Factor Xa inhibitors

#This application claims the benefit of European Application No. 05107992.9, filed Sep. 1, 2005, which is hereby incorporated by reference in its entirety.

#The invention is concerned with novel cycloalkane carboxamides of formula (I),

#wherein R1′ is selected from the group consisting of carboxyl, cyano, C1-6 alkoxycarbonyl, hydroxy C1-6 alkyl-NH—C(O)—, N(C1-6 alkyl)(hydroxy C1-6 alkyl)-C(O)—, C1-6 alkyl-NH—C(O)—, halo C1-6 alkyl-NH—C(O)—, C3-7 cycloalkyl-C1-6 alkyl-NH—C(O)—, C3-7 cycloalkyl-C1-6 alkyl, C3-7 cycloalkyl-C1-6 alkoxy, hydroxy C1-6 alkyl-, hydroxy, C1-6 alkoxy, C1-6 alkoxy-C1-6 alkyl-, halo C1-6 alkoxy-C1-6 alkyl-, halo C1-6 alkyl, halo C1-6 alkoxy, heterocyclyl-C1-6 alkyl-, C1-6 alkyl-sulfonylamino-C1-6 alkyl-, (C1-6 alkyl-sulfonyl)(C1-6 alkyl)amino-C1-6 alkyl-, C1-6 alkyl-carbonylamino-C1-6 alkyl-, hydroxy-C2-6 alkoxy-, amino, mono- or di-C1-6 alkyl substituted amino, mono- or di-C1-6 alkyl substituted amino-C1-6 alkyl-, mono- or di-halo C1-6 alkyl substituted amino, C3-7 cycloalkylamino-C1-6 alkyl-, heterocyclyl-amino-C1-6 alkyl-, (C3-7 cycloalkyl)(C1-6 alkyl)amino-C1-6 alkyl-, (heterocyclyl)(C1-6 alkyl)amino-C1-6 alkyl-, C1-6 alkyl-sulfonylamino-, C1-6 alkyl-carbonylamino-, (C1-6 alkylcarbonyl)(C1-6 alkyl)amino-, (C1-6 alkoxy-carbonyl)(C1-6 alkyl)amino-, (C1-6 alkylsulfonyl)(C1-6 alkyl)amino-, optionally halogenated heterocyclyl, where one or two carbon atoms of the heterocyclyl are optionally replaced with a carbonyl group, halogen, (C1-6 alkyl-carbonyl)(C1-6 alkyl)amino-C1-6 alkyl-, C1-6 alkyl-O—CO—NH—, C1-6 alkyl-O—CO—NH—C1-6 alkyl-, C1-6 alkyl-NH—CO—NH—, C1-6 alkyl-NH—CO—NH—C1-6 alkyl-, aryl, heteroaryl, aryloxy, heteroaryloxy, aryl-C1-6-alkoxy, heteroaryl-C1-6-alkoxy, and NR′R″-C1-6 alkyl- where R′ and R″, together with the nitrogen atom to which they are attached, form a lactam containing one to six carbon atoms; R1″ is hydrogen; or R1′ and R1″ form, together with the same carbon atom to which they are attached, a member selected from the group consisting of —C(═O)—, —C═CH2, C3-7 cycloalkyl, and heterocyclyl, where one or two carbon atoms of the heterocyclyl are optionally replaced with a carbonyl group; R2 is selected from the group consisting of hydrogen and C1-6 alkyl; R3 is selected from the group consisting of hydrogen and C1-6 alkyl; X and X1 are independently selected from the group consisting of arylene, heteroarylene and heterocyclylene, wherein the arylene, heteroarylene and heterocyclylene are optionally substituted by one or more substituents independently selected from the group consisting of C1-6 alkyl, C1-6 alkoxy, halogen, cyano, nitro, amino, —N(R′)—CO—(C1-6 alkyl optionally substituted by one or more fluorine atoms), in which R′ is selected from the group consisting of hydrogen, C1-6 alkyl and fluoro C1-6 alkyl, —N(R′)—CO—O—(C1-6 alkyl optionally substituted by one or more fluorine atoms), in which R′ is hydrogen, C1-6 alkyl or fluoro C1-6 alkyl, —N(R′)—CO—N(R″) (R′″), in which R′, R″ and R′″ are independently hydrogen, C1-6 alkyl or fluoro C1-6 alkyl, —C(O)—N(R′)(R″), in which R′ and R″ are independently hydrogen, C1-6 alkyl or fluoro C1-6 alkyl, or R′ and R″, together with the nitrogen atom to which they are attached, form heterocyclyl, —NR′R″, in which R′ and R″ are independently hydrogen, C1-6 alkyl or fluoro C1-6 alkyl, or R′ and R″, together with the nitrogen atom to which they are attached, form heterocyclyl,

