HETEROARYL-SUBSTITUTED IMIDAZO[1,2-A]PYRIDINES AND THEIR USE

Abstract

The present application relates to novel heteroaryl-substituted imidazo[1,2-a]pyridines, to processes for preparation thereof, to the use thereof, alone or in combinations, for the treatment and/or prophylaxis of diseases, and to the use thereof for production of medicaments for the treatment and/or prophylaxis of diseases, especially for the treatment and/or prophylaxis of cardiovascular disorders.

Claims

1. A compound of the formula (I) ##STR00082## in which A represents CH.sub.2, CD.sub.2 or CH(CH.sub.3), R.sup.1 represents (C.sub.3-C.sub.7)-cycloalkyl, phenyl or pyridyl, where (C.sub.3-C.sub.7)-cycloalkyl may be substituted by 1 to 4 substituents independently of one another selected from the group consisting of fluorine, trifluoromethyl and (C.sub.1-C.sub.4)-alkyl, where phenyl is substituted by 1 to 4 substituents independently of one another selected from the group consisting of halogen, cyano, monofluoromethyl, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl, (C.sub.1-C.sub.4)-alkoxy and difluoromethoxy and where pyridyl is substituted by 1 or 2 substituents independently of one another selected from the group consisting of halogen, cyano and (C.sub.1-C.sub.4)-alkyl, R.sup.2 represents (C.sub.1-C.sub.4)-alkyl, cyclopropyl, cyclobutyl, monofluoromethyl, difluoromethyl or trifluoromethyl, R.sup.3 represents phenyl or 6-membered heteroaryl, where phenyl is substituted by —NR.sup.7R.sup.8, in which R.sup.7 represents hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.8 represents (C.sub.1-C.sub.6)-alkyl or (C.sub.1-C.sub.4)-alkylcarbonyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by amino and up to three times by fluorine, or R.sup.7 and R.sup.8 together with the nitrogen atom to which they are attached form a 4- to 6-membered heterocycle, where phenyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine, chlorine, bromine, methyl and ethyl, where 6-membered heteroaryl is up to disubstituted by —NR.sup.9R.sup.10, in which R.sup.9 represents hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.10 represents hydrogen, (C.sub.1-C.sub.6)-alkyl, phenyl or (C.sub.1-C.sub.4)-alkylcarbonyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of (C.sub.1-C.sub.4)-alkoxy, amino and phenyl, and up to pentasubstituted by fluorine, or R.sup.9 and R.sup.10 together with the nitrogen atom to which they are attached form a 4- to 6-membered heterocycle, in which the 4- to 6-membered heterocycle may be substituted by 1 or 2 (C.sub.1-C.sub.4)-alkyl substituents, where 6-membered heteroaryl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine, chlorine, bromine, cyclopropyl, methyl and ethyl, R.sup.4 represents hydrogen, R.sup.5 represents hydrogen, fluorine, chlorine, cyano, (C.sub.1-C.sub.4)-alkyl, cyclopropyl, difluoromethyl or trifluoromethyl, R.sup.6 represents hydrogen or fluorine, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof except for N-(3-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}phenyl)acetamide, and 8-[(2,6-difluorobenzyl)oxy]-2-methyl-3-[3-(piperidin-1-yl)phenyl]imidazo[1,2-a]pyridine.

2. The compound of the formula (I) as claimed in claim 1 in which A represents CH.sub.2, R.sup.1 represents cyclohexyl, phenyl or pyridyl, where phenyl is substituted by 1 to 4 substituents independently of one another selected from the group consisting of fluorine, chlorine, cyano and methyl, and where pyridyl is substituted by 1 or 2 fluorine substituents, R.sup.2 represents methyl, cyclopropyl or trifluoromethyl, R.sup.3 represents phenyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 5-pyrimidyl or 1,3,5-triazinyl, where phenyl is substituted in the 3-position by —NR.sup.7R.sup.8, in which R.sup.7 represents hydrogen, R.sup.8 represents methyl, ethyl, methylcarbonyl or ethylcarbonyl, or R.sup.7 and R.sup.8 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, where phenyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine and chlorine, where 3-pyridyl, 4-pyridyl, 2-pyrimidyl and 5-pyrimidyl are substituted by —NR.sup.9R.sup.10, in which R.sup.9 represents hydrogen or methyl, R.sup.10 represents (C.sub.1-C.sub.6)-alkyl or phenyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of (C.sub.1-C.sub.4)-alkoxy, amino and phenyl, and up to trisubstituted by fluorine, or R.sup.9 and R.sup.10 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, in which the 5- or 6-membered heterocycle may be substituted by 1 or 2 methyl substituents, where 3-pyridyl, 4-pyridyl, 2-pyrimidyl and 5-pyrimidyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine, chlorine and methyl, where 1,3,5-triazinyl is up to disubstituted by —NR.sup.9AR.sup.10A, in which R.sup.9A represents hydrogen or methyl, R.sup.10A represents hydrogen, (C.sub.1-C.sub.6)-alkyl or phenyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by amino and up to three times by fluorine, or R.sup.9A and R.sup.10A together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, in which the 5- or 6-membered heterocycle may be substituted by 1 or 2 methyl substituents, where 1,3,5-triazinyl may be substituted by chlorine, R.sup.4 represents hydrogen, R.sup.5 represents hydrogen, chlorine, methyl or cyclopropyl, R.sup.6 represents hydrogen, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof except for N-(3-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}phenyl)acetamide, and 8-[(2,6-difluorobenzyl)oxy]-2-methyl-3-[3-(piperidin-1-yl)phenyl]imidazo[1,2-a]pyridine.

3. The compound of the formula (I) as claimed in claim 1 in which A represents CH.sub.2, R.sup.1 represents cyclohexyl, phenyl or pyridyl, where phenyl is substituted by 1 to 3 fluorine substituents, where pyridyl is substituted by 1 fluorine substituent, R.sup.2 represents methyl, R.sup.3 represents phenyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 5-pyrimidyl or 1,3,5-triazinyl, where phenyl is substituted in the 3-position by —NR.sup.7R.sup.8, in which R.sup.7 represents hydrogen, R.sup.8 represents methyl, ethyl, methylcarbonyl or ethylcarbonyl, or R.sup.7 and R.sup.8 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, where 3-pyridyl, 4-pyridyl, 2-pyrimidyl and 5-pyrimidyl are substituted by —NR.sup.9R.sup.10, in which R.sup.9 represents hydrogen or methyl, R.sup.10 represents (C.sub.1-C.sub.6)-alkyl or phenyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of (C.sub.1-C.sub.4)-alkoxy, amino and phenyl, and up to trisubstituted by fluorine, or R.sup.9 and R.sup.10 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle, in which the 5- or 6-membered heterocycle may be substituted by 1 or 2 methyl substituents, where 3-pyridyl, 4-pyridyl, 2-pyrimidyl and 5-pyrimidyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of fluorine, chlorine and methyl, where 1,3,5-triazinyl is up to disubstituted by —NR.sup.9AR.sup.10A, in which R.sup.9A represents hydrogen or methyl, R.sup.10A represents hydrogen, (C.sub.1-C.sub.6)-alkyl or phenyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by amino and up to three times by fluorine, where 1,3,5-triazinyl may be substituted by chlorine, R.sup.4 represents hydrogen, R.sup.5 represents hydrogen, chlorine, methyl or cyclopropyl, R.sup.6 represents hydrogen, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts except for N-(3-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}phenyl)acetamide, and 8-[(2,6-difluorobenzyl)oxy]-2-methyl-3-[3-(piperidin-1-yl)phenyl]imidazo[1,2-a]pyridine.

