NOVEL ARYL-CYANOGUANIDINE COMPOUNDS

Abstract

The present invention relates to protein-lysine N-methyltransferase SMYD2 (SET and MYND domain-containing protein 2) inhibitors, in particular SMYD2-inhibitory substituted cyanoguanidine-pyrazolines of general formula (I) wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, X and r have the meaning as described and defined herein, as well as to pharmaceutical compositions comprising compounds according to the invention and to their prophylactic and therapeutic use for hyper-proliferative disorders, in particular for cancer, respectively tumour disorders. The present invention furthermore relates to the use of SMYD2 inhibitors for benign hyperplasias, atherosclerotic disorders, sepsis, autoimmune disorders, vascular disorders, viral infections, neurodegenerative disorders, inflammatory disorders, atherosclerotic disorders and the control of male fertility.

##STR00001##

Claims

1: Compounds of general formula (I) ##STR00161## in which R.sup.1 represents a C.sub.1-C.sub.6-alkyl group, which is substituted with one substituent selected from —OH, —NH.sub.2 or —NHCH.sub.3, R.sup.2 represents a hydrogen atom, a methyl or an ethyl group, R.sup.3 represents a fluorine or a chlorine atom or a methyl group, R.sup.4 represents a group selected from: —CF.sub.3, —CH.sub.2CF.sub.3, —OCH.sub.3, —OCHF.sub.2, —OCF.sub.3, —OCH.sub.2CF.sub.3 or —OCH.sub.2CH.sub.2N(CH.sub.3).sub.2, R.sup.5 represents a fluorine or a chlorine atom or a group selected from: —OCH.sub.3, —OCF.sub.3, ##STR00162## X represents CH or N and r represents 0 or 1, as well as their polymorphs, enantiomers, diastereomers, racemates, E/Z-isomers, tautomers, solvates, physiological acceptable salts and solvates of these salts.

2: Compounds of general formula (I) according to claim 1 in which R.sup.1 represents the group —CH.sub.2—OH, —CH(OH)—CH.sub.3, —C(CH.sub.3).sub.2—OH, —CH.sub.2—NH.sub.2, —CH.sub.2—NH—CH.sub.3, —CH(CH.sub.3)—NH.sub.2, —CH.sub.2—CH.sub.2—NH.sub.2, —CH.sub.2—CH.sub.2—CH.sub.2—NH.sub.2, —CH(NH—CH.sub.3)—CH.sub.3, —CH(CH(CH.sub.3).sub.2)—NH.sub.2, —C(CH.sub.3)(CH(CH.sub.3).sub.2)—NH.sub.2 or —CH—(CH.sub.2—CH(CH.sub.3).sub.2)—NH.sub.2 R.sup.2 represents a hydrogen atom, a methyl or an ethyl group, R.sup.3 represents a fluorine or a chlorine atom or a methyl group, R.sup.4 represents a group selected from: —CF.sub.3, —CH.sub.2CF.sub.3, —OCH.sub.3, —OCHF.sub.2, —OCF.sub.3, —OCH.sub.2CF.sub.3 or —OCH.sub.2CH.sub.2N(CH.sub.3).sub.2, R.sup.5 represents a fluorine or a chlorine atom or a group selected from: —OCH.sub.3, —OCF.sub.3, ##STR00163## X represents CH or N and r represents 0 or 1, as well as their polymorphs, enantiomers, diastereomers, racemates, E/Z-isomers, tautomers, solvates, physiological acceptable salts and solvates of these salts.

3: Compounds of general formula (I) according to claim 1: (2S)—N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxypropanamide (1:1 mixture of diastereomers); Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-N-methylacetamide; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylglycinamide; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-2-methylpropanamide; N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-2-methylpropanamide Isomer 1; N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-2-methylpropanamide Isomer 2; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-beta-alaninamide; N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-alaninamide (1:1 mixture of diastereomers); N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-alaninamide Isomer 1; N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-alaninamide Isomer 2; (2S)—N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxypropanamide (1:1 mixture of diastereomers); (2R)—N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxypropanamide (1:1 mixture of diastereomers); Rac-4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide; 4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide Isomer 1; 4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide Isomer 2; Rac-N-[1-{N-[4-chloro-3-(difluoromethoxy)phenyl]-N′-cyanocarbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N-[4-chloro-3-(difluoromethoxy)phenyl]-N′-cyanocarbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N-[4-chloro-3-(difluoromethoxy)phenyl]-N′-cyanocarbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[4-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-{1-[N′-cyano-N-(3-methoxyphenyl)carbamimidoyl]-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl}-N-ethyl-2-hydroxyacetamide; N-{1-[N′-cyano-N-(3-methoxyphenyl)carbamimidoyl]-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl}-N-ethyl-2-hydroxyacetamide Isomer 1; N-{1-[N′-cyano-N-(3-methoxyphenyl)carbamimidoyl]-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl}-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylglycinamide; Rac-N-[1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[3-(2,2,2-trifluoroethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[3-(2,2,2-trifluoroethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[3-(2,2,2-trifluoroethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)-4-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)-2-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[3-fluoro-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[3-fluoro-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[3-fluoro-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-{N′-cyano-N-[2-(pyrrolidin-1-ylmethyl)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[1-{N′-cyano-N-[2-(pyrrolidin-1-ylmethyl)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[1-{N′-cyano-N-[2-(pyrrolidin-1-ylmethyl)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; Rac-N-[1-{N′-cyano-N-[2-(trifluoromethoxy)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-(N′-cyano-N-{5-(difluoromethoxy)-2-[3-(dimethylamino)propoxy]phenyl}-carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-(N′-cyano-N-{2-[2-(pyrrolidin-1-yl)ethoxy]-5-(trifluoromethyl)phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-(N′-cyano-N-{2-[(1-methylpiperidin-4-yl)oxy]-5-(trifluoromethyl)phenyl}-carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[1-(N′-cyano-N-{2-[(1-methylpiperidin-4-yl)oxy]-5-(trifluoromethyl)phenyl}-carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]-carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-L-alaninamide (1:1 mixture of diastereomers); N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-L-alaninamide Isomer 1; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-L-alaninamide Isomer 2; Rac-N-[3-(4-chloro-3-fluorophenyl)-1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]-carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide; Rac-N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N.sup.2-methylglycinamide; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N.sup.2-methylglycinamide Isomer 1; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N.sup.2-methylglycinamide Isomer 2; N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N.sup.2-methyl-D-alaninamide; Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamide; N-[1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamide Isomer 1; N-[1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamide Isomer 2; N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-leucinamide; N-[1-{N′-Cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-valinamide and N-[1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-valinamide; as well as their polymorphs, enantiomers, diastereomers, racemates, E/Z isomers, tautomers, solvates, physiological acceptable salts and solvates of these salts.

4: A method for the prophylaxis or treatment of hyperproliferative disorders, the method comprising administering to a patient in need thereof, a pharmaceutically effective amount of a compound according to claim 1, or a polymorph, an enantiomer, a diastereomer, a racemate, an E/Z-isomer, a tautomer, a solvate, a physiological acceptable salt, or a solvate of a physiological acceptable salt thereof.

5: A method for the prophylaxis or treatment of cancer, respectively tumour disorders, the method comprising administering to a patient in need thereof, a pharmaceutically effective amount of a compound according to claim 1, or a polymorph, an enantiomer, a diastereomer, a racemate, an E/Z-isomer, a tautomer, a solvate, a physiological acceptable salt, or a solvate of a physiological acceptable salt thereof.

6: A method for the prophylaxis or treatment of a benign hyperplasia, an atherosclerotic disorder, sepsis, an autoimmune disorder, a vascular disorder, a viral infection, a neurodegenerative disorder, or an inflammatory disorder, the method comprising administering to a patient in need thereof, a pharmaceutically effective amount of a compound according to claim 1, or a polymorph, an enantiomer, a diastereomer, a racemate, an E/Z-isomer, a tautomer, a solvate, a physiological acceptable salt, or a solvate of a physiological acceptable salt thereof.

7-8. (canceled)

9: Compounds of general formula (I) according to claim 1 in combination together with one or more pharmaceutical active compounds.

10: A pharmaceutical formulation comprising a compound general formula (I) according to claim 1.

11: A method for controlling male fertility, the method comprising administering to a patient in need thereof, a pharmaceutically effective amount of a compound according to claim 1, or a polymorph, an enantiomer, a diastereomer, a racemate, an E/Z-isomer, a tautomer, a solvate, a physiological acceptable salt, or a solvate of a physiological acceptable salt thereof.

Description

SPECIFIC EXPERIMENTAL DESCRIPTIONS

[0445] NMR peak forms in the following specific experimental descriptions are stated as they appear in the spectra, possible higher order effects have not been considered. Reactions employing microwave irradiation may be run with a Biotage Initator® microwave oven optionally equipped with a robotic unit. The reported reaction times employing microwave heating are intended to be understood as fixed reaction times after reaching the indicated reaction temperature. The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. from Separtis such as Isolute® Flash silica gel or Isolute® Flash NH.sub.2 silica gel in combination with a Isolera® autopurifier (Biotage) and eluents such as gradients of e.g. hexane/ethyl acetate or DCM/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia. In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc) of a compound of the present invention as isolated as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity.

[0446] The percentage yields reported in the following examples are based on the starting component that was used in the lowest molar amount. Air and moisture sensitive liquids and solutions were transferred via syringe or cannula, and introduced into reaction vessels through rubber septa. Commercial grade reagents and solvents were used without further purification. The term “concentrated in vacuo” refers to use of a Buchi rotary evaporator at a minimum pressure of approximately 15 mm of Hg. All temperatures are reported uncorrected in degrees Celsius (° C.).

[0447] In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.

Flash Column Chromatography Conditions

[0448] “Purification by (flash) column chromatography” as stated in the subsequent specific experimental descriptions refers to the use of a Biotage Isolera purification system. For technical specifications see “Biotage product catalogue” on www.biotage.com.

Representation of Stereochemistry

[0449] All example structures have been synthesized as racemates or 1:1 mixtures of diastereomers, whereas one stereocenter is formed racemic during the synthesis and a second stereocenter is in some cases introduced by amide coupling with an enantiopure carboxylic acid. The racemic stereocenter is indicated as follows:

##STR00016##

[0450] After separation of the stereoisomers, the chiral center with an unknown absolute configuration is indicated as follows:

##STR00017##

[0451] In this case, the two different stereoisomers are specified by the terms Isomer 1 and Isomer 2.

[0452] The cyanoguanidine moiety can formally adopt E- or Z-configuration:

##STR00018##

[0453] It is assumed, that at relevant temperatures, the two isomers are present in a fast equilibrium, and cannot be analytically or preparatively distinguished, as similarly described for N,N,N′,N′-tetramethylcyanoguanidines (C. Gordon McCarty and Donald M. Wieland: Syn-Anti Isomerization Involving the N-Cyanoimino Group; Tetrahedron Letters No. 22, PP. 1787-1790, 1969). Therefore, any representation of the cyanoguanidine used herein represents both isomers.

EXPERIMENTAL SECTION

Methods:

Method 1:

Column: XBridge C18 IS 5 μm 2.1×30 mm

[0454] Eluents: A: 10 mM ammonium bicarbonate pH 10, B: MeCN
Gradient: 0-95% A in 3.10 min, hold @ 95% A to 3.9 min
Flow: 1 mL/min

Method 2:

Column: Acquity UPLC BEH C18 1.7 μm 50×2.1 mm

[0455] Eluents: A: H.sub.2O+0.2% Vol. NH.sub.3 (32%); B: acetonitrile

Gradient: 0-1.6 min 1-99% B; 1.6-2.0 min 99% B

[0456] Flow: 0.8 mL/min

Method 3:

Column: XBridge C18 2.5 μm 2.1×20 mm

[0457] Eluents: A: 10 mM ammonium bicarbonate pH 10; B: acetonitrile
Gradient: 0% B to 0.18 min, 0-95% B to 2.00 min, hold @ 95% B to 2.60 min
Flow: 1 mL/min

Method 4:

Column: Acquity BEH C18 1.7 μm 2.1×50 mm

[0458] Eluents: A: 0.05% aqueous formic acid; B: 0.05% formic acid in acetonitrile
Gradient: 30-80% B to 4.00 min, 80% 5.00 min, 80-50% B to 5.01 min
Flow: 0.4 mL/min

Method 5:

Column: Acquity UPLC BEH C18 1.7 μm 50×2.1 mm

[0459] Eluents: A: 0.1% aqueous formic acid; B: acetonitrile

Gradient: 0-1.6 min 1-99% B; 1.6-2.0 min 99% B

[0460] Flow: 0.8 mL/min

Method 6:

Column: XBridge BEH C18 2.5 μm 2.1×50 mm

[0461] Eluents: A: 10 mM ammonium bicarbonate pH 10; B: acetonitrile
Gradient: 2-98% B in 0.80 min, hold at 98% B to 1.30 min
Flow: 0.8 mL/min

Method 7:

Column: XBridge BEH C18 2.5 μm 2.1×50 mm

[0462] Eluents: A: 10 mM ammonium bicarbonate pH 10; B: acetonitrile
Gradient: 2-98% B in 4.00 min, hold @ 98% B to 4.70 min
Flow: 0.8 mL/min

Optical Rotation Values:

[0463] Instrument: JASCO P2000 Polarimeter; wavelength 589 nm; temperature: 20° C.; integration time 10 s; path length 100 mm.

INTERMEDIATES

Intermediate 1

2-Bromo-1-(3,4-dichlorophenyl)ethanone

[0464] ##STR00019##

[0465] The reaction was carried out twice on 135 g scale.

[0466] To a stirred solution of 3,4-dichloroacetophenone, 135 g (0.714 mol) in acetic acid (675 mL) cooled to 17° C. was added bromine, 37.0 mL (0.722 mol) in acetic acid (360 mL) dropwise. After approximately a third of the bromine had been added no reaction had occurred therefore the reaction mixture was warmed to 25° C. at which point an exotherm to 35° C. occurred. The remainder of the bromine was added and the reaction mixture stirred at room temperature for 30 minutes. The mixture was poured into ice water (1.5 L) while stirring vigorously. The precipitate was collected by filtration and the two batches combined and washed with water. The solid was triturated in diethyl ether (300 mL) to give the desired product 2-bromo-1-(3,4-dichlorophenyl)ethanone, 230 g. The filtrate was washed with brine, dried over magnesium sulfate and concentrated to give a brown oil. The oil was poured into ice/water (1 L) and stirred. The precipitate was collected by filtration to give a second batch of the desired product, 157 g, which were used directly without further purification.

[0467] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=4.95 (s, 2H), 7.81 (d, 1H), 7.91 (dd, 1H), 8.18 (d, 1H).

[0468] LC (method 1): R.sub.t 2.82 min

Intermediate 2

2-Bromo-1-(4-chloro-3-methylphenyl)ethanone

[0469] ##STR00020##

[0470] 1, 2-bromo-1-(4-chloro-3-methylphenyl)ethanone (intermediate 2) was prepared in analogy to intermediate 1, starting from 1-(4-chloro-3-methylphenyl)ethanone.

[0471] .sup.1H NMR (300 MHz, CDCl.sub.3): δ [ppm]=2.44 (s, 3H), 4.40 (s, 2H), 7.45 (d, 1H), 7.73 (dd, 1H), 7.85 (d, 1H).

[0472] LCMS (method 2): R.sub.t 1.28 min

Intermediate 3

2-Amino-1-(3,4-dichlorophenyl)ethanone hydrochloride (1:1)

[0473] ##STR00021##

[0474] To a stirred solution of 2-bromo-1-(3,4-dichlorophenyl)ethanone (Intermediate 1), 155 g (0.590 mol) in dichloromethane (600 mL) was added a suspension of hexamethylenetetramine, 113 g (0.810 mol) in dichloromethane (600 mL). The reaction mixture was stirred for 2 hours and the resulting precipitate was filtered and washed with dichloromethane (2×150 mL) before being re-suspended in ethanol (1 L). Concentrated hydrochloric acid (600 mL, 37 wt %) was added cautiously and resulted in dissolution of the suspension over 10 minutes. The reaction mixture was stirred for a further 2 hours after which time a precipitate formed, which was collected by filtration, washed with acetone (2×100 mL) and allowed to dry overnight to yield 2-amino-1-(3,4-dichlorophenyl)ethanone hydrochloride, 157 g as a white solid. Excess ammonium chloride was present therefore product was overweight.

[0475] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=4.57 (s, 2H), 7.84 (d, 1H), 7.94 (dd, 1H), 8.22 (d, 1H).

[0476] LC (method 1): R.sub.t 2.13 min

Intermediate 4

2-Amino-1-(4-chloro-3-methylphenyl)ethanone hydrochloride (1:1)

[0477] ##STR00022##

[0478] 2-amino-1-(4-chloro-3-methylphenyl)ethanone hydrochloride was prepared in analogy to intermediate 3, starting from intermediate 2.

[0479] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=2.39 (s, 3H), 4.52 (s, 2H), 7.61 (d, 1H), 7.82 (dd, 1H), 8.00 (dd, 1H).

[0480] LCMS (method 2): R.sub.t 0.93 min

[0481] MS (ESI): [M+H].sup.+=184.0

Intermediate 5

Allyl [2-(3,4-dichlorophenyl)-2-oxoethyl]carbamate

[0482] ##STR00023##

[0483] To a stirred solution of 2-Amino-1-(3,4-dichlorophenyl)ethanone hydrochloride (1:1) (intermediate 3), 116 g (0.480 mol) in water (500 mL) was added allyl chloroformate, 56.5 mL (0.530 mol) in dichloromethane (800 mL). The reaction mixture was cooled to 0° C. and potassium carbonate, 207 g (1.49 mol) in water (1 L) was added dropwise to the reaction mixture over 1 hour. The reaction mixture was allowed to warm to room temperature and was stirred overnight. The reaction mixture was diluted with dichloromethane (500 mL) and the organic phase was extracted and washed with saturated ammonium chloride solution (400 mL) followed by brine solution (500 mL). The organic phase was collected, dried over magnesium sulfate, filtered and the solvent evaporated in vacuo. The crude reaction mixture was purified by dry flash column chromatography (eluent: dichloromethane-heptane 2:1, 3:1, 4:1; dichloromethane; ethyl acetate) to yield allyl [2-(3,4-dichlorophenyl)-2-oxoethyl]carbamate, 120 g (46% over 3 steps) as a white crystalline solid.

