David W. Cushman

Azetidine-2-carboxylic acid derivatives

An improved method for the preparation of DL-azetidine-2-carboxylic acid is reported. The reaction of thionyl chloride with azetidine-2-carboxylic acid in methanol gave rise to 2-carbomethoxyazetidine hydrochloride, which upon treatment with triethylamine yielded 2-carbomethoxyazetidine. 2-Carbomethoxyazetidine proved to be unstable and upon standing formed azetidine-2-carboxylic acid anhydride. The nmr and ir spectra of 2-carbomethoxyazetidine are discussed in terms of its conformation. The conversion of azetidine-2-carboxylic acid to azetidine-2-carboxamide is also described.

Design of angiotensin converting enzyme inhibitors

Specific inhibitors of angiotensin-converting enzyme, known to the lay public as ACE inhibitors, have now been embraced by the medical community as first-line therapy for hypertensive disease and congestive heart failure. However, it is important for historical and scientific perspective to note that in the late 1960s, the so-called renin–angiotensin system, of which ACE is a component, was a poorly understood enzyme system that was not widely accepted as being important for blood pressure regulation. Renin was discovered in the late 19th century as a hypertensive substance in blood, and was shown in 1934 to be an enzyme that was elevated in the blood of renal hypertensive rats. The potent blood pressure-elevating octapeptide angiotensin II, produced in blood by the action of renin, was isolated in the late 1930s (ref. 2). But ACE, which cleaves a His–Leu dipeptide from the inactive decapeptide renin product angiotensin I to form angiotensin II, was not identified until 1954 (ref. 3). Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Angiotensin II Design of angiotensin converting enzyme inhibitors

2 Inhibitiors of Angiotensin-Converting Enzyme

Publisher Summary This chapter focuses on the development, mechanism of action, and biological activity of two distinct classes of therapeutically useful inhibitors of angiotensin-converting enzyme: the snake venom peptides and a newly developed class of orally active inhibitors that were designed for specific binding to the active site of this enzyme. No other class of drugs has been as logically developed as the antihypertensive inhibitors of angiotensin-converting enzyme, whose primary mechanism of action, enzyme inhibition, has been clear from the first preclinical animal studies. The orally-active inhibitor captopril has been designed for optimal and specific binding to the active center of this enzyme. It is not then too surprising to find that such drugs have remarkable therapeutic ratios and relative freedom from side effects. The logical development of this class of therapeutic agents serve as a model for development of specific therapeutic agents in certain other disease states where pathophysiological mechanisms are beginning to be unravelled.

Design of new antihypertensive drugs: Potent and specific inhibitors of angiotensin-converting enzyme

Abstract The similarity of the biologically important enzyme angiotensin-converting enzyme to the structurally characterized digestive enzyme carboxypeptidase A has led us to develop a hypothetical model of the mechanism of binding of substrates to its active site. In this model, a positively charged group on the enzyme forms an ionic bond with the negatively charged carboxyl group of the substrate; a hydrogen bonding group of the enzyme binds with the terminal peptide bond of the substrate, and the tightly bound zinc ion of the enzyme binds to the penultimate (scissile) peptide bond of the substrate. Succinyl- l -proline (SQ 13,745) was synthesized as a potential inhibitor of angiotensin-converting enzyme by analogy to d -2-benzylsuccinic acid, an inhibitor of carboxypeptidase A; it was a moderately potent but specific inhibitor of the enzyme. Structure-activity studies carried out using the hypothetical model as a guide led to the synthesis of d -2-methyl-succinyl- l -proline (SQ 13,-297) and d -2-methylglutaryl- l -proline (SQ 14,-102), more potent inhibitors of the enzyme that were shown to be orally active in rats. Attempts to replace the zinc-binding carboxyl group of these compounds with groups with greater affinity for zinc have led to the synthesis of extremely potent inhibitors such as 3-mercapto-propanoyl- l -proline (SQ 13,863) and d -3-mercapto-2-methylpropanoyl- l -proline (SQ 14,225). The most active compound, SQ 14,225, is a purely competitive inhibitor of angiotensin-converting enzyme with an enzyme-inhibitor dissociation constant (Ki) of 1.7 × 10−9M. It is an extremely potent and specific inhibitor of angiotensin-converting enzyme and appears to have great potential for the treatment of hypertensive disease.

Angiotensin-converting enzyme inhibitors: biochemical properties and biological actions

The review will cover the chemistry and biochemistry of angiotensin-converting enzyme inhibitors with emphasis on data published since the publication of previous reviews. The relative merits of each contribution will be evaluated, as well as their potential for leading to new discoveries. The biology of angiotensin-converting enzyme inhibitors will be brought up-to-date to give the reader an appreciation of the medical implications of this new type of antihypertensive agent.

Development and design of specific inhibitors of angiotensin-converting enzyme

Captopril is a remarkably effective new antihypertensive drug designed and developed as a potent and specific inhibitor of angiotensin-converting enzyme, a zinc metallopeptidase that participates in the synthesis of a hypertensive peptide, angiotensin II, and in the degradation of a hypotensive peptide, bradykinin. Earlier studies with a snake venom peptide (teprotride or SQ 20881) that could be administered only by injection demonstrated that specific inhibitors of angiotensin-converting enzyme could be highly effective as antihypertensive drugs, and helped to clarify the specificity and mechanism of action of the enzyme. A hypothetical model of the active center of angiotensin-converting enzyme based on its presumed analogy to the well characterized zinc metallopeptidase carboxypeptidase A was used to guide logical sequential improvements of a weakly active prototype inhibitor that led eventually to the highly optimized structure of captopril. The hypothetical working model of the active site of angiotensin-converting enzyme used to develop captopril continues to provide a firm basis for development of new types of specific inhibitors of this biologically important enzyme.