4. The compound of the formula (I) as claimed in claim 1, in which A represents CH.sub.2, R.sup.1 represents cyclohexyl, or represents a phenyl group of the formula ##STR00083## where ## represents the point of attachment to A, and R.sup.11 represents hydrogen or fluorine, or represents a pyridyl group of the formula ##STR00084## where # represents the point of attachment to A, R.sup.2 represents methyl, R.sup.3 represents phenyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 5-pyrimidyl or 1,3,5-triazinyl, where phenyl is substituted in the 3-position by —NR.sup.7R.sup.8, in which R.sup.7 represents hydrogen, R.sup.8 represents methylcarbonyl, or R.sup.7 and R.sup.8 together with the nitrogen atom to which they are attached form a pyrrolidinyl ring, where 3-pyridyl is substituted in the 4-position by —NR.sup.9R.sup.10, where 4-pyridyl is substituted in the 3-position by —NR.sup.9R.sup.10, where 2-pyrimidyl is substituted in the 4-position by —NR.sup.9R.sup.10, where 5-pyrimidyl is substituted in the 2-position by —NR.sup.9R.sup.10, in which in each case R.sup.9 represents hydrogen or methyl, R.sup.10 represents (C.sub.1-C.sub.6)-alkyl or phenyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 or 2 substituents independently of one another selected from the group consisting of (C.sub.1-C.sub.4)-alkoxy, amino and phenyl, and up to trisubstituted by fluorine, or R.sup.9 and R.sup.10 together with the nitrogen atom to which they are attached form a pyrrolidinyl, morpholinyl or piperazinyl ring, in which the pyrrolidinyl, morpholinyl or piperazinyl ring may be substituted by 1 or 2 methyl substituents, where 4-pyridyl may be substituted in the 2-position by fluorine, where 1,3,5-triazinyl is up to disubstituted by —NR.sup.9AR.sup.10A, in which R.sup.9A represents hydrogen or methyl, R.sup.10A represents hydrogen, (C.sub.1-C.sub.6)-alkyl or phenyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by amino and up to three times by fluorine, where 1,3,5-triazinyl may be substituted by chlorine, R.sup.4 represents hydrogen, R.sup.5 represents hydrogen, chlorine or methyl, R.sup.6 represents hydrogen, and the N-oxides, salts, solvates, salts of the N-oxides and solvates of the N-oxides and salts thereof except for N-(3-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}phenyl)acetamide, and 8-[(2,6-difluorobenzyl)oxy]-2-methyl-3-[3-(piperidin-1-yl)phenyl]imidazo[1,2-a]pyridine.

5. A process for preparing compounds of the formula (I) as defined in claim 1, comprising [A] reacting a compound of the formula (II) ##STR00085## in which A, R.sup.1, R.sup.2, R.sup.4, R.sup.5 and R.sup.6 are each as defined in claim 1 and T.sup.1 represents (C.sub.1-C.sub.4)-alkyl or benzyl, in an inert solvent in the presence of a suitable base or acid to give a carboxylic acid of the formula (III) ##STR00086## in which A, R.sup.1, R.sup.2, R.sup.4, R.sup.5 and R.sup.6 each have the meanings given in claim 1, and reacting the carboxylic acid of formula (III) in the presence of a suitable acid to give an imidazo[1,2-a]-pyridine of the formula (IV) ##STR00087## in which A, R.sup.1, R.sup.2, R.sup.4, R.sup.5 and R.sup.6 each have the meanings given in claim 1, and converting the imidazo[1,2-a]-pyridine of the formula (IV) with a halogen equivalent into a compound of the formula (V) ##STR00088## in which A, R.sup.1, R.sup.2, R.sup.4, R.sup.5 and R.sup.6 are each as defined in claim 1 and X.sup.1 represents chlorine, bromine or iodine, reacting the compound of the formula (V) in an inert solvent, in the presence of a suitable transition metal catalyst, with a compound of the formula (VI) ##STR00089## in which R.sup.3A has the meanings given above for R.sup.3 and T.sup.2 represents hydrogen or (C.sub.1-C.sub.4)-alkyl, or the two T.sup.2 radicals together form a —C(CH.sub.3).sub.2—C(CH.sub.3).sub.2— bridge, detaching any protective groups that may be present, and optionally converting the resulting compounds of the formula (I) with the appropriate (i) solvents and/or (ii) acids or bases to the solvates, salts and/or solvates of the salts thereof.

6. (canceled)

7. The use of a compound of the formula (I) as defined in claim 1 for producing of a medicament for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischaemias, vascular disorders, renal insufficiency, thromboembolic disorders and arteriosclerosis.

8. A medicament comprising the compound of the formula (I) as defined in claim 1 in combination with an inert, non-toxic, pharmaceutically suitable excipient.

9. A medicament comprising the compound of the formula (I) as defined in claim 1 in combination with a further active compound selected from the group consisting of organic nitrates, NO donors, cGMP-PDE inhibitors, antithrombotic agents, hypotensive agents and lipid metabolism modifiers.

10. (canceled)

11. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischaemias, vascular disorders, renal insufficiency, thromboembolic disorders and arteriosclerosis in humans and animals comprising administering an effective amount of at least one compound of the formula (I) as defined in claim 1 to the human or animal in need thereof.

12. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischaemias, vascular disorders, renal insufficiency, thromboembolic disorders and arteriosclerosis in humans and animals comprising administering an effective amount of the medicament of claim 8 to the human or animal in need thereof.

13. A method for the treatment and/or prophylaxis of heart failure, angina pectoris, hypertension, pulmonary hypertension, ischaemias, vascular disorders, renal insufficiency, thromboembolic disorders and arteriosclerosis in humans and animals comprising administering an effective amount of the medicament of claim 9 to the human or animal in need thereof.

Description

WORKING EXAMPLES

Example 1

N-Benzyl-5-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}pyridine-2-amine

[0725] ##STR00062##

[0726] 35 mg (0.10 mmol) of 3-bromo-8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridine (Example 19A), 5.8 mg (0.005 mmol) of tetrakis(triphenylphosphine)palladium(0), 22 mg (0.20 mmol) of sodium carbonate and 0.2 ml of water were added to 31 mg (0.10 mmol) of N-benzyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-amine in 0.6 ml of 1,4-dioxane, and the mixture was shaken at 85° C. overnight. After the reaction had ended, the reaction solution was filtered, the 1,4-dioxane was removed under reduced pressure and the residue was dissolved in a little DMSO and purified by preparative HPLC (Method 11). This gave 0.7 mg (2% of theory) of the title compound.

[0727] LC-MS (Method 12): R.sub.t=0.89 min

[0728] MS (ESpos): m/z=457.3 (M+H).sup.+

[0729] Analogously to Example 1, the example compounds shown in Table 1 were prepared by reacting 3-bromo-8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridine (Example 19A) with the appropriate boronic acids or boronic ester [4,4,5,5-tetramethyl-1,3,2-dioxaborolanyl=boronic acid pinacol ester].

