropwise to a mixture of N-(2-ethyl-6-methylphenyl)-3,3-dimethylbutanamide_(1.88 g, 8 mmol) in acetic acid (5 ml) at 0° C. The reaction mixture was progressively warmed to room temperature and stirred for 12 hours and then poured onto cold water (50 ml). The mixture was extracted with ethyl acetate (100 ml). The organic layer was washed twice with NaOH aq., 1N (30 ml), dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to yield N-(6-ethyl-2-methyl-3-nitrophenyl)-3,3-dimethylbutanamide (0.654 g, 2.35 mmol, 29%) as off-white powder and N-(2-ethyl-6-methyl-3-nitrophenyl)-3,3-dimethylbutanamide (0.258 g, 0.93 mmol, 11%) as off-white powder.
A suspension of Raney-Nickel (1 g) in a solution of N-(6-ethyl-2-methyl-3-nitrophenyl)-3,3-dimethylbutanamide (0.654 g, 2.35 ml) in methanol (20 ml) was vigorously stirred under hydrogen atmosphere (1 atm) at room temperature for 3 hours. The reaction mixture was filtered and then the filtrate was concentrated and chromatographed (EtOAc:Hexanes, 1:1) to yield to N-(6-ethyl-3-(4-fluorobenzylamino)-2-methylphenyl)-3,3-dimethylbutanamide ( 0.358 g, 1.44 mmol, 61%) as white powder.
A mixture of N-(3-amino-6-ethyl-2-methylphenyl)-3,3-dimethylbutanamide (0.2 g, 0.8 mmol) and 4-fluorobenzaldehyde (0.149 g, 1.2 mmol) in THF (5 ml) was stirred at room temperature for 3 hours and then NaBH4 (0.151 g, 4.0 mmol) and MeOH (1 ml) were added into the mixture simultaneously. The mixture was stirred for an additional 3 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and purified by chromatography on silica (EtOAc:Hexanes, 1:3) to yield to the desired product (0.183 g, 0.514 mmol, 64%) as white powder. 1H NMR (DMSO-d6, 500 MHz) δ 0.97 (t, J=7.5 Hz, 3H), 1.05 (s, 9H), 1.95 (s, 3H), 2.19 (s, 2H), 2.32 (q, J=7.5 Hz, 2H), 4.24 (d, J=5.5 Hz, 2H), 5.51 (t, J=5.5 Hz, 1H), 6.24 (d, J=8.2 Hz, 1H), 6.73 (d, J=8.2 Hz, 1H), 7.10 (dd, J=8.8, 8.8 Hz, 2H), 7.37 (dd, J=8.3, 5.9 Hz, 2H), 8.96 (s, 1H).
A suspension of Raney-Nickel (1 g) in a solution of N-(2-ethyl-6-methyl-3-nitrophenyl)-3,3-dimethylbutanamide (see example 4, 0.258 g, 0.93 mmol) in methanol (15 ml) was vigorously stirred under hydrogen atmosphere (1 atm) at room temperature for 3 hours. The reaction mixture was filtered and then the filtrate was concentrated and chromatographed (EtOAc:Hexanes, 1:1) to yield to the title product (0.189 g, 0.76 mmol, 81%) as off-white powder.
A solution of N-(3-amino-2-ethyl-6-methylphenyl)-3,3-dimethylbutanamide (0.046 g, 0.185 mmol) and 4-fluorobenzaldehyde (0.03 g, 2.4 mmol) in THF (2 ml) was stirred at room temperature for 3 hours and then NaBH4 (0.038 g, 1.0 mmol) and MeOH (0.5 ml) were added simultaneously. The resulting mixture was stirred for an additional 3 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to yield the title product (0.035 g, 0.098 mmol, 53%) as white powder. 1H NMR (CDCl3, 500 MHz) δ 0.92 (t, J=7.5 Hz, 3H), 1.13 (s, 9H), 2.03 (s, 3H), 2.40 (s, 2H), 2.42 (q, J=7.5 Hz, 2H), 4.15 (s, 2H), 6.91 (d, J=8.2 Hz, 1H), 6.98-7.09 (m, 3H), 7.23=7.27 (m, 2H), 7.45 (s, 1H). (rotomer effect)
T-butyl-acetyl chloride (1.83 ml, 13 mmol) was added to a mixture of 2,6-dimethylaniline (1.21 g, 10 mmol) and triethylamine ( 1.8 ml, 13 mmol) in THF at 0° C. The reaction mixture was progressively warmed to room temperature and stirred overnight. Ethyl acetate (200 ml) was added to the reaction mixture which was washed twice with HCl aq., 1N (100 ml). The organic layer was dried over MgSO4 and concentrated. The oily crude product was subjected to crystallization form hexanes to yield the title compound (1.805 g, 8.24 mmol, 82%) as white crystal.
