NEW DUAL INHIBITORS OF NEUTRAL ENDOPEPTIDASE AND ANGIOTENSIN-CONVERTING ENZYME - RATIONAL DESIGN, BIOAVAILABILITY, AND PHARMACOLOGICAL RESPONSES IN EXPERIMENTAL-HYPERTENSION

In the treatment of cardiovascular diseases, it could be of therapeutic interest to associate the hypotensive effects resulting from the inhibition of angiotensin II formation, ensured by endothelial angiotensin-converting enzyme (ACE), with the diuretic and natriuretic responses due to the protection of the endogenous atrial natriuretic peptide (ANP) from inactivation by epithelial neutral endopeptidase (NEP). However, an investigation of this hypothesis requires an orally active compound able to jointly inhibit ACE and NEP. Dual inhibitors have therefore been designed by a rational approach, based on the characteristics of the active sites of both enzymes, which belong to the same family of zinc metallopeptidases, and on the structures of their most potent and selective inhibitors. As both NEP and ACE contain a large S'(1)-S'(2) domain able to accommodate aromatic residues, the cyclic ACE inhibitor 3-(mercaptomethyl)-3,4,5,6-tetrahydro-2-oxo-1H-1-benzazocine-1-acetic acid was selected as a template. Various aliphatic constraints were introduced on the benzyl moiety of the potent NEP inhibitor N-[2-(mercaptomethyl)-3-phenylpropanoyl]-L-tyrosine (IC50 NEP = 2 nM, IC50 ACE = 25 nM) to improve the fit between the computed most stable conformers of these molecules and the ACE template. New dual inhibitors, of general formula, N-[2(R,S)-(mercaptomethyl)-3(R,S)-phenylbutanoyl]-L-amino acid with IC50 values in the nanomolar range for both enzymes were generated by this approach. The separation of the four stereoisomers using chiral amines and the stereoselective synthesis of the 2-(mercaptomethyl)3-phenylbutanoyl moiety showed that inhibitors with the 2S,3R configuration are the most potent on both NEP and ACE. The ''in vivo'' potency of various prodrugs of these inhibitors to inhibit ACE activity in lung and NEP activity in kidney was measured after oral administration in mice. From this pharmacokinetical study the most potent dual inhibitor RB 105 (N-[(2S,3R)-2(mercaptomethyl)-3-phenylbutanoyl-L-alanine (compound 44c) (K-I NEP 1.7 nM, K-I ACE 4.5 nM) and its most efficient in vivo prodrug mixanpril, [N-[(2S,3R)-2-[(benzoylthio)methyl]-3-phenylbutanoyl]-L-alanine (compound 18) (ED(50) NEP similar to 1 mg/kg, ED(50) ACE similar to 7 mg/kg) were selected. Competition experiments with a tritiated inhibitor of ACE or NEP bound to mouse lung and kidney membranes respectively showed that mixanpril has a long duration of action (>8 h). As expected, after iv administration in the spontaneously hypertensive rat (SHR), RB 105 decreased blood pressure and increased diuresis and natriuresis. Both effects were also observed after chronic oral administration of 50 mg/kg mixanpril twice a day in SHR, These results indicate that an efficient and orally active dual inhibitor of NEP and ACE produces beneficial changes in hemodynamics and could represent a therapeutic progress in the treatment of cardiovascular diseases.