[0484] .sup.1H NMR (400 MHz, CDCl3): δ [ppm]=4.46 (d, 2H), 4.51 (d, 2H), 5.15 (dd, 1H), 5.27 (dd, 1H), 5.81-5.92 (m, 1H), 7.54 (t, 1H), 7.79 (d, 1H), 7.90 (dd, 1H), 8.16 (d, 1H).

[0485] LCMS (method 3): R.sub.t 1.59 min

[0486] MS (ESI): [M+H].sup.+=288.06

Intermediate 6

Allyl [2-(4-chloro-3-methylphenyl)-2-oxoethyl]carbamate

[0487] ##STR00024##

[0488] Allyl [2-(4-chloro-3-methylphenyl)-2-oxoethyl]carbamate was prepared in analogy to intermediate 5, starting from intermediate 4.

[0489] .sup.1H NMR (400 MHz, CDCl3): δ [ppm]=2.44 (s, 3H), 4.62 (d, 2H), 4.67 (d, 2H), 5.23 (dd, 1H), 5.33 (dd, 1H), 5.72 (br s, 1H), 5.94 (ddt, 1H), 7.45 (d, 1H), 7.71 (dd, 1H), 7.83 (dd, 1H).

[0490] LCMS (method 2): R.sub.t 1.19 min

[0491] MS (ESI): [M+H].sup.+=268.0

Intermediate 7

Rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate

[0492] ##STR00025##

Step 1

Allyl [3-(3,4-dichlorophenyl)-3-oxoprop-1-en-2-yl]carbamate

[0493] ##STR00026##

[0494] To a stirred suspension of allyl [2-(3,4-dichlorophenyl)-2-oxoethyl]carbamate (intermediate 5), 50.0 g (0.174 mol) in ethanol (390 mL) was added formaldehyde solution, 20 mL (0.261 mol, 37 wt % in water) followed by the dropwise addition of piperidine, 26 mL (0.261 mol) in ethanol (130 mL) over 30 minutes. The reaction mixture was stirred overnight and thin layer chromatography indicated consumption of the starting material. The solvent was removed by evaporation to yield an orange oil, no further purification was performed and the crude product was used in the subsequent step as isolated.

Step 2

Rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate

[0495] ##STR00027##

[0496] To a solution of allyl [3-(3,4-dichlorophenyl)-3-oxoprop-1-en-2-yl]carbamate, (˜0.174 mol) in ethanol (480 mL) was added hydrazine monohydrate, 29.6 mL (0.609 mol) and the reaction mixture was heated to reflux for 2.5 hours. The reaction mixture was allowed to cool to room temperature then concentrated before pouring over ice cooled saturated ammonium chloride solution (300 mL). The crude product was extracted with ethyl acetate (1.5 L) and the organic layers were combined and washed with brine solution (300 mL). The collected organic phase was dried over magnesium sulfate, filtered and the solvent evaporated to yield rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate, 50.0 g (91%) as a pale yellow solid.

[0497] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=3.24 (m partially masked by H.sub.2O peak), 3.59 (td, 1H), 4.39-4.54 (m, 2H), 5.08-5.25 (m, 3H), 5.79-5.90 (m, 1H), 7.52 (dd, 1H), 7.57 (br s, 1H), 7.59 (d, 1H), 7.68 (d, 1H), 7.84 (d, 1H).

[0498] LCMS (method 3): R.sub.t 1.55 min

[0499] MS (ESI): [M+H].sup.+=314.1

Intermediate 8

Rac-allyl [3-(4-chloro-3-methylphenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate

[0500] ##STR00028##

[0501] Rac-allyl [3-(4-chloro-3-methylphenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate was prepared in analogy to intermediate 7, starting from intermediate 6.

[0502] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=2.28 (s, 3H), 3.20 (dd, 1H), 3.55 (td, 1H), 4.45 (qd, 2H), 5.11 (d, 1H), 5.14-5.24 (m, 2H), 5.85 (ddt, 1H), 3.30-3.39 (m, 3H), 7.52 (s, 1H), 7.80 (d, 1H).

[0503] LCMS (method 2): R.sub.t 1.14 min

[0504] MS (ESI): [M+H].sup.+=294.2

Intermediate 9

Rac-phenyl 4-{[(allyloxy)carbonyl]amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate

[0505] ##STR00029##

[0506] To a stirred suspension of rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate (intermediate 7), 50.0 g (0.159 mol) in 2-propanol (860 mL) was added diphenyl N-cyanocarbonimidate, 38.0 g (0.159 mol). The reaction mixture was heated to reflux at which point the suspension dissolved into solution after a further 10 minutes at reflux a white precipitate formed. The reaction mixture was stirred at reflux for a further 1 hour before allowing to slowly cool to room temperature overnight. The precipitate was filtered, washing with diethyl ether (2×250 mL) and the resulting white solid was allowed to dry to yield rac-phenyl 4-{[(allyloxy)carbonyl]amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate as a white solid, 48.6 g (67%).

[0507] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=4.13 (apparent d, 1H), 4.47 (m, 3H), 5.14 (dd, 2H), 5.51-5.63 (m, 1H), 5.79-5.90 (m, 1H), 7.23 (d, 2H), 7.30 (t, 1H), 7.45 (t, 2H), 7.79 (br m, 2H), 7.97 (br s, 1H), 8.19 (d, 1H).

[0508] LCMS (method 3): R.sub.t 1.75 min

[0509] MS (ESI): [M+H].sup.+=458.0

Intermediate 10

Rac-phenyl 4-{[(allyloxy)carbonyl]amino}-3-(4-chloro-3-methylphenyl)-N-cyano-4,5-dihydro-1H-pyrazole-1-carboximidate

[0510] ##STR00030##

[0511] Rac-phenyl 4-{[(allyloxy)carbonyl]amino}-3-(4-chloro-3-methylphenyl)-N-cyano-4,5-dihydro-1H-pyrazole-1-carboximidate was prepared in analogy to intermediate 9, starting from intermediate 8.

[0512] .sup.1H NMR (400 MHz, CDCl3): δ [ppm]=2.18 (s, 3H), 4.30 (d, 2H), 4.64 (d, 2H), 5.24 (d, 1H), 5.34 (d, 1H), 5.60-5.70 (m, 1H), 5.94 (ddt, 1H), 6.89-7.35 (m, 7H), 7.48 (dd, 1H), 7.54 (d, 1H).

[0513] LCMS (method 2): R.sub.t 1.30 min

[0514] MS (ESI): [M+H].sup.+=438.2

Intermediate 11

Rac-phenyl 4-{[(allyloxy)carbonyl]amino}-3-(4-chloro-3-methylphenyl)-N-cyano-4,5-dihydro-1H-pyrazole-1-carboximidate

[0515] ##STR00031##

[0516] Rac-phenyl 4-{[(allyloxy)carbonyl]amino}-3-(4-chloro-3-methylphenyl)-N-cyano-4,5-dihydro-H-pyrazole-1-carboximidate was prepared as described for intermediate 9, starting from 1-(4-chloro-3-fluorophenyl)ethanone.

[0517] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=4.17 (d, 1H), 4.49-4.60 (m, 3H), 5.10-5.27 (m, 2H), 5.52-5.67 (m, 1H), 5.79-5.96 (m, 1H), 7.26 (d, 2H), 7.30-7.38 (m, 1H), 7.44-7.54 (m, 2H), 7.69 (br. s., 1H), 7.79 (d, 2H), 8.21 (d, 1H).

[0518] MS (ESI): [M+H].sup.+=442

Intermediate 12

Rac-allyl [1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate

[0519] ##STR00032##

[0520] To a stirred solution of m-difluoromethoxy aniline, 8.20 mL (65.5 mmol) in anhydrous tetrahydrofuran (100 mL) at −78° C. was added n-butyl lithium, 33.0 mL (65.5 mmol, 2 M in hexane) dropwise maintaining the reaction temperature below −65° C. during the addition. The reaction mixture was stirred for 1 hour at −78° C. before Rac-phenyl 4-{[(allyloxy)carbonyl]amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate (intermediate 9), 10.0 g (21.8 mmol) in anhydrous tetrahydrofuran (600 mL) was added dropwise maintaining the reaction temperature below −65° C. The reaction mixture was stirred for 2 hours at −78° C. before slowly pouring over saturated ammonium chloride solution (700 mL). The crude product was extracted into ethyl acetate (700 mL) and the organic layers were combined and washed with brine solution (350 mL). The collected organic phase was dried over magnesium sulfate, filtered and the solvent evaporated to yield an off-white crude solid. The crude solid was precipitated from a minimum volume of ethyl acetate and filtered, washing with diethyl ether to yield rac-allyl [1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate, 7.6 g (67%) as a white solid.

[0521] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=4.08 (dd, 1H), 4.36-4.53 (m, 3H), 5.11 (dd, 1H), 5.17 (dd, 1H), 5.50-5.59 (m, 1H), 5.77-5.90 (m, 1H), 6.99 (dd, 1H), 7.16 (t, 1H), 7.21 (t, 1H), 7.23 (dd, 1H), 7.39 (t, 1H), 7.73-7.81 (m, 2H), 8.15 (d, 1H), 8.17 (d, 1H), 9.79 (br s, 1H).

[0522] LCMS (method 3): R.sub.t 1.78 min

[0523] MS (ESI): [M+H].sup.+=523.2

[0524] The following intermediates were prepared according to the method described for intermediate 12, by addition of the respective aniline derivatives to intermediate 9, intermediate 10 or intermediate 11.

TABLE-US-00002 Intermediate Structure No IUPAC name Analytical data 13 [00033] LMCS (method 2): R.sub.t 1.35 min MS (ESI): [M + H].sup.+ = 503.2 14 [00034] LMCS (method 2): R.sub.t 1.40 min MS (ESI): [M + H].sup.+ = 557.2 15 [00035] LMCS (method 2): R.sub.t 1.24 min MS (ESI): [M + H].sup.+ = 559.2 16 [00036] LMCS (method 2): R.sub.t 1.43 min MS (ESI): [M + H].sup.+ = 541.1 17 [00037] LMCS (method 2): R.sub.t 1.35 min MS (ESI): [M + H].sup.+ = 559.0 18 [00038] LMCS (method 2): R.sub.t 1.33 min MS (ESI): [M + H].sup.+ = 487.1 19 [00039] LMCS (method 2): R.sub.t 1.31 min MS (ESI): [M + H].sup.+ = 544.0 20 [00040] LMCS (method 3): R.sub.t 1.05 min MS (ESI): [M + H].sup.+ = 524.1 21 [00041] LMCS (method 2): R.sub.t 1.39 min MS (ESI): [M + H].sup.+ = 539.1 22 [00042] LMCS (method 2): R.sub.t 1.32 min MS (ESI): [M + H].sup.+ = 540.8 23 [00043] LMCS (method 2): R.sub.t 1.29 min MS (ESI): [M + H].sup.+ = 541.2 24 [00044] LMCS (method 2): R.sub.t 1.33 min MS (ESI): [M + H].sup.+ = 559.2 25 [00045] LMCS (method 2): R.sub.t 1.61 min MS (ESI): [M + H].sup.+ = 608.2 26 [00046] LMCS (method 2): R.sub.t 1.30 min MS (ESI): [M + H].sup.+ = 608.7 27 [00047] LMCS (method 2): R.sub.t 1.44 min MS (ESI): [M + H].sup.+ = 637.8 28 [00048] LMCS (method 2): R.sub.t 1.33 min MS (ESI): [M + H].sup.+ = 653.8 29 [00049] LMCS (method 2): R.sub.t 1.33 min MS (ESI): [M + H].sup.+ = 524.8

Intermediate 30

Rac-allyl [1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate

[0525] ##STR00050##

[0526] Intermediate 30 was prepared from intermediate 44 and intermediate 7 according to the scheme below.

##STR00051##

Step 1

[0527] To a solution of 5-(difluoromethoxy)-2-methoxyaniline (intermediate 44), 5.17 g (27.3 mmol) in dichloromethane (100 mL) was added an aqueous solution of sodium hydrogen carbonate 100 mL. Thiophosgene 2.2 mL (28.7 mmol) was added dropwise to the vigorously stirred mixture at room temperature and stirring continued for 1 hour. The reaction mixture was diluted with dichloromethane and washed with water. The aqueous layer was washed with further dichloromethane and the combined organic extracts were washed with brine, dried over sodium sulfate and concentrated to give 4-(difluoromethoxy)-2-isothiocyanato-1-methoxybenzene as a dark red oil 5.59 g (89%).

[0528] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=3.90 (s, 3H), 6.40 (t, 1H), 6.85 (d, 1H), 6.91 (d, 1H), 7.01 (dd, 1H)

[0529] UPLC (method 6) 0.89 min

Step 2

[0530] A solution of 4-(difluoromethoxy)-2-isothiocyanato-1-methoxybenzene 5.59 g (24.2 mmol) and mono sodium cyanamide 1.55 g (24.2 mmol) in ethanol 50 mL was stirred at reflux for 1 hour. The suspension was allowed to cool and concentrated in vacuo. The resulting residue was triturated with diethyl ether to give a light purple solid which was collected by filtration and washed with further diethyl ether to give sodium cyano {[5-(difluoromethoxy)-2-methoxyphenyl]carbamothioyl}azanide as a light purple solid 5.79 g (81%).

[0531] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=3.80 (s, 3H), 6.65 (dd, 1H), 6.92 (d, 1H), 6.99 (t, 1H), 7.87 (s, 1H), 8.20 (d, 1H)

[0532] UPLC (method 6) 0.56 min

[0533] MS (ESI): [M−Na].sup.−=272.01

Step 3

[0534] To a solution of rac-allyl (3-[3,4-dichlorophenyl]-4,5-dihydro-1H-pyrazol-4-yl)carbamate (intermediate 7) 8.16 g (27.6 mmol) in N,N-dimethylformamide 100 mL was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride 7.07 g (36.9 mmol) and sodium cyano {[5-(difluoromethoxy)-2-methoxyphenyl]carbamothioyl}azanide (intermediate 44) 5.79 g (18.4 mmol) were added sequentially. The dark brown solution was stirred at room temperature overnight. The solution was diluted with ethyl acetate 50 mL and washed with 10% citric acid aqueous solution 25 mL then brine (3×25 mL). The organic layer was dried over sodium sulfate and concentrated to a brown solid. Trituration with dichloromethane and diethyl ether gave a solid which was collected by filtration and washed with further diethyl ether to give rac-allyl [1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate as a cream solid 3.66 g (36%).

[0535] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=3.79 (s, 3H), 3.93 (dd, 1H), 4.31 (t, 1H), 4.43-4.49 (m, 2H), 5.10-5.19 (m, 1H), 5.49-5.57 (m, 1H), 5.79-5.89 (m, 1H), 7.08-7.11 (m, 3H), 7.13 (s, 1H), 7.74-7.75 (m, 2H), 8.14 (s, 1H), 8.15-8.18 (m, 2H), 9.53 (br s, 1H)

[0536] UPLC (method 6) 0.90 min

[0537] MS (ESI): [M−Na].sup.−=553.13

Intermediate 31

Rac-allyl [1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate

[0538] ##STR00052##

[0539] Intermediate 31 was prepared as described for intermediate 30 starting from 2-methoxy-5-(trifluoromethyl)aniline and intermediate 7, to obtain rac-allyl [1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate, in 42% over three steps.

[0540] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=3.88 (s, 3H), 3.90-3.96 (m, 1H), 4.31 (t, 1H), 4.43-4.51 (m, 2H), 5.10-5.19 (m, 1H), 5.51-5.58 (m, 1H), 5.80-5.89 (m, 1H), 7.26 (d, 1H), 7.57 (d, 1H), 7.65 (dd, 1H), 7.73-7.78 (m, 2H), 8.14 (s, 1H), 8.17 (d, 1H), 9.62 (br s, 1H)

[0541] UPLC (method 6): R.sub.t 0.92 min

[0542] MS (ESI): [M−H].sup.−=553.09

Intermediate 32

Rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]ethylcarbamate

[0543] ##STR00053##

[0544] Intermediate 32 was synthesized starting from intermediate 2 following the scheme below.

##STR00054##

Step 1

[0545] To 2-bromo-1-(4-chloro-3-methylphenyl)ethanone (intermediate 2) (20 g, 74.6 mmol), ethyl amine (2M in tetrahydrofuran) (187 ml) was added. The mixture was cooled to −50° C., and allyl chloroformate (18 g) was added. The reaction was stirred at room temperature for 16 h.

[0546] Step 2 and 3 were performed as described for intermediate 7, to obtain the N-ethylated analogue Rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]ethylcarbamate.

[0547] MS (ESI): [M+H].sup.+=342.1

Intermediate 33

Rac-phenyl 4-{[(allyloxy)carbonyl](ethyl)amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate

[0548] ##STR00055##

[0549] Intermediate 33 was prepared from intermediate 32 in analogy to the preparation of intermediate 9 from intermediate 7. Rac-phenyl 4-{[(allyloxy)carbonyl](ethyl)amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate was obtained as an off-white solid.

[0550] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=1.02 (m, 3H), 3.21 (m, 1H), 3.48 (m, 1H), 4.24 (d, 1H), 4.50-4.64 (m, 3H), 5.12-5.32 (m, 2H), 5.78-5.95 (m, 2H), 7.24-7.37 (m, 3H), 7.48 (t, 2H), 7.71 (m, 1H), 7.84 (d, 1H), 7.93 (m, 1H).

[0551] LCMS (method 4): R.sub.t 3.32 min

[0552] MS (ESI): [M+H].sup.+=486.1

Intermediate 34

Rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]methylcarbamate

[0553] ##STR00056##

[0554] Intermediate 32 was synthesized starting from intermediate 2 following the scheme below.

##STR00057##

[0555] Step 1 and 2 were performed as similarly described in Org. Process Res. Dev. 2012, 16, 982-1002 (page 989, scheme 10), starting with Alloc-protected instead of Boc-protected sarcosine and using 4-bromo-1,2-dichlorobenzene instead of 4-bromo-1-fluoro-2-(trifluoromethyl)benzene for the preparation of the grignard reagent.