Fuming nitric acid (3 ml) was added dropwise to a mixture of N-(2,6-dimethylphenyl)-3,3-dimethylbutanamide (1.524 g, 6.95 mmol) in acetic acid (4 ml) at 0° C. The reaction mixture was progressively warmed to room temperature and stirred for 2 hours and then poured onto cold water (50 ml). The mixture was extracted with ethyl acetate (100 ml). The organic layer was washed twice with NaOH aq., 1N (30 ml) and then dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to yield to the title compound (1.62 g, 6.13 mmol, 88%) as off-white powder.
A suspension of Raney-Nickel (1 g) in a solution of N-(2,6-dimethyl-3-nitrophenyl)-3,3-dimethylbutanamide (1.06 g, 4.0 ml) in methanol (20 ml) was vigorously stirred under hydrogen atmosphere (1 atm) at room temperature for 12 hours. The reaction mixture was filtered and then the filtrate was concentrated and chromatographed (EtOAc:Hexanes, 3:1) to yield to the title compound (0.664 g, 2.84 mmol, 71%) as white powder.
A solution of N-(3-amino-2,6-dimethylphenyl)-3,3-dimethylbutanamide (0.327 g, 1.4 mmol) and 4-fluorobenzaldehyde (0.226 g, 1.82 mmol) in THF (3 ml) was stirred at room temperature for 2 hours and then NaBH4 (0.211 g, 5.6 mmol) and MeOH (1 ml) were added simultaneously. The resulting mixture was stirred for an additional 2 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to yield to the title compound (0.271 g, 0.792 mmol, 56%) as white powder (0.113 g, 0.33 mmol, 24%) as white powder. 1H NMR (DMSO-d6, 500 MHz) δ 1.06 (s, 9H), 1.98 (s, 6H), 2.20 (s, 2H), 4.31 (d, J=5.7 Hz, 2H), 5.52 (t, J=5.7 Hz, 1H), 6.21 (d, J=8.2 Hz, 1H), 6.73 (d, J=8.2 Hz, 1H), 7.10 (dd, J=8.8, 8.8 Hz, 2H), 7.37 (dd, J=8.3, 5.9 Hz, 2H), 9.01 (s, 1H).
A solution of N-(3-amino-2,6-dimethylphenyl)-3,3-dimethylbutanamide (0.327 g, 1.4 mmol) and 4-(trifluoromethyl)benzaldehyde (0.337 g, 1.82 mmol) in THF (5 ml) was stirred in the presence of MS 4A at room temperature for 3 hours and then NaBH4 (0.2.11 g, 5.6 mmol) and MeOH (0.5 ml) were added simultaneously. The resulting mixture was stirred for an additional 24 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to yield to the title compound (0.159 g, 0.4 mmol, 28%) as white powder. 1H NMR (DMSO-d6, 500 MHz) δ 1.07 (s, 9H), 1.98 (s, 3H), 2.00 (s, 3H), 2.21 (s, 2H), 4.43 (d, J=5.7 Hz, 2H), 5.66 (t, J=5.7 Hz, 1H), 6.16 (d, J=8.2 Hz, 1H), 6.73 (d, J=8.2 Hz, 1H), 7.56 (d, J=8.3 Hz, 2H), 7.67 (d, J=8.3 Hz, 2H), 9.03 (s, 1H).
