A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M. tuberculosis have reaffirmed tuberculosis as a primary public health threat. However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years. Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M. tuberculosis. Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M. tuberculosis and promising oral activity in animal infection models. We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.

A simple, inexpensive apparatus for performance of preparative scale solution phase multiple parallel synthesis of drug analogs. II. Biological evaluation of a retrospective library of quinolone antiinfective agents.

A series of pure fluoroquinolone antiinfective agents was prepared by multiple parallel synthesis using a simple new apparatus. These compounds were evaluated biologically against Gram-positive and Gram-negative microorganisms and against a BCG strain transfected with luciferase in a fluorescence-based antitubercular assay. Activity against relatively fast growing, acid-fast Mycobacterium smegmatis was determined in part by agar-dilution streak assays. Data obtained against Escherichia coli-derived DNA gyrase does not correlate well with whole cell assays against E. coli. These compounds were assayed by a convenient glass-fiber filter binding method modified for high throughput screening. In these analogs, the results with a N-1 cyclopropyl substituent were often inferior to those obtained with a N-1 2',4'-difluorophenyl substituent. None of the new compounds prepared was superior in its antimycobacterial potency to ciprofloxacin or temafloxacin.

Characterization of the pH titration shifts of ribonuclease A by one- and two-dimensional nuclear magnetic resonance spectroscopy.

One- and two-dimensional 1H NMR experiments were used to determine the chemical shifts of resonances arising from 29 residues of RNase A in H2O at 17 pH values ranging from 1.2 to 7.9 Nearly all resonances displaying pH-induced changes in chemical shift greater than 0.1 ppm were monitored. Individual plots of the chemical shift as a function of pH were fit by nonlinear least squares methods to Henderson-Hasselbalch models yielding pK alpha values which were then analyzed using a set of criteria to determine their reliability. The criteria included statistics from the curve fitting analysis as well as the distance of the reporter proton to ionizing groups. Only the most reliable pK alpha values were assigned to specific ionizing groups within RNase A based upon the proximity of the reporter proton to the ionizing group as determined from the X-ray crystal structure. Only 2 of the 15 groups expected to undergo ionization within the pH range investigated could not be assigned pK alpha values within the highest two levels of reliability. Of the 11 carboxylate groups, 5 have pK alpha values less than 3.0. Many of the low pK alpha values can be interpreted as resulting from favorable hydrogen bonds between the carboxylate group and other moieties within the protein. The pK alpha values for the four histidine residues are similar to earlier literature reports. Two resonances underwent particularly large pH-induced shifts of approximately 2.3 ppm and corresponded to nitrogen-bound protons involved in hydrogen bonds with carboxylate groups.

Renin inhibitors: C-terminal oxetanes as potent transition-state mimics.

A novel transition-state mimic containing a C-terminal oxetane has been developed. Renin inhibitors incorporating this fragment exhibit enhanced potency against human plasma renin at physiological pH. The binding affinity of this new species has allowed size reductions at other sites.

Nonpeptide renin inhibitors with good intraduodenal bioavailability and efficacy in dog.

The aim of this study was the discovery of nonpeptide renin inhibitors with much improved oral absorption, bioavailability, and efficacy, for use as antihypertensive agents. Our prior efforts led to the identification of A-74273 [1,R = 3-(4-morpholino)propyl], with a bioavailability of 26 +/- 10% [10 mg/kg intraduodenally (id), dog]. In vivo metabolism studies of A-74273 showed that the morpholino moiety underwent metabolic degradation. Computer modeling of A-74273 bound to renin indicated that the C-terminus was involved in a hydrogen-bonding network. New C-terminal groups were examined in two series of nonpeptides for effects on renin binding potency, lipophilicity (log P), and aqueous solubility. Those groups which possessed multiple hydrogen-bonding ability (3,5-diaminotriazole, cyanoguanidines, morpholino) provided particularly potent renin binding. Intraduodenal bioavailabilities of selected compounds, evaluated in rats, ferrets, and dogs, were higher for inhibitors with moderate solubility as well as moderate lipophilicity, in general. Although the absolute values varied substantially among species, the relative ordering of the inhibitors in terms of absorption and bioavailability was reasonably consistent. Such well absorbed inhibitors (e.g. 41, 44, and 51) were demonstrated as highly efficacious hypotensive agents in the salt-depleted dog. We report here the discovery of a series of efficacious nonpeptide renin inhibitors based on the 3-azaglutaramide P2-P4 replacement, the best of which showed id bioavailabilities > 50% in dog.

Cardiovascular effects and hemodynamic mechanism of action of the novel, nonpeptidic renin inhibitor A-74273 in dogs.

