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We previously described the design and synthesis of rigid tricyclic phenylalanylleucine (PheLeu) mimetic 1 and its incorporation into 2, an inhibitor of angiotensin I-converting enzyme (ACE, EC 3.4.15.1).' Mimetic 1 was designed2 to closely resemble the anti orientation (XI = 180°, X2 = Oo) of the carboxy terminal histidylleucine (HisLeu) portion of angiotensin I (Chart I). The replacement of His by Phe in mimetic 1 was made with knowledge that His is not essential to ACE bindin? and that neutral endopeptidase 24.11 (NEP, EC 3.4.24.111, a related zinccontaining proteinase, cleaves the PheLeu dipeptide from Leu-enke~halin.~ The cleavage of bradykinin adjacent to Phe(8) by both ACE and NEP6 further suggests that these two metalloproteinases could have shared active-site characteristics. We speculated early on that side chain constrained peptidomimetics would be useful tools to study the conformational preferences in peptide-protein interactions. The similarities and biological significance of these two enzymes made them ideal choices for evaluation by this approach. More recently, NEP has been shown to play a role in the degradation of the natriuretic peptides! a family of hormones, some of which are secreted by the heart into the circulation in increased amounts in patienta with congestive heart failure (CHF).' Because the reninangiotensin-aldosterone system opposess the beneficial natriuretic and diuretic actions of atrial natriuretic peptide (ANP), inhibition of ACE during NEP inhibition should be advantageous. The feasibility of simultaneous ACE and NEP inhibition was investigated utilizing our dipeptide mimic approach. Gros et al. have described close analogs of thiorphan which exhibit equipotent nanomolar ACE and NEP inhibition in vitro and demonstrate enzyme occupation upon oral dosing of a prodrug form.Q We now report the design rationale and synthesis of a new class of subnanomolar dual ACE/NEP inhibitor, a member of which produces blood pressure lowering in animal models of both essential and salt dependent hypertension when orally administered in prodrug form.Derivatives of 1 were used to probe the active site requirements of both ACE and NEP (Chart 11). Whereas 2 was found to be an extremely selective inhibitor of ACE, epimeric tricyclic mercaptomethylene derivatives 3 and 4'0 gave the indication that simultaneous inhibition of ACE and NEP was feasible in spite of the intrinsic conformational constraints of mimetic 1. The mercaptoacetyl derivative of phenylalanylglycine had been reChart I Anglotensln I n Leu-Enkephalln TyrGlyGlj 1 , n Leu-Enkephalln U Chart I1 3 4 K,(ACE) nM 2 r7 2 130 KI(NEP) nM r10,OOO r300 45 5 2ported to inhibit NEP (Ki = 20 nM).I1 The mercaptoacetyl derivative 5 of mimetic 1 was found to be a potent inhibitor of ACE as well as NEP. This result, combined wfth the structural rigidity of mimetic 1, indicates that significant similarities in the SI' and S2' binding domains of these two enzymes exist and that an internal unsubstituted CO-NH function is not essential for binding to this ...

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1 The effects of several doses of labetalol (0.03 to 1 mg/kg) given intravenously and into the vertebral artery were examined in anaesthetized dogs. Labetalol produced no immediate (5 min) change in blood pressure or heart rate when given by either route, with one exception. Heart rate increased after the first dose (0.03 mg/kg i.v.) of labetalol. By contrast, clonidine (1 jg/kg) elicited an immediate and prolonged fall in blood pressure and heart rate when given into the vertebral artery, but not intravenously. 2 In the isolated perfused gracilis muscle of the dog, following a-and ,B-adrenoceptor blockade, intra-arterial injections of labetalol (0.3 to 10 mg) or diazoxide (0.3 to 1 mg) produced decreases in perfusion pressure that were dose-related in both magnitude and duration. The doses of labetalol and diazoxide required to produce a half-maximal vasodilatation were 1.5 mg and 0.7 mg respectively. 3 In adrenalectomized, vagotomized spinal dogs, both labetalol (0.1 to 1 mg/kg i.v.) and hydralazine (1 mg/kg i.v.) elicited a fall in blood pressure without changing heart rate or cardiac output.4 These results suggest that the hypotension produced by systemically administered labetalol does not involve an action in the brain. It may involve instead a direct vasodilatation of resistance blood vessels, since labetalol in sufficient amounts, directly dilates resistance vessels and lowers blood pressure in dogs devoid of adrenergic tone. Direct vasodilatation may be a component of the hypotensive action of labetalol.