3-Cyclopentylpropionyl chloride (3.25 g, 19.5 mmol) was added to a mixture of 2,6-diethylaniline (2.239 g, 15 mmol) and triethylamine (2.02 g, 20 mmol) in THF at 0° C. The reaction mixture was progressively warmed to room temperature and stirred overnight. Ethyl acetate (200 ml) was added to the reaction mixture which was washed twice with HCl aq., 1N (100 ml). The organic layer was dried over MgSO4 and concentrated. The oily crude product was subjected to crystallization form hexanes to yield to the title compound (3.74 g, 13.4 mmol, 89%) as white crystal.
Fuming nitric acid (5.0 ml) was added dropwise to a mixture of 3-cyclopentyl-N-(2,6-diethylphenyl)propanamide (3.74 g, 13.4 mmol) in acetic acid (10 ml) at 0° C. The reaction mixture was progressively warmed to room temperature and stirred for 5 hours and then poured onto cold water (100 ml). The mixture was extracted with ethyl acetate (200 ml). The organic layer was washed twice with NaOH aq., 1N (50 ml) and then dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 2:3) to yield the title compound (2.515 g, 7.9 mmol, 59%) as off-white powder.
A suspension of Raney-Nickel (1 g) in a solution of 3-cyclopentyl-N-(2,6-diethyl-3-nitrophenyl)propanamide (1.0 g, 3.0 mmol) in methanol (15 ml) was vigorously stirred under hydrogen atmosphere (1 atm) at room temperature for 12 hours. The reaction mixture was filtered and then the filtrate was concentrated and chromatographed (EtOAc:Hexanes, 1:1) to yield the title compound (0.774 g, 2.63 mmol, 88%) as white powder.
A mixture of N-(3-amino-2,6-diethylphenyl)-3-cyclopentylpropanamide (0.383 g, 1.3 mmol) and 4-fluorobenzaldehyde (0.242 g, 1.95 mmol) in THF (4 ml) was stirred at room temperature for 3 hours and then NaBH4 (6.5 g, 0245 mmol) and MeOH (1 ml) were added simultaneously. The resulting mixture was stirred for an additional 2 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and chromatographed (EtOAc:Hexanes, 1:3) to yield to the title compound (0.195 g, 0.492 mmol, 38%) as white powder. 1H NMR (DMSO-d6, 500 MHz) δ 0.97 (t, J=7.5 Hz, 3H), 1.01 (t, J=7.5 Hz, 3H), 1.04-1.10 (m, 2H), 1.40-1.50 (m, 2H), 1.55-1.65 (m, 4H), 1.70-1.82 (m, 3H), 1.93 (s, 6H), 2.28 (q, J=7.5 Hz, 4H), 2.35-2.45 (m, 2H), 4.29 (s, 2H), 5.67 (bs, 1H), 6.23 (d, J=8.2 Hz, 1H), 6.72 (d, J=8.2 Hz, 1H), 7.10 (dd, J=8.8, 8.8 Hz, 2H), 7.37 (dd, J=8.3, 5.8 Hz, 2H), 8.99 (s, 1H).
3-cyclopentylpropionyl chloride (2.24 g, 15.31 mmol) was added to a mixture of 2-methoxy-6-methylaniline (2.0 g, 14.58 mmol) and triethylamine (2.24 mL, 16.04 mmol) in DCM at 0° C. The reaction mixture was progressively warmed to room temperature and stirred overnight. Ethyl acetate (200 ml) was added to the reaction mixture which was washed twice with HCl aq., 1N (100 ml). The organic layer was dried over MgSO4 and concentrated. The oily crude product was subjected to crystallization form hexanes to yield to the title compound (3.55 g, 14.37 mmol, 99%) as white crystal.
Fuming nitric acid (2 ml) was added dropwise to a mixture of 2-cyclopentyl-N-(2-methoxy-6-methylphenyl)acetamide (1.0 g, 4.04 mmol) in acetic acid (3 ml) at 0° C. The reaction mixture was progressively warmed to room temperature and stirred for 2 hours and then poured onto cold water (50 ml). The mixture was extracted with ethyl acetate (100 ml). The organic layer was washed twice with NaOH aq., 1N (30 ml) and then dried over MgSO4, concentrated and isolated by column chromatography on silica gel (EtOAc:Hexanes, 2:3) to yield to the desired product (0.505 g, 1.73 mmol, 43%) as a mixture of two isomers.