#The inhibition of the coagulation factor Xa by the compounds of the present invention can be demonstrated with the aid of a chromogenic peptide substrate assay as described hereinafter.

#Factor Xa activity was measured spectrophotometrically in microtiter plates in a final volume of 150 μl using the following conditions: Inhibition of human factor Xa (Enzyme Research Laboratories) was tested at an enzyme concentration of 3 nM using the chromogenic substrate S-2222 (Chromogenix AB, Mölndal, Sweden) at 200 nM. The reaction kinetics of the enzyme and the substrate were linear with both time and the enzyme concentration. The inhibitors were dissolved in DMSO and tested at various concentrations up to 100 μM. The inhibitors were diluted using HNPT buffer consisting of HEPES 100 mM, NaCl 140 mM, PEG 6000 0.1% and Tween 80 0.02%, pH 7.8. The cleavage of S-2222 by human factor Xa was followed at 405 nm for 5 minutes at room temperature. The velocity of the reaction was determined by the autoreader from the slope of the linear regression fit to 7 time points (1 minute). The initial velocity for each inhibitor concentration was determined by the slope of at least 4 time points in the linear phase by a linear regression fit (mOD/min2). Apparent dissociation constants Ki were calculated according to Cheng and Prusoff [Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of the inhibitor that causes 50 percent inhibition (IC50) of an enzyme reaction. Biochem. Pharmacol. 1973, 22, 3099-3108.] based on the IC50 and the respective Km, determined previously (Ki=IC50/(1+S/Km)). The Km for the substrate used was determined under the conditions of the test with at least 5 substrate concentrations ranging from 0.5 to 15 times Km. [Lottenberg R, Hall J A, Blinder M, Binder E P, Jackson C M., The action of thrombin on peptide p-nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions. Biochim Biophys Acta. 1983 Feb. 15; 742(3):539-57]. According to Eadie [Eadie G. S. The inhibition of cholinesterase by physostigmine and prostigmine. J. Biol. Chem. 1942, 146, 85-93.], the Km for S-2222 amounted to 613 μM.

#The activity of the low molecular weight substances can, moreover, be characterized in the “prothrombin time” (PT) clotting test. The substances are prepared as a 10 mM solution in DMSO and thereafter made up to the desired dilution in the same solvent. Thereafter, 0.25 ml of human plasma (obtained from whole blood anticoagulated with 1/10 volume of 108 mM Na citrate) was placed in the instrument-specific sample container. In each case 5 μl of each dilution of the substance-dilution series was then mixed with the plasma provided. This plasma/inhibitor mixture was incubated at 37° C. for 2 minutes. Thereafter, there were pipetted to the semi-automatic device (ACL, Automated Coagulation Laboratory (Instrument Laboratory)) 50 μl of plasma/inhibitor mixture in the measurement container. The clotting reaction was initiated by the addition of 0.1 ml of Dade® Innovin® (recombinant human tissue factor combined with calcium buffer and synthetic phospholipids, Dade Behring, Inc., Cat. B4212-50). The time up to the fibrin cross-linking was determined photooptically from the ACL. The inhibitor concentration, which brought about a doubling of the PT clotting time, was determined by fitting the data to an exponential regression (XLfit).