[0556] Step 3 and 4 were performed as described for intermediate 7, to obtain the N-methylated analogue Rac-allyl [3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]methylcarbamate.

[0557] MS (ESI): [M+H].sup.+=328.1

Intermediate 35

Rac-phenyl 4-{[(allyloxy)carbonyl](methyl)amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate

[0558] ##STR00058##

[0559] Intermediate 35 was prepared from intermediate 34 in analogy to the preparation of intermediate 9 from intermediate 7. Rac-phenyl 4-{[(allyloxy)carbonyl](methyl)amino}-N-cyano-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-1-carboximidate was obtained as an off-white solid.

[0560] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=2.75 (s, 3H), 4.35 (dd, 1H), 4.48 (t, 1H), 4.55-4.70 (m, 2H), 5.14-5.40 (m, 2H), 5.93 (m, 1H), 6.17 (m, 1H), 7.27-7.37 (m, 3H), 7.48 (t, 2H), 7.70 (m, 1H), 7.84 (d, 1H), 7.90 (m, 1H).

[0561] LCMS (method 4): R.sub.t 3.19 min

[0562] MS (ESI): [M+H].sup.+=472.1

Intermediate 36

Rac-allyl [1-{N′-cyano-N-[3-(difluoromethoxy)-2-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]ethylcarbamate

[0563] ##STR00059##

[0564] Intermediate 36 was prepared from intermediate 33, following the procedure described for the synthesis of intermediate 12. In this case, 3-(difluoromethoxy)-2-fluoroaniline was used as the aniline instead of 3-(difluoromethoxy)aniline. The crude product was directly used without purification.

[0565] LCMS (method 2): R.sub.t 1.19 min

[0566] MS (ESI): [M+H].sup.+=568.7

Intermediate 37

Rac-allyl [1-{N′-cyano-N-[2-methoxy-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]ethylcarbamate

[0567] ##STR00060##

[0568] Intermediate 37 was prepared from intermediate 33, following the procedure described for the synthesis of intermediate 12. In this case, 2-methoxy-5-(trifluoromethoxy)aniline was used as the aniline instead of 3-(difluoromethoxy)aniline. The crude product was treated with diethyl ether. The suspension was stirred for 10 min, and then the solid was filtered and dried to give the desired product.

[0569] LCMS (method 2): R.sub.t 1.53 min

[0570] MS (ESI): [M+H].sup.+=599.3

Intermediate 38

Rac-allyl [1-(N′-cyano-N-{5-(difluoromethoxy)-2-[3-(dimethylamino)propoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]ethylcarbamate

[0571] ##STR00061##

[0572] Intermediate 38 was prepared from intermediate 33, following the procedure described for the synthesis of intermediate 12. In this case, 5-(difluoromethoxy)-2-[3-(dimethylamino)propoxy]aniline was used as the aniline instead of 3-(difluoromethoxy)aniline. The crude product was directly used without purification.

[0573] Analytical data of subsequent alloc-deprotected rac-N′-cyano-3-(3,4-dichlorophenyl)-N-{5-(difluoromethoxy)-2-[3-(dimethylamino)propoxy]phenyl}-4-(ethylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide:

[0574] LCMS (method 2): R.sub.t 1.50 min

[0575] MS (ESI): [M+H].sup.+=569.9

Intermediate 39

Rac-allyl [1-(N′-cyano-N-{2-[(1-methylpiperidin-4-yl)oxy]-5-(trifluoromethyl)phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]ethylcarbamate

[0576] ##STR00062##

[0577] Intermediate 39 was prepared from intermediate 33, following the procedure described for the synthesis of intermediate 12. In this case, 2-[(1-methylpiperidin-4-yl)oxy]-5-(trifluoromethyl)aniline was used as the aniline instead of 3-(difluoromethoxy)aniline. The crude product was directly used

[0578] LCMS (method 2): R.sub.t 1.50 min

[0579] MS (ESI): [M+H].sup.+=666.4

Intermediate 40

Rac-allyl [1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]methylcarbamate

[0580] ##STR00063##

[0581] Intermediate 40 was prepared from intermediate 34, following the procedure described for the synthesis of intermediate 12.

[0582] LCMS (method 1): R.sub.t 1.36 min

[0583] MS (ESI): [M+H].sup.+=536.8

Intermediate 41

4-methoxy-3-nitrophenyl acetate

[0584] ##STR00064##

[0585] Acetic anhydride 100 mL was added to a solution of 4-methoxyphenol (CAS: 150-76-5) 24.8 g (0.2 mol) in acetic acid 100 mL at room temperature. The pale yellow solution was stirred at 100° C. for 3.5 hours. The solution was cooled to 0° C. and 70% nitric acid 20 mL was added slowly over 10 minutes, solution became warm. The orange solution was stirred at 0° C. for 1 hour, a solid precipitated. Water 100 mL was added and the solid collected by filtration and washed with further water to give 4-methoxy-3-nitrophenyl acetate as a cream solid, 47.25 g (112%, still water present). Taken onto next step without further drying.

[0586] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=2.30 (s, 3H), 3.95 (s, 3H), 7.08 (d, 1H), 7.30 (dd, 1H), 7.65 (d, 1H)

[0587] UPLC (method 6): R.sub.t 0.69 min

Intermediate 42

4-methoxy-3-nitrophenol

[0588] ##STR00065##

[0589] 4-Methoxy-3-nitrophenyl acetate 47.25 g (200 mmol) was suspended in ethanol 800 mL and cooled to 0° C. A 1 M aqueous solution of sodium hydroxide 220 mL was added slowly and the light yellow suspension became a dark red solution. Stirred for 1 hour and allowed to warm to room temperature. The reaction was quenched with acetic acid to give a light orange solution. Partitioned between brine and ethyl acetate, the aqueous was washed with further ethyl acetate. The combined organic extracts were washed with further brine, dried over sodium sulfate and concentrated to a dark orange oil which crystallised on standing to give 4-methoxy-3-nitrophenol, 30.97 g (92%).

[0590] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=3.90 (s, 3H), 5.54 (br s, 1H), 6.98 (d, 1H), 7.07 (dd, 1H), 7.38 (d, 1H)

[0591] UPLC (method 6): R.sub.t 0.47 min

[0592] MS (ESI): [M−H].sup.−=167.95

Intermediate 43

4-(difluoromethoxy)-1-methoxy-2-nitrobenzene

[0593] ##STR00066##

[0594] A solution of 4-methoxy-3-nitrophenol 10.0 g (59.1 mmol) in N,N-dimethylformamide 150 mL was degassed with argon for 20 minutes. Cesium carbonate 38.5 g (118.2 mmol) was added and the orange solution became a dark red suspension. Sodium chlorodifluoroacetate 18.0 g (118.2 mmol) was added and the suspension was stirred at 100° C. for 1.5 hours, a light brown suspension formed. Diluted with water and extracted with ethyl acetate twice. The combined organic extracts were washed with brine three times, dried over sodium sulfate and concentrated to a light brown solid. The solid was triturated with methanol and the cream solid collected by filtration. The filtrate was concentrated to a dark brown oil (6.60 g). Purification by dry-flash column chromatography on silica gel 60 (heptanes:ethyl acetate 4:1 to 1:1) to give a yellow oil which solidified on standing to give 4-(difluoromethoxy)-1-methoxy-2-nitrobenzene, 4.65 g (36%).

[0595] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=3.96 (s, 3H), 6.48 (t, 1H), 7.08 (d, 1H), 7.36 (dd, 1H), 7.67 (d, 1H)

[0596] UPLC (method 6): R.sub.t 0.74 min

Intermediate 44

5-(difluoromethoxy)-2-methoxyaniline

[0597] ##STR00067##

[0598] A suspension of 4-(difluoromethoxy)-1-methoxy-2-nitrobenzene 6.43 g (29.3 mmol) and tin(II) chloride dihydrate, 33.1 g (146.7 mmol) in ethyl acetate 100 mL was heated to reflux for 2.5 hours. The reaction mixture was poured into a potassium carbonate aqueous solution 200 mL. The resulting suspension was filtered through a pad of celite and washed with ethyl acetate. The biphasic filtrate was separated and the aqueous was washed with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated to give 5-(difluoromethoxy)-2-methoxyaniline as a purple oil 5.17 g (93%).

[0599] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=3.83 (s, 3H), 3.88 (br s, 2H), 6.38 (t, 1H), 6.46 (dd, 1H), 6.50 (d, 1H), 6.69 (d, 1H)

[0600] UPLC (method 6): R.sub.t 0.68 min

[0601] MS (ESI): [M+H].sup.+=190.05

Examples for the Production of the Inventive Compounds

Example 1

(2S)—N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxypropanamide (1:1 Mixture of Diastereomers)

[0602] ##STR00068##

[0603] Example 1 was prepared starting from intermediate 12 according to the following scheme:

##STR00069##

Step 1

[0604] To a stirred solution of rac-allyl [1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]carbamate (intermediate 12), 14.2 g (27.0 mmol) in degassed tetrahydrofuran (370 mL) was added 1,3-dimethylbarbituric acid, 17.0 g (108 mmol) followed by tetrakis(triphenylphosphine) palladium 0, 2.40 g (2.16 mmol). The reaction mixture was stirred under argon for 15 minutes then cautiously quenched with saturated sodium hydrogen carbonate solution (400 mL) and extracted into ethyl acetate (400 mL). The organic layer was washed with brine solution (200 mL) before being dried over magnesium sulfate, filtered and the solvent evaporated to yield a crude orange oil. The crude material was purified by dry flash column chromatography (eluent: ethyl acetate-heptane 1:1, 2:1; ethyl acetate; methanol-ethyl acetate 0.01:1) to yield 4-amino-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1H-pyrazole-1-carboximidamide, 9.3 g (78%) as an orange oil.

[0605] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=3.95-4.02 (m, 1H under ethyl acetate signal), 4.35 (dd, 1H), 4.80 (dd, 1H), 6.98 (dd, 1H), 7.19 (t, 1H), 7.21 (t, 1H), 7.23 (dd, 1H), 7.38 (t, 1H), 7.48-7.61 (m, 1H), 7.72 (d, 1H), 8.00 (dd, 1H), 8.31 (d, 1H), 9.67 (br s, 1H);

[0606] LCMS (method 3): R.sub.t 1.65 min

[0607] MS (ESI): [M+H].sup.+=439.1

Step 2

[0608] To the solution of L-lactic acid (61.5 mg, 683 μmol) in N,N-dimethylformamide (2 ml) was added 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, CAS No. 148893-10-1), 260 mg (683 μmol), followed by N-methylmorpholine (150 yl, 1.37 mmol), and the mixture was stirred for 30 min. rac-4-amino-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1H-pyrazole-1-carboximidamide (150 mg, 341 μmol) dissolved in N,N-dimethylformamide (1 ml) was added and stirred for 12 h at room temperature. The reaction mixture was treated with potassium carbonate (25 mg) and methanol (2 ml) for 1 h. The solids were filtered off and the filtrate was concentrated in vacuo. The residue was purified by preparative HPLC (gradient of acetonitrile in water) to yield 5 mg (3%) of the desired product.

[0609] LCMS (method 5): R.sub.t 1.18 min

[0610] MS (ESI): [M+H].sup.+=510.9

Example 2

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-N-methylacetamide

[0611] ##STR00070##

[0612] Example 2 was prepared starting from intermediate 40 according to the following scheme:

##STR00071##

Step 1

[0613] To a stirred solution of rac-allyl [1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]methylcarbamate (intermediate 40), 875 mg (1.6 mmol) in tetrahydrofuran (12 mL) was added 1,3-dimethylbarbituric acid, 508 mg (3.3 mmol) followed by tetrakis(triphenylphosphine) palladium 56.5 mg (0.05 mmol). The reaction mixture was stirred under argon for 1 hour then cautiously quenched with saturated sodium hydrogen carbonate solution (400 mL) and extracted into ethyl acetate (400 mL). The organic layer was washed with brine solution (200 mL) before being dried over magnesium sulfate, filtered and the solvent evaporated. The crude material was stirred in diethyl ether (10 ml), filtered, and dried to yield crude rac-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4-(methylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide (902 mg), which was used in the next step without further purification.

[0614] LCMS (method 5): R.sub.t 1.29 min

[0615] MS (ESI): [M+H].sup.+=452.8

Step 2

[0616] To rac-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4-(methylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide 246 mg (0.54 mmol) in dichloromethane (12 mL) was added saturated sodium hydrogen carbonate solution (12 mL). The biphasic mixture was stirred vigorously and cooled to 0° C., acetoxyacetyl chloride (111 mg, 0.81 mmol) in dichloromethane, 3 mL was added dropwise over 15 min. The reaction mixture was stirred for 30 minutes at 0° C. Dichloromethane was removed by evaporation to yield an oily aqueous suspension, to which potassium carbonate, 150 mg (1.1 mmol) was added followed by methanol (4 mL). The reaction mixture was brought to reflux for 30 min then allowed to cool to room temperature. Upon cooling rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-N-methylacetamide precipitated out of solution as a white solid which was filtered, and purified by column chromatography (reversed phase, water, acetonitrile), to yield 37 mg (13%) of the desired product.

[0617] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=2.69 (s, 3H), 4.09 (d, 2H), 4.20 (dd, 1H), 4.39 (t, 1H), 4.77 (t, 1H), 6.37 (br. s., 1H), 6.98 (d, 1H), 7.19 (s, 1.25H), 7.20-7.26 (m, 1.5H), 7.39-7.44 (m, 1.25H), 7.63 (dd, 1H), 7.76 (d, 1H), 8.05-8.15 (m, 1H), 9.25 (br. s., 1H).

[0618] MS (ESI): [M+H].sup.+=511

Example 3

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylglycinamide

[0619] ##STR00072##

[0620] Example 3 was prepared starting from intermediate 12 according to the following scheme:

##STR00073## ##STR00074##

Step 1

[0621] As described for example 1.

Step 2

[0622] To a stirred solution of rac-4-amino-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1H-pyrazole-1-carboximidamide, 9.30 g (21.2 mmol) in methanol (170 mL) at 0° C. was added acetaldehyde, 1.12 g (25.4 mmol) followed by the portion wise addition of sodium borohydride, 0.96 g (25.4 mmol) over 20 minutes. The reaction mixture was stirred for 30 minutes before pouring over saturated sodium hydrogen carbonate solution (100 mL). The methanol was removed by evaporation and the resulting aqueous slurry was extracted with ethyl acetate (2×100 mL). The organic layers were combined and washed with brine solution (100 mL) dried over magnesium sulfate, filtered and the solvent evaporated to yield a crude black oil. The crude material was purified by dry flash column chromatography (eluent: ethyl acetate-heptane 1:1, 2.1; ethyl acetate) to yield a black oil, which was triturated with diethyl ether to yield rac-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-H-pyrazole-1-carboximidamide, 7.40 g (75%) as a grey solid.

[0623] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=0.98 (t, 3H), 2.40-2.64 (m, 2H partially under DMSO signal), 4.16-4.27 (m, 2H), 4.83 (dd, 1H), 6.98 (dd, 1H), 7.20 (d, 1H), 7.21 (t, 1H), 7.25 (dd, 1H), 7.39 (t, 1H), 7.70 (d, 1H), 7.97 (dd, 1H), 8.29 (d, 1H), 9.71 (br s, 1H)

[0624] LCMS (method 3): R.sub.t 1.80 min

[0625] MS (ESI): [M+H].sup.+=467.18

Step 3

[0626] To a solution of rac-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide (300 mg, 0.64 mmol) in DMF (15 mL) was added Fmoc-glycine (382 mg, 1.3 mmol), HATU (488 mg, 1.3 mmol), and 4-methylmorpholine (0.28 mL, 2.6 mmol). The solution was stirred at room temperature for 4 h. The reaction mixture was poured into water (25 mL), and the mixture was extracted with ethyl acetate (3×50 mL). The organic phases were dried over magnesium sulphate and concentrated in vacuo. The residue was directly used in the following reaction.

Step 4

[0627] The crude product from step 3 was dissolved in dichloromethane (20 mL) and piperidine (0.8 mL) was added. The reaction was stirred at room temperature for 1.5 h. Water (10 mL) was added, and the aqueous was extracted with ethyl acetate (3×20 mL). The combined organic phases were washed with brine, dried over magnesium sulphate and concentrated. The crude product was purified by chromatography (RP, water+0.1% ammonia, acetonitrile) to give rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylglycinamide (150 mg, 45%).

[0628] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.07-1.22 (m, 3H), 3.77-3.98 (m, 2H), 4.15 (dd, 1H), 4.55 (t, 1H), 7.02-7.07 (m, 1.25H), 7.21 (t, 1H), 7.23-7.29 (m, 1.5H), 7.41-7.47 (m, 1.25H), 7.68 (dd, 1H), 7.75 (m, 1H), 7.90-8.03 (m, 3H), 8.16 (d, 1H), 9.86 (s, 1H); 3H obscured by solvent and water signals.

[0629] LCMS (method 2): R.sub.t 1.12 min

[0630] MS (ESI): [M+H].sup.+=523.8

Example 4

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0631] ##STR00075##

[0632] Example 4 was prepared starting from intermediate 12 according to the following scheme:

##STR00076##

Step 1

[0633] As described for example 1.

Step 2

[0634] As described for example 3.

Step 3

[0635] To rac-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide, 7.24 g (15.5 mmol) in dichloromethane, 62 mL was added saturated sodium hydrogen carbonate solution, 72 mL. The biphasic mixture was stirred vigorously and cooled to 5° C., acetoxyacetyl chloride, 2.50 mL (23.2 mmol) in dichloromethane, 10 mL was added dropwise over 15 min. The reaction mixture was stirred for 10 mins at 5° C. after which time LC analysis indicated total consumption of starting material with only one major peak. Dichloromethane was removed by evaporation to yield an oily aqueous suspension to which potassium carbonate, 4.28 g (31.0 mmol) was added followed by methanol, 110 mL. The reaction mixture was brought to reflux for 5 min then allowed to cool to room temperature. Upon cooling rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide precipitated out of solution as a white solid which was filtered, washing with water, 50 mL and diethyl ether, 50 mL. The precipitate was dried in vacuo to yield the desired product, 7.58 g (93%) as a white powder.

