An orally active inhibitor of renin.

A potent renin inhibitor, U-71038 (Boc-Pro-Phe-N-MeHis-Leu psi[CHOHCH2]Val-Ile-Amp), was tested for oral effectiveness. Enzyme kinetic studies indicated that U-71038 was a competitive inhibitor of hog renin with an inhibitor constant (Ki) value of 12 nM. Intravenous as well as oral administration of U-71038 to anesthetized, ganglion-blocked rats infused with hog renin elicited dose-related hypotensive responses. Intravenous administration of U-71038 to conscious, sodium-depleted monkeys caused dose-related decreases of blood pressure and plasma renin activity without affecting heart rate. Similarly, the oral administration of U-71038 at 50 mg/kg to conscious, sodium-depleted monkeys elicited a pronounced hypotension and decrease in plasma renin activity that persisted for 5 hours. The hypotensive responses elicited by intravenous and oral administration of U-71038 to hog renin-infused rats and sodium-depleted monkeys were shown to be due entirely to inhibition of the renin-angiotensin system. A comparison of the results obtained after the intravenous administration of U-71038 with the results obtained after the oral administration of U-71038 implied that at least 10% of the orally administered U-71038 must have been absorbed to cause the observed effects in hog renin-infused rats and sodium-depleted monkeys. The studies demonstrated that an inhibitor of renin with a long duration of action and with oral effectiveness is a feasible entity.

Conformationally constrained renin inhibitory peptides: gamma-lactam-bridged dipeptide isostere as conformational restriction.

A model of the conformation of the enzyme-bound inhibitor of human renin suggested the possibility of a gamma-lactam conformational restriction at the P2-P3 site. Synthetic routes to these gamma-lactam dipeptide isosteres and their incorporation into potential renin inhibitors are described. Peptide VIa,b with a gamma-lactam conformational constraint and a hydroxyethylene isostere at the cleavage site inhibited human plasma renin with an IC50 value of 6.5 nM. The flexibility of these syntheses should make available a number of potential enzyme inhibitors with this structural feature for the study of enzyme-bound conformers.

alpha,alpha-Difluoro-beta-aminodeoxystatine-containing renin inhibitory peptides.

The preparations of sodium 4(S)-[(tert-butyloxycarbonyl)amino]-2,2-difluoro-3(S)- and -3(R)-[(4-methoxyphenyl)amino]-6-methylheptanoates (7a and 7b) from sodium 4(S)-[(tert-butyloxycarbonyl)amino]-2,2-difluoro-3(R)- and -3(S)-hydroxy-6-methylheptanoates (1a and 1b) are described. The key step involves the stereospecific intramolecular displacement via a Mitsunobu reaction for the conversion of a beta-hydroxy hydroxamate to a beta-lactam ring. Compounds 7a and 7b are useful as synthetic intermediates for the preparation of enzyme inhibitors that contain 3(S),4(S)- and 3(R),4(S)-diamino-2,2-difluoro-6-methylheptanoic acid inserts. Angiotensinogen analogues VII and VIII that contain these novel amino analogues of difluorostatine were shown to be inhibitors of the enzyme renin. The alpha,alpha-difluoro-beta-aminodeoxystatine-containing compounds were shown to be weaker inhibitors than the corresponding difluorostatine-containing congeners.

Conformationally constrained renin inhibitory peptides: cyclic (3-1)-1-(carboxymethyl)-L-prolyl-L-phenylalanyl-L-histidinamide as a conformational restriction at the P2-P4 tripeptide portion of the angiotensinogen template.

Interest in conformationally constrained peptides as potential inhibitors of renin led us to examine an N-terminal cycle of linear renin inhibitory peptides. A cyclic structure was prepared by joining the N-terminal proline at the P4 site to the imidazole ring of histidine at the P2 site via a carboxymethylene fragment. An efficient synthetic route to this 14-membered macrocycle was developed and this N-terminal cyclic tripeptide could be readily incorporated into renin inhibitory peptides. Monte Carlo molecular modeling methods were used to generate bound conformations of a representative inhibitor in a model of the renin active site, suggesting possible modes of binding of these inhibitors to renin. Two representative compounds that contain this 14-membered macrocycle were evaluated for their inhibitory activities against human plasma renin and they were found to exhibit very high binding affinity with IC50 values in the nanomolar and subnanomolar range.