TABLE-US-00001 TABLE 1 IUPAC name Example Structure No. (Yield) Analytical data 2 [00063] LC-MS (Method 12): R.sub.t = 0.75 min MS (ESpos): m/z = 425 (M + H).sup.+ 3 [00064] LC-MS (Method 12): R.sub.t = 0.80 min MS (ESpos): m/z = 409.2 (M + H).sup.+ 4 [00065] LC-MS (Method 12): R.sub.t = 0.94 min MS (ESpos): m/z = 465.2 (M + H).sup.+ 5 [00066] LC-MS (Method 12): R.sub.t = 0.94 min MS (ESpos): m/z = 443.2 (M + H).sup.+ [1] Preparation analogous to ASTRAZENECA, patent: WO2007/86800 A1, 2007 from 5-bromo-N-(2-methoxyethyl)pyridine-2-amine. [2] Preparation analogous to Majo, Vattoly J.; Prabhakaran, Jaya; Mann, J. John; Kumar, J.S. Dileep, Advanced Synthesis and Catalysis, 2003, vol. 345, p. 620-624 from 5-bromo-N-ethyl-N-methylpyridine-2-amine. [3] Preparation analogous to XCOVERY, INC. patent: WO2008/88881 A1, 2008 from 4-(5-bromopyridin-2-yl)-2,6-dimethylmorpholine. [4] Preparation analogous to BioMarin IGA, Ltd.; Wren, Stephen Paul; Wynne, Graham Michael; Lecci, Cristina; Wilson, Francis Xavier; Davis, Paul James; patent: WO2010/57833 A1, 2010 from 5-bromo-N-phenylpyridine-2-amine.

Example 6

8-[(2,6-Difluorobenzyl)oxy]-2-methyl-3-[2-(4-methylpiperazin-1-yl)pyrimidin-5-yl]imidazo[1,2-a]pyridine

[0730] ##STR00067##

[0731] 125 mg (0.35 mmol) of 3-bromo-8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridine (Example 19A), 118 mg (0.39 mmol) of 2-(4-methylpiperazin-1-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine and 119 mg (1.42 mmol) of sodium bicarbonate were initially charged with 14 mg (0.02 mmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride/dichloromethane complex, and a degassed 3:1 mixture of 1,2-dimethoxyethane and water was added. The mixture was stirred overnight at 80° C. The reaction solution was diluted with acetonitrile/water, filtered through a Millipore filter and purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated and the residue was taken up in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted two more times with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, concentrated and lyophilized. This gave 77 mg of the target compound (48% of theory).

[0732] LC-MS (Method 16): R.sub.t=0.46 min

[0733] MS (ESpos): m/z=451 (M+H).sup.+

Example 7

6-{8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridin-3-yl}-1,3,5-triazine-2,4-diamine

[0734] ##STR00068##

[0735] Under argon, 43 mg (0.31 mmol) of imidodicarbonimidediamide hydrochloride [biguanide hydrochloride] were initially charged in 0.93 ml abs. methanol, 148 mg (0.16 ml, 0.69 mmol) of sodium methoxide (25% in methanol) were added and the mixture was stirred at 50° C. for 30 min. Subsequently, 75 mg (0.21 mmol) of ethyl 8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxylate from Example 10A were added and the mixture was stirred under reflux overnight. After cooling, the reaction mixture was poured onto water and the solid formed was filtered off and dried. The crude product was dissolved in acetonitrile/water/TFA and purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product-containing fractions were concentrated, dissolved in dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted twice with dichloromethane with addition of a little methanol. The combined organic phases were dried over sodium sulfate and filtered, the filtrate was concentrated and the residue was stirred with water. The precipitate was filtered off and dried under high vacuum. This gave 6.3 mg of the target compound (7.6% of theory).

[0736] LC-MS (Method 1): R.sub.t=0.73 min

[0737] MS (ESpos): m/z=398 (M+H).sup.+

[0738] .sup.1H-NMR (400 Mhz, DMSO-d.sub.6) δ=2.38 (s, 3H), 2.73 (s, 3H), 5.29 (s, 2H), 6.72 (br. s, 4H), 6.92 (s, 1H), 7.22 (t, 2H), 7.54-7.64 (m, 1H), 9.60 (s, 1H).

Example 8

6-{8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridin-3-yl}-N, N-dimethyl-1,3,5-triazine-2,4-diamine

[0739] ##STR00069##

[0740] Under argon, 103 mg (0.62 mmol) of N,N-dimethylimidodicarbonimidediamide hydrochloride [methformin hydrochloride] were initially charged in 1.87 ml abs. methanol, 297 mg (0.31 ml, 1.38 mmol) of sodium methoxide (25% in methanol) were added and the mixture was stirred at 50° C. for 30 min. Subsequently, 150 mg (0.42 mmol) of ethyl 8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxylate from Example 10A were added and the mixture was stirred under reflux overnight. After cooling, the reaction mixture was poured onto water and the solid formed was filtered off with suction and dried. The crude product was dissolved in acetonitrile/water/TFA and purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The crude product was purified again by silica gel chromatography (mobile phase: cyclohexane/ethyl acetate: 2/1 then 1/1). This gave 31 mg of the target compound (16% of theory, purity 92%).

[0741] LC-MS (Method 1): R.sub.t=0.85 min

[0742] MS (ESpos): m/z=426 (M+H).sup.+

[0743] .sup.1H-NMR (400 Mhz, DMSO-d.sub.6) δ=2.37 (s, 3H), 2.72 (s, 3H), 3.05-3.20 (m, 6H), 5.29 (s, 2H), 6.78-7.09 (m, 3H), 7.22 (t, 2H), 7.54-7.64 (m, 1H), 9.62 (s, 1H).

Example 9

6-{8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridin-3-yl}-N-propyl-1,3,5-triazine-2,4-diamine

[0744] ##STR00070##

[0745] Under argon, 89 mg (0.62 mmol) of 1-amino(propylamino)methylene]guanidine were initially charged in 1.87 ml abs. methanol, 297 mg (0.31 ml, 1.38 mmol) of sodium methoxide (25% in methanol) were added and the mixture was stirred at 50° C. for 30 min. Subsequently, 150 mg (0.42 mmol) of ethyl 8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridine-3-carboxylate from Example 10A were added and the mixture was stirred under reflux overnight. After cooling, the reaction mixture was poured onto water and the solid formed was filtered off with suction and dried. The crude product was dissolved in acetonitrile/water/TFA and purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The crude product was purified again by silica gel chromatography (mobile phase: cyclohexane/ethyl acetate: 2/1 then 1/1). This gave 36 mg of the target compound (18% of theory, purity 93%).

[0746] LC-MS (Method 1): R.sub.t=0.87 min

[0747] MS (ESpos): m/z=440 (M+H).sup.+

[0748] .sup.1H-NMR (400 Mhz, DMSO-d.sub.6) δ=0.91 (t, 3H), 1.49-1.63 (2H), 2.38 (s, 3H), 2.72 (s, 3H), 3.20-3.32 (m, 2H), 5.30 (s, 2H), 6.60-6.90 (m, 2H), 6.91-6.98 (m, 1H), 7.20-7.32 (m, 2H), 7.55-7.63 (m, 1H), 9.58-9.64 (m, 1H).