A-74273 is a nonpeptidic, potent inhibitor of human and canine renin (IC50 = 3.1 and 43 nM, respectively, in plasma at pH 7.4) and has been shown to be orally active in dogs. To determine the hemodynamic mechanism underlying this renin inhibitor's hypotensive activity, the cardiac and hemodynamic effects of A-74273 were studied in sodium-depleted and sodium-replete pentobarbital-anesthetized dogs. Vehicle [5% dextrose in water (V, D5W), n = 8] or a single dose of A-74273 was administered intravenously (i.v.) as a bolus followed by a 30-min infusion (one tenth the bolus dose per minute). Baseline mean arterial pressure (MAP) was similar among all treatment groups, but baseline plasma renin activity (PRA) was increased in the sodium-depleted dogs as compared with the sodium-replete dogs. In sodium-depleted dogs (n = 7-8/dose), MAP decreased maximally as compared with baseline by 4 +/- 1, 19 +/- 3, and 23 +/- 3% during infusion of A-74273 at doses of 0.001, 0.01, and 0.1 mg/kg/min, respectively (p < 0.05 vs. baseline or V). The two highest infusion doses also produced significant reductions (p < 0.05 vs. baseline and V) in systemic vascular resistance (SVR, 21 +/- 2 and 25 +/- 2%) and left ventricular end-diastolic pressure (LVEDP, 40 +/- 8 and 47 +/- 12%). In sodium-replete dogs (n = 4/dose), an infusion dose of 0.01 mg/kg/min elicited no hemodynamic response, whereas 0.1 mg/kg/min reduced MAP by 13 +/- 2% (p < 0.05 vs. baseline) and SVR by 7 +/- 6%.(ABSTRACT TRUNCATED AT 250 WORDS)

Ameloblastic fibro-odontoma of the anterior maxilla. Report of a case.

The ameloblastic fibro-odontoma is an infrequently encountered mixed odontogenic tumor. There has been much discussion in the literature regarding its proper classification. It is characteristically slow growing and asymptomatic, and occurs most commonly in the posterior region. It shows a slight predilection for boys with a mean age of roughly 10 years. A case in a 9-month-old boy of an exophytic, rapidly growing ameloblastic fibro-odontoma of the anterior maxilla is reported.

Effects of high doses of A-74273, a novel nonpeptidic and orally bioavailable renin inhibitor.

Previous studies with peptidic renin inhibitors have shown that high intravenous (i.v.) doses can induce unexpectedly large decreases in blood pressure (BP) that appear to be independent of plasma renin inhibition. A-74273 represents a new class of potent and orally bioavailable nonpeptidic renin inhibitors. We evaluated the BP effects of this renin inhibitor administered orally (p.o.) or i.v. at high doses to conscious salt-depleted dogs. Administration of A-74273 at 30 and 60 mg/kg p.o. (n = 6 per dose) produced similar maximum reductions in BP (-40 +/- 4 vs. -46 +/- 5 mm Hg) despite the occurrence of greater plasma drug concentrations at the higher dose. Duration of hypotension, however, was increased (p < 0.05) from 9 h at 30 mg/kg to 18 h at 60 mg/kg. The initial depressor response to 10 and 30 mg/kg i.v. doses of A-74273 (n = 6 per dose) was comparable, although duration and overall BP response was greater at 30 mg/kg i.v. No BP responses to A-74273 were noted in salt-replete dogs (n = 5). The hypotension produced by 30 mg/kg p.o. A-74273 was completely reversed by norepinephrine (NE 5 micrograms/kg/min; n = 5) or isotonic saline (4 ml/min/kg, n = 5) infusion. These studies demonstrate that high doses of A-74273 result in predictable BP responses that are renin-dependent and reversible. Therefore, large decreases in BP with high doses is not an attribute common to all renin inhibitors but appears to be a function of the structural characteristics specific to a particular compound.

Discovery of a well-absorbed, efficacious renin inhibitor, A-74273.

The development of orally active renin inhibitors has been plagued by limited bioavailability in animals and humans. A-74273 is a novel, potent nonpeptide inhibitor of human renin (IC50 = 3.1 nM). This compound was absorbed into the portal and systemic circulations of anesthetized rats, ferrets, monkeys, and dogs after intraduodenal dosing. This favorable pattern also was observed after oral dosing in conscious animals, except in monkeys. Hepatic extraction of A-74273 was more efficient in rats and monkeys than in dogs or ferrets. A-74273 modestly inhibits dog renin, and when given orally as the base (0, 0.3, 1, 3, 10, and 30 mg/kg; n = 8 per dose) to conscious, salt-depleted dogs it induced dose-related reductions in mean arterial pressure and plasma renin activity. Peak falls in mean arterial pressure from normotensive baselines were -14 +/- 1, -26 +/- 3, and -44 +/- 3 mm Hg for the 3, 10, and 30 mg/kg groups, respectively (p < 0.05). Baseline plasma renin activity values (10.9 +/- 1.1-12.7 +/- 1.1 ng angiotensin I/ml/hr) were maximally inhibited, ranging from 43 +/- 8% at 0.3 mg/kg to 98 +/- 1% at 30 mg/kg. Bioavailability in this model was estimated to be 54 +/- 13% when plasma drug levels were determined by a renin inhibitory activity assay, but bioavailability was lower when compared with high-performance liquid chromatographic analysis of A-74273. This discrepancy was accounted for by the identification of structurally similar metabolites that are as active as the parent drug against human renin but much less potent against dog renin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of angiotensinase inhibitors on plasma protein binding and IC50 determinations of renin inhibitors.