[0636] .sup.1H NMR (400 MHz, DMSO-d6): δ [ppm]=0.98-1.10 (m, 3H), 3.42-3.17 (m, 2H partially under water signal), 3.94-4.17 (m, 3H), 4.46 (dd, 1H), 4.75 (dd, 1H), 6.98 (dd, 1H), 7.18 (t, 1H), 7.22 (dd, 1H), 7.23 (t, 1H), 7.40 (t, 1H), 7.62 (dd, 1H), 7.72 (d, 1H), 8.11 (d, 1H), 9.85 (br s, 1H)

[0637] UPLC (method 6): R.sub.t 0.58 min

[0638] MS (ESI): [M+H].sup.+=525

[0639] Example 4 was separated into its enantiomers by chiral SFC:

TABLE-US-00003 System: Sepiatec: Prep SFC100, Column: Chiralpak ID 5 μm 250 × 20 mm Solvent: CO2/propan-2-ol 7/3 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 4.1 3.35-4.40 4.2 7.31-9.00

Example 4.1

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0640] ##STR00077##

[0641] Chiralpak ID 5 m 100×4.6 mm (CO2/2-Propanol 7/3), R.sub.t 2.41 min

[0642] [α].sub.D=−102° (c: 0.44, MeOH)

Example 4.2

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0643] ##STR00078##

[0644] Chiralpak ID 5 m 100×4.6 mm (CO2/2-Propanol 7/3), R.sub.t 5.66 min

[0645] [α].sub.D=+96° (c: 0.25, MeOH)

Example 5

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-2-methylpropanamide

[0646] ##STR00079##

[0647] Example 5 was prepared from intermediate 12 according to the following scheme:

##STR00080##

Step 1

[0648] As described for example 1.

Step 2

[0649] Step 2 was performed in analogy to example 4 (step 3), by reacting rac-4-amino-N′-cyano-3-(3,4-dichlorophenyl)-N-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1H-pyrazole-1-carboximidamide with acetoxy isobutyryl chloride, followed by removal of the acetyl group, to obtain the desired product.

[0650] .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ [ppm]=1.16 (s, 3H), 1.17 (s, 3H), 3.74-3.82 (m, 1H), 4.11-4.22 (m, 1H), 5.64-5.75 (m, 1H), 6.78-6.84 (m, 1H), 6.95 (s, 0.25H), 7.19 (s, 0.5H), 7.29-7.37 (m, 1H), 7.44 (s, 0.25H), 7.51-7.63 (m, 3H), 7.70-7.75 (m, 1H), 7.82 (d, J=2.07 Hz, 1H), 8.11 (d, J=1.88 Hz, 1H), 8.71 (d, J=9.23 Hz, 1H), 9.28 (s, 1H).

[0651] LCMS (method 2): R.sub.t=1.22 min

[0652] MS (ESI): [M+H].sup.+=525.1

[0653] Example 5 was separated into its enantiomers by chiral SFC:

TABLE-US-00004 System: Sepiatec: Prep SFC100, Column: Chiralpak ID 5 μm 250 × 20 mm Solvent: CO2/2-Propanol + 0.4% DEA 8/2 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 5.1 2.60-3.00 5.2 3.40-4.40

Example 5.1

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-2-methylpropanamide Isomer 1

[0654] ##STR00081##

[0655] Chiralpak ID 5 μm 100×4.6 mm (CO2/2-Propanol+0.2% Diethylamine 8:2) R.sub.t=3.02 min

[0656] [α].sub.D=+14.9° (c: 1.0, DMSO)

Example 5.2

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-2-hydroxy-2-methylpropanamide Isomer 2

[0657] ##STR00082##

[0658] Chiralpak ID 5 μm 100×4.6 mm (CO2/2-Propanol+0.2% Diethylamine 8:2) R.sub.t=7.09 min

[0659] [α].sub.D=−23.2° (c: 1.0, DMSO)

Example 6

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-beta-alaninamide

[0660] ##STR00083##

[0661] Example 6 was prepared analogously to example 3 using Fmoc-beta-alanine instead of Fmoc-glycine for the amide coupling.

[0662] LCMS (method 2): R.sub.t=0.97

[0663] MS (ESI): [M+H].sup.+=538.0

Example 7

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-alaninamide (1:1 Mixture of Diastereomers)

[0664] ##STR00084##

[0665] Example 7 was prepared analogously to example 3 using Fmoc-D-alanine instead of Fmoc-glycine for the amide coupling.

[0666] .sup.1H-NMR (400 MHz, DMSO-d6): d [ppm]=0.13-0.26 (m, 2H), 0.35-0.55 (m, 6H), 2.65 (d, 1H), 2.85-2.99 (m, 1H), 3.40 (dd, 1H), 3.67-3.80 (m, 1H), 6.03 (t, 1H), 6.19-6.27 (m, 1H), 6.41 (t, 1H), 6.46 (ddd, 1H), 6.60 (t, 1H), 6.79 (d, 1H), 6.85 (dd, 1H), 7.19-7.29 (m, 1H).

[0667] MS (ESI): [M+H].sup.+=538

[0668] Example 7 was separated into its diastereomers by chiral SFC:

TABLE-US-00005 System: Sepiatec: Prep SFC100, Column: Chiralpak IC 5 μm 250 × 20 mm Solvent: CO2/Ethanol + 0.4% DEA 7/3 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 7.1 2.2-4.0 7.2 7.0-10.2

Example 7.1

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-alaninamide Isomer 1

[0669] ##STR00085##

[0670] Chiralpak IC 5 μm 100×4.6 mm (CO2/Ethanol+0.2% DEA 7/3), R.sub.t 1.80 min

[0671] [α].sub.D=−60° (c: 0.30, DMSO)

Example 7.2

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-alaninamide Isomer 2

[0672] ##STR00086##

[0673] Chiralpak IC 5 μm 100×4.6 mm (CO2/Ethanol+0.2% DEA 7/3), R.sub.t 4.77 min

[0674] [α].sub.D=+56° (c: 0.20, DMSO)

Example 8

(2S)—N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxypropanamide (1:1 Mixture of Diastereomers)

[0675] ##STR00087##

[0676] Example 8 was prepared analogously to example 4 using (2S)-2-{[tert-butyl(dimethyl)silyl]-oxy}propanoyl chloride instead of acetoxyacetyl chloride for the amide coupling. The silyl group was removed by treating the crude protected amide with 2 eq N,N,N-tributylbutan-1-aminium fluoride in tetrahydrofuran at room temperature for 45 min. The reaction mixture was concentrated and the residue was purified by HPLC (gradient of acetonitrile in 0.1% aqueous ammonia).

[0677] .sup.1H-NMR (400 MHz, DMSO-d6): d [ppm]=1.08-1.17 (m, 6H), 3.34-3.48 (m, 1H), 3.63 (br. s., 1H), 4.08 (m, 1H), 4.27-4.41 (m, 1H), 4.44-4.58 (m, 1H), 4.98 (d, 0.5H), 5.18 (d, 0.5H), 6.99-7.07 (m, 1.25H), 7.20-7.30 (m, 2.5H), 7.39-7.48 (m, 1.25H), 7.62-7.70 (m, 1H), 7.75 (t, 1H), 8.09 (dd, 1H), 9.84 (br. s., 1H).

[0678] LCMS (method 2): R.sub.t=1.22

[0679] MS (ESI): [M+H].sup.+=539.1

Example 9

(2R)—N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxypropanamide (1:1 Mixture of Diastereomers)

[0680] ##STR00088##

[0681] Example 9 was prepared analogously to example 8 using (2R)-2-{[tert-butyl(dimethyl)silyl]oxy}propanoyl chloride instead of acetoxyacetyl chloride for the amide coupling.

[0682] .sup.1H-NMR corresponds to example 8.

[0683] LCMS (method 2): R.sub.t=1.22

[0684] MS (ESI): [M+H].sup.+=539.1

Example 10

Rac-4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide

[0685] ##STR00089##

[0686] Example 10 was prepared analogously to example 3 using Fmoc-4-aminobutanoic acid instead of Fmoc-glycine for the amide coupling.

[0687] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.03 (t, 3H), 1.57 (m, 2H), 2.26-2.44 (m, 3H), 3.17-3.53 (m, 4H), 4.05 (dd, 1H), 4.36 (m, 1H), 6.84-6.88 (m, 1H), 7.01 (s, 0.25H), 7.09 (m, 2H), 7.19 (s, 0.5H), 7.32 (t, 1H), 7.38 (s, 0.25H), 7.61 (m, 3H), 7.72 (d, 1H), 7.99 (d, 1H).

[0688] LCMS (method 2): R.sub.t=1.25 min

[0689] MS (ESI): [M+H].sup.+=552.1

[0690] Example 10 was subsequently separated into its enantiomers by the following sequence:

##STR00090## ##STR00091##

Step 1

Rac-tert-butyl (4-{[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl](ethyl)amino}-4-oxobutyl)carbamate

[0691] ##STR00092##

[0692] To a cold (0° C.) stirred solution of 4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide (280 mg, 0.51 mmol) in dichloromethane (50 mL) was added N,N-diisopropylethylamine (0.265 mL, 1.5 mmol), followed by dropwise addition of di-tert-butyldicarbonate (0.116 mL, 0.51 mmol). The reaction was allowed to warm slowly to room temperature and stirred for 16 hours. After this time, water was added and the layers were separated. The aqueous phase was extracted with ethyl acetate and the combined organic phases were washed with brine, dried (MgSO.sub.4) and evaporated to give the crude boc-protected amine. Purification of the crude material by silica gel chromatography gave tert-butyl (4-{[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl](ethyl)amino}-4-oxobutyl)carbamate (280 mg, 79% yield) as a white solid.

[0693] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.05 (br. s., 3H), 1.55 (br. s., 2H), 2.21-2.39 (m, 2H), 2.88 (br. d, 2H), 3.28 (br. s., 1H), 3.46 (br. s., 1H), 4.07-4.14 (m, 1H), 4.47 (t, 1H), 6.79 (br. t, 1H), 7.03 (dd, 1H), 7.21-7.29 (m, 3H), 7.41-7.46 (m, 1H), 7.52-7.68 (m, 2H), 7.76 (d, 1H), 8.12 (d, 1H), 9.86 (s, 1H).

[0694] LCMS (method 2): R.sub.t=1.43 min

[0695] MS (ESI): [M+H].sup.+=652.3

Separation:

[0696] Separation of the racemic material by chiral preparative HPLC (conditions below) gave 105 mg (R.sub.t=4.3-5.1 min) of one enantiomer and 108 mg (R.sub.t=5.1-6.4 min) of the second enantiomer.

Step 2:

[0697]

TABLE-US-00006 System: Agilent: Prep 1200, 2xPrep Pump, DLA, MWD, Gilson: Liquid Handler 215 Column: Chiralpak IE 5 μm 250 × 20 mm Solvent: Acetonitrile 100% + 0.1% Diethylamine Flow: 30 mL/min Temperature: RT Detection: UV 325 nm Fraction Rt in min 1 4.3-5.1 2 5.1-6.4

[0698] Deprotection of the Boc-protected amines was carried out according to the following procedure: To a stirred solution of tert-butyl (4-{[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl](ethyl)amino}-4-oxobutyl)carbamate (108 mg, 0.17 mmol) in 1,2-dichloroethane (10 mL) was added zinc bromide (75 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature. After this time, the reaction mixture was diluted with dichloromethane, pH 10 buffer was added and the layers were separated. The aqueous phase was extracted with dichloromethane (3 times) and the combined organic phases were washed with brine, dried (MgSO.sub.4) and evaporated to give the crude amine. Purification of the crude material by preparative HPLC gave 4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide (25 mg, 27% yield) as a white solid. Analytical data for both isomers can be found in below.

Example 10.1 (from Separation Fraction 2 Above)

4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide Isomer 1

[0699] ##STR00093##

[0700] [α].sub.D=−52.5° (c: 1.0, DMSO)

Example 10.2 (from Separation Fraction 1 Above)

4-amino-N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylbutanamide Isomer 2

[0701] ##STR00094##

[0702] [α].sub.D=+59.6° (c: 1.0, DMSO)

Example 11

Rac-N-[1-{N-[4-chloro-3-(difluoromethoxy)phenyl]-N′-cyanocarbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0703] ##STR00095##

[0704] Example 11 was prepared analogously to example 4 starting from intermediate 14 instead intermediate 12.

[0705] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=0.81 (br. s., 1H), 0.99 (br. s., 3H), 2.92 (br. s., 0.4H), 3.01-3.29 (m, 3H), 3.94-4.33 (m, 6H), 4.40 (br. s., 0.4H), 4.72 (t, 1H), 5.13 (br. s., 0.3H), 5.73 (br. s., 0.4H), 5.94 (br. s., 1H), 6.93-7.42 (m, 6H), 7.52-7.72 (m, 3H), 7.86-7.96 (m, 1H).

[0706] LCMS (method 2): R.sub.t=1.22 min

[0707] MS (ESI): [M+H].sup.+=559.2

[0708] Example 11 was separated into its diastereomers by chiral SFC:

TABLE-US-00007 System: Sepiatec: Prep SFC100 Column: Chiralpak IC 5 μm 250 × 20 mm Solvent: CO.sub.2/Ethanol 70:30 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 11.1 4.75-5.60 11.2 6.25-7.50

[0709] Example 11.2 was further purified by chiral SFC:

TABLE-US-00008 System: Sepiatec: Prep SFC100 Column: Chiralpak IC 5 μm 250 × 20 mm Solvent: CO.sub.2/Ethanol 70:30 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 11.2 4.50-5.60

Example 11.1

N-[1-{N-[4-chloro-3-(difluoromethoxy)phenyl]-N′-cyanocarbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0710] ##STR00096##

[0711] Chiralpak IC 5 μm 100×4.6 mm (CO.sub.2/Ethanol, 70:30, 4.0 mL/min) R.sub.t=2.99 min

[0712] [α].sub.D=−37.4° (c: 1.0, DMSO)

Example 11.2

N-[1-{N-[4-chloro-3-(difluoromethoxy)phenyl]-N′-cyanocarbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0713] ##STR00097##

[0714] Chiralpak IC 5 μm 100×4.6 mm (CO.sub.2/Ethanol, 70:30, 4.0 mL/min) R.sub.t=3.96 min

[0715] [α].sub.D=+41.2° (c: 1.0, DMSO)

Example 12

Rac-N-[1-{N′-cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0716] ##STR00098##

[0717] Example 12 was prepared analogously to example 4 starting from intermediate 15 instead intermediate 12.

[0718] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.09 (br. s., 3H), 4.00-4.14 (m, 3H), 4.44 (t, 1H), 4.77 (t, 1H), 7.34-7.40 (m, 1H), 7.47-7.59 (m, 2H), 7.63 (d, 1H), 7.76 (d, 1H), 8.13 (s, 1H), 9.95 (br. s., 1H).

[0719] LCMS (method 2): R.sub.t=1.03 min

[0720] MS (ESI): [M+H].sup.+=561.0

[0721] Example 12 was separated into its diastereomers by chiral HPLC:

TABLE-US-00009 System: Agilent: Prep 1200, 2xPrep Pump, DLA, MWD, Prep FC Column: Chiralpak ID 5 μm 250 × 30 mm Nr. 018 Solvent: Hexan/Ethanol/Diethylamin 70:30:0.1 (v/v/v) Flow: 40 mL/min Temperature: RT Detection: UV 325 nm Example No Rt in min 12.1 5.4-7.2 12.2 7.2-9.4

Example 12.1

N-[1-{N′-cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0722] ##STR00099##

[0723] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol/Diethylamine 70:30:0.1 (v/v/v), 1.0 mL/min) R.sub.t=2.61 min

[0724] [α].sub.D=+27.4° (c: 1.0, DMSO)

Example 12.2

N-[1-{N′-cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0725] ##STR00100##

[0726] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol/Diethylamine 70:30:0.1 (v/v/v), 1.0 mL/min) R.sub.t=3.46 min

[0727] [α].sub.D=−22.9° (c: 1.0, DMSO)

Example 13

Rac-N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0728] ##STR00101##

[0729] Example 13 was prepared analogously to example 4 starting from intermediate 16 instead intermediate 12.

[0730] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.03 (t, 3H), 3.3-3.5 (m, 2H), 3.9-4.1 (m, 3H), 4.4-4.5 (m, 1H), 4.7-4.8 (m, 1H), 7.15 (d, 1H), 7.3-7.8 (m, 6H), 8.09 (s, 1H), 9.88 (s, 1H)

[0731] LCMS (method 3): R.sub.t=2.63

[0732] MS (ESI): [M+H].sup.+=542.92

[0733] Example 13 was separated into its enantiomers by chiral SFC:

TABLE-US-00010 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Gilson: Liquid Handler 215 Column: Chiralpak ID 5 μm 250 × 30 mm Nr.018 Solvent: Hexan/Ethanol 70:30 (v/v) Flow: 50 mL/min Temperature: RT Detektion: UV 254 nm Example No Rt in min 13.1 10.8-13.8 13.2 16.8-21.4

Example 13.1

N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0734] ##STR00102##

[0735] Chiralpak ID 3 m 100×4.6 mm (Hexan/Ethanol 70:30 (v/v); 1.0 mL/min) R.sub.t=2.67 min

[0736] [α].sub.D=890 (C: 0.93, MeOH)

Example 13.2

N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0737] ##STR00103##

[0738] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol 70:30 (v/v); 1.0 mL/min) R.sub.t=3.66 min

[0739] [α].sub.D=−79° (c: 0.83, MeOH)

Example 14

Rac-N-[1-{N′-cyano-N-[4-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0740] ##STR00104##

[0741] Example 14 was prepared analogously to example 4 starting from intermediate 17 instead intermediate 12.

[0742] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.06 (m, 3H), 3.16-3.30 (m, 2H, partially obscured by water signal), 3.98-4.20 (m, 3H), 4.37-4.52 (m, 1H), 4.76 (t, 1H), 5.83 (br. s., 1H), 7.34-7.60 (m, 3H), 7.60-7.68 (m, 1H), 7.73-7.77 (m, 1H), 8.10 (s, 1H), 9.87 (br. s., 1H).