Example 10

N′-(4-{8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridin-3-yl}-5-fluoropyridin-2-yl)-2-methylpropane-1,2-diamine

[0749] ##STR00071##

[0750] 30 mg (0.072 mmol) of 8-[(2,6-difluorobenzyl)oxy]-3-(2,5-difluoropyridin-4-yl)-2,6-dimethylimidazo[1,2-a]pyridine from Example 33A were initially charged in 0.27 ml of NMP. At room temperature, 32 mg (0.36 mmol) of 2-methylpropane-1,2-diamine were added and the mixture was stirred at 150° C. in a closed vessel in an oil bath for 2 h. Another 13 mg (0.15 mmol) of 2-methylpropane-1,2-diamine were added and the mixture was stirred at 150° C. in a closed vessel in the microwave for 3 h. Another 19 mg (0.22 mmol) of 2-methylpropane-1,2-diamine were added and the mixture was stirred at 150° C. in a closed vessel in the microwave for 3 h. 0.27 ml of NMP was added, and the mixture was stirred in the microwave at 180° C. for 5 h. The reaction solution was diluted with acetonitrile/water, TFA was added and the mixture was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated, dissolved in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by rotary evaporation. This gave 9 mg of the target compound (27% of theory).

[0751] LC-MS (Method 1): R.sub.t=0.64 min

[0752] MS (ESpos): m/z=470 (M+H).sup.+

[0753] .sup.1H-NMR (400 Mhz, DMSO-d.sub.6) δ=1.05 (s, 6H), 1.75 (br. s, 2H), 2.24-2.29 (m, 6H), 3.18 (d, 2H), 5.30 (s, 2H), 6.53 (t, 1H), 6.68 (d, 1H), 6.84 (s, 1H), 7.19-7.28 (m, 2H), 7.55-7.65 (m, 2H), 8.08 (s, 1H).

Example 11

N′-(4-Chloro-6-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-1,3,5-triazin-2-yl)-2-methylpropane-1,2-diamine

[0754] ##STR00072##

[0755] 25 mg (0.058 mmol) of 3-(4,6-dichloro-1,3,5-triazin-2-yl)-8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridine from Example 35A were initially charged in 0.22 ml of NMP. At room temperature, 5.2 mg (0.058 mmol) of 2-methylpropane-1,2-diamine were added and the mixture was stirred at RT in a closed vessel for 1.5 h. The reaction solution was diluted with acetonitrile/water, TFA was added and the mixture was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated, dissolved in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated by rotary evaporation. This gave 15 mg of the target compound (55% of theory).

[0756] LC-MS (Method 1): R.sub.t=0.70 min

[0757] MS (ESpos): m/z=474 (M+H).sup.+

[0758] .sup.1H-NMR (400 Mhz, DMSO-d.sub.6) δ=1.07 (s, 6H), 1.69 (br. s, 2H), 2.73-2.77 (m, 3H), 3.23-3.40 (m, 2H; superposed by solvent peak), 5.33 (s, 2H), 7.05-7.28 (m, 4H), 7.55-7.64 (m, 1H), 8.48 (br. s, 1H), 9.48 and 9.68 (each d, together 1H).

Example 12

ent-N.SUP.1.-(4-{8-[(2,6-Difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-1,3,5-triazin-2-yl)-5,5,5-trifluoro-2-methylpentane-1,2-diamine (enantiomer B)

[0759] ##STR00073##

[0760] 54 mg (0.07 mmol) of ent-benzyl {1-[(4-chloro-6-{8-[(2,6-difluorobenzyl)oxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-1,3,5-triazin-2-yl)amino]-5,5,5-trifluoro-2-methylpentan-2-yl}carbamate trifluoroacetate (enantiomer B) from Example 36A were dissolved in 1.7 ml of ethanol, and 5 μl (0.07 mmol) of trifluoroacetic acid were added. Under argon, 2 mg of palladium/activated carbon (10%) were added. The reaction mixture was hydrogenated at RT under standard pressure for 4.5 h. The reaction mixture was filtered through a Millipore filter, washed well with ethanol and concentrated using a rotary evaporator. The residue was dissolved again in 1.7 ml of ethanol, 5 μl (0.07 mmol) of trifluoroacetic acid and 2 mg of palladium/activated carbon (10%) were added under argon and the mixture was hydrogenated at standard pressure for 1.5 min. The reaction mixture was filtered through a Millipore filter, washed well with ethanol and concentrated using a rotary evaporator. The residue was taken up in acetonitrile, water/TFA was added and the mixture was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were combined and concentrated on a rotary evaporator. The residue was dissolved in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated. This gave 19 mg of the target compound (54% of theory).

[0761] LC-MS (Method 1): R.sub.t=0.68 min

[0762] MS (ESpos): m/z=522 (M+H).sup.+

[0763] .sup.1H-NMR (400 Mhz, DMSO-d.sub.6) δ=1.01 (s, 3H), 1.48-1.69 (m, 4H), 2.21-2.48 (m, 2H), 2.77 (s, 3H), 3.26-3.42 (m, 2H; superposed by solvent peak), 5.33 (s, 2H), 7.00-7.09 (m, 1H), 7.10-7.16 (m, 1H), 7.19-7.28 (m, 2H), 7.55-7.64 (m, 1H), 7.96-8.08 (m, 1H), 8.58 (d, 1H), 9.61 and 9.76 (each d, together 1H).

Example 13

8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethyl-3-[2-(piperazin-1-yl)pyridin-4-yl]imidazo[1,2-a]pyridine

[0764] ##STR00074##

[0765] Under argon, 71 mg (0.25 mmol) of 1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl]piperazine, 104 mg (0.49 mmol) of potassium phosphate and 8 mg (0.016 mmol) of bis(tri-tert-butylphosphine)palladium(0) were added in succession to 60 mg (0.16 mmol) of 3-bromo-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridine (Example 21A) in a mixture of 1.2 ml of ethanol, 0.6 ml of water and 0.6 ml of toluene. The suspension was degassed with argon and then stirred at 120° C. for 40 min. After the reaction had ended, the reaction mixture was concentrated and the residue was taken up in ethyl acetate/water and extracted. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, concentrated on a rotary evaporator and dried under high vacuum. The residue was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were combined and concentrated on a rotary evaporator. The residue was dissolved in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated. This gave 31 mg of the target compound (41% of theory).

[0766] LC-MS (Method 1): R.sub.t=0.54 min

[0767] MS (ESpos): m/z=450 (M+H).sup.+

Example 14

8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethyl-3-[3-(pyrrolidin-1-yl)phenyl]imidazo[1,2-a]pyridine

[0768] ##STR00075##

[0769] Under argon, 78 mg (0.41 mmol) of [3-(pyrrolidin-1-yl)phenyl]boric acid, 104 mg (0.49 mmol) of potassium phosphate and 8 mg (0.016 mmol) of bis(tri-tert-butylphosphine)palladium(0) were added in succession to 60 mg (0.16 mmol) of 3-bromo-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridine (Example 21A) in a mixture of 1.2 ml of ethanol, 0.6 ml of water and 0.6 ml of toluene. The suspension was degassed with argon and then stirred at 120° C. for 30 min. After the reaction had ended, the reaction mixture was concentrated and the residue was taken up in ethyl acetate/water and extracted. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, concentrated on a rotary evaporator and dried under high vacuum. The residue was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were combined and concentrated on a rotary evaporator. The residue was dissolved in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated. This gave 32 mg of the target compound (44% of theory).