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.

Discovery of a peptide-based renin inhibitor with oral bioavailability and efficacy.

Peptidic renin inhibitors have been poorly absorbed across the intestine or rapidly eliminated by the liver and have been reported to have oral bioavailabilities of less than 2%. A peptide-based renin inhibitor, A-72517 (molecular mass of 706 daltons), was devised that has oral bioavailabilities of 8, 24, 32, and 53% in the monkey, rat, ferret, and dog, respectively. Dose-related reductions in blood pressure, plasma renin activity, and plasma angiotensin II in parallel with increased plasma drug concentrations were observed after oral administration of A-72517 to conscious, salt-depleted dogs. Thus, peptide-based molecules of sizable molecular mass can be absorbed intact into the systemic circulation of animals. These findings support the potential of peptide-based drugs for oral administration.

1,2,3-trisubstituted cyclopropanes as conformationally restricted peptide isosteres: application to the design and synthesis of novel renin inhibitors.

The 1,2,3-trisubstituted cyclopropanes 6 and 7 are the first members of a novel class of isosteric replacements for peptide linkages that are more generally represented by the dipeptide mimics 2 and 3. These unique peptide surrogates are specifically designed to lock a section of a peptide backbone in an extended beta-strand conformation (phi-angle restriction) while simultaneously enforcing one of two specifically defined orientations for the amino acid side chain (chi 1-angle restriction). Methods were first developed for the stereoselective, asymmetric synthesis of the trisubstituted cyclopropanes 15a-d, 18a-d, 22a-d, and 23a-d (Scheme II), by an efficient approach featuring the Rh2(S-MEPY)4 (11) and Rh2(R-MEPY)4 (20) catalyzed cyclization of the allylic diazoacetates 10a-d to give the optically active lactones 12a-d and 21a-d, respectively, in up to greater than or equal to 94% enantiomeric excess. Nucleophilic opening of the lactone ring of 12a-d gave the corresponding morpholine amides 14a-d. By exploiting tactics that allowed for selective epimerization of one of the two functionalized side chains on the cyclopropane nucleus, 14a-d were transformed into the two series of diastereoisomeric morpholine amide carboxylic acids 15a-d and 18a-d. Epimerization of the morpholine amide group on 14a-d followed by Jones oxidation of the intermediate alcohols gave 15a-d. Alternatively, initial oxidation of the primary alcohol groups in 14a-d followed by selective, base-catalyzed inversion alpha to the aldehyde function and then Jones oxidation gave the diastereomeric dicarboxylic acid derivatives 18a-d. In a similar fashion, the enantiomeric lactones 21a-d were converted into the two corresponding enantiomeric series of dicarboxylic acid derivatives 22a-d and 23a-d. Inhibitors of aspartic proteinases, of which renin is a typical example, are known to bind to the enzyme active site cleft in an extended conformation. Thus, in order to evaluate the efficacy of 1,2,3-trisubstituted cyclopropanes as rigid replacements of beta-strand secondary structure in pseudopeptidic ligands, 15a-d, 18a-d, 22a-d, and 23a-d were incorporated at the P3 subsite of the potential renin inhibitors 24a-h and 25a-h by coupling with the tripeptide replacement 8. A significant number of substances inhibited renin at nanomolar concentrations. On the basis of this preliminary test, 1,2,3-trisubstituted cyclopropanes do appear to constitute a viable new class of peptide mimics. Since the stereochemistry at each carbon on the cyclopropane ring may be altered, these novel replacements may also function as stereochemical probes to establish the conformation of pseudopeptide ligands bound to their macromolecular targets.

C-terminal modifications of nonpeptide renin inhibitors: improved oral bioavailability via modification of physicochemical properties.