[0743] LCMS (method 2): R.sub.t=1.46

[0744] MS (ESI): [M+H].sup.+=561

Example 15

Rac-N-{1-[N′-cyano-N-(3-methoxyphenyl)carbamimidoyl]-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl}-N-ethyl-2-hydroxyacetamide

[0745] ##STR00105##

[0746] Example 15 was prepared analogously to example 4 starting from intermediate 18 instead intermediate 12.

[0747] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.04 (br. s., 3H), 3.14-3.27 (m, 1H), 3.74 (s, 3H), 3.99-4.14 (m, 4H), 4.38 (t, 1H), 4.69-4.75 (m, 1H), 5.85 (br. s., 1H), 6.84 (br. s., 4H), 7.20 (t, 1H), 7.51-7.64 (m, 2H), 7.69-7.73 (m, 1H), 8.07 (br. s., 1H), 9.75 (br. s., 1H).

[0748] LCMS (method 2): R.sub.t=1.20 min

[0749] MS (ESI): [M+H].sup.+=489.2

[0750] Example 15 was separated into its diastereomers by chiral HPLC:

TABLE-US-00011 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Prep FC Column: Chiralpak IC 5 μm 250 × 20 mm Nr. 009 Solvent: Methanol/Ethanol/Diethylamin 50:50:0.1 (v/v/v) Flow: 20 mL/min Temperature: RT Detection: UV 325 nm Example No Rt in min 15.1 6.75-8.0 15.2  5.0-6.0

Example 15.1

N-{1-[N′-cyano-N-(3-methoxyphenyl)carbamimidoyl]-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl}-N-ethyl-2-hydroxyacetamide Isomer 1

[0751] ##STR00106##

[0752] Chiralpak IC 5 μm 150×4.6 mm (Methanol/Ethanol/Diethylamine 50:50:0.1 (v/v/v), 1.0 mL/min) R.sub.t=3.47 min

[0753] [α].sub.D=+105.0° (c: 1.0, MeOH)

Example 15.2

N-{1-[N′-cyano-N-(3-methoxyphenyl)carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0754] ##STR00107##

[0755] Chiralpak IC 5 μm 150×4.6 mm (Methanol/Ethanol/Diethylamine 50:50:0.1 (v/v/v), 1.0 mL/min) R.sub.t=2.65 min

[0756] [α].sub.D=−88.1° (c: 1.0, MeOH)

Example 16

Rac-N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0757] ##STR00108##

[0758] Example 16 was prepared analogously to example 4 starting from intermediate 19 instead intermediate 12.

[0759] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ [ppm]=1.02 (br. s., 3H), 2.19 (s, 6H), 2.60 (t, 2H), 3.94-4.45 (m, 8H), 5.84 (br. s., 1H), 6.46-6.85 (m, 4H), 7.16 (t, 1H), 7.50-7.75 (m, 7H), 8.02 (s, 1H).

[0760] LCMS (method 2): R.sub.t=1.20 min

[0761] MS (ESI): [M+H].sup.+=546.24

[0762] Example 16 was separated into its diastereomers by chiral HPLC:

TABLE-US-00012 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Prep FC Column: Chiralpak IA 5 μm 250 × 20 mm Solvent: Methanol/Ethanol/Diethylamine 50:50:0.1 (v/v/v) Flow: 20 mL/min Temperature: RT Detection: UV 325 nm Example No Rt in min 16.1 8.1-9.7 16.2 6.5-7.7

Example 16.1

N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0763] ##STR00109##

[0764] Chiralpak IC 5 μm 150×4.6 mm (Methanol/Ethanol/Diethylamine 50:50:0.1 (v/v/v), 1.0 mL/min) R.sub.t=4.51 min

[0765] [α].sub.D=+39.2° (c: 1.0, DMSO)

Example 16.2

N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0766] ##STR00110##

[0767] Chiralpak IC 5 μm 150×4.6 mm (Methanol/Ethanol/Diethylamine 50:50:0.1 (v/v/v), 1.0 mL/min) R.sub.t=3.53 min

[0768] [α].sub.D=−35.6° (c: 1.0, DMSO)

Example 17

Rac-N-[1-(N′-cyano-N-{3-[2-(dimethylamino)ethoxy]phenyl}carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethylglycinamide

[0769] ##STR00111##

[0770] Example 17 was prepared analogously to example 3 starting from intermediate 19 instead intermediate 12.

[0771] .sup.1H-NMR (400 MHz, METHANOL-D.sub.3): d [ppm]=1.05-1.27 (m, 3H), 2.35 (s, 6H), 2.80 (t, 2H), 3.35 (m, 1H), 3.42-3.54 (m, 2H), 4.14 (t, 2H), 4.21 (dd, 1H), 4.40-4.50 (m, 1H), 6.87 (m, 1H), 6.98 (dd, 1H), 7.00-7.04 (m, 1H), 7.30 (t, 1H), 7.59 (d, 1H), 7.65 (dd, 1H), 8.11 (d, 1H); two hydrogens obscured by solvent or water signal.

[0772] LCMS (method 7): R.sub.t=1.84

[0773] MS (ESI): [M].sup.+=545.18

Example 18

Rac-N-[1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0774] ##STR00112##

[0775] Example 18 was prepared analogously to example 4 starting from intermediate 30 instead intermediate 12.

[0776] LCMS (method 7): R.sub.t=2.49

[0777] MS (ESI): [M+H].sup.+=555.2

[0778] Example 18 was separated into its enantiomers by chiral SFC:

TABLE-US-00013 System: Sepiatec: Prep SFC100, Column: Chiralpak ID 5 μm 250 × 20 mm Solvent: CO2/2-propanol 64/36 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 18.1 5.0-7.0 18.2 8.0-11.0

Example 18.1

N-[1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0779] ##STR00113##

[0780] Chiralpak ID 5 μm 100×4.6 mm (CO2/2-Propanol 64/36, 4.0 mL/min) R.sub.t=2.32 min

[0781] [α].sub.D=+52.5° (c: 0.3, DMSO)

Example 18.2

N-[1-{N′-cyano-N-[5-(difluoromethoxy)-2-methoxyphenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0782] ##STR00114##

[0783] Chiralpak ID 5 m 100×4.6 mm (CO2/2-Propanol 64/36, 4.0 mL/min) R.sub.t=3.93 min

[0784] [α].sub.D=−58.4° (c: 0.22, DMSO)

Example 19

Rac-N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0785] ##STR00115##

[0786] Example 19 was prepared analogously to example 4 starting from intermediate 31 instead intermediate 12.

[0787] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.06 (br. s., 3H), 3.91 (s, 3H), 3.96-4.16 (m, 3H), 4.37 (t, 1H), 4.78 (t, 1H), 7.29 (d, 1H), 7.61 (d, 1H), 7.64-7.72 (m, 2H), 7.75 (d, 1H), 8.13 (br. s., 1H), 9.68 (br. s., 1H). (2H obscured by water signal)

[0788] LCMS (method 6): R.sub.t=0.84

[0789] MS (ESI): [M+H].sup.+=557.2

[0790] Example 19 was separated into its enantiomers by chiral SFC:

TABLE-US-00014 System: Sepiatec: Prep SFC100, Column: Chiralpak ID 5 μm 250 × 20 mm Solvent: CO2/2-propanol 65/35 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 19.1 4.0-5.5 19.2 6.5-8.0

Example 19.1

N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0791] ##STR00116##

[0792] Chiralpak ID 5 μm 100×4.6 mm (CO2/2-Propanol 65/35, 4.0 mL/min) R.sub.t=1.90 min

[0793] [α].sub.D=+90.7° (c: 0.31, MeOH)

Example 19.2

N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0794] ##STR00117##

[0795] Chiralpak ID 5 m 100×4.6 mm (CO2/2-Propanol 65/35, 4.0 mL/min) R.sub.t=3.28 min

[0796] [α].sub.D=−91.2° (c: 0.37, MeOH)

Example 20

Rac-N-[1-{N′-cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0797] ##STR00118##

[0798] Example 20 was prepared analogously to example 4 starting from intermediate 20 instead intermediate 12.

[0799] LCMS (method 6): R.sub.t=0.87

[0800] MS (ESI): [M+H].sup.+=526.14

[0801] Example 20 was separated into its enantiomers by chiral SFC:

TABLE-US-00015 System: Sepiatec: Prep SFC100, Column: Chiralpak ID 5 μm 250 × 20 mm Solvent: CO2/2-Propanol 71/29 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 20.1 3.5-5.0 20.2 5.0-7.0

Example 20.1

N-[1-{N′-cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0802] ##STR00119##

[0803] Chiralpak ID 5 m 100×4.6 mm (CO2/2-Propanol 71/29, 4.0 mL/min) R.sub.t=2.74 min

[0804] [α].sub.D=+69.7° (c: 0.29, DMSO)

Example 20.2

N-[1-{N′-cyano-N-[6-(difluoromethoxy)pyridin-2-yl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0805] ##STR00120##

[0806] Chiralpak ID 5 μm 100×4.6 mm (CO2/2-Propanol 71/29, 4.0 mL/min) R.sub.t=3.82 min

[0807] [α].sub.D=−56.2° (c: 0.53, MeOH)

Example 21

Rac-N-[1-{N′-cyano-N-[3-(2,2,2-trifluoroethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0808] ##STR00121##

[0809] Example 21 was prepared analogously to example 4 starting from intermediate 21 instead intermediate 12.

[0810] MS (ESI): [M+H].sup.+=541

[0811] Example 21 was separated into its enantiomers by chiral SFC:

TABLE-US-00016 System: Sepiatec: Prep SFC100, Column: Chiralpak IC 5 μm 250 × 30 mm Solvent: CO2/Methanol 66/34 Flow: 100 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 21.1 6.25-7.75 21.2 8.30-9.55

Example 21.1

N-[1-{N′-cyano-N-[3-(2,2,2-trifluoroethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0812] ##STR00122##

[0813] Chiralpak IC 5 μm 100×4.6 mm (CO2/Methanol 66/34, 4.0 mL/min) R.sub.t=2.42 min

Example 21.2

N-[1-{N′-cyano-N-[3-(2,2,2-trifluoroethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0814] ##STR00123##

[0815] Chiralpak IC 5 μm 100×4.6 mm (CO2/Methanol 66/34, 4.0 mL/min) R.sub.t=3.04 min

Example 22

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)-4-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0816] ##STR00124##

[0817] Example 22 was prepared analogously to example 4 starting from intermediate 22 instead intermediate 12.

[0818] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.05 (m, 3H), 3.94-4.23 (m, 3H), 4.23-4.52 (m, 1H), 4.75 (m, 1H), 5.86 (br. s., 1H), 7.01-7.49 (m, 4H), 7.63 (d, 1H), 7.74 (d, 1H), 8.09 (br. s., 1H), 9.86 (br. s., 1H), two hydrogens obscured by solvent or water signal.

[0819] MS (ESI): [M+H].sup.+=543

Example 23

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)-2-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0820] ##STR00125##

[0821] Example 23 was prepared analogously to example 4 starting from intermediate 36 instead intermediate 12.

[0822] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.07 (m, 3H), 3.89-4.18 (m, 3H), 4.32-4.48 (m, 1H), 4.74 (t, 1H), 5.79 (br. s., 1H), 7.15-7.36 (m, 4H), 7.61 (d, 1H), 7.74 (d, 1H), 8.10 (br. s., 1H). 9.82 (br. s., 1H)

[0823] MS (ESI): [M+H].sup.+=543

Example 24

Rac-N-[1-{N′-cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0824] ##STR00126##

[0825] Example 24 was prepared analogously to example 4 starting from intermediate 23 instead intermediate 12.

[0826] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.08 (br. s., 3H), 3.41 (br. s., 1H), 3.99-4.17 (m, 3H), 4.50 (t, 1H), 4.77 (t, 1H), 6.95 (d, 1H), 7.09 (br. s., 1H), 7.13 (s, 0.25H), 7.19 (d, 1H), 7.31 (s, 0.5H), 7.49 (s, 0.25H), 7.66 (d, 1H), 7.77 (d, 1H), 8.11 (s, 1H), 9.94 (br. s., 1H).

[0827] LCMS (method 2): R.sub.t=1.06 min

[0828] MS (ESI): [M+H].sup.+=543.2

[0829] Example 24 was separated into its diastereomers by chiral HPLC:

TABLE-US-00017 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Prep FC Column: Chiralpak ID 5 μm 250 × 30 mm Nr. 018 Solvent: Hexane/Ethanol/Diethylamine 70:30:0.1 (v/v/v) Flow: 40 mL/min Temperature: RT Detection: UV 325 nm Example No Rt in min 24.1 8.1-9.7 24.2 6.5-7.7

Example 24.1

N-[1-{N′-cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0830] ##STR00127##

[0831] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol/Diethylamine 70:30:0.1 (v/v/v), 1.0 mL/min) R.sub.t=2.68 min

[0832] [α].sub.D=+38.2° (c: 1.0, DMSO)

Example 24.2

N-[1-{N′-cyano-N-[3-(difluoromethoxy)-5-fluorophenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0833] ##STR00128##

[0834] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol/Diethylamine 70:30:0.1 (v/v/v), 1.0 mL/min) R.sub.t=3.81 min

[0835] [α].sub.D=−38.1° (c: 1.0, DMSO)

Example 25

Rac-N-[1-{N′-cyano-N-[3-fluoro-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0836] ##STR00129##

[0837] Example 25 was prepared analogously to example 4 starting from intermediate 24 instead intermediate 12.

[0838] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=0.79 (br. s., 1H), 0.99 (br. s., 3H), 2.99-3.21 (m, 1H), 3.96-4.16 (m, 3H), 4.18-4.44 (m, 2H), 4.70 (t, 1H), 5.12 (br. s., 1H), 5.80 (d, 1H), 6.59-6.88 (m, 1H), 6.92-7.12 (m, 2H), 7.52-7.64 (m, 1H), 7.68 (d, 1H), 7.86-7.95 (m, 1H).

[0839] LCMS (method 2): R.sub.t=1.14 min

[0840] MS (ESI): [M+H].sup.+=561.3

[0841] Example 25 was separated into its diastereomers by chiral HPLC:

TABLE-US-00018 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Prep FC Column: Chiralpak IC 5 μm 250 × 30 mm Nr. 009 Solvent: Hexane/Ethanol/Diethylamine 70:30:0.1 (v/v/v) Flow: 40 mL/min Temperature: RT Detection: UV 325 nm Example No Rt in min 25.1 11.4-13.4 25.2 20.9-23.6

Example 25.1

N-[1-{N′-cyano-N-[3-fluoro-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0842] ##STR00130##

[0843] Chiralpak IC 3 μm 100×4.6 mm (Hexan/Ethanol/Diethylamine 70:30:0.1 (v/v/v), 1 mL/min) R.sub.t=4.09 min

[0844] [α].sub.D=−35.8° (c: 1.0, DMSO)

Example 25.2

N-[1-{N′-cyano-N-[3-fluoro-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0845] ##STR00131##

[0846] Chiralpak IC 3 μm 100×4.6 mm (Hexan/Ethanol/Diethylamine 70:30:0.1 (v/v/v), 1 mL/min) R.sub.t=7.66 min

[0847] [α].sub.D=+32.7° (c: 1.0, DMSO)

Example 26

Rac-N-[1-{N′-cyano-N-[2-methoxy-5-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0848] ##STR00132##

[0849] Example 26 was prepared analogously to example 4 starting from intermediate 37 instead intermediate 12.

[0850] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.07 (m, 3H), 3.17-3.47 (m, 2H, overlain by water signal), 3.85 (s, 3H), 3.94-4.16 (m, 3H), 4.38 (t, 1H), 4.75 (t, 1H), 5.79 (br. s., 1H), 7.15-7.21 (m, 1H), 7.29-7.35 (m, 1H), 7.37 (br. s., 1H), 7.61 (d, 1H), 7.75 (d, 1H), 8.12 (s, 1H), 9.61 (br. s., 1H).

[0851] MS (ESI): [M+H].sup.+=573

Example 27

Rac-N-[1-{N′-cyano-N-[2-(pyrrolidin-1-ylmethyl)-5-(trifluoromethyl)phenyl]carbamimidoy}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0852] ##STR00133##

[0853] Example 27 was prepared analogously to example 4 starting from intermediate 25 instead intermediate 12.

[0854] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): d [ppm]=1.09 (m, 3H), 1.68 (m, 4H), 3.16-3.55 (m, 3H), 3.78 (s, 2H), 3.95-4.26 (m, 3H), 4.52 (t, 1H), 5.83 (br. s., 1H), 7.39-7.65 (m, 3H), 7.78 (d, 1H), 7.91 (d, 2H); 10.95 (br. s., 1H); four hydrogens obscured by solvent or water signals.

[0855] MS (ESI): [M+H].sup.+=610

[0856] Example 27 was separated into its enantiomers by chiral SFC:

TABLE-US-00019 System: Sepiatec: Prep SFC100, Column: Chiralpak IC 5 μm 250 × 20 mm Solvent: CO2/Ethanol 63/37 Flow: 80 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 27.1 3.10-3.85 27.2 4.25-5.20

Example 27.1

N-[1-{N′-cyano-N-[2-(pyrrolidin-1-ylmethyl)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0857] ##STR00134##

[0858] Chiralpak IC 5 μm 100×4.6 mm (CO2/Ethanol 63:37, 4.0 mL/min) R.sub.t=3.69 min

Example 27.2

N-[1-{N′-cyano-N-[2-(pyrrolidin-1-Ylmethyl)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0859] ##STR00135##

[0860] Chiralpak IC 5 μm 100×4.6 mm (CO2/Ethanol 63:37, 4.0 mL/min) R.sub.t=5.62 min

Example 28

Rac-N-[1-{N′-cyano-N-[2-(trifluoromethoxy)-5-(trifluoromethyl)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0861] ##STR00136##

[0862] Example 28 was prepared analogously to example 4 starting from intermediate 26 instead intermediate 12.