[0770] LC-MS (Method 1): R.sub.t=1.00 min

[0771] MS (ESpos): m/z=434 (M+H).sup.+

[0772] .sup.1H-NMR (500 Mhz, DMSO-d.sub.6) δ=1.93-2.02 (m, 4H), 2.24 (s, 3H), 2.37 (s, 3H), 3.23-3.32 (m, 4H; superposed by solvent peak), 5.29 (s, 2H), 6.54 (s, 1H), 6.62 (d, 1H), 6.66 (d, 1H), 6.72 (s, 1H), 7.19-7.28 (m, 2H), 7.32 (t, 1H), 7.55-7.63 (m, 1H), 7.71 (s, 1H).

Example 15

N-(3-{8-[(2,6-Difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridin-3-yl}phenyl)acetamide

[0773] ##STR00076##

[0774] Under argon, 44 mg (0.25 mmol) of (3-acetamidophenyl)boric acid, 104 mg (0.49 mmol) of potassium phosphate and 8 mg (0.016 mmol) of bis(tri-tert-butylphosphine)palladium(0) were added in succession to 60 mg (0.16 mmol) of 3-bromo-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[1,2-a]pyridine (Example 21A) in a mixture of 1.2 ml of ethanol, 0.6 ml of water and 0.6 ml of toluene. The suspension was degassed with argon and then stirred at 120° C. for 30 min. After the reaction had ended, the reaction mixture was concentrated and the residue was taken up in ethyl acetate/water and extracted. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, concentrated on a rotary evaporator and dried under high vacuum. The residue was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were combined and concentrated on a rotary evaporator. The residue was dissolved in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated. This gave 35 mg of the target compound (48% of theory).

[0775] LC-MS (Method 1): R.sub.t=0.76 min

[0776] MS (ESpos): m/z=422 (M+H).sup.+

Example 16

2,6-Dimethyl-3-[4-(pyrrolidin-1-yl)pyrimidin-2-yl]-8-[(2,3,6-trifluorobenzyl)oxy]imidazo[1,2-a]pyridine trifluoroacetate

[0777] ##STR00077##

[0778] 80 mg (0.26 mmol) of 2,6-dimethyl-8-[(2,3,6-trifluorobenzyl)oxy]imidazo[1,2-a]pyridine (Example 22A), 39 mg (0.21 mmol) of 2-chloro-4-(pyrrolidin-1-yl)pyrimidine and 26 mg (0.26 mmol) of potassium acetate were initially charged in 0.5 ml of NMP. Argon was passed through the reaction mixture for 5 min. 15 mg (0.01 mmol) of tetrakis(triphenylphosphine)palladium(0) were then added, and the mixture was stirred in the microwave at 150° C. for 12 h. The mixture was once more gassed with argon, 15 mg (0.01 mmol) of tetrakis(triphenylphosphine)palladium(0) were added and the mixture was stirred in the microwave at 150° C. for 4.5 h. The reaction mixture was cooled, water/TFA was added and the product was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). This gave 5.5 mg of the target compound (3.4% of theory, purity 92%).

[0779] LC-MS (Method 1): R.sub.t=0.98 min

[0780] MS (ESpos): m/z=454 (M+H).sup.+

Example 17

2-{2,6-Dimethyl-8-[(2,3,6-trifluorobenzyl)oxy]imidazo[1,2-a]pyridin-3-yl}-N,N-dimethylpyrimidine-4-amine

[0781] ##STR00078##

[0782] 140 mg (0.46 mmol) of 2,6-dimethyl-8-[(2,3,6-trifluorobenzyl)oxy]imidazo[1,2-a]pyridine (Example 22A), 58 mg (0.37 mmol) of 2-chloro-N,N-dimethylpyrimidine-4-amine and 45 mg (0.46 mmol) of potassium acetate were initially charged in 0.7 ml of NMP. Argon was passed through the reaction mixture for 5 min. 26 mg (0.02 mmol) of tetrakis(triphenylphosphine)palladium(0) were then added, and the mixture was stirred in the microwave at 150° C. for 12 h. The reaction mixture was cooled, water/TFA was added and the product was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated and purified by silica gel chromatography (mobile phase: dichloromethane/methanol gradient). The product fractions were concentrated and re-purified by thick-layer chromatography (mobile phase: dichloromethane/methanol/ethyl acetate=10/1/2). This gave 1 mg of the target compound (0.3% of theory, purity 55%).

[0783] LC-MS (Method 1): R.sub.t=0.75 min

[0784] MS (ESpos): m/z=428 (M+H).sup.+

Example 18

N-Benzyl-2-{2,6-dimethyl-8-[(2,3,6-trifluorobenzyl)oxy]imidazo[1,2-a]pyridin-3-yl}pyrimidine-4-amine

[0785] ##STR00079##

[0786] 100 mg (0.33 mmol) of 2,6-dimethyl-8-[(2,3,6-trifluorobenzyl)oxy]imidazo[1,2-a]pyridine (Example 22A), 57 mg (0.26 mmol) of N-benzyl-2-chloropyrimidine-4-amine and 32 mg (0.33 mmol) of potassium acetate were initially charged in 0.95 ml of NMP. Argon was passed through the reaction mixture for 5 min. 38 mg (0.03 mmol) of tetrakis(triphenylphosphine)palladium(0) were then added, and the mixture was stirred in the microwave at 150° C. for 8 h. The reaction mixture was cooled, acetonitrile/water/TFA was added and the product was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated and purified by thick-layer chromatography (mobile phase: dichioromethane/methanol/ethyl acetate=10/1/2). The product fractions were concentrated and re-purified by preparative HPLC (Kinetix, 5μ, C18 column, mobile phase: acetonitrile/water (50/50) with addition of 0.2% TFA). This gave 1.5 mg of the target compound (0.6% of theory, purity 60%).

[0787] LC-MS (Method 1): R.sub.t=0.92 min

[0788] MS (ESpos): m/z=490 (M+H).sup.+

Example 19

N-{3-[8-(Cyclohexylmethoxy)-2-methylimidazo[1,2-a]pyridin-3-yl]phenyl}acetamide

[0789] ##STR00080##

[0790] Under argon, 46 mg (0.26 mmol) of (3-acetamidophenyl)boric acid, 110 mg (0.52 mmol) of potassium phosphate and 9 mg (0.017 mmol) of bis(tri-tert-butylphosphine)palladium(0) were added in succession to 60 mg (0.17 mmol; purity 93%) of 3-bromo-8-(cyclohexylmethoxy)-2-methylimidazo[1,2-a]pyridine (Example 37A) in a mixture of 1.2 ml of ethanol, 0.6 ml of water and 0.6 ml of toluene. The suspension was degassed with argon and then stirred at 120° C. for 30 min. After the reaction had ended, the reaction mixture was concentrated and the residue was taken up in ethyl acetate/water and extracted. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, concentrated on a rotary evaporator and dried under high vacuum. The residue was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were combined and concentrated on a rotary evaporator. The residue was dissolved in dichloromethane and a little methanol and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated. The product fractions were purified once more by thick-layer chromatography (mobile phase: dichloromethane/methanol=15/1). This gave 34 mg of the target compound (51% of theory).