Rat brain enkephalinase: Characteristion of the active site using mercaptopropanoyl amino acid inhibitors, and comparison with angiotensin-converting enzyme

Over fifty mercaptopropanoyl amino acids and related derivatives were synthesized to define the steric, electronic and stereochemical requirements for binding to the active site of enkephalinase (ENKASE), and also for their ability to inhibit angiotensin-converting enzyme (ACE). In this way the character of ENKASE and ACE active sites were compared.

Purification and characterization of enkephalinase, angiotensin converting enzyme, and a third peptidyldipeptidase from rat brain

Three distinct peptidyldipeptidases (exopeptidases releasing carboxyl terminal dipeptide residues) can be solubilized from nerve terminal membrane fractions from whole rat brain or striatum, and separated by ion exchange chromatography. Brain angiotensin-converting enzyme (PDP-1) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin, and is markedly inhibited by several specific inhibitors such as captopril, teprotide, and MK-422. Enkephalinase (PDP-2) cleaves 80 nM [3H-Tyr1, Leu5]-enkephalin, but not Hip-His-Leu; it is not inhibited by any of the standard competitive inhibitors of angiotensin-converting enzyme (all analogs of carboxyl-terminal peptide sequences Phe-Ala-Pro or Ala-Pro), but is strongly inhibited by captopril analogs such as thiorphan (Phe-Gly analog). A third peptidyldipeptidase (PDP-3) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin; it is inhibited by dipeptide analog inhibitors such as captopril and thiorphan, but not by longer peptides such as teprotide or tripeptide analog inhibitors such as MK-422. Both PDP-2 (enkephalinase) and PDP-3 are apparently present in nerve terminal membranes predominantly as inactive proenzyme precursors, which elute from DEAE-cellulose at high salt concentration, and are activated very slowly by a process involving one or more trypsin-like enzymes. Rechromatography of activated PDP-2 and PDP-3 achieves a nearly complete separation of the two enzymes, both markedly purified, since each is much less acidic than its proenzyme precursor. Purified enkephalinase does not appear to have any significant endopeptidase activity. It cleaves Hip-Phe-Arg 200 times more effectively than Hip-Phe-Arg-NH2, and appears to be quite selective for cleaving the terminal dipeptide residue, Phe-Arg, from bradykinin, with no release of the second dipeptide and no cleavage of the Gly4-Phe5 interior peptide bond.

Chapter 9. Inhibitors of the Renin-Angiotensin System

Publisher Summary Pharmacological interruption of the renin-angiotensin system is a powerful tool for elucidating its role in hypertensive disease. This chapter discusses various approaches to the inhibition of different components of this system, but concentrates on the recent developments concerning those agents that have the greatest potential for therapeutic use. Renin is inhibited in vitro by high concentrations of naturally occurring renal lysophosphatidylethanolamines, synthetic phosphatidylethanolamines, and lysophosphatidylethanolamine analogs, and also by neutral lipids and phosphatidylcholine derivatives. In spite of their weak inhibitory activity in vitro , several of these compounds significantly lower blood pressure in the Goldblatt two-kidney renal hypertensive rat (RHR), a renin-mediated hypertensive model, in which one renal artery is constricted and the other kidney is left intact. Renin inhibitors have a unique potential for studying the pathophysiological function of the renin-angiotensin system, as they lack the ambiguity of angiotensin antagonists (partial agonism) or angiotensin-converting enzyme (ACE) inhibitors (bradykinin potentiation). However, none of the inhibitors of renin that have been developed till date has been thoroughly evaluated in vivo . Parenterally administered, AII antagonists and ACE inhibitors have contributed significantly to the understanding of the renin-angiotensin system and its role in human hypertension. The orally active ACE inhibitors show great promise as a novel type of antihypertensive drug.

Active-Site Specific Inhibitors of Angiotensin-Converting Enzyme

Angiotensin-converting enzyme (kininase II) con-verts the inactive decapeptide angiotensin I (Asp- Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) to the vasopressor and sodium-retaining octapeptide angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and inactivates the vasodepressor, sodium-deplet- ing nonapeptide bradykinin (Arg-Pro-Pro-Gly- Phe-Ser-Pro-Phe-Arg) (1, 2). Inhibition of this enzyme is the most effective method now employed for interrupting the pathophysiologic function of the renin-angiotensin-aldosterone system. Con-verting enzyme inhibitors do not have the agonistic activity encountered with presently available angiotensin II receptor antagonists (3); they bind to their receptor (the enzyme’s active site) much more effectively than the peptide (angiotensin I) with which they compete (4), and their target enzyme, unlike renin (5), is not subject to great increases in functional activity in response to reduced blood levels of angiotensin II. The ability of converting enzyme inhibitors to increase blood or tissue levels of bradykinin may be a diagnostic liability, but it is at least potentially a therapeutic asset.