[0863] LCMS (method 2): R.sub.t=1.07

[0864] MS (ESI): [M+H].sup.+=610.7

Example 29

Rac-N-[1-(N′-cyano-N-{5-(difluoromethoxy)-2-[3-(dimethylamino)propoxy]phenyl}-carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0865] ##STR00137##

[0866] Example 29 was prepared analogously to example 4 starting from intermediate 38 instead intermediate 12.

[0867] LCMS (method 2): R.sub.t=1.31

[0868] MS (ESI): [M+H].sup.+=626

Example 30

Rac-N-[r-(N′-cyano-N-[2-[2-(pyrrolidin-1-yl)ethoxy]-5-(trifluormethyl)phenyl]carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0869] ##STR00138##

[0870] Example 30 was prepared analogously to example 4 starting from intermediate 27 instead intermediate 12.

[0871] LCMS (method): R.sub.t=1.34

[0872] MS (ESI): [M+H].sup.+=640.3

Example 31

Rac-N-[1-(N′-cyano-N-{2-[(1-methylpiperidin-4-yl)oxy]-5-(trifluoromethyl)phenyl}-carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0873] ##STR00139##

[0874] Example 31 was prepared analogously to example 4 starting from intermediate 39 instead intermediate 12.

[0875] LCMS (method 2): R.sub.t=1.27

[0876] MS (ESI): [M+H].sup.+=640.4

Example 32

Rac-N-[1-(N′-cyano-N-{2-[(1-methylpiperidin-4-yl)oxy]-5-(trifluoromethyl)phenyl}-carbamimidoyl)-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0877] ##STR00140##

[0878] Example 32 was prepared analogously to example 4 starting from intermediate 28 instead intermediate 12.

[0879] LCMS (method 2): R.sub.t=1.25

[0880] MS (ESI): [M+H].sup.+=656.3

Example 33

Rac-N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]-carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0881] ##STR00141##

[0882] Example 33 was prepared analogously to example 4 starting from intermediate 13 instead intermediate 12.

[0883] LCMS (method 7): R.sub.t=2.59

[0884] MS (ESI): [M+H].sup.+=505.0

[0885] Example 33 was separated into its enantiomers by chiral HPLC:

TABLE-US-00020 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Gilson: Liquid Handler 215 Column: Chiralpak ID 5 μm 250 × 30 mm Nr.: 018 Solvent: Hexan/Ethanol 70:30 (v/v) Flow: 50 mL/min Temperature: RT Detection: UV 280 nm Example No Rt in min 33.1  7.2-9.0 33.2 10.9-12.9

Example 33.1

N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 1

[0886] ##STR00142##

[0887] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol 70:30 (v/v), 1.0 mL/min) R.sub.t=4.00 min

[0888] [α].sub.D=+97.7° (c: 0.82, MeOH)

Example 33.2

N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide Isomer 2

[0889] ##STR00143##

[0890] Chiralpak ID 3 μm 100×4.6 mm (Hexan/Ethanol 70:30 (v/v), 1.0 mL/min) R.sub.t=6.22 min

[0891] [α].sub.D=−96.8° (c: 0.88, MeOH)

Example 34

N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-L-alaninamide (1:1 Mixture of Diastereomers)

[0892] ##STR00144##

[0893] Example 34 was prepared analogously to example 3 starting from intermediate 13 instead intermediate 12 and using Fmoc-L-alanine instead Fmoc-glycine for the amide coupling.

[0894] LCMS (method 2): R.sub.t=0.94 and 0.96 (two diastereomers)

[0895] MS (ESI): [M+H].sup.+=518.1

[0896] Example 34 was separated into its diastereomers by chiral SFC:

TABLE-US-00021 System: Sepiatec: Prep SFC100, Column: Chiralpak IB 5 μm 250 × 30 mm Solvent: CO2/ethanol + 0.4% DEA 8/2 Flow: 100 mL/min Temperature: 40° C. Detection: UV 254 nm Example No Rt in min 34.1 2.0-2.5 34.2 4.0-5.0

Example 34.1

N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-L-alaninamide Isomer 1

[0897] ##STR00145##

[0898] Chiralpak IB 5 μm 100×4.6 mm (CO2/Ethanol+0.2% DEA, 4.0 mL/min) R.sub.t=2.33 min

Example 34.2

N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-L-alaninamide Isomer 2

[0899] ##STR00146##

[0900] Chiralpak TB 5 μm 100×4.6 mm (CO2/Ethanol+0.2% DEA, 4.0 mL/min) R.sub.t=3.24 min

Example 35

Rac-N-[3-(4-chloro-3-fluorophenyl)-1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]-carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-2-hydroxyacetamide

[0901] ##STR00147##

[0902] Example 35 was prepared analogously to example 4 starting from intermediate 29 instead intermediate 12.

[0903] LCMS (method 2): R.sub.t=1.18

[0904] MS (ESI): [M+H].sup.+=526.8

[0905] Further, the compounds of formula (I) of the present invention can be converted to any salt as described herein, by any method which is known to the person skilled in the art. Similarly, any salt of a compound of formula (I) of the present invention can be converted into the free compound, by any method which is known to the person skilled in the art.

Example 36

rac-N-[3-(4-Chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N.SUP.2.-methylglycinamide

[0906] ##STR00148##

[0907] Example 36 was prepared starting from intermediate 13 according to the following scheme:

##STR00149##

Step 1:

[0908] Step 1 was carried out analogously to step 1 described for example 3, starting from intermediate 13 to yield rac-4-amino-3-(4-chloro-3-methylphenyl)-N′-cyano-N-[3-(difluoromethoxy)phenyl]-4,5-dihydro-1H-pyrazole-1-caboximidamide.

[0909] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=2.43 (s, 3H), 4.37 (dd, 1H), 4.46 (dd, 1H), 4.87 (dd, 1H), 6.55 (t, 1H), 6.96 (dd, 1H), 7.22-7.29 (m, 2H), 7.29-7.56 (m, 3H), 7.60-7.69 (m, 2H), 7.75 (d, 1H), 8.17 (br s, 1H).

[0910] LCMS (method 3): R.sub.t=1.63 min

[0911] MS (ESI): [M+H].sup.+=419.1

Step 2:

[0912] Step 1 was carried out analogously to step 2 described for example 3, to obtain rac-3-(4-chloro-3-methylphenyl)-N′-cyano-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide.

[0913] .sup.1H NMR (400 MHz, CDCl.sub.3): δ [ppm]=1.11 (t, 3H), 2.43 (s, 3H), 2.61-2.71 (m, 2H), 4.32 (dd, 1H), 4.54 (dd, 1H), 4.81 (dd, 1H), 6.56 (t, 1H), 6.97 (dd, 1H), 7.21-7.43 (m, 4H), 7.67 (dd, 1H), 7.77 (d, 1H), 8.12 (br s, 1H).

[0914] LCMS (method 3): R.sub.t=1.79 min

[0915] MS (ESI): [M+H].sup.+=445.1

Step 3:

[0916] To a solution of N-(tert-butoxycarbonyl)-N-methylglycine in DMF (3.0 mL) were added 1-[Bis-(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU), 255 mg (671 μmol) and 4-methylmorpholine (148 μL). After stirring for 30 min at room temperature was added rac-3-(4-chloro-3-methylphenyl)-N′-cyano-N-[3-(difluoromethoxy)phenyl]-4-(ethylamino)-4,5-dihydro-1H-pyrazole-1-carboximidamide (150 mg, 366 μmol). The reaction mixture stirred overnight at room temperature and was then purified by preparative HPLC (Waters XBrigde C18 5 μm 100×30 mm, 0.2% aqueous ammonia, acetonitrile) to give rac-tert-butyl-(2-{[(4S)-3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl](ethyl)amino}-2-oxoethyl)methylcarbamate, 120 mg (56%) as a white solid.

[0917] UPLC-MS (method 2): R.sub.t=1.43 min

[0918] MS (ESI): [M+H].sup.+=618.4

Step 4:

[0919] To a solution of tert-butyl (2-{[(4S)-3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoro-methoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl](ethyl)amino}-2-oxoethyl)methylcarbamate, 120 mg (194 μmol) in 1,2-dichloroethane (6 mL) was added zinc bromide, 87 mg (389 μmol). The reaction mixture stirred 3 h at 60° C. and was then diluted with an aqueous pH 10 buffer and dichloromethane. The organic layer was washed with brine, dried and concentrated under reduced pressure. The residue was purified by preparative HPLC (Waters XBrigde C18 5 μm 100×30 mm, 0.2% aqueous ammonia, acetonitrile) to give rac-N-[3-(4-Chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N2-methylglycinamide, 40 mg (41%) as a white solid.

[0920] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.756 (0.47), 0.775 (0.84), 0.793 (0.52), 1.052 (2.76), 1.329 (0.54), 2.128 (0.59), 2.168 (6.94), 2.323 (1.06), 2.327 (1.46), 2.331 (1.53), 2.347 (16.00), 2.361 (7.49), 2.363 (6.97), 2.523 (7.80), 2.539 (2.63), 2.665 (0.71), 2.669 (0.93), 2.674 (0.65), 3.219 (1.68), 3.260 (3.74), 3.362 (2.99), 3.464 (0.71), 3.502 (0.55), 4.082 (0.84), 4.094 (1.02), 4.113 (1.15), 4.125 (1.20), 4.431 (1.08), 4.460 (2.18), 4.490 (1.01), 6.980 (1.85), 6.987 (2.32), 7.001 (2.22), 7.008 (2.55), 7.056 (2.89), 7.205 (3.28), 7.210 (4.64), 7.216 (2.92), 7.241 (6.99), 7.250 (2.58), 7.265 (2.80), 7.267 (2.92), 7.392 (2.95), 7.412 (4.90), 7.426 (3.50), 7.433 (2.89), 7.452 (0.89), 7.497 (2.27), 7.518 (3.70), 7.560 (0.79), 7.578 (2.39), 7.580 (2.59), 7.583 (2.56), 7.599 (1.46), 7.604 (1.50), 7.774 (3.16), 7.776 (3.36), 7.780 (3.00), 7.923 (0.93), 7.925 (0.95).

[0921] UPLC-MS (method 2): R.sub.t=1.20 min

[0922] MS (ESI): [M+H].sup.+=518.3

[0923] Example 36 was separated into its isomers by chiral preparative HPLC:

TABLE-US-00022 System: Agilent: Prep 1200, 2 × Prep Pump, DLA, MWD, Preparative FC, Column: Chiralpak IE 5 μm 250 × 20 mm Solvent: acetonitrile/ethanol 90:10 + 0.1% diethylamine Flow: 15 mL/min Temperature: room temperature Detection: UV 254 nm solution 36 mg/1.5 mL dichloromethane/methanol 1:1 injection 8 × 0.2 mL Example No R.sub.t in min 36.1  8.0-9.2 36.2 10.4-12.5

[0924] Analytical chiral HPLC method: Instrument: Agilent 1260/Agilent 1290; column: Chiralpak IE 3 μm 100×4.6 mm; eluent: acetonitrile/ethanol 90:10+0.1% diethylamine; flow 1.0 mL/min; temperature: 25° C.; solution: 1.0 mg/mL ethanol/methanol 1:1; injection: 5 μL; detection: DAD 254 nm.

Example 36.1

N-[3-(4-Chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N2-methylglycinamide Isomer 2

[0925] ##STR00150##

[0926] Analytical chiral HPLC: R.sub.t=2.72 min

[0927] [α].sub.D.sup.20=−+61.6° (c: 0.33 DMSO)

Example 36.2

N-[3-(4-Chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N2-methylglycinamide Isomer 1

[0928] ##STR00151##

[0929] Analytical chiral HPLC: R.sub.t=3.55 min

[0930] [α].sub.D.sup.20=−64.0° (c: 0.37, DMSO)

Example 37

N-[3-(4-chloro-3-methylphenyl)-1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-N2-methyl-D-alaninamide

[0931] ##STR00152##

[0932] Example 37 was prepared analogously to example 3 starting from intermediate 13 instead intermediate 12 and using N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-alanine instead N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine for the amide coupling.

[0933] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.754 (0.43), 0.772 (0.83), 0.790 (0.54), 0.811 (0.41), 0.863 (0.45), 0.970 (1.08), 1.014 (5.72), 1.020 (3.18), 1.030 (5.97), 1.036 (2.95), 1.110 (4.87), 1.128 (1.62), 1.147 (1.15), 1.160 (1.19), 1.176 (1.01), 1.197 (0.47), 1.235 (1.10), 1.278 (0.43), 1.292 (0.52), 1.295 (0.52), 1.775 (0.47), 1.808 (0.47), 1.907 (0.70), 1.919 (0.79), 1.951 (0.74), 2.045 (0.41), 2.131 (11.88), 2.140 (3.90), 2.145 (1.42), 2.162 (2.77), 2.264 (0.56), 2.322 (2.95), 2.336 (16.00), 2.355 (3.81), 2.358 (3.99), 2.417 (0.52), 2.523 (4.33), 2.660 (0.50), 2.664 (0.99), 2.669 (1.44), 2.674 (1.06), 2.679 (0.61), 2.693 (0.45), 2.938 (1.08), 2.956 (1.10), 3.090 (1.31), 3.103 (1.22), 3.204 (0.52), 3.386 (0.99), 3.405 (1.60), 3.422 (1.78), 3.437 (1.60), 3.453 (1.24), 3.474 (0.59), 3.975 (0.47), 4.163 (0.41), 4.471 (0.95), 4.501 (1.60), 4.529 (0.72), 5.371 (0.77), 5.435 (0.77), 6.991 (1.71), 6.998 (2.37), 7.012 (2.25), 7.019 (2.61), 7.053 (4.21), 7.214 (3.49), 7.220 (6.35), 7.226 (3.90), 7.238 (8.65), 7.254 (2.79), 7.255 (3.27), 7.261 (2.43), 7.274 (3.43), 7.279 (3.70), 7.281 (3.15), 7.300 (1.13), 7.303 (1.10), 7.319 (0.79), 7.322 (0.74), 7.355 (0.61), 7.370 (0.86), 7.389 (0.47), 7.400 (3.47), 7.420 (6.11), 7.423 (7.35), 7.441 (2.70), 7.493 (2.93), 7.514 (4.15), 7.551 (0.41), 7.572 (0.56), 7.581 (1.44), 7.587 (1.55), 7.602 (1.04), 7.608 (1.10), 7.623 (1.22), 7.644 (0.88), 7.696 (0.99), 7.716 (1.98), 7.718 (2.05), 7.723 (1.76), 7.756 (1.87), 7.762 (1.78), 7.843 (1.15), 7.860 (1.15), 7.891 (0.61), 7.893 (0.59).

[0934] UPLC-MS (method 2): R.sub.t=1.20 min

[0935] MS (ESI): [M+H].sup.+=518.3

Example 38

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamide

[0936] ##STR00153##

[0937] Example 38 was prepared analogously to example 3, omitting step 2 and using N-[(9H-fluoren-9-yl-methoxy)carbonyl]-3-methyl-D-isovaline instead of N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine to give N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamid as a white solid.

[0938] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.564 (6.16), 0.581 (6.49), 0.600 (5.47), 0.617 (5.57), 0.718 (6.76), 0.727 (6.51), 0.735 (7.61), 0.744 (6.04), 1.021 (16.00), 1.025 (13.30), 1.109 (2.48), 1.251 (0.56), 1.852 (0.51), 1.870 (1.30), 1.887 (1.99), 1.905 (1.88), 1.923 (1.13), 1.940 (0.42), 2.073 (1.02), 2.523 (2.19), 2.532 (0.97), 2.537 (0.78), 4.016 (1.00), 4.030 (1.07), 4.044 (1.23), 4.054 (1.25), 4.058 (1.40), 4.066 (0.99), 4.082 (0.98), 4.095 (0.95), 4.429 (0.93), 4.436 (1.10), 4.457 (2.15), 4.464 (2.53), 4.486 (0.93), 4.493 (1.05), 5.741 (0.85), 5.754 (1.01), 5.763 (1.07), 5.769 (1.27), 5.777 (1.17), 5.783 (1.06), 5.792 (0.92), 5.805 (0.73), 6.998 (1.90), 7.000 (1.97), 7.003 (1.98), 7.005 (2.05), 7.019 (2.22), 7.025 (2.26), 7.061 (3.00), 7.202 (1.94), 7.208 (4.12), 7.213 (3.97), 7.219 (1.68), 7.246 (6.08), 7.255 (1.67), 7.261 (2.25), 7.275 (1.89), 7.280 (2.78), 7.407 (3.32), 7.427 (6.05), 7.448 (2.37), 7.733 (3.16), 7.736 (3.51), 7.754 (3.93), 7.758 (4.69), 7.846 (2.32), 7.851 (2.26), 7.868 (1.72), 7.872 (1.87), 7.876 (2.09), 7.881 (1.84), 7.897 (1.36), 7.902 (1.38), 8.142 (3.27), 8.147 (3.19), 8.154 (2.89), 8.159 (2.69).

[0939] LC-MS (method 9): R.sub.t=1.05 min

[0940] MS (ESI): [M+H].sup.+=552.0

[0941] Example 38 was separated into its isomers by chiral SFC:

TABLE-US-00023 System: Sepiatec: Prep SFC100, Column: Chiralpak ID 5 μm 250 × 30 mm Solvent: carbon dioxide/2-propnaol + 0.2% diethylamine 70/30 Flow: 100 mL/min Temperature: 40° C. Detection: UV 254 nm Pressure 150 bar solution 240 mg/4 mL methanol/DMSO 3:1 injection 8 × 0.5 mL Example No R.sub.t in min 38.1  6.5-9.0 38.2 14.0-18.0

[0942] Analytical chiral HPLC method: Instrument: Agilent: 1260 AS, MWD, Aurora SFC-Modul; column: Chiralpak IC 5 μm 100×4.6 mm; eluent: carbon dioxide/2-propnaol+0.2% diethylamine 70:30; flow 4.0 mL/min; temperature: 37.5° C.; solution: 1.0 mg/mL ethanol/methanol 1:1; injection: 10 μL; detection: DAD 254 nm.