[0791] LC-MS (Method 1): R.sub.t=0.78 min

[0792] MS (ESpos): m/z=378 (M+H).sup.+

Example 20

N-(3-{6-Chloro-8-[(3-fluoropyridin-2-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}phenyl)acetamide

[0793] ##STR00081##

[0794] Under argon, 17 mg (0.09 mmol) of (3-acetamidophenyl)boric acid, 65 mg (0.31 mmol) of potassium phosphate and 5.2 mg (0.01 mmol) of bis(tri-tert-butylphosphine)palladium(0) were added in succession to 40 mg (0.10 mmol) of 3-bromo-6-chloro-8-[(3-fluoropyridin-2-yl)methoxy]-2-methylimidazo[1,2-a]pyridine (Example 43A) in a mixture of 0.75 ml of ethanol, 0.37 ml of water and 0.37 ml of toluene. The suspension was degassed with argon and then stirred at 100° C. for 30 min. After the reaction had ended, the reaction mixture was taken up in ethyl acetate/water and extracted. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and concentrated by rotary evaporation. The residue was purified by preparative HPLC (RP18 column, mobile phase: acetonitrile/water gradient with addition of 0.1% TFA). The product fractions were concentrated and purified by thick-layer chromatography (mobile phase: dichloromethane/methanol=10/1). The product fractions were dissolved in dichloromethane and washed twice with saturated aqueous sodium bicarbonate solution. The combined aqueous phases were extracted twice with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered and concentrated. This gave 3 mg of the target compound (6% of theory).

[0795] LC-MS (Method 1): R.sub.t=0.71 min

[0796] MS (ESpos): m/z=425 (M+H).sup.+

B. ASSESSMENT OF PHARMACOLOGICAL EFFICACY

[0797] The following abbreviations are used: [0798] ATP adenosine triphosphate [0799] Brij 35 polyoxyethylene(23) lauryl ether [0800] BSA bovine serum albumin [0801] DTT dithiothreitol [0802] TEA triethanolamine

[0803] The pharmacological action of the compounds of the invention can be demonstrated in the following assays:

B-1. Measurement of sGC Enzyme Activity by Means of PPi Detection

[0804] Soluble guanylyl cyclase (sGC) converts GTP to cGMP and pyrophosphate (PPi) when stimulated. PPi is detected with the aid of the method described in WO 2008/061626. The signal that arises in the assay increases as the reaction progresses and serves as a measure of the sGC enzyme activity. With the aid of a PPi reference curve, the enzyme can be characterized in a known manner, for example in terms of conversion rate, stimulability or Michaelis constant.

Conduct of the Test

[0805] To conduct the test, 29 μl of enzyme solution (0-10 nM soluble guanylyl cyclase (prepared according to Honicka et al., Journal of Molecular Medicine 77 (1999) 14-23), in 50 mM TEA, 2 mM magnesium chloride, 0.1% BSA (fraction V), 0.005% Brij 35, pH 7.5) were initially charged in the microplate, and 1 μl of the stimulator solution (0-10 μM 3-morpholinosydnonimine, SIN-1, Merck in DMSO) was added. The microplate was incubated at RT for 10 min. Then 20 μl of detection mix (1.2 nM Firefly Luciferase (Photinus pyralis luciferase, Promega), 29 μM dehydroluciferin (prepared according to Bitler & McElroy, Arch. Biochem. Biophys. 72 (1957) 358), 122 μM luciferin (Promega), 153 μM ATP (Sigma) and 0.4 mM DTT (Sigma) in 50 mM TEA, 2 mM magnesium chloride, 0.1% BSA (fraction V), 0.005% Brij 35, pH 7.5) were added. The enzyme reaction was started by adding 20 μl of substrate solution (1.25 mM guanosine 5′-triphosphate (Sigma) in 50 mM TEA, 2 mM magnesium chloride, 0.1% BSA (fraction V), 0.005% Brij 35, pH 7.5) and analysed continuously in a luminometer.

B-2. Effect on a Recombinant Guanylate Cyclase Reporter Cell Line

[0806] The cellular activity of the compounds according to the invention is determined using a recombinant guanylate cyclase reporter cell line, as described in F. Wunder et al., Anal. Biochem. 339, 104-112 (2005).

[0807] Representative MEC values (MEC=minimum effective concentration) for the compounds of the invention are shown in the table below (in some cases as mean values from individual determinations):

TABLE-US-00002 TABLE A Example No. MEC [μM] 1 0.1 2 0.1 3 0.2 4 1.0 5 3.0 6 10 7 0.3 8 0.3 9 0.3 10 0.1 11 0.3 12 1.0 13 0.2 14 6.5 15 0.02 16 3.0 19 0.2 20 0.3

B-3. Vasorelaxant Effect In Vitro

[0808] Rabbits are stunned by a blow to the neck and exsanguinated. The aorta is removed, freed from adhering tissue and divided into rings of width 1.5 mm, which are placed individually under prestress into 5 ml organ baths with carbogen-sparged Krebs-Henseleit solution at 37° C. having the following composition (each in mM): sodium chloride: 119; potassium chloride: 4.8; calcium chloride dihydrate: 1; magnesium sulfate heptahydrate: 1.4; potassium dihydrogenphosphate: 1.2; sodium bicarbonate: 25; glucose: 10. The contractile force is determined with Statham UC2 cells, amplified and digitalized using A/D transducers (DAS-1802 HC, Keithley Instruments Munich), and recorded in parallel on linear recorders. To generate a contraction, phenylephrine is added to the bath cumulatively in increasing concentration. After several control cycles, the substance to be studied is added in increasing dosage each time in every further run, and the magnitude of the contraction is compared with the magnitude of the contraction attained in the last preceding run. This is used to calculate the concentration needed to reduce the magnitude of the control value by 50% (IC.sub.50 value). The standard administration volume is 5 μl; the DMSO content in the bath solution corresponds to 0.1%.

B-4. Blood Pressure Measurement on Anaesthetized Rats

[0809] Male Wistar rats having a body weight of 300-350 g are anaesthetized with thiopental (100 mg/kg i.p.). After tracheotomy, a catheter is introduced into the femoral artery to measure the blood pressure. The substances to be tested are administered as solutions, either orally by means of a gavage or intravenously via the femoral vein (Stasch et al. Br. J. Pharmacol. 2002; 135: 344-355).

B-5. Radiotelemetry Measurement of Blood Pressure in Conscious, Spontaneously Hypertensive Rats

[0810] A commercially available telemetry system from DATA SCIENCES INTERNATIONAL DSI, USA, is employed for the blood pressure measurement on conscious rats described below.

[0811] The system consists of 3 main components:

implantable transmitters (Physiotel® telemetry transmitter)
receivers (Physiotel® receiver) which are linked via a multiplexer (DSI Data Exchange Matrix) to a
data acquisition computer.

[0812] The telemetry system makes it possible to continuously record blood pressure, heart rate and body motion of conscious animals in their usual habitat.

Animal Material

[0813] The studies are conducted on adult female spontaneously hypertensive rats (SHR Okamoto) with a body weight of >200 g. SHR/NCrl from the Okamoto Kyoto School of Medicine, 1963, were a cross of male Wistar Kyoto rats having greatly elevated blood pressure and female rats having slightly elevated blood pressure, and were handed over at F13 to the U.S. National Institutes of Health.