Renin inhibitors containing psi[CH2O] pseudopeptide inserts.

Renin inhibitors 2-4 with the D-Lys renin inhibitory peptide (RIP) sequence, but containing Leu psi[CH2O]Ala (2), Leu psi[CH2O]Val (3), and Leu psi[CH2O]Leu (4) at the P1-P1' site, were of a comparable potency to RIP. N-Terminal Boc-protected inhibitors containing Pro psi[CH2O]Phe in positions P4-P3 were potent inhibitors of renin, with Boc-Phe-Pro psi[CH2O]Phe-His-Leu psi[CH(OH)CH2]Val-Ile-(2-aminomethyl) pyridine (17) having an IC50 of 1.6 X 10(-9) M.

Renin inhibitory peptides. A beta-aspartyl residue as a replacement for the histidyl residue at the P-2 site.

In an effort to decrease the size and to increase the hydrophilicity of the previously prepared renin inhibitory peptides, it was postulated that one might be able to take advantage of the polar Thr-84 on the flap region of the enzyme renin by potential hydrogen bonding to polar functionality on the inhibitory peptide at the P-2 site. A beta-aspartyl residue with a carboxylic acid group was proposed to be a possible replacement for the histidyl residue at the P-2 site. A series of renin inhibitory peptides were prepared with the beta-aspartyl residue to probe the structure-activity relationship of the resulting peptides. Potent inhibitory peptides could be realized with activity in the subnanomolar range. Molecular modeling was also undertaken to investigate the interactions between the enzyme active site and the new inhibitors and to suggest a possible mode of binding of these ligands to the enzyme. From this modeling study, the role of Ser-229 at the active site in the bound conformer of the inhibitors was suggested. It was further noted in the analogue study that a malic acid residue, which is the oxygen analogue of the beta-aspartic acid residue, could lead to further enhancement of inhibitory potency of congeneric peptides. Small renin inhibitors, such as compound XII with molecular weight 535 and with no alpha-amino acid residue, could be prepared and exhibited renin inhibitory activity in the nanomolar range.

alpha-Methylproline-containing renin inhibitory peptides: in vivo evaluation in an anesthetized, ganglion-blocked, hog renin infused rat model.

A structure-activity analysis of peptides containing backbone C alpha-methyl modification at the P4 site of the angiotensinogen sequence led to the discovery of potent renin inhibitors with apparent in vitro metabolic stability. Boc-alpha-MePro-Phe-His-Leu psi[CHOHCH2]Val-Ile-Amp dicitrate (Va) is a potent inhibitor of human plasma renin with an IC50 value of 1.8 nM. This peptide was shown not to be degraded in vitro by chymotrypsin, elastase, pepsin, and a rat liver homogenate preparation. It is also a potent inhibitor of hog renin with an IC50 value of 1.6 nM and was shown to elicit in vivo activity and cause dose-dependent hypotensive responses when given intravenously to anesthetized ganglion-blocked, hog renin infused rats.

Design and synthesis of potent and specific renin inhibitors containing difluorostatine, difluorostatone, and related analogues.

Peptides that contain difluorostatine and difluorostatone residues have been shown to be potent inhibitors of the aspartyl protease renin. The readily hydrated fluoro ketone is proposed to mimic the tetrahedral intermediate that forms during the enzyme-catalyzed hydrolysis of a peptidic bond. It is suggested that the sp3-hybridized ketal acts as a transition-state analogue renin inhibitor. The fluoro ketone is shown to be a much more effective inhibitor than the corresponding nonfluorinated ketone, which acts as a pseudosubstrate. More lipophilic side chains at the P1 site can enhance the inhibitory potency of the difluorostatine analogue, but this cannot be demonstrated in the difluorostatone series. Additionally, high renin specificity has been shown for a difluorostatone-containing peptide.

