Ion channel modulating agents

The present invention relates to ion channel modulating agents. More particularly, the present invention relates to a particular class of chemical compounds represented by general Formula (I) and a pharmaceutically acceptable salt or an oxide or a hydrate thereof, that has proven useful as modulators of SK Ca , IK Ca and BK Ca channels. In further aspects, the present invention relates to the use of these SK/IK/BK channel modulating agents for the manufacture of medicaments, and pharmaceutical compositions comprising the SK/IK/BK channel modulating agents. The SK/IK/BK channel modulating agents of the invention are useful for the treatment or alleviation of diseases and conditions associated with the SK/IK/BK channels.

EXAMPLES The invention is further illustrated with reference to the following examples which are not intended to be in any way limiting to the scope of the invention as claimed. 
 Example 1 3 1-iodo-2,6-diisopropylbenzene (Compound 1A). A suspension of 2,6-diisopropylaniline (5.3 ml, 28.2 mmol) in conc. hydrochloric acid (25 ml) was cooled to −10° C. A cooled solution of sodium nitrite (2.14 g; 31.0 mmol) in water (5 ml) was added dropwise, keeping the temperature below 0° C. At the end of the addition the mixture was stirred for additionally 20 minutes. The resulting mixture was carefully added to a solution of potassium iodide (5.9 g; 35.5 mmol) in water (10 ml) and stirred at ambient temperature until the evolution of nitrogen had ceased. Aqueous sodium sulphite (1M) was added to afford a light-coloured solution, which was extracted three times with ethyl acetate. The combined organic extracts were dried over magnesium sulphate, concentrated under reduced pressure and chromatographied on silica gel using a mixture of ethyl acetate and ligroin (1:4 v/v) as the eluent. Yield: 4.67 g (57.5%). 3-(2,6-diisopropylphenyl)-pyridine (Compound 1B). A mixture of 1-iodo-2,6-diisopropylbenzene (0.70 g; 2.43 mmol), diethyl 3-pyridylborane (0.54 g; 3.65 mmol), potassium carbonate (1.0 g; 7.3 mmol) and tetrakis(triphenylphosphine)palladium (80 mg) in a mixture of water (10 ml) and dimethoxyethane (20 ml) was stirred at reflux in a nitrogen atmosphere for two hours. The cooled mixture was diluted with two volumes of water and extracted twice with ethyl acetate. The combined extracts were dried over magnesium sulphate and concentrated under reduced pressure. The residue was chromatographied on silica gel using a mixture of ethyl acetate and ligroin (1:4 v/v) as the eluent to yield the product (70 mg) as a yellow oil. M &plus; : 239. 
 Example 2 4 2-((2,6-di-tertbutyl-4-methylphenyl)-oxymethyl)-pyridine (Compound 2A). A solution of 2,6-di-tertbutyl-4-methylphenol (0.58 g; 2.65 mmol) in anhydrous DMF (2.5 ml) was kept in an inert atmosphere. Sodium hydride (2.92 mmol; 0.12 g 60% dispersion in mineral oil) was added and the mixture was stirred for 15 min. A solution of 2-picolylchloride (0.33 g; 2.65 mmol) in anhydrous DMF was added and the reaction mixture was heated to 80° C. overnight. After cooling the mixture was diluted with four volumes of water and extracted thrice with ethyl acetate. The combined extracts were dried over magnesium sulphate and concentrated under reduced pressure. Column-chromatographic work-up on silica gel with a mixture of ethyl acetate and ligroin (1:4 v/v) as the eluent afforded the product as a yellow oil (0.34 g; 41%), which crystallised upon standing. Mp. 79.3-83.6° C. 
 Example 3 5 N-(2-picolyl)-2,6-diisopropylaniline (Compound 3A). A mixture of 2-picolylchloride (0.33 g; 2.65 mmol), 2,6-diisopropylaniline (0.50 ml; 2.65 mmol) and triethylamine (0.37 ml; 2.65 mmol) in anhydrous DMF (2.5 ml) was stirred in an inert atmosphere while heated to 80° C. overnight. The cooled mixture was diluted with four volumes of water and extracted twice with ethyl acetate. The combined extracts were washed with brine, dried over magnesium sulphate and concentrated under reduced pressure. The residue was chromatographied on silica gel using a mixture of ethyl acetate and ligroin (1:4 v/v) as the eluent. The product (0.14 g; 20%) was isolated as a brownish oil. M &plus; : 268. 
 Example 4 6 N-(2,6-diisopropylaniline)-4-pyridinecarbimine (Compound 4A). A mixture of 2,6-diisopropylaniline (1.88 ml, 10.0 mmol), 4-pyridinecarboxaldehyde (0.96 ml, 10.0 mmol) and a catalytic amount of p-toluenesulphonic acid in toluene (10 ml) was heated to 100° C. for one hour and subsequently left with stirring at ambient temperature overnight. The resulting mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over magnesium sulphate and evaporated to dryness. Trituration of the residue with ligroin afforded the crystalline product. Yield: 0.91 g (34%). Mp: 100.5-101.6° C. 