[0814] After transmitter implantation, the experimental animals are housed singly in type 3 Makrolon cages. They have free access to standard feed and water.

[0815] The day/night rhythm in the experimental laboratory is changed by the room lighting at 6:00 am and at 7:00 pm.

Transmitter Implantation

[0816] The TA11 PA-C40 telemetry transmitters used are surgically implanted under aseptic conditions in the experimental animals at least 14 days before the first experimental use. The animals instrumented in this way can be used repeatedly after the wound has healed and the implant has settled.

[0817] For the implantation, the fasted animals are anaesthetized with pentobarbital (Nembutal, Sanofi: 50 mg/kg i.p.) and shaved and disinfected over a large area of their abdomens. After the abdominal cavity has been opened along the linea alba, the liquid-filled measuring catheter of the system is inserted into the descending aorta in the cranial direction above the bifurcation and fixed with tissue glue (VetBonD™, 3M). The transmitter housing is fixed intraperitoneally to the abdominal wall muscle, and the wound is closed layer by layer.

[0818] An antibiotic (Tardomyocel COMP, Bayer, 1 ml/kg s.c.) is administered postoperatively for prophylaxis of infection.

Substances and Solutions

[0819] Unless stated otherwise, the substances to be studied are administered orally by gavage to a group of animals in each case (n=6). In accordance with an administration volume of 5 ml/kg of body weight, the test substances are dissolved in suitable solvent mixtures or suspended in 0.5% tylose.

[0820] A solvent-treated group of animals is used as control.

Experimental Procedure

[0821] The telemetry measuring unit present is configured for 24 animals. Each experiment is recorded under an experiment number (Vyear month day).

[0822] Each of the instrumented rats living in the system is assigned a separate receiving antenna (1010 Receiver, DSI).

[0823] The implanted transmitters can be activated externally by means of an incorporated magnetic switch. They are switched to transmission in the run-up to the experiment. The signals emitted can be detected online by a data acquisition system (Dataquest™ A.R.T. for WINDOWS, DSI) and processed accordingly. The data are stored in each case in a file created for this purpose and bearing the experiment number.

[0824] In the standard procedure, the following are measured for 10-second periods in each case:

systolic blood pressure (SBP)
diastolic blood pressure (DBP)
mean arterial pressure (MAP)
heart rate (HR)
activity (ACT).

[0825] The acquisition of measurements is repeated under computer control at 5-minute intervals. The source data obtained as absolute values are corrected in the diagram with the currently measured barometric pressure (Ambient Pressure Reference Monitor; APR-1) and stored as individual data. Further technical details are given in the extensive documentation from the manufacturer company (DSI).

[0826] Unless indicated otherwise, the test substances are administered at 9:00 am on the day of the experiment. Following the administration, the parameters described above are measured over 24 hours.

Evaluation

[0827] After the end of the experiment, the acquired individual data are sorted using the analysis software (DATAQUEST™ A.R.T.™ ANALYSIS). The blank value is assumed here to be the time 2 hours before administration, and so the selected data set encompasses the period from 7:00 am on the day of the experiment to 9:00 am on the following day.

[0828] The data are smoothed over a predefinable period by determination of the average (15-minute average) and transferred as a text file to a storage medium. The measured values presorted and compressed in this way are transferred to Excel templates and tabulated. For each day of the experiment, the data obtained are stored in a dedicated file bearing the number of the experiment. Results and test protocols are stored in files in paper form sorted by numbers.

LITERATURE

[0829] Klaus Witte, Kai Hu, Johanna Swiatek, Claudia Miissig, Georg Ertl and Björn Lemmer: Experimental heart failure in rats: effects on cardiovascular circadian rhythms and on myocardial β-adrenergic signaling. Cardiovasc Res 47 (2): 203-405, 2000; Kozo Okamoto: Spontaneous hypertension in rats. Int Rev Exp Pathol 7: 227-270, 1969; Maarten van den Buuse: Circadian Rhythms of Blood Pressure, Heart Rate, and Locomotor Activity in Spontaneously Hypertensive Rats as Measured With Radio-Telemetry. Physiology & Behavior 55(4): 783-787, 1994.

B-6. Determination of Pharmacokinetic Parameters Following Intravenous and Oral Administration

[0830] The pharmacokinetic parameters of the compounds according to the invention are determined in male CD-1 mice, male Wistar rats and female beagles. Intravenous administration in the case of mice and rats is carried out by means of a species-specific plasma/DMSO formulation, and in the case of dogs by means of a water/PEG400/ethanol formulation. In all species, oral administration of the dissolved substance is performed via gavage, based on a water/PEG400/ethanol formulation. The removal of blood from rats is simplified by inserting a silicone catheter into the right Vena jugularis externa prior to substance administration. The operation is carried out at least one day prior to the experiment with isofluran anaesthesia and administration of an analgesic (atropine/rimadyl (3/1) 0.1 ml s.c.). The blood is taken (generally more than 10 time points) within a time window including terminal time points of at least 24 to a maximum of 72 hours after substance administration. The blood is removed into heparinized tubes. The blood plasma is then obtained by centrifugation; if required, it is stored at −20° C. until further processing.

[0831] An internal standard (which may also be a chemically unrelated substance) is added to the samples of the compounds of the invention, calibration samples and qualifiers, and there follows protein precipitation by means of acetonitrile in excess. Addition of a buffer solution matched to the LC conditions, and subsequent vortexing, is followed by centrifugation at 1000 g. The supernatant is analysed by LC-MS/MS using C18 reversed-phase columns and variable mobile phase mixtures. The substances are quantified via the peak heights or areas from extracted ion chromatograms of specific selected ion monitoring experiments.

[0832] The plasma concentration/time plots determined are used to calculate the pharmacokinetic parameters such as AUC, C.sub.max, t.sub.1/2 (terminal half-life), F (bioavailability), MRT (mean residence time) and CL (clearance), by means of a validated pharmacokinetic calculation program.

[0833] Since the substance quantification is performed in plasma, it is necessary to determine the blood/plasma distribution of the substance in order to be able to adjust the pharmacokinetic parameters correspondingly. For this purpose, a defined amount of substance is incubated in heparinized whole blood of the species in question in a rocking roller mixer for 20 min. After centrifugation at 1000 g, the plasma concentration is measured (by means of LC-MS/MS; see above) and determined by calculating the ratio of the C.sub.blood/C.sub.plasma value.

B-7. Metabolic Study

[0834] To determine the metabolic profile of the inventive compounds, they are incubated with recombinant human cytochrome P450 (CYP) enzymes, liver microsomes or primary fresh hepatocytes from various animal species (e.g. rats, dogs), and also of human origin, in order to obtain and to compare information about a very substantially complete hepatic phase I and phase II metabolism, and about the enzymes involved in the metabolism. The compounds of the invention were incubated with a concentration of about 0.1-10 μM. To this end, stock solutions of the compounds of the invention having a concentration of 0.01-1 mM in acetonitrile were prepared, and then pipetted with a 1:100 dilution into the incubation mixture. The liver microsomes and recombinant enzymes were incubated at 37° C. in 50 mM potassium phosphate buffer pH 7.4 with and without NADPH-generating system consisting of 1 mM NADP.sup.+, 10 mM glucose-6-phosphate and 1 unit glucose-6-phosphate dehydrogenase. Primary hepatocytes were incubated in suspension in Williams E medium, likewise at 37° C. After an incubation time of 0-4 h, the incubation mixtures were stopped with acetonitrile (final concentration about 30%) and the protein was centrifuged off at about 15 000×g. The samples thus stopped were either analyzed directly or stored at −20° C. until analysis.