Example 38.1

N-[1-{N′-cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamide Isomer 2

[0943] ##STR00154##

[0944] Analytical chiral HPLC: R.sub.t=3.78 min

[0945] [α].sub.D.sup.20=+34.0° (c: 0.22, DMSO)

Example 38.2

N-[1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-3-methyl-D-isovalinamide Isomer 1

[0946] ##STR00155##

[0947] Analytical chiral HPLC: R.sub.t=7.41 min

[0948] [α].sub.D.sup.20=+16.6° (c: 0.28, DMSO)

Example 39

N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-leucinamide

[0949] ##STR00156##

[0950] Example 38 was prepared analogously to example 3, starting from intermediate 16 using N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-leucine instead of N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine to give N-[1-{N′-cyano-N-[3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-leucinamide.

[0951] UPLC-MS (method 2): R.sub.t=1.41 min

[0952] MS (ESI): [M+H].sup.+=598.3

Example 40

N-[1-{N′-Cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-valinamide

[0953] ##STR00157##

[0954] Example 40 was prepared analogously to example 3, starting from intermediate 15 and using N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-valine instead of N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine to give N-[1-{N′-Cyano-N-[2-fluoro-3-(trifluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-valinamide.

[0955] .sup.1H-NMR (400 MHz, CHLOROFORM-d) 6 [ppm]: 0.658 (0.58), 0.675 (0.57), 0.895 (5.94), 0.912 (6.12), 0.952 (0.73), 0.974 (5.65), 0.991 (5.61), 1.193 (1.44), 1.212 (2.63), 1.229 (1.45), 1.261 (1.37), 1.825 (0.69), 1.842 (1.06), 1.859 (0.99), 1.875 (0.61), 2.015 (16.00), 3.259 (2.00), 3.275 (2.07), 3.299 (0.61), 3.319 (0.50), 3.328 (0.40), 4.531 (0.41), 4.615 (0.53), 7.192 (0.90), 7.207 (3.10), 7.216 (0.88), 7.223 (1.52), 7.226 (1.52), 7.247 (0.49), 7.502 (2.22), 7.523 (3.68), 7.577 (1.52), 7.582 (1.55), 7.597 (0.90), 7.603 (0.96), 7.813 (2.55), 7.818 (2.48), 7.920 (0.74), 7.925 (0.61), 7.938 (0.80), 7.943 (0.86), 7.945 (0.87), 7.954 (0.46), 7.962 (0.43).

[0956] UPLC-MS (method 2): R.sub.t=1.06

[0957] MS (ESI): [M+H].sup.+=602.3

Example 41

N-[1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-valinamide

[0958] ##STR00158##

[0959] Example 41 was prepared analogously to example 3 using N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-valine instead of N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine to give N-[1-{N′-Cyano-N-[3-(difluoromethoxy)phenyl]carbamimidoyl}-3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-4-yl]-N-ethyl-D-valinamide.

[0960] .sup.1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.466 (3.32), 0.528 (0.67), 0.545 (0.58), 0.629 (0.44), 0.682 (4.75), 0.699 (4.81), 0.770 (0.70), 0.793 (0.64), 0.811 (0.52), 0.868 (1.08), 0.886 (2.10), 0.903 (1.52), 0.920 (3.18), 0.932 (13.29), 0.940 (15.80), 0.949 (14.43), 0.958 (14.05), 1.036 (0.99), 1.054 (1.54), 1.071 (3.91), 1.088 (7.72), 1.109 (4.93), 1.126 (1.17), 1.140 (0.96), 1.157 (0.79), 1.210 (2.30), 1.229 (4.23), 1.246 (2.91), 1.754 (0.85), 2.034 (1.17), 2.051 (1.81), 2.066 (1.75), 2.071 (2.56), 2.084 (1.25), 2.101 (0.55), 2.322 (1.19), 2.327 (1.72), 2.332 (1.22), 2.336 (0.61), 2.523 (5.60), 2.665 (1.17), 2.669 (1.72), 2.674 (1.14), 2.679 (0.58), 3.355 (1.14), 3.373 (1.31), 3.395 (1.11), 3.412 (0.79), 3.727 (0.96), 3.745 (1.14), 3.769 (0.99), 3.787 (0.73), 4.100 (2.74), 4.114 (3.35), 4.137 (3.96), 4.152 (2.94), 4.166 (2.68), 4.181 (2.71), 4.239 (1.84), 4.268 (2.21), 4.301 (1.31), 4.469 (2.68), 4.498 (4.49), 4.528 (2.42), 4.571 (1.95), 4.600 (3.44), 4.629 (1.98), 6.991 (0.55), 7.019 (2.56), 7.026 (2.80), 7.034 (3.03), 7.041 (5.74), 7.046 (3.85), 7.055 (3.38), 7.060 (8.66), 7.063 (9.59), 7.149 (0.41), 7.177 (0.50), 7.189 (3.35), 7.195 (6.18), 7.201 (3.67), 7.228 (4.26), 7.233 (6.91), 7.238 (5.07), 7.245 (15.07), 7.248 (16.00), 7.257 (1.63), 7.265 (3.64), 7.271 (6.35), 7.276 (3.44), 7.290 (3.67), 7.296 (3.50), 7.415 (5.01), 7.425 (5.80), 7.430 (6.38), 7.432 (7.87), 7.436 (8.54), 7.446 (8.28), 7.456 (3.93), 7.466 (3.64), 7.473 (0.79), 7.581 (0.44), 7.586 (0.52), 7.602 (0.70), 7.607 (0.64), 7.700 (3.61), 7.706 (2.65), 7.712 (3.82), 7.717 (4.08), 7.721 (6.99), 7.726 (6.59), 7.733 (5.62), 7.738 (6.12), 7.747 (11.77), 7.769 (4.43), 7.784 (0.70), 7.798 (9.62), 7.819 (5.89), 7.990 (4.26), 8.021 (6.82), 8.026 (6.64), 8.064 (0.70), 8.127 (7.46), 8.132 (7.69), 8.168 (4.23), 8.254 (1.60), 8.259 (1.84), 8.278 (0.85), 9.832 (3.32), 9.951 (4.02), 10.040 (1.02).

[0961] LC-MS (method 5): R.sub.t=1.02 and 1.04 min (2 diastereomers)

[0962] MS (ESI): [M+H].sup.+=566.1

Comparison Example

[0963] To show superiority of the inventive compounds over the closest state of the art compounds that have been disclosed in WO 2006/072350, the following comparison example has been done:

TABLE-US-00024 IC.sub.50 Example No. Structure [mol/l] (SPA Assay) 4.1 of the instant invention [00159] 2.82E−8 WO 2006/072350 Example 67 more active isomer [00160] 5.48E−7
Purification, Crystallization and Crystal Structure Determination of Human SMYD2 in Complex with SAM and Example 4.1

Purification of Human SMYD2

[0964] Recombinant human SMYD2 (Uniprot Q9NRG4; amino acids 2-433) was expressed in insect cells (Sf9) containing a N-terminal TEV-cleavable 6×His-tag. Cell pellets were re-suspended in lysis buffer (40 mM Tris, pH8; 500 mM NaCl; 0.1% IGEPAL; 5 mM imidazole; 1 mM DTT) supplemented with complete EDTA-free protease inhibitor tablets and 50 U/mL benzonase. The cell lysate was loaded onto a Ni-NTA column, eluted with imidazole and concentrated using an ultra centrifugal filter unit. Subsequently SMYD2 was gel filtrated on a Superdex S200 column equilibrated in 20 mM Tris (pH 8), 100 mM NaCl, 5% glycerol, 1 mM DTT. The 6×His-tag was cleaved with TEV protease in solution overnight at 6° C. Uncleaved SMYD2 and TEV protease were separated from the cleaved product by applying a second Ni-NTA affinity step. The cleaved SMYD2 protein was further purified by a second gel filtration step using a Superdex 200 equilibrated in 20 mM Tris (pH 8), 150 mM NaCl, 5% glycerol, 1 mM TCEP. The protein was concentrated to 15.5 mg/mL (313 μM) (UV-Vis) using an ultra centrifugal filter unit and shock frozen in liquid nitrogen.

Crystallization of Human SMYD2

[0965] For crystallization, the co-factor S-adenosyl methionine (SAM) was added to a final concentration of 3.8 mM as follows: 1.2 μl of a SAM stock solution (100 mM in DMSO) were added to 30 μl of concentrated SMYD2 solution and incubated for 2 hours at 4° C. Crystals grew within 3 days at 20° C. using the hanging drop method. Drops were made from 1 μl SMYD2:SAM solution and 0.8 μl reservoir solution (20-24% (w/v) PEG 3350, 100 mM HEPES pH 7.0). 30 min after drop set-up, 0.2 μl of a seed solution were added. The seed solution was made from SMYD2:SAM crystals (obtained with same reservoir conditions in a previous experiment) which were crashed manually (using Seed Beads, Hampton Research), diluted in reservoir solution, shock frozen and stored at −80° C.

Complex Formation of Human SMYD2:SAM and Example 4.1 in the Crystal

[0966] For complex formation, a crystal was transferred into a new drop of 1.5 yl reservoir solution. A stock solution of Example 4.1 (100 mM in DMSO) was 10-fold diluted with reservoir solution. Over the course of 2 hours, 1.5 μl of this diluted stock solution were added in three steps of 0.5 μl to the drop containing the SMYD2:SAM crystal, resulting in a final concentration of 5 mM Example 4.1 in the soaking drop. The crystal was soaked in this drop for 4 days at 20° C.

Data Collection and Processing

[0967] The soaked crystal was briefly immerged in cryo buffer (0.1 M HEPES pH 7.0, 22% PEG 3350, 20% glycerol and 2 mM Example 4.1) and shock frozen in liquid nitrogen. A diffraction data set was collected at beamline 14.1 at Helmholtz-Zentrum Berlin at 100 K using a wavelength of 0.91841 Å and a PILATUS detector. The diffraction images were processed using the program XDS. The crystal diffracted to a resolution of 2.0 Å and belonged to space group P2.sub.12.sub.12.sub.1 with unit cell dimensions of a=52.3 Å and b=69.6 Å, c=131.1 Å with one molecule per asymmetric unit.

Structure Determination and Refinement

[0968] The crystal form described here was first solved for a SMYD2:SAM crystal in the absence of an inhibitor, using the Molecular Replacement method with the program PHASER from the CCP4 program suite and 3TG5 (PDB entry code) as search model. The data set for SMYD2:SAM:Example 4.1 was then solved by rigid body refinement using the SMYD2:SAM structure as starting model and the program REFMAC as part of the CCP4 program suite. A 3D model for Example 4.1 was generated using the program Discovery Studio and parameter files for crystallographic refinement and model building were generated using software PRODRG. Example 4.1 was manually built into the electron density maps using the program COOT, followed by several cycles of refinement (using program REFMAC) and rebuilding in COOT. The final co-complex structure features a R(work) of 23.0% and R(free) of 27.3%. The statistics of the data collection and refinement are summarized in Table 1.

TABLE-US-00025 TABLE 1 Data collection and refinement statistics for human SMYD2 in complex with SAM and Example 4.1 SMYD2:SAM:Example 4.1 Data Collection: Source BL 14.1 (Helmholtz-Zentrum Berlin) Wavelength [Å] 0.9841 Space group (no.) P2(1)2(1)2(1) (19) Unit cell parameters, a, b, c [Å] 52.3, 69.6, 131.1 Resolution limit [Å] 48.61-1.99 (2.11-1.99) No. of reflections 221439 No. of uniques 33656 Multiplicity 6.58 I/sigI 14.39 (2.41) R_meas [%] 10.1 (81.5) Completeness [%] 99.9 (99.5) B (Wilson) [Å.sup.2] 35.09 Mosaicity [deg] 0.129 Refinement Resolution limit [Å] 1.99-47.72 (1.99-2.04) Completeness [%] 99.9 (99.0) No. of reflections 31972 R (work)/R (free) [%] 23.0/27.3 (33.2/38.1) Mean B value [Å.sup.2] 55.5 RMSD bond length [Å] 0.017 RMSD bond angles [deg] 2.03 Values in brackets refer to the highest resolution shell.

Absolute Configuration of Example 4.1 in Human SMYD2

[0969] The complex of human SMYD2, SAM and Example 4.1 (FIG. X3) crystallizes with one molecule in the asymmetric unit. The stereo chemistry of Example 4.1 is unambiguously defined by the knowledge of the stereo chemistry of the protein human SMYD2. Example 4.1 unambiguously features the S configuration on carbon atom C1. (FIG. X3). (Wang L1, Li L, Zhang H, Luo X, Dai J, Zhou S, Gu J, Zhu J, Atadja P, Lu C, Li E, Zhao K. Structure of human SMYD2 protein reveals the basis of p53 tumor suppressor methylation.) [0970] References for the crystallographic software tools CCP4: M. D. Winn et al. Acta. Cryst. D67, 235-242 (2011) [0971] “Overview of the CCP4 suite and current developments” Phaser: J. Appl. Cryst. (2007). 40, 658-674. [0972] Phaser crystallographic software. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., & Read, R. J. [0973] Refmac: “Refinement of Macromolecular Structures by the Maximum-Likelihood method” G. N. Murshudov, A. A. Vagin and E. J. Dodson, (1997) in Acta Cryst. D53, 240-255. [0974] ProDrg: A. W. Schüttelkopf and D. M. F. van Aalten (2004). “PRODRG: a tool for high-throughput crystallography of protein-ligand complexes”, Acta Crystallogr D60, 1355-1363. [0975] COOT: Paul Emsley, Bernhard Lohkamp, William G. Scott, Kevin Cowtan, “Features and Development of Coot”, (2010) Acta Cryst. D66:486-501

Pharmaceutical Compositions of the Compounds

[0976] This invention also relates to pharmaceutical compositions containing one or more compounds of the present invention. These compositions can be utilised to achieve the desired pharmacological effect by administration to a patient in need thereof. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease. Therefore, the present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound, or salt thereof, of the present invention. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. A pharmaceutically effective amount of compound is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The compounds of the present invention can be administered with pharmaceutically-acceptable carriers well known in the art using any effective conventional dosage unit forms, including immediate, slow and timed release preparations, orally, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.

[0977] For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions. The solid unit dosage forms can be a capsule that can be of the ordinary hard- or soft-shelled gelatine type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.

[0978] In another embodiment, the compounds of this invention may be tableted with conventional tablet bases such as lactose, sucrose and cornstarch in combination with binders such as acacia, corn starch or gelatine, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, gum tragacanth, acacia, lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example talc, stearic acid, or magnesium, calcium or zinc stearate, dyes, colouring agents, and flavouring agents such as peppermint, oil of wintergreen, or cherry flavouring, intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent or emulsifying agent. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance tablets, pills or capsules may be coated with shellac, sugar or both.

[0979] Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example those sweetening, flavouring and colouring agents described above, may also be present.

[0980] The pharmaceutical compositions of this invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived form fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

[0981] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil such as, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as, for example, beeswax, hard paraffin, or cetyl alcohol. The suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more colouring agents; one or more flavouring agents; and one or more sweetening agents such as sucrose or saccharin.

[0982] Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, and preservative, such as methyl and propyl parabens and flavouring and colouring agents.

[0983] The compounds of this invention may also be administered parenterally, that is, subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly, or interperitoneally, as injectable dosages of the compound in preferably a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or, a fatty acid glyceride, or an acetylated fatty acid glyceride, with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent and other pharmaceutical adjuvants.

[0984] Illustrative of oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty acid alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; non-ionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and poly(oxyethylene-oxypropylene)s or ethylene oxide or propylene oxide copolymers; and amphoteric detergents, for example, alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures.

[0985] The parenteral compositions of this invention will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. In order to minimise or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) preferably of from about 12 to about 17. The quantity of surfactant in such formulation preferably ranges from about 5% to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB.

[0986] Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

[0987] The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadeca-ethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate.

[0988] The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents that may be employed are, for example, water, Ringer's solution, isotonic sodium chloride solutions and isotonic glucose solutions. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

[0989] A composition of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritation excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are, for example, cocoa butter and polyethylene glycol.

[0990] Another formulation employed in the methods of the present invention employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (see, e.g., U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, incorporated herein by reference). Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0991] Controlled release formulations for parenteral administration include liposomal, polymeric microsphere and polymeric gel formulations that are known in the art.

[0992] It may be desirable or necessary to introduce the pharmaceutical composition to the patient via a mechanical delivery device. The construction and use of mechanical delivery devices for the delivery of pharmaceutical agents is well known in the art. Direct techniques for, for example, administering a drug directly to the brain usually involve placement of a drug delivery catheter into the patient's ventricular system to bypass the blood-brain barrier. One such implantable delivery system, used for the transport of agents to specific anatomical regions of the body, is described in U.S. Pat. No. 5,011,472, issued Apr. 30, 1991.

[0993] The compositions of the invention can also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al., “Compendium of Excipients for Parenteral Formulations” PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311; Strickley, R. G “Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1” PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349; and Nema, S. et al., “Excipients and Their Use in Injectable Products” PDA Journal of Pharmaceutical Science & Technology 1997, 51(4), 166-171.