A suspension of Raney-Nickel (100 mg) in a solution of 2-cyclopentyl-N-(6-methoxy-2-methyl-3-nitrophenyl)acetamide (0.8 g, 2.75 mmol) in ethanol (10 ml) was vigorously stirred under hydrogen atmosphere (1 atm) at room temperature for 6 hours. The reaction mixture was filtered and then the filtrate was concentrated and isolated by column chromatography on silica gel (EtOAc:Hexanes, 1:1) to yield to the desired compound (0.385 g, 1.47 mmol, 85%) as a mixture of two isomers.
A mixture of the previous reaction (0.385 g, 1.47 mmol) and 4-fluorobenzaldehyde (0.24 mL, 2.21 mmol) in THF (3 ml) were stirred at room temperature for 3 hours and then NaBH4 (0.280 mg, 7.35 mmol) and MeOH (1 ml) were added simultaneously. The resulting mixture was stirred for an additional 2 hours. The mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was dried over MgSO4, concentrated and isolated by column chromatography on silica gel (EtOAc:Hexanes, 1:3) to yield the title compound (0.165 g, 0.73 mmol, 30%) as off-white powder. 1H NMR (CDCl3, 300 MHz) δ 1.23-1.30 (m, 2H), 1.60-1.70 (m, 4H), 1.89-1.96 (m, 2H), 2.03 (s, 3H), 2.32-2.37 (m, 1H), 2.43 (d, J=6.3 Hz, 2H), 3.64 (bs, 1H), 3.72 (s, 3H), 4.28 (s, 2H), 6.44 (d, J=9.0 Hz, 1H), 6.66 (d, J=8.7, Hz, 1H), 6.95 (s, 1H), 7.05 (t, J=8.7 Hz, 2H), 7.36 (dd, J=8.1, 5.4 Hz, 2H).
N-chlorosuccinimide ( 0.359 g, 2.69 mmol) was added to a solution of 3-bromo-2-methyl aniline (0.5 g, 2.69 mmol) in acetonitrile (5 mL) at room temperature. The reaction mixture was then stirred at 60° C. for 24 hours, cooled to room temperature and concentrated. Purification by column chromatography (5-20% EtOAc:Hexanes) afforded compound 1a (0.135 g, 0.61 mmol) in 23% yield.
3,3-Dimethylbutanoyl chloride (0.085 mL, 0.61 mmol) was added to a mixture 3-bromo-6-chloro-2-methylaniline (0.135 g, 0.61 mmol) and triethylamine (0.09 mL, 0.64 mmol) in DCM (2.5 mL) at 0° C. The reaction mixture was then stirred at 50° C. overnight. Ethyl acetate (50 ml) was added to the reaction mixture which was washed twice with HCl aq., 1N (20 ml). The organic layer was dried over MgSO4 and concentrated. Purification by column chromatography (15-30% EtOAc:Hexanes) afforded N-(3-bromo-6-chloro-2-methylphenyl)-3,3-dimethylbutanamide and N-(3-bromo-4-chloro-2-methylphenyl)-3,3-dimethylbutanamide as white solids.
Bis(dibenzylidineacetone)palladium (13 mg, 0.022 mmol) and (2′-dicyclohexylphosphanyl-biphenyl-2-yl)-dimethylamine (14 mg, 0.035 mmol) were added to dry toluene (1.5 mL purged with argon) and stirred for 15 minutes under argon. Potassium tert-butoxide (74 mg, 0.66 mmol), 4-fluorobenzyl amine (100 μL, 0.91 mmol) and N-(6-chloro-3-(4-fluorobenzylamino)-2-methylphenyl)-3,3-dimethylbutanamide (100 mg, 0.314 mmol) were then added and the reaction mixture was stirred at 90° C. overnight. The reaction mixture was then cooled to room temperature, concentrated and purified by thin layer chromatography (Dichloromethane:Methanol 10%) to afford compound 1c as a solid. (67 mg, 60% yield). 1H NMR (CDCl3, 300 MHz) δ 1.15 (s, 9H), 2.05 (s, 3H), 2.32 (s, 2H), 3.88 (bs, 1H), 4.31 (s, 2H), 6.44 (d, J=9.0 Hz, 1H), 6.92 (s, 1H), 7.06 (t, J=8.4, Hz, 2H), 7.12 (d, J=9 Hz, 1H), 7.34 (dd, J=8.7, 5.4 Hz, 2H).