[0835] The analysis is carried out by high-performance liquid chromatography with ultraviolet and mass spectrometry detection (HPLC-UV-MS/MS). To this end, the supernatants of the incubation samples are chromatographed with suitable C18 reversed-phase columns and variable mobile phase mixtures of acetonitrile and 10 mM aqueous ammonium formate solution or 0.05% formic acid. The UV chromatograms in conjunction with mass spectrometry data serve for identification, structural elucidation and quantitative estimation of the metabolites, and for quantitative metabolic reduction of the compound of the invention in the incubation mixtures.

B-8. Caco-2 Permeability Test

[0836] The permeability of a test substance was determined with the aid of the Caco-2 cell line, an established in vitro model for permeability prediction at the gastrointestinal barrier (Artursson, P. and Karlsson, J. (1991). Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. 175 (3), 880-885). The Caco-2 cells (ACC No. 169, DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were sown in 24-well plates having an insert and cultivated for 14 to 16 days. For the permeability studies, the test substance was dissolved in DMSO and diluted to the final test concentration with transport buffer (Hanks Buffered Salt Solution, Gibco/Invitrogen, with 19.9 mM glucose and 9.8 mM HEPES). In order to determine the apical to basolateral permeability (P.sub.appA-B) of the test substance, the solution comprising the test substance was applied to the apical side of the Caco-2 cell monolayer, and transport buffer to the basolateral side. In order to determine the basolateral to apical permeability (P.sub.appB-A) of the test substance, the solution comprising the test substance was applied to the basolateral side of the Caco-2 cell monolayer, and transport buffer to the apical side. At the start of the experiment, samples were taken from the respective donor compartment in order to ensure the mass balance. After an incubation time of two hours at 37° C., samples were taken from the two compartments. The samples were analyzed by means of LC-MS/MS and the apparent permeability coefficients (P.sub.app) were calculated. For each cell monolayer, the permeability of Lucifer Yellow was determined to ensure cell layer integrity. In each test run, the permeability of atenolol (marker for low permeability) and sulfasalazine (marker for active excretion) was also determined as quality control.

B-9. hERG Potassium Current Assay

[0837] The hERG (human ether-a-go-go related gene) potassium current makes a significant contribution to the repolarization of the human cardiac action potential (Scheel et al., 2011). Inhibition of this current by pharmaceuticals can in rare cases cause potentially lethal cardiac arrhythmias, and is therefore studied at an early stage during drug development.

[0838] The functional hERG assay used here is based on a recombinant HEK293 cell line which stably expresses the KCNH2(HERG) gene (Zhou et al., 1998). These cells are studied by means of the “whole-cell voltage-clamp” technique (Hamill et al., 1981) in an automated system (Patchliner™; Nanion, Munich, Germany), which controls the membrane voltage and measures the hERG potassium current at room temperature. The PatchControlHT™ software (Nanion) controls the Patchliner system, data capture and data analysis. The voltage is controlled by 2 EPC-10 quadro amplifiers controlled by the PatchMasterPro™ software (both: HEKA Elektronik, Lambrecht, Germany). NPC-16 chips with moderate resistance (˜2 MΩ; Nanion) serve as the planar substrate for the voltage clamp experiments.

[0839] NPC-16 chips are filled with intra- and extracellular solution (cf. Himmel, 2007) and with cell suspension. After forming a gigaohm seal and establishing whole-cell mode (including several automated quality control steps), the cell membrane is clamped at the −80 mV holding potential. The subsequent voltage clamp protocol changes the command voltage to +20 mV (for 1000 ms), −120 mV (for 500 ms), and back to the −80 mV holding potential; this is repeated every 12 s. After an initial stabilization phase (about 5-6 minutes), test substance solution is introduced by pipette in rising concentrations (e.g. 0.1, 1, and 10 mol/1) (exposure about 5-6 minutes per concentration), followed by several washing steps.

[0840] The amplitude of the inward “tail” current which is generated by a change in potential from +20 mV to −120 mV serves to quantify the hERG potassium current, and is described as a function of time (IgorPro™ Software). The current amplitude at the end of various time intervals (for example stabilization phase before test substance, first/second/third concentration of test substance) serves to establish a concentration/effect curve, from which the half-maximum inhibiting concentration IC.sub.50 of the test substance is calculated. [0841] Hamill O P, Marty A, Neher E, Sakmann B, Sigworth F J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 1981; 391:85-100. [0842] Himmel H M. Suitability of commonly used excipients for electrophysiological in-vitro safety pharmacology assessment of effects on hERG potassium current and on rabbit Purkinje fiber action potential. J Phannacol Toxicol Methods 2007; 56:145-158. [0843] Scheel O, Himmel H, Rascher-Eggstein G, Knott T. Introduction of a modular automated voltage-clamp platform and its correlation with manual human ether-a-go-go related gene voltage-clamp data. Assay Drug Dev Technol 2011; 9:600-607. [0844] Zhou Z F, Gong Q, Ye B, Fan Z, Makielski J C, Robertson G A, January C T. Properties of hERG channels stably expressed in HEK293 cells studied at physiological temperature. Biophys J 1998; 74:230-241.

C. WORKING EXAMPLES OF PHARMACEUTICAL COMPOSITIONS

[0845] The compounds of the invention can be converted to pharmaceutical preparations as follows:

Tablet:

Composition:

[0846] 100 mg of the compound of the invention, 50 mg of lactose (monohydrate), 50 mg of corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25) (BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.

[0847] Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.

Production:

[0848] The mixture of compound of the invention, lactose and starch is granulated with a 5% solution (w/w) of the PVP in water. The granules are dried and then mixed with the magnesium stearate for 5 minutes. This mixture is compressed using a conventional tableting press (see above for format of the tablet). The guide value used for the pressing is a pressing force of 15 kN.

Suspension for Oral Administration:

Composition:

[0849] 1000 mg of the compound of the invention, 1000 mg of ethanol (96%), 400 mg of Rhodigel® (xanthan gum from FMC, Pennsylvania, USA) and 99 g of water.

[0850] 10 ml of oral suspension correspond to a single dose of 100 mg of the compound of the invention.

Production:

[0851] The Rhodigel is suspended in ethanol; the compound of the invention is added to the suspension. The water is added while stirring. The mixture is stirred for about 6 h until the swelling of the Rhodigel is complete.

Solution for Oral Administration:

Composition:

[0852] 500 mg of the compound of the invention, 2.5 g of polysorbate and 97 g of polyethylene glycol 400. 20 g of oral solution correspond to a single dose of 100 mg of the compound of the invention.

Production:

[0853] The compound of the invention is suspended in the mixture of polyethylene glycol and polysorbate with stirring. The stirring operation is continued until dissolution of the compound of the invention is complete.

i.v. solution:

[0854] The compound of the invention is dissolved in a concentration below the saturation solubility in a physiologically acceptable solvent (e.g. isotonic saline solution, glucose solution 5% and/or PEG 400 solution 30%). The resulting solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection vessels.