Design and synthesis of a potent and specific renin inhibitor with a prolonged duration of action in vivo.

A structure-activity analysis of peptides containing backbone C alpha-methyl and N alpha-methyl modifications led to the discovery of potent renin inhibitors with high metabolic stability. In vitro, Boc-Pro-Phe-N alpha-MeHis-Leu psi-[CHOHCH2]Val-Ile-Amp (XII) is a potent inhibitor of human plasma renin with IC50 of 0.26 nM. It is a much weaker inhibitor of other aspartic proteases such as porcine pepsin or bovine cathepsin D (IC50 = 6 microM). It was shown not to be degraded by a rat liver homogenate preparation. In vivo, it inhibited plasma renin activity and lowered blood pressure of furosemide-treated cynomolgus monkeys. At a dose of 5 mg/kg iv, the pronounced hypotensive response persisted for greater than 3 h postinfusion.

Renin inhibitors. Design of angiotensinogen transition-state analogues containing novel. (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid.

A highly stereoselective synthesis of 2(R)-[5(R)-[1(S)-[(tert-butyloxycarbonyl)amino]-3-methylbutyl]-2,2- dimethyl-4(R)-dioxolanyl]-3-methylbutanoic acid is described. This is a suitably protected carboxylic acid useful as an intermediate for the preparation of renin inhibitory peptides. Angiotensinogen analogues such as peptides IX and X that contain the dipeptide isostere (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid residue at the scissile site are shown to be potent inhibitors of human plasma renin. The glycol moiety in this novel acid, dihydroxyethylene isostere, is suggested to act as a transition-state analogue and mimics the tetrahedral intermediate formed during the enzyme-catalyzed hydrolysis of the peptidic bond.

Synthesis and renin inhibitory activity of angiotensinogen analogues having dehydrostatine, Leu psi [CH2S]Val, or Leu psi [CH2SO]Val at the P1-P1' cleavage site.

The synthesis and in vitro renin inhibitory potencies of angiotensinogen (ANG) analogues having amide (CONH) bond replacements at P1-P1', the Leu-Val cleavage site, corresponding to Leu psi[CH2SO]Val, and the trans olefinic analogue of statine (Sta), 4(S)-amino-6-methyl-2(E)-heptenoic acid (dehydrostatine, Dhs), are reported. These are compared to P1-P1' Leu psi[CH2NH]Val-, Sta-, or Phe-Phe-substituted analogues of the same template. The Dhs pseudodipeptide was found to be an adequate mimic of a trans CONH bond and gave a peptide, H-Pro-His-Pro-Phe-His-Dhs-Ile-His-D-Lys-OH, approximately equal in potency to a Phe-Phe-containing inhibitor, but 200-fold less potent than its Sta-substituted congener. That the enhanced potency of the Sta-containing peptide most likely depends on hydrogen bonding as well as tetrahedral geometry is indicated by the 50-100-fold lower potency of the tetrahedral Leu psi[CH2S]Val and Leu psi[CH2SO]Val analogues as compared to the Leu psi[CH2NH]Val-containing congener.

Potent renin inhibitory peptides containing hydrophilic end groups.

A previously reported renin inhibitor, Boc-Pro-Phe-N(Me)His-Leu psi [CHOHCH2]Val-Ile-Amp (U-71038), was altered by the incorporation of polar, hydrophilic moieties at either end, e.g., tris(hydroxymethyl)aminomethane (THAM) or glucosamine urea groups at the N-terminus, and the THAM amide or aminomethylpyridine N-oxide at the C-terminus. These modified analogues, with dramatically improved water solubility, all retained the potent renin inhibitory activity of U-71038 in vitro. The fact that good activity was maintained in these new analogues, which possess hydrophilicity and steric bulk considerably different from the parent compound, suggests that neither end of these molecules is critical for recognition and binding of the inhibitors by renin. These modified analogues were evaluated in a rat model, and several exhibited hypotensive activity after both oral and iv administration which was greater in magnitude and longer in duration than that caused by equimolar doses of U-71038. Furthermore, unlike U-71038, the oral activity of these analogues was not dependent upon administration in a citric acid vehicle.

