The clinical syndromes of thromboembolism are evoked by an excessive stimulation of the coagulation cascade. In this context, the serine protease thrombin plays a key role. Considerable efforts have therefore been devoted to the discovery of safe, orally active inhibitors of this enzyme. On the basis of the X-ray crystal structure of the peptidelike thrombin inhibitor NAPAP complexed with bovine thrombin, we have designed a new structural class of nonpeptidic inhibitors employing a 1,2,5-trisubstituted benzimidazole as the central scaffold. Supported by a series of X-ray structure analyses, we optimized the activity of these compounds. Thrombin inhibition in the lower nanomolar range could be achieved although the binding energy mainly results from nonpolar, hydrophobic interactions. To improve in vivo potency, we increased the overall hydrophilicity of the molecules by introducing carboxylate groups. The very polar compound 24 (BIBR 953) exhibited the most favorable activity profile in vivo. This zwitterionic molecule was converted into the double-prodrug 31 (BIBR 1048), which showed strong oral activity in different animal species. On the basis of these results, 31 was chosen for clinical development.
Starting from the recently reported nonpeptidic angiotensin II (AII) receptor antagonists DuP753 (1) and Exp 7711 (2), we have designed and investigated novel substituted benzimidazoles. Systemic variation of several substituents at the benzimidazole ring positions 4-7 led to the finding that substitution in position 6 with acylamino groups results in highly active AII antagonists. Compounds with 6-membered lactam or sultam substituents in position 6 of benzimidazole showed receptor activities in the low nanomolar range but were only weakly active when given orally to rats. In contrast, analogous substitution of the benzimidazole moiety with basic heterocycles resulted in potent AII antagonists which were also well absorbed after oral application. The most active compound of this series, 33 (BIBR 277), was selected as a candidate for clinical development. On the basis of molecular modeling studies a binding model of this new class of AII antagonists to the AT1 receptor is proposed.
The pharmacological profile of BIBR 277, 4′‐[(1,4′‐dimethyl‐2′‐propyl[2,6′‐bi‐1H‐benzimidazol]‐1′‐yl)methyl]‐[1,1′‐biphenyl]‐2‐carboxylic acid, a novel, nonpeptide angiotensin II receptor antagonist has been investigated by use of receptor binding studies, enzymatic assays, functional in vitro assays in rabbit aorta as well as in vivo experiments in pithed, anaesthetized and conscious rats. BIBR 277 potently interacted with rat AT1 receptors (Ki 3.7 nm). Competitive receptor interaction was shown by radioligand saturation experiments performed in the presence of BIBR 277. The failure to inhibit radioligand binding to AT2 sites demonstrates the selectivity of BIBR 277 for AT1 receptors. This is further substantiated by the findings that BIBR 277 neither interacted with other receptor systems investigated nor affected the activity of components of the human renin‐angiotensin system, such as plasma renin or serum converting enzyme. In rabbit aorta, BIBR 277 had no agonistic properties and was shown to be an insurmountable antagonist of angiotensin II‐induced contractions (KB 0.33 nm). The antagonistic effect persisted even after several wash‐out procedures. However, this interaction was not irreversible since the insurmountable antagonism was concentration‐dependently reversed when BIBR 277 (0.1 μm) and the surmountable antagonist, losartan (0.1 and 1.0 μm) were incubated simultaneously. The specificity of BIBR 277 for the AT1 receptor was further substantiated in this preparation since micromolar concentrations of BIBR 277 neither affected potassium chloride and noradrenaline‐induced contractions nor acetylcholine‐mediated tissue relaxation. In pithed rats, i.v. administration of BIBR 277 (0.1, 0.3 and 1.0 mg kg−1) shifted the dose‐pressor response curve to angiotensin II dose‐dependently to the right with ED50 values of 0.23 μg kg−1 (control) and 1.4 μg kg−1, 4.7 μg kg−1 and 20 μg kg−1, respectively. As observed in the in vitro experiments no agonistic effect was detected and the maximum of the blood pressure response to angiotensin II at the highest dose of BIBR 277 was decreased by 29%. In anaesthetized rats, bolus i.v. administration of 0.1, 0.3 and 1.0 mg kg−1 BIBR 277 attenuated the blood pressure response to bolus i.v. injections of angiotensin II (0.1 μg kg−1). At the highest dose an almost complete blockade was observed even after 2 h. Single oral administration of BIBR 277 (0.3 and 1.0 mg kg−1) to conscious, chronically instrumented renovascular hypertensive rats dose‐dependently decreased the mean arterial blood pressure by 15 and 30 mmHg, respectively. At the higher dose a significant antihypertensive effect was maintained for more than 24 h. Moreover, consecutive daily dosing of 1 mg kg−1 orally resulted in a sustained reduction in blood pressure over the 4 day observation period. It is concluded that BIBR 277 is an effective and selective angiotensin II antagonist with antihypertensive activity after oral administration.
The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.
Summary. Background: Oral anticoagulant therapies targeted at thrombin are being developed to overcome limitations associated with current standard therapies. Objectives: This study was undertaken to assess and compare the antithrombotic and anticoagulant effects of the novel, selective and reversible, direct thrombin inhibitor (DTI), dabigatran, and its oral prodrug dabigatran etexilate, to that of unfractionated heparin (UFH), hirudin and melagatran using a rabbit model of venous thrombosis. Methods: A rabbit model of venous thrombosis consisting of endothelial damage with blood flow reduction was used with minor modifications. Results: All compounds demonstrated a dose-dependent reduction in thrombus formation following i.v. administration with complete or almost complete inhibition at the highest doses. Dabigatran (in the dose range 0.03-0.5 mg kg ) inhibited thrombus formation in a dose-dependent manner. Maximum inhibition was achieved within 1 h of administration, suggesting a rapid onset of action. For both routes of administration, inhibition of thrombus formation directly correlated with prolongation of the activated partial thromboplastin time. Conclusions: These findings demonstrate the potent anticoagulant and antithrombotic activity of dabigatran as a selective thrombin inhibitor in a rabbit model of venous thrombosis. Notably, dose-dependent and long-lasting antithrombotic efficacy was observed after application of its oral form dabigatran etexilate, which is currently undergoing phase III clinical development.
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