Angiotensin-converting enzyme inhibitors: synthesis and biological activity of acyl tripeptide analogues of enalapril.

The synthesis and biological activity of a series of inhibitors of angiotensin-converting enzyme (EC 3.4.15.1) are described. Incorporation of the substituted N-carboxymethyl dipeptide design of enalapril (MK-421) into acyl tripeptides and larger peptides yielded potent inhibitors of the enzyme. These can be viewed as substrate analogues in which the carbonyl of the scissile peptide bond is replaced by a CHCO2H group. Several of the analogues described possess inhibitory potency equal to that of enalaprilat (MK-422), but none achieves an increase in potency which would demonstrate additional binding interactions contributed by the extended peptide chain. Application of the design described may be useful for inhibition of other metallopeptidases.

Azapeptides: a new class of angiotensin-converting enzyme inhibitors.

A class of potent inhibitors of angiotensin-converting enzyme (dipeptidyl carboxypeptidase, E.C. 3.4.15.1) is reported, in which an alpha-aza substitution into the substituted N-carboxymethyl dipeptide structure of enalapril is made. The inhibitors 2 exhibit striking alterations in their conformational and acid-base properties due to the aza substitution, as is clear from pKa data and the x-ray crystal structure of a model azapeptide. In spite of this, they bind tightly to the enzyme, with inhibitor potency comparable to that of captopril.

Novel renin inhibitors containing the amino acid statine.

The proteolytic enzyme renin (EC3.4.99.19) cleaves the protein substrate angiotensinogen to yield angiotensin I, the decapeptide substrate transformed by converting enzyme into the pressor substance angiotensin II. Although the contribution of this pathway to the maintenance of normal blood pressure is unclear, it seems to be a major factor in various hypertensive states. Important progress in the control of hypertension has been achieved by development of the potent inhibitors SQ-14,225 (captopril) and MK-421 (enalapril maleate), which block the generation of angiotensin II by the inhibition of angiotensin converting enzyme. An attractive alternative to the inhibition of converting enzyme would be the blockade of the preceding step in the cascade, the renin reaction. We report here new highly potent (IC50 = 10(-9)-10(-8) M) competitive inhibitors of renin in which statine, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, is incorporated into analogues of the pig renin substrate (Fig. 1).

Enalapril maleate and a lysine analogue (MK-521): disposition in man.

1 The disposition of two angiotensin converting-enzyme inhibitor drugs was studied in normal volunteers. One drug was enalapril maleate (MK-421), which requires in vivo esterolysis to yield active inhibitor (MK-422). The other was a lysine analogue of MK-422 (MK-521), which requires no bioactivation. 2 Absorption of enalapril maleate (10 mg, p.o.) was rapid, with peak serum concentrations of enalapril observed 0.5-1.5 h after administration. Based upon urinary recovery of total drug (enalapril plus MK-422), absorption was at least 61%. Bioactivation appeared to be largely post-absorptive. From the ratio of MK-422 to total drug in urine, the minimum extent of bioactivation was estimated at 0.7. 3 A similar dose of MK-521 was absorbed more slowly, reaching peak serum concentrations 6-8 h following drug administration. Minimum absorption, based upon urinary recovery, was 29%. 4 Serum concentration v time profiles for both drugs were polyphasic and exhibited prolonged terminal phases. 5 Recovery in urine and faeces of administered enalapril maleate (intact and as MK-422) was 94%. Recovery of MK-521 was 97%. These results indicate lack of significant metabolism of these agents, apart from the bioactivation of enalapril.

Phosphorus-containing inhibitors of angiotensin-converting enzyme.

Several phosphonamides, phosphoramides, and phosphates having the general structure R-Y-P(O)(OH)-X-CH(CH3)-CO-Pro have been synthesized and tested for inhibition of angiotensin-converting enzyme (dipeptidyl carboxypeptidase; peptidyl-dipeptide hydrolase, EC 3.4.15.1). Inhibition was found to depend on the nature of R, Y, and X such that the maximal effect was observed when X = NH, Y = CH2, and R = phi CH2 (50% inhibition at 7 nM). Substitution of CH2 or O at X and O at Y produced significantly less potent inhibitors. Groups shorter or longer than R = phi CH2 led to less active inhibitors, presumably due to nonoptimal interaction of the side chain with the S1 subsite.