We describe the development of a series of soluble, potent, and bioavailable nonpeptide renin inhibitors. These inhibitors derived from a series of novel nonpeptide renin inhibitors which were recently identified in our laboratories, by alteration of the nature of the C-terminus (P2') of the molecules. Introduction of basic substituents into modified hydroxyethylene dipeptide isosteres gave inhibitors with improved solubility as well as improved potency against human plasma renin. In addition, these modifications produced inhibitors which displayed markedly improved intraduodenal bioavailability in both the ferret and cynomolgus monkey. We also present data which demonstrate excellent efficacy in the monkey for A-74273 (65), with an intraduodenal bioavailability of 16 +/- 4% in the monkey, compared to 1.7 +/- 0.5% for the dipeptide renin inhibitor enalkiren (A-64662, 75). A-74273 is an example of a nonpeptide inhibitor which possesses a good balance of the desirable properties of potency, solubility, and lipophilicity and which is well absorbed into the intestine.

Nonpeptide renin inhibitors employing a novel 3-aza(or oxa)-2,4-dialkyl glutaric acid moiety as a P2/P3 amide bond replacement.

A new series of renin inhibitors has been developed. The inhibitors feature a novel replacement for the P2/P3 dipeptide moiety normally associated with renin inhibitors. The dipeptide replacement was a (2S,4S)-3-aza(or oxa)-2,4-dialkylglutaric acid amide. Extensive structure-activity relationship studies determined that optimum potency was achieved when inhibitors employed a benzyl and butyl group at the C(4) and C(2) carbon position, respectively. In addition, maximum in vitro potency was obtained when the N-terminus was functionalized by incorporating a 4-(1,3-dioxabutyl)piperidine amide. SAR data suggested that the 1,3-dioxabutyl group (methoxymethyl ether) interacted by hydrogen bonding to groups in the S4 domain of renin. This hypothesis was strengthened when a 4-butylpiperidine amide was substituted and inhibitor potency decreased dramatically. Inhibitors employing this novel dipeptide mimic were prepared by coupling the glutaric acid amides with either the transition-state mimic (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6- methylheptane (18) or the hydroxyethylene dipeptide isostere. The glutaric acid amides were prepared by two general procedures. The first procedure involved the reductive amination of alpha-amino acid esters with alpha-keto esters. The second procedure involved the displacement reaction of alpha-bromo esters or acids with alpha-amino acid amides.

Slow, tight binding to human renin of some nonpeptidic renin inhibitors containing a 4-methoxymethoxypiperidinylamide at the P4 position.

A series of nonpeptidic human renin inhibitors with a 4-methoxymethoxypiperidinylamide at the P4 position of the molecule exhibited slow tight binding to the enzyme. Replacement of the methoxymethoxy moiety on the piperidine ring with H, OH, methoxyethyl, propyloxy or n-butyl eliminated the effect. The inhibition was partially reversed by prolonged dialysis at 4 degrees C, arguing against formation of a covalent bond in the tightened complex.

New macrolides active against Streptococcus pyogenes with inducible or constitutive type of macrolide-lincosamide-streptogramin B resistance.

Macrolide-resistant bacteria can be classified as inducibly resistant or constitutively resistant. Inducibly resistant bacteria are resistant to 14-membered macrolides, such as erythromycin and clarithromycin (A-56268), but are susceptible to the 16-membered macrolides, such as tylosin and spiramycin, as well as to clindamycin. Constitutively resistant bacteria are resistant to macrolide-lincosamide-streptogramin B antibiotics. In this study, the MICs of several erythromycin and clarithromycin analogs against macrolide-susceptible and macrolide-resistant Streptococcus pyogenes strains were determined. Four 11,12-carbamate analogs of clarithromycin had lower MICs than erythromycin did against S. pyogenes with the inducible or constitutive type of macrolide-lincosamide-streptogramin B resistance. Five 11,12-carbonate analogs of erythromycin with modifications at the 4" position of cladinose had lower MICs than did erythromycin against S. pyogenes with the constitutive type of resistance, and one of these compounds, which had a naphthyl-glycyl substitution at the 4" position, had a lower MIC than erythromycin against both the inducibly resistant and constitutively resistant strains. Two analogs of erythromycin with a modification on the 4" position of cladinose had lower MICs than erythromycin did against the constitutively resistant organisms but not against the inducibly resistant organisms. Thus, 14-membered macrolides can be modified so as to confer a low MIC when tested in vitro.

An interactive data management and analysis system for clinical investigators.

An interactive minicomputer-based system has been developed that enables the clinical research investigator to personally explore and analyze his research data and, as a consequence of these explorations, to acquire more information. This system, which does not require extensive training or computer programming, enables the investigator to describe his data interactively in his own terms, enter data values while having them checked for validity, store time-oriented patient data in a carefully controlled on-line data base, retrieve data by patient, variable, and time, create subsets of patients with common characteristics, perform statistical analyses, and produce tables and graphs. It also permits data to be transferred to and from other computers. The system is well accepted and is being used by a variety of medical specialists at the three clinical research centers where it is operational. Reported benefits include less elapsed and nonproductive time, more thorough analysis of more data, greater and earlier insight into the meaning of research data, and increased publishable results.