Renin inhibitory peptides. Incorporation of polar, hydrophilic end groups into an active renin inhibitory peptide template and their evaluation in a human renin infused rat model and in conscious sodium-depleted monkeys.

We previously reported that Boc-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]-Ile-Amp (1) is a potent and specific inhibitor of human renin in vitro. It was shown to resist degradation by selected proteases and a rat liver homogenate. It was shown to inhibit plasma renin activity and to reduce blood pressure in renin-dependent-animal models both by the intravenous and by the oral routes using dilute citric acid as vehicle. In an effort to discover compounds with improved pharmacological efficacy, we set out to modify the physical characteristics of this highly lipophilic renin inhibitor by incorporation of hydrophilic end groups. We report here a variety of water-solubilizing groups and the resulting structure-activity relationship of these compounds. They all maintain an extremely high level of enzyme inhibitory activity in vitro. Evaluation of these potent renin inhibitors in a human renin infused rat model suggests that some of these compounds exhibit improved pharmacological efficacy in vivo. This observation was further confirmed in the conscious sodium-depleted cynomolgus monkey. Importantly, the oral efficacy was demonstrated in a water vehicle in the absence of citric acid.

Tetrahydro-2-benzoxepins: a novel family of hypotensives.

A novel series of 1-(alkylamino)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins shows hypotensive activity. A typical example is 1-[2-(1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepin-1-yl)ethyl]-4-(4-fluorophenyl)piperazine. This compound is an alpha blocker with peripheral and central activities.

Design, structure-activity, and molecular modeling studies of potent renin inhibitory peptides having N-terminal Nin-For-Trp (Ftr): angiotensinogen congeners modified by P1-P1' Phe-Phe, Sta, Leu psi[CH(OH)CH2]Val or leu psi[CH2NH]Val substitutions.

A structure-conformation-activity investigation of several angiotensinogen (ANG) based inhibitors of human renin modified by either Phe-Phe, Sta, Leu psi[CH2NH]Val, or Leu psi[CH(OH)CH2]Val at the P1-P1' clevage site and P5 Trp(Nin-For) (Ftr) was performed. Specifically, Ac-Ftr-Pro-Phe-His-Phe-Phe-Val-Ftr-NH2 (1) provided a potent (KI = 2.7 X 10(-8) M) P1-P1' Phe-Phe substituted renin inhibitor to initiate these studies. Substitution of the P1-P1' Phe-Phe in compound 1 by Sta effected a 1,000-fold increase in biological potency for the resultant octapeptide Ac-Ftr-Pro-Phe-His-Sta-Val-Ftr-NH2 (10; KI = 6.7 X 10(-11) M). Kinetic analysis of compound 10 showed it to be a competitive inhibitor of human renin catalyzed proteolysis of human ANG. Chemical modifications of the compounds 1 and 10 were evaluated on the basis of comparative human plasma renin inhibitory activities (IC50 values) in vitro. Carboxy-terminal truncation studies on compound 10 showed that the P2' Val and P3' Ftr residues could both be eliminated without significant loss (ca. 10-fold) in renin inhibitory activity as exemplified by the pentapeptide Ac-Ftr-Pro-Phe-His-Sta-NH2 (12; IC50 = 3.8 X 10(-9) M). In addition, the corresponding P1-P1' Leu psi[CH(OH)CH2]Val and Leu psi[CH2NH]Val derivatives of compound 12 were potent renin inhibitors: Ac-Ftr-Pro-Phe-His-Leu psi[CH(OH)CH2]Val-NH2 (13; IC50 = 3.1 X 10(-10) M) and Ac-Ftr-Pro-Phe-His-Leu psi[CH2NH]Val-NH2 (14; IC50 = 2.1 X 10(-8) M). The structure-conformation-activity properties of the N-terminal Ftr substitution of these human renin inhibitors was examined by (1) comparative analysis of several analogues of 1 and Ac-Ftr-Pro-Phe-His-Sta-Ile-NH2 (17; IC50 = 1.0 X 10(-10) M) having P5 site modifications by Trp, His, D-Ftr, and D-His, (2) deletion of the N-terminal Ftr residue from compounds 12 and 17, to provide Ac-Pro-Phe-His-Sta-Ile-NH2 (16; IC50 = 3.1 X 10(-8) M) and Ac-Pro-Phe-His-Sta-NH2 (15; IC50 = 5.6 X 10(-6) M), and (3) computer modeling and dynamics studies of compounds 1 and 17 bound to CKH-RENIN, a simulated human renin model, which were focused on identifying potential intermolecular interactions of their common P5-P2 sequence, Ac-Ftr-Pro-Phe-His, at the enzyme active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Historical development of saralasin.