Total serum angiotensin converting enzyme activity in rats and dogs after enalapril maleate (MK-421).

A centrifugal gel filtration separation of serum angiotensin converting enzyme (ACE) from a potent stable inhibitor is described. This, together with a 20 hr assay incubation of very dilute enzyme, permitted the assessment of the effects of enalapril maleate treatment on total serum ACE in rats and dogs. Total serum ACE increased in both species after 1 or 2 weeks at 10 mg/kg/day. Serum ACE in rats was more than doubled; whereas the increase was modest in dogs (48 +/- 9% minimum). The effect of the drug on serum ACE combined with inherent variability of ACE precluded use of serum ACE activity as an accurate measure of inhibitor concentration in animals receiving enalapril maleate.

The physiological disposition and metabolism of enalapril maleate in laboratory animals.

N-[1-(S)-carboxy-3-phenylpropyl]-L-alanyl-L-proline (MK-422), is a potent angiotensin I-converting enzyme (ACE) inhibitor, but as a diacid is poorly absorbed in laboratory animals. Enalapril maleate, the monoethyl ester of MK-422, proved to be significantly better absorbed in both rats and dogs. Peak levels of radioactivity in plasma occurred in 30 min in rats and 2 hr in dogs after a single dose of 14C-enalapril maleate (1 mg/kg, po). Rats excreted 26% of the dose in the urine and 72% in the feces in 72 hr; dogs excreted 40% of the dose in the urine and 36% in the feces. After the intravenous dose, the presence of radioactivity in the feces of both species suggested that some biliary excretion had occurred. Absorption was estimated to be 34% in the rat and 61% in the dog. The major metabolite of enalapril maleate in dogs, accounting for 86% of the urine radioactivity, was identified as MK-422 by GC/MS. A procedure was developed for the quantitation of MK-422 and enalapril in plasma and urine by their inhibition of purified ACE. The assays showed that enalapril was absorbed intact in dogs and converted to MK-422 after absorption.

Effect of N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro and its ethyl ester (MK-421) on angiotensin converting enzyme in vitro and angiotensin I pressor responses in vivo.

The parent diacid (N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro of MK-421 inhibited hog plasma angiotensin converting enzyme (ACE) by 50% (I50) at a concentration of 1.2 nM and was 17 times more potent than captopril. In vitro the I50 for MK-421, an ethyl ester, was 1200 nM because de-esterification did not occur. Similarly in the guinea-pig ileum, the diacid inhibitor and MK-421 potentiated the contractile effects of bradykinin at an AC50 of 77 pM and 18 nM, respectively. Inhibition of the pressor effects of angiotensin I by the diacid ACE inhibitor occurred at an ID50 of 8.2 micrograms/kg i.v. in rats and 6.4 micrograms/kg i.v. in dogs. Thus, the diacid was approximately 12 times more potent than captopril. The ID50 for MK-421 was 14 and 278 micrograms/kg i.v. in rats and dogs, respectively, because of differences in the rates of de-esterification. Oral ACE inhibitory activity was determined by blockade of the pressor effects of angiotensin I in conscious rats and dogs. In rats, but not in dogs, the diacid inhibitor was poorly absorbed, whereas MK-421 was well absorbed in both species. MK-421 inhibited the pressor effects of angiotensin I at 0.1 to 3.0 mg/kg p.o. for at least 6 hr in rats and dogs, and compared to captopril was 8.6 times more potent in rats and 4.6 times more potent in dogs. These data demonstrate that MK-421 and its parent diacid are potent, long-lasting orally active inhibitors of ACE. In addition, the low activity of MK-421 in vitro contrasts with its substantial in vivo activity, and supports the hypothesis that MK-421 is a prodrug that first must be de-esterified to permit full expression of its significant in vivo pharmacological activity.

A new class of angiotensin-converting enzyme inhibitors.

Much current attention focuses on the renin-angiotensin system in relation to mechanisms controlling blood pressure and renal function. Recent demonstrations (ref. 1, ref. 2 and refs therein) that angiotensin-converting enzyme inhibitors show promising clinical antihypertensive properties have been of particular interest. We now report on the design of a novel series of substituted N-carboxymethyl-dipeptides which are active in inhibiting angiotensin-converting enzyme at nanomolar levels. We suggest that these compounds are transition-state inhibitors and that extensions of this design to other metalloendopeptidases merit further study.