[0994] Commonly used pharmaceutical ingredients that can be used as appropriate to formulate the composition for its intended route of administration include:

acidifying agents (examples include but are not limited to acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid);
alkalinizing agents (examples include but are not limited to ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine);
adsorbents (examples include but are not limited to powdered cellulose and activated charcoal);
aerosol propellants (examples include but are not limited to carbon dioxide, CCl.sub.2F.sub.2, F.sub.2ClC—CClF.sub.2 and CClF.sub.3)
air displacement agents (examples include but are not limited to nitrogen and argon);
antifungal preservatives (examples include but are not limited to benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate);
antimicrobial preservatives (examples include but are not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal);
antioxidants (examples include but are not limited to ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite);
binding materials (examples include but are not limited to block polymers, natural and synthetic rubber, polyacrylates, polyurethanes, silicones, polysiloxanes and styrene-butadiene copolymers);
buffering agents (examples include but are not limited to potassium metaphosphate, dipotassium phosphate, sodium acetate, sodium citrate anhydrous and sodium citrate dihydrate)
carrying agents (examples include but are not limited to acacia syrup, aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orange syrup, syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection and bacteriostatic water for injection)
chelating agents (examples include but are not limited to edetate disodium and edetic acid)
colourants (examples include but are not limited to FD&C Red No. 3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8, caramel and ferric oxide red);
clarifying agents (examples include but are not limited to bentonite);
emulsifying agents (examples include but are not limited to acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyoxyethylene 50 monostearate);
encapsulating agents (examples include but are not limited to gelatin and cellulose acetate phthalate)
flavourants (examples include but are not limited to anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin);
humectants (examples include but are not limited to glycerol, propylene glycol and sorbitol);
levigating agents (examples include but are not limited to mineral oil and glycerin);
oils (examples include but are not limited to arachis oil, mineral oil, olive oil, peanut oil, sesame oil and vegetable oil);
ointment bases (examples include but are not limited to lanolin, hydrophilic ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment, and rose water ointment);
penetration enhancers (transdermal delivery) (examples include but are not limited to monohydroxy or polyhydroxy alcohols, mono- or polyvalent alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalin, terpenes, amides, ethers, ketones and ureas)
plasticizers (examples include but are not limited to diethyl phthalate and glycerol);
solvents (examples include but are not limited to ethanol, corn oil, cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection and sterile water for irrigation);
stiffening agents (examples include but are not limited to cetyl alcohol, cetyl esters wax, microcrystalline wax, paraffin, stearyl alcohol, white wax and yellow wax);
suppository bases (examples include but are not limited to cocoa butter and polyethylene glycols (mixtures));
surfactants (examples include but are not limited to benzalkonium chloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan mono-palmitate);
suspending agents (examples include but are not limited to agar, bentonite, carbomers, carboxymethylcellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, kaolin, methylcellulose, tragacanth and veegum);
sweetening agents (examples include but are not limited to aspartame, dextrose, glycerol, mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose);
tablet anti-adherents (examples include but are not limited to magnesium stearate and talc);
tablet binders (examples include but are not limited to acacia, alginic acid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, non-crosslinked polyvinyl pyrrolidone, and pregelatinized starch);
tablet and capsule diluents (examples include but are not limited to dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate, sorbitol and starch);
tablet coating agents (examples include but are not limited to liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, cellulose acetate phthalate and shellac);
tablet direct compression excipients (examples include but are not limited to dibasic calcium phosphate);
tablet disintegrants (examples include but are not limited to alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polacrillin potassium, cross-linked polyvinylpyrrolidone, sodium alginate, sodium starch glycollate and starch);
tablet glidants (examples include but are not limited to colloidal silica, corn starch and talc);
tablet lubricants (examples include but are not limited to calcium stearate, magnesium stearate, mineral oil, stearic acid and zinc stearate);
tablet/capsule opaquants (examples include but are not limited to titanium dioxide);
tablet polishing agents (examples include but are not limited to carnuba wax and white wax);
thickening agents (examples include but are not limited to beeswax, cetyl alcohol and paraffin);
tonicity agents (examples include but are not limited to dextrose and sodium chloride);
viscosity increasing agents (examples include but are not limited to alginic acid, bentonite, carbomers, carboxymethylcellulose sodium, methylcellulose, polyvinyl pyrrolidone, sodium alginate and tragacanth); and
wetting agents (examples include but are not limited to heptadecaethylene oxycetanol, lecithins, sorbitol monooleate, polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

[0995] Pharmaceutical compositions according to the present invention can be illustrated as follows:

Sterile IV Solution: A 5 mg/mL solution of the desired compound of this invention can be made using sterile, injectable water, and the pH is adjusted if necessary. The solution is diluted for administration to 1-2 mg/mL with sterile 5% dextrose and is administered as an IV infusion over about 60 minutes.
Lyophilised powder for IV administration: A sterile preparation can be prepared with (i) 100-1000 mg of the desired compound of this invention as a lyophilised powder, (ii) 32-327 mg/mL sodium citrate, and (iii) 300-3000 mg Dextran 40. The formulation is reconstituted with sterile, injectable saline or dextrose 5% to a concentration of 10 to 20 mg/mL, which is further diluted with saline or dextrose 5% to 0.2-0.4 mg/mL, and is administered either IV bolus or by IV infusion over 15-60 minutes.
Intramuscular suspension: The following solution or suspension can be prepared, for intramuscular injection:
50 mg/mL of the desired, water-insoluble compound of this invention
5 mg/mL sodium carboxymethylcellulose
4 mg/mL TWEEN 80
9 mg/mL sodium chloride
9 mg/mL benzyl alcohol
Hard Shell Capsules: A large number of unit capsules are prepared by filling standard two-piece hard galantine capsules each with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesium stearate.
Soft Gelatin Capsules: A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into molten gelatin to form soft gelatin capsules containing 100 mg of the active ingredient. The capsules are washed and dried. The active ingredient can be dissolved in a mixture of polyethylene glycol, glycerin and sorbitol to prepare a water miscible medicine mix.
Tablets: A large number of tablets are prepared by conventional procedures so that the dosage unit is 100 mg of active ingredient, 0.2 mg. of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg. of starch, and 98.8 mg of lactose. Appropriate aqueous and non-aqueous coatings may be applied to increase palatability, improve elegance and stability or delay absorption.
Immediate Release Tablets/Capsules: These are solid oral dosage forms made by conventional and novel processes. These units are taken orally without water for immediate dissolution and delivery of the medication. The active ingredient is mixed in a liquid containing ingredient such as sugar, gelatin, pectin and sweeteners. These liquids are solidified into solid tablets or caplets by freeze drying and solid state extraction techniques. The drug compounds may be compressed with viscoelastic and thermoelastic sugars and polymers or effervescent components to produce porous matrices intended for immediate release, without the need of water.

[0996] In accordance with another aspect therefore, the present invention covers a compound of general formula (I), or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, as described and defined herein, for use in the treatment or prophylaxis of a disease, as mentioned above.

[0997] The term “pharmaceutically acceptable salt” refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. For example, see S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci. 1977, 66, 1-19.

[0998] A suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethanesulfonic, dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluenesulfonic, methansulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, hemisulfuric, or thiocyanic acid, for example.

[0999] Further, another suitably pharmaceutically acceptable salt of a compound of the present invention which is sufficiently acidic, is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with N-methyl-glucamine, dimethyl-glucamine, ethyl-glucamine, lysine, dicyclohexylamine, 1,6-hexadiamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxy-methyl-aminomethane, aminopropandiol, sovak-base, 1-amino-2,3,4-butantriol. Additionally, basic nitrogen containing groups may be quaternised with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.

[1000] Those skilled in the art will further recognise that acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the invention are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods.

[1001] The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.

[1002] As used herein, the term “in vivo hydrolysable ester” is understood as meaning an in vivo hydrolysable ester of a compound of the present invention containing a carboxy or hydroxy group, for example, a pharmaceutically acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters for carboxy include for example alkyl, cycloalkyl and optionally substituted phenylalkyl, in particular benzyl esters, C.sub.1-C.sub.6 alkoxymethyl esters, e.g. methoxymethyl, C.sub.1-C.sub.6 alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl esters, C.sub.3-C.sub.8 cycloalkoxy-carbonyloxy-C.sub.1-C.sub.6 alkyl esters, e.g. 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters, e.g. 5-methyl-1,3-dioxolen-2-onylmethyl; and C.sub.1-C.sub.6-alkoxycarbonyloxyethyl esters, e.g. 1-methoxycarbonyloxyethyl, and may be formed at any carboxy group in the compounds of this invention.

[1003] An in vivo hydrolysable ester of a compound of the present invention containing a hydroxy group includes inorganic esters such as phosphate esters and [alpha]-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group. Examples of [alpha]-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. The present invention covers all such esters.

[1004] Another particular aspect of the present invention is therefore the use of a compound of general formula (I), described above, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for the prophylaxis or treatment of a disease.

[1005] Another particular aspect of the present invention is therefore the use of a compound of general formula (I) described above for manufacturing a pharmaceutical composition for the treatment or prophylaxis of a disease.

[1006] The diseases referred to in the two preceding paragraphs are diseases of uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses, or diseases which are accompanied with uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses, particularly in which the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses is mediated by the Wnt pathway, such as, for example, haematological tumours, solid tumours, and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumours including brain tumours and brain metastases, tumours of the thorax including non-small cell and small cell lung tumours, gastrointestinal tumours, endocrine tumours, mammary and other gynaecological tumours, urological tumours including renal, bladder and prostate tumours, skin tumours, and sarcomas, and/or metastases thereof.

[1007] The term “inappropriate” within the context of the present invention, in particular in the context of “inappropriate cellular immune responses, or inappropriate cellular inflammatory responses”, as used herein, is to be understood as preferably meaning a response which is less than, or greater than normal, and which is associated with, responsible for, or results in, the pathology of said diseases.

[1008] Preferably, the use is in the treatment or prophylaxis of diseases, wherein the diseases are haemotological tumours, solid tumours and/or metastases thereof.

DESCRIPTION OF THE FIGURES

[1009] FIG. X1 shows the sequence of human SMYD2 with N-terminal His tag before cleavage by TEV protease.

[1010] FIG. X2 shows the sequence of human SMYD2 after cleavage by TEV protease.

[1011] FIG. X3 shows the Example 4.1 in complex with human SMYD2 and SAM. [1012] Hydrogen atoms, SMYD2 and SAM are not shown. Carbon atom C1 unambiguously features S configuration.

Biological Activity of the Compounds According to the Invention

[1013] The following assays can be used to illustrate the commercial utility of the compounds according to the present invention.

[1014] Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein [1015] the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and [1016] the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values.

[1017] Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values calculated utilizing data sets obtained from testing of one or more synthetic batch.

1. Assays

[1018] The in vitro pharmacological properties of the compounds can be determined according to the following assays:

1.1 Scintillation Proximity Assay (SPA) for Detection of SMYD2 Enzymatic Inhibition

[1019] SMYD2 inhibitory activities of the compounds described in the present invention were quantified using a scintillation proximity assay (SPA) which measures methylation by the enzyme of the synthetic, biotinylated peptide Btn-Ahx-GSRAHSSHLKSKKGQSTSRH-Amid×TFA (Biosyntan) derived from p53 and referred to from here on as “p53 Peptide”. The SMYD2 full length enzyme was produced in-house by expression (with an N-terminal 6×His tag) in E. coli and purification by affinity chromatography on a Ni-NTA Sepharose column followed by a size exclusion chromatography step on a Superdex 200 16/60 column (GE Healthcare).

[1020] In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One), from which 50 nl of compound solutions were transferred into a white low volume test microtiter plate from the same supplier. Subsequently, 2.5 μl SMYD2 in aqueous assay buffer [50 mM Tris/HCl pH 9.0 (AppliChem), 1 mM dithiothreitol (DTT, Sigma), 0.01% (w/v) bovine serum albumine (BSA, Sigma), 0.0022% (v/v) Pluronic (Sigma)] were added to the compounds in the test plate to a final enzyme concentration of—typically—3 nM (this parameter was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay). The samples were then incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the methylation reaction, which was initiated by the addition of 2.5 μl 2-fold concentrated solution (in assay buffer) of titrated S-Adenosyl-L-Methionine (3H-SAM, Perkin Elmer, final concentration: 60 nM) and p53 Peptide substrate (final concentration: 1.0 μM). The resulting mixture (5 μl final volume) was shortly centrifuged (2 min., 1500 rpm) and incubated at 22° C. during 30 min. Thereupon the reaction was stopped by adding 3 μl of Streptavidin PS SPA imaging beads (Perkin Elmer, final concentration of 3.12 μg/μl) and “cold” SAM (AK Scientific, 25 μM final concentration) for non-specific binding reduction. Plates containing the stopped reaction were sealed with transparent adhesive foil (Perkin Elmer), centrifuged (2 min., 1500 rpm), and further incubated for—at least—1 h at RT (or overnight at 4° C.) in order to allow the SPA signals to develop. Subsequently, the amount of product was evaluated by measuring the energy transfer from the β-particles emitted by the 3H-labeled substrate to the Europium scintillator co-polymerized in the polystyrene matrix of the PS imaging beads, using the standard settings for this purpose of a Viewlux (Perkin-Elmer) CCD plate imaging device (emission filter 613/55 (IFP). The resulting scintillation counts were taken as indicator for the amount of methylated peptide per well. The data were normalised using two sets of control wells (typically 16 each) for high- (=enzyme reaction with DMSO instead of test compound=0%=Minimum inhibition) and low- (=all assay components without enzyme=100%=Maximum inhibition) SMYD2 activity. IC.sub.50 values were calculated by fitting the normalized inhibition data to a 4-parameter logistic equation using the “Screener” analysis software from Genedata.

1.2 Cell-Based Assay for Detection of SMYD2 Methylation Activity

[1021] For the detection of SMYD2 cellular methylation activity an In Cell Western (ICW) assay was established. This assay allows rapid processing of multiple samples for SMYD2 methylation derived immunofluorescence signals, with normalization to cell number via the use of the nucleic acid dye DRAQ5. KYSE-150 cells (human esophageal carcinoma cell line; DSMZ-German Collection of Microorganisms and Cell Cultures; No: ACC 375) have been stably transfected with a construct expressing wild-type SMYD2 (NCBI Reference Sequence: NP_064582.2). To detect SMYD2-mediated methylation signals in cells, a customized antibody directed against mono-methylated lysine 370 on protein p53 (p53K370me1) was used. The polyclonal antibody was generated (Eurogentec) against a p53 peptide containing the mono-methylated K370 epitope as described elsewhere (Huang et al., Nature, 2006, 444(7119):629-32).

[1022] For conducting the ICW assay 5000 SMYD2 overexpressing KYSE-150 cells/well were seeded in 96-well plates (SIGMA) and cultivated for 24 h. As a control for maximal inhibition of ectopic methylation activity, non-transfected KYSE-150 cells were used. Cells were grown in 49% RPMI 1640 with 49% Ham's F12 media supplemented with 2% heat inactivated fetal calf serum (FCS). For determination of SMYD2 inhibitory activity, cells were treated for 72 h in the presence of compounds or with DMSO. Cells were treated with compounds to be tested at a final concentration range varying from 3.9×10.sup.−8 to 5×10.sup.−6 M. Media was removed and 3.7% (w/v) formaldehyde in PBS was added for 20 min. After two washes with phosphate buffered saline (PBS), 0.25% (v/v) Triton X100 in PBS was added for 15 minutes of permeabilization. After one washing step with PBS, cells were blocked for 1 h with 5% (w/v) non-fat dry milk in PBS. Fixed cells were exposed to primary p53K270me1 antibody in 5% non-fat dry milk in PBS for 24 h. One row of cells on each plate was not exposed to p53K370me1 antibody and was reserved for background control measurements. The wells were washed three times with PBS, then secondary IR800 conjugated antibody (LI-COR) and DNA-intercalating dye, 5 μM DRAQ5 (LI-COR) were added for 3 h. After 5 washes with PBS, the fluorescence in each well was measured on an Odyssey (LI-COR) scanner at 800 nm (p53K370me1 signal; 764 nm excitation) and 700 nm (DRAQ5 signal; 683 nm excitation). Fluorescence intensity was quantified and normalized to background and DRAQ5 signals. IC.sub.50 values were calculated by fitting the normalized inhibition data to a 4-parameter logistic equation (Minimum, Maximum, IC.sub.50, Hill; Y=Max+(Min−Max)/(1+(X/IC.sub.50)Hill)) for each tested compound. For IC.sub.50 determination C0 (=no inhibition) was defined as the signal measured for DMSO treated controls. Ci (maximal inhibition) was defined as the signal measured for non SMYD2 overexpressing KYSE150 cells.

Measurement of the Inhibitory Activity of Selected Compounds on the SMYD2 Methylation Activity

[1023]

TABLE-US-00026 TABLE 2 IC.sub.50 IC.sub.50 Example No [mol/l] (SPA Assay) [mol/l] (ICW assay) 1 5.18E−07 2 8.33E−08 3 1.50E−08 1.01E−07 4 8.25E−08 1.76E−07 4.1 2.82E−08 4.67E−08 4.2 1.32E−06 1.39E−06 5 7.85E−07 4.14E−06 5.1 >2.00E−05 5.2 2.96E−07 6 2.86E−07 7.1 1.35E−08 1.32E−08 7.2 1.05E−06 8 9.01E−08 9 1.30E−08 2.64E−09 10 4.66E−07 8.00E−06 10.1 1.99E−07 10.2 3.83E−06 11 1.44E−07 3.76E−07 11.1 4.41E−08 11.2 2.98E−06 12 1.38E−07 12.1 2.66E−06 12.2 5.41E−08 13 8.67E−08 1.96E−07 13 2.54E−07 13.1 4.61E−06 13.2 5.00E−08 2.58E−07 14 1.47E−07 15 1.21E−07 15.1 1.76E−05 15.2 7.10E−08 9.96E−08 16 3.58E−07 16.1 4.79E−06 16.2 1.59E−07 2.58E−06 17 5.95E−08 18 1.25E−08 2.77E−08 18.1 >2.00E−5 18.2 3.00E−08 2.87E−08 19 9.98E−09 2.13E−08 19.1 7.68E−07 19.2 6.65E−09 2.36E−08 20 2.47E−07 4.03E−07 20.1 1.74E−05 20.2 1.19E−07 21 1.07E−06 21.1 5.30E−08 21.2 7.63E−07 22 6.48E−08 23 6.74E−08 24 6.31E−08 24.1 2.51E−06 24.2 1.92E−08 25 1.08E−07 25.1 2.06E−07 25.2 1.03E−05 26 3.06E−08 27 3.55E−07 9.59E−08 27.1 7.32E−08 27.2 1.62E−06 28 1.40E−07 29 5.55E−09 2.14E−07 30 1.58E−08 31 9.09E−09 1.26E−07 32 2.61E−08 33 8.45E−08 33.1 1.84E−05 33.2 3.76E−08 9.64E−08 34.1 1.54E−05 34.2 4.88E−07 35 1.57E−07 36 4.07E−8 1.66E−7 36.1 7.54E−7 36.2 2.29E−8 37 2.31E−7 38 3.20E−8 3.72E−8 38.1 1.35E−5 38.2 3.75E−8 3.56E−8 39 2.40E−6 40 4.18E−7 41 7.35E−8