The research environment in which saralasin was discovered has been described, and illustrations of the rationale which contributed to its synthesis, selection for development, and eventual development have been presented. As an example, the synthesis of [Sar1, Val5, Ala8]-AII (P113, saralasin) was an attempt to make an AII antagonist which would be resistant to metabolism by aminopeptidases. Subsequent evaluation, however, indicated that the substitution of sarcosine had not only protected against aminopeptidase degradation but unexpectedly also had greatly increased the octapeptide's affinity for vascular smooth muscle receptors. Finally, the laboratory demonstration of saralasin as a potential therapeutic and diagnostic entity and the clinical confirmation of use of saralasin in hypertensive patients are reviewed.

Renin inhibitory peptides: a study of structural modifications in the peptide backbone.

We studied the peptide backbone modifications that improve the metabolic stability of the resulting peptides and yet retain high inhibitory activity against human plasma renin. A systematic investigation of N-alpha-methyl and C-alpha-methyl modifications at the P2 and P3 sites of renin-inhibitory peptides that contain part of the human angiotensinogen sequence led to the discovery of N-alpha-methyl amino acids at the P2 site as a useful structural modification. U-71,038 (11) inhibited human plasma renin with an in vitro potency (IC50) of 2.6 x 10(-10) mol/l. It is highly selective for renin and, as anticipated, resistant to proteolytic degradation. Additional study based on molecular graphic modelling has led us to propose a gamma-lactam conformational constraint at the P2-P3 site. This pseudo-dipeptide has proved useful in the preparation of active renin inhibitors. Compound 18a inhibited human plasma renin with an in vitro potency (IC50) of 2.1 x 10(-9) mol/l. This class of compounds also offers structural features for the study of enzyme-bound conformers.

Systemic and renal haemodynamic effects of renin or angiotensin converting enzyme inhibition in non-human primates.

The cardiovascular actions of a renin inhibitor, U-71038 (Boc-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]Val-Ile-Amp), and of an angiotensin converting enzyme (ACE) inhibitor, captopril, were determined in conscious sodium-depleted cynomolgus monkeys. Cardiac output was measured with a thermodilution technique. The hypotension induced by U-71038 was associated with a significant reduction in total peripheral resistance without alteration in cardiac output or the heart rate. A similar reduction in total peripheral resistance was observed after captopril at a dose which caused hypotension equivalent to that elicited by U-71038. The latter effects were not accompanied by significant alterations in cardiac output or the heart rate. The glomerular filtration rate was measured by the plasma disappearance of 125I-sodium iothalamate. Renin or ACE inhibition adequate to cause equivalent hypotensive responses did not change the glomerular filtration rate to a significantly different degree. The systemic and renal haemodynamic profiles of U-71038 and captopril appear to be similar, suggesting that renin and ACE inhibition elicit fundamentally similar cardiovascular effects in conscious sodium-depleted cynomolgus monkeys via a decreased formation of angiotensin II (Ang II).