This compound was prepared analogously (example 10) from N-(3-bromo-4-chloro-2-methylphenyl)-3,3-dimethylbutanamide (step B, example 10) and 4-fluorobenzyl amine. Yield: 53% (Step C). 1H NMR (CDCl3, 300 MHz) δ 1.12 (s, 9H), 2.56 (s, 3H), 2.27 (s, 2H), 3.8 (bs, 1H), 4.07 (d, J=5.7 Hz, 2H), 6.84 (m, 1H), 7.05 (m, 2H), 7.17 (m, 1H), 7.34 (m, 2H).
This compound was prepared analogously (example 10) from N-(3-bromo-2-chloro-6-methylphenyl)-3,3-dimethylbutanamide and 4-fluorobenzyl amine. Yield: 62% (Step C). 1H NMR (CDCl3, 300 MHz) δ 1.15 (s, 9H), 2.16 (s, 3H), 2.31 (s, 2H), 4.35 (d, J=5.7 Hz, 2H), 4.60 (bs, 1H), 6.48 (d, J=8.1 Hz, 1H), 6.79 (bs, 1H), 6.98 (d, J=8.7, Hz, 1H), 7.05 (t, J=8.4 Hz, 2H), 7.33 (dd, J=8.7, 5.7 Hz, 2H).
This compound was prepared analogously (example 10) from N-(3-bromo-2-chloro-6-methylphenyl)-3,3-dimethylbutanamide and 4-fluorobenzyl amine. Yield: 37% (Step C). 1H NMR (CDCl3, 300 MHz) δ 1.09 (s, 9H), 2.12 (s, 3H), 2.21 (s, 2H), 4.34 (s, 2H), 6.75 (m,1H), 7.04 (m, 2H), 7.36(m, 3H).
This compound was prepared analogously (example 10, Step C) from N-(5-bromo-2-methylphenyl)-3,3-dimethylbutanamide and 4-fluorobenzyl amine. Yield: 35%. 1H NMR (CDCl3, 300 MHz) δ 1.03 (s, 9H), 2.17 (s, 3H), 2.26 (s, 2H), 4.29 (bs, 2H), 4.43 (bs, 1H), 6.65 (dd, J=8.1, 2.1 Hz, 1H), 7.01 (q, J=7.8, Hz, 2H), 7.20 (s, 1H), 7.29 (m, 3H), 7.42 (s, 1H).
This compound was prepared analogously (example 10, Step C) from N-(3-bromo-2-methylphenyl)-3,3-dimethylbutanamide and 4-fluorobenzyl amine Yield: 57%. 1H NMR (CDCl3, 300 MHz) δ 1.13 (s, 9H), 2.04 (s, 3H), 2.25 (s, 2H), 3.89 (bs, 1H), 4.34 (d, J=4.2 Hz, 2H), 6.46 (d, J=7.8, Hz, 1H), 6.88 (d, J=7.2, Hz, 2H), 7.09 (q, J=8.7 Hz, 3H), 7.35 (m, 2H).
This compound was prepared analogously (example 10, Step C) from N-(5-bromo-2,4-difluorophenyl)-3,3-dimethylbutanamide (example 10, Step B) and 4-fluorobenzyl amine Yield: 20%. 1H NMR (CDCl3, 300 MHz) δ 1.09 (s, 9H), 2.23 (s, 2H), 3.89 (bs, 1H), 4.30 (d, J=4.2 Hz, 2H), 6.85 (t, 1H), 7.05 (m, 3H), 7.34 (m, 2H), 7.35 (t, 1H).
This compound was prepared by a method analogous to the synthesis of example 10, Step C, from N-(3-bromo-4-fluorophenyl)-3,3-dimethylbutanamide (see example 10, Step B) and 4-fluorobenzyl amine Yield: 47%. 1H NMR (CDCl3, 300 MHz) δ 1.08 (s, 9H), 2.17 (s, 2H), 4.26 (bs, 1H), 4.33 (d, J=4.2 Hz, 2H), 6.63 (m, 1H), 6.89 (m, 2H), 7.03 (m, 3H), 7.34 (m, 1H).
Compounds of formulas IB, IC, ID, IE, IF, IG, and IJ can be prepared by analogous reaction schemes, using the appropriate amine precursors in Schemes 1 and 2.
Compounds of this invention formula were evaluated as KCNQ 2/3 modulators by measuring rhubidium release in the following assay.
Methods: PC-12 cells were grown at 37° C. and 5% CO2 in DMEM/F12 Medium supplemented with 10% horse serum, 5% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin. They were plated in poly-D-lysine-coated 96-well cell culture microplates at a density of 40,000 cells/well and differentiated with 100 ng/ml NGF-7s for 2-5 days. For the assay, the medium was aspirated and the cells were washed once with 0.2 ml in wash buffer (25 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.8 mM NaH2PO4, 2 mM CaCl2). The cells were then loaded with 0.2 ml Rb+ loading buffer (wash buffer plus 5.4 mM RbCl2, 5 mM glucose) and incubated at 37° C. for 2 h. Attached cells were quickly washed three times with buffer (same as Rb+ loading buffer, but containing 5.4 mM KCl instead of RbCl) to remove extracellular Rb+. Immediately following the wash, 0.2 ml of depolarization buffer (wash buffer plus 15 mM KCl) with or without compounds was added to the cells to activate efflux of potassium ion channels. After incubation for 10 min at room temperature, the supernatant was carefully removed and collected. Cells were lysed by the addition of 0.2 ml of lysis buffer (depolarization buffer plus 0.1% Triton X-100) and the cell lysates were also collected. If collected samples were not immediately analyzed for Rb+ contents by atomic absorption spectroscopy (see below), they were stored at 4° C. without any negative effects on subsequent Rb+ analysis.
The concentration of Rb+ in the supernatants (Rb+Sup) and cell lysates (Rb+Lys) was quantified using an ICR8000 flame atomic absorption spectrometer (Aurora Biomed Inc., Vancouver, B.C.) under conditions defined by the manufacturer. One 0.05 ml samples were processed automatically from microtiter plates by dilution with an equal volume of Rb+ sample analysis buffer and injection into an air-acetylene flame. The amount of Rb+ in the sample was measured by absorption at 780 nm using a hollow cathode lamp as light source and a PMT detector. A calibration curve covering the range 0-5 mg/L Rb+ in sample analysis buffer was generated with each set of plates. The percent Rb+ efflux (F) was defined by
F=[Rb
+
Sup/(Rb+Sup+Rb+Lys)]×100%.
The effect (E) of a compound was defined by:
E=[(Fc−Fb)/(Fs−Fb)]×100%
where the Fc is the efflux in the presence of compound in depolarization buffer, Fb is the efflux in basal buffer, and Fs is the efflux in depolarization buffer, and Fc is the efflux in the presence of compound in depolarization buffer. The effect (E) and compound concentration relationship was plotted to calculate an EC50 value, a compound's concentration for 50% of maximal Rb+ efflux. The results are shown below. Legend: A: EC50<50 nM; B: EC50=50 nM-200 nM; C: EC50=200 nM-1.0 μM; D: EC50=1.0 μM-10 μM; E: EC50>10 μM
This application claims benefit of U.S. Provisional Application Nos. 60/838,808, filed Aug. 18, 2006, and 60/839,642, filed Aug. 22, 2006.
| Number | Date | Country | |
|---|---|---|---|
| 60838808 | Aug 2006 | US | |
| 60839642 | Aug 2006 | US |
