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SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

The sirtuins SIRT1, SIRT2, and SIRT3 are NAD(+) dependent deacetylases that are considered potential targets for metabolic, inflammatory, oncologic, and neurodegenerative disorders. Encoded library technology (ELT) was used to affinity screen a 1.2 million heterocycle enriched library of DNA encoded small molecules, which identified pan-inhibitors of SIRT1/2/3 with nanomolar potency (e.g., 11c: IC50 = 3.6, 2.7, and 4.0 nM for SIRT1, SIRT2, and SIRT3, respectively). Subsequent SAR studies to improve physiochemical properties identified the potent drug like analogues 28 and 31. Crystallographic studies of 11c, 28, and 31 bound in the SIRT3 active site revealed that the common carboxamide binds in the nicotinamide C-pocket and the aliphatic portions of the inhibitors extend through the substrate channel, explaining the observable SAR. These pan SIRT1/2/3 inhibitors, representing a novel chemotype, are significantly more potent than currently available inhibitors, which makes them valuable tools for sirtuin research.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

Carba-NAD is a synthetic compound identical to NAD except for one substitution, where an oxygen atom adjacent to the anomeric linkage bearing nicotinamide is replaced with a methylene group. Because it is inert in nicotinamide displacement reactions, carba-NAD is an unreactive substrate analogue for NAD-consuming enzymes. SIRT3 and SIRT5 are NAD-consuming enzymes that are potential therapeutic targets for the treatment of metabolic diseases and cancers. We report an improved carba-NAD synthesis, including a pyrophosphate coupling method that proceeds in approximately 60% yield. We also disclose the X-ray crystal structures of the ternary complexes of SIRT3 and SIRT5 bound to a peptide substrate and carba-NAD. These X-ray crystal structures provide critical snapshots of the mechanism by which human sirtuins function as protein deacylation catalysts.

O-Acetyl-ADP-ribose (OAADPR) is a metabolite produced from nicotinamide adenine dinucleotide (NAD) as a product of sirtuin-mediated protein deacetylation. We present here a simple, one-step, nonenzymatic synthesis of OAADPR from NAD and sodium acetate in acetic acid. We extended the reaction to other carboxylic acids, demonstrating that the reaction between NAD and nonaqueous carboxylate buffers produces mixtures of the corresponding 2'- and 3'-carboxylic esters.

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340-8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.

Telaprevir 2 (VX-950), an inhibitor of the hepatitis C virus (HCV(a)) NS3-4A protease, is in phase 3 clinical trials. One of the major metabolites of 2 is its P1-(R)-diastereoisomer, 3 (VRT-394), containing an inversion at the chiral center next to the alpha-ketoamide on exchange of a proton with solvent. Compound 3 is approximately 30-fold less active against HCV protease. In an attempt to suppress the epimerization of 2 without losing activity against the HCV protease, the proton at that chiral site was replaced with deuterium (d). The compound 1 (d-telaprevir) is as efficacious as 2 in in vitro inhibition of protease activity and viral replication (replicon) assays. The kinetics of in vitro stability of 1 and 2 in buffered pH solutions and plasma samples, including human plasma, suggest that 1 is significantly more stable than 2. Oral administration (10 mg/kg) in rats resulted in a approximately 13% increase of AUC for 1.

SIRT3 is a major mitochondrial NAD(+)-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD(+). In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD(+). These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.

SIRT1 is an NAD(+)-dependent protein deacetylase that appears to produce beneficial effects on metabolic parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to and more potent than resveratrol.

A series of imidazo[1,2-b]thiazole derivatives is shown to activate the NAD(+)-dependent deacetylase SIRT1, a potential new therapeutic target to treat various metabolic disorders. This series of compounds was derived from a high throughput screening hit bearing an oxazolopyridine core. Water-solubilizing groups could be installed conveniently at either the C-2 or C-3 position of the imidazo[1,2-b]thiazole ring. The SIRT1 enzyme activity could be adjusted by modifying the amide portion of these imidazo[1,2-b]thiazole derivatives. The most potent analogue within this series, namely, compound 29, has demonstrated oral antidiabetic activity in the ob/ob mouse model, the diet-induced obesity (DIO) mouse model, and the Zucker fa/fa rat model.

SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C-terminus of human SIRT3 but lacks an N-terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5' rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full-length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD(+). Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.

A series of triamide derivatives bearing a benzothiazole core is shown to be potent microsomal triglyceride transfer protein (MTP) inhibitors. In order to minimize liver toxicity, these compounds have been optimized to have activity only in the enterocytes and have limited systemic bioavailability. Upon oral administration, selected analogs within this series have been further demonstrated to reduce food intake along with body weight and thereby improve glucose homeostasis and insulin sensitivity in a 28-day mice diet-induced obesity (DIO) model.

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

Reversible tetrapeptide-based compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease. Inhibition of viral replicon RNA production in Huh-7 cells has also been demonstrated. We show herein that the inclusion of hydrogen bond donors on the P4 capping group of tetrapeptide-based inhibitors result in increased binding potency to the NS3.4A protease. The capping groups also impart significant effects on the pharmacokinetic profile of these inhibitors.

Hepatitis C virus (HCV) NS3.4A protease inhibitors have potential for treating chronic HCV disease. Robust antiviral effects have been reported for the three HCV NS3.4A inhibitors (BILN-2061 (ciluprevir), telaprevir (VX-950; Vertex Pharmaceuticals Inc./Janssen Pharnmaceutica NV/Mitsubishi Pharma Corp.) and SCH-503034; Schering-Plough Research Institute) that have been studied in clinical trials to date in HCV