 Example 5 7 2,6-diisopropyl-4-nitroacetanilide (Compound 5A). To a cooled (2° C.) suspension of 2,6-diisopropylacetanilide (prepared from 2,6-diisopropylaniline using acetic anhydride under standard conditions) (11.0 g; 50.2 mmol) in conc. sulphuric acid (10 ml) was added potassium nitrate (5.58 g; 55.3 mmol) portion-wise over 2 hours, during which time the reaction mixture was allowed to reach ambient temperature. Stirring was continued for 1.5 hours. The mixture was poured into ice-water, the precipitate was filtered off, washed with water and air-dried to afford the desired product, quantitatively. Mp. 170-175° C. 
 Example 6 8 2,6-diphenyl-4-nitrophenol (Compound 6A). To a solution of 2,6-dibromo-4-nitrophenol (2.0 g; 6.7 mmol) in a mixture of dimethoxyethane (40 ml) and water (20 ml) was added phenyl boronic acid (2.5 g; 20.2 mmol), potassium carbonate (2.8 g; 20.2 mmol) and bis(triphenylphosphine)palladiumdichloride. The mixture was heated to reflux for 1 hour. After cooling the volatile solvent was removed by evaporation and the residual aqueous phase was extracted twice with ethyl acetate. The extract was dried over magnesium sulphate, evaporated to dryness and eluted through silica gel with a mixture of ethyl acetate and ligroin (1:9 v/v). The pure fractions were evaporated to dryness to leave the pure product (0.7 g; 36%). Mp. 110-112° C. 1-(2,6-diphenyl-4-chlorophenyl)-2,5-dimethylpyrrole (Compound 6B) was prepared analogously from 1-(2,6-dibromo-4-chlorophenyl)-2,5-dimethylpyrrole. Yield 56%. Mp. 178-180° C. 
 Example 7 9 1-(2,6-diisopropyl-4-nitrophenyl)-2,5-dimethylpyrrole (Compound 7A). A mixture of 2,6-diisopropyl-4-nitroaniline (0.5 g; 2.25 mmol), 2,5-hexadione (0.53 ml; 4.5 mmol) and a catalytic amount of p-TSA in toluene (10 ml) was heated to reflux for two hours. After cooling the solvent was removed under reduced pressure and the residue was eluted through silica gel with dichloromethane. The pure fractions were evaporated to dryness to leave the desired product (0.15 g; 22%). Mp. 125-132° C. 1-(2,6-dibromo-4-chlorophenyl)-2,5-dimethylpyrrole (Compound 7B) was prepared analogously from 4-chloro-2,6-dibromoaniline. Yield 72%. 
 Example 8 10 1-(2,6-dimethylphenyl)imidazole (Compound 8A). A mixture of 2-bromo-m-xylene (1.0 g; 5.4 mmol), imidazole (1.1 g; 16.2 mmol), potassium carbonate (0.75 g; 5.4 mmol) and catalytic amoumts of copper powder and cuprous iodide in NMP (1 ml) was heated with stirring to 180° C. in a nitrogen atmosphere for four days. After cooling the reaction mixture was partitioned between ethyl acetate and water. The organic phase was washed with water, dried over magnesium sulfate and evaporated to dryness. The residue was triturated with water to leave the desired product (0.23 g; 25%). Mp. 79-80° C. 
 Example 9 Inhibition of T Cell Proliferation The chemical compounds used according to the invention prevent immunological proliferation by selective inhibition of the Ca 2&plus; -activated K-channels in T- and B-lymphocytes. This effect may be verified using various proliferation assays. In this experiment the proliferative assay described by Ødum et al &lsqb;Ødum N, Kanner S B, Ledbetter J A, & Svejgaard A; J. Immunol. 1993 150 (12) 5289-5298&rsqb; was used. A chemical compound representative for the invention tested in this experiment is 3-(2,6-diisopropylphenyl)-pyridine (Compound 1B). Assays were performed in culture medium (RPMI 1640; available from Gibco, Grand Island, N.Y.) supplemented with 10% pooled human serum, 2 mM L-glutamine, 100 &mgr;g/ml penicillin, and 100 &mgr;g/ml streptomycin (available from Novo Nordisk, Copenhagen, Denmark) in 96-well round bottom tissue culture plates (available from Nunc, Roskilde, Denmark) with a final volume of 200 &mgr;l. T cells were pre-incubated for three hours with the test compounds before addition of antigen (PPD, purified protein derivative, 10 &mgr;g/ml). T cells were cultured at 5×10 4 cells/well for 144 hours. Twelve hours before harvest, &lsqb; 3 H&rsqb;thymidine (1× Ci/well) was added. The cells were harvested onto glass fibre filters, and the &lsqb; 3 H&rsqb;thymidine incorporation was measured in a scintillation counter. The results were expressed as mean counts per minute (cpm) from triplicate cultures. The results are presented in Table 1, below. 1 TABLE 1 Inhibition of T Cell Proliferation T Cell Proliferation (cpm × 10 −3 ) Test Medium Antigen, PPD Compound Solvent Solvent 2.5 &mgr;M 10 &mgr;M 25 &mgr;M 1B 0.2 26.1 19.5 19 14 These results show that the number of T cells decreases in the presence of increasing concentrations of the chemical compound of the invention, and support the fact that the chemical compounds of the invention inhibit the antigen induced T cell proliferation and thus are useful for the reduction or inhibition of undesired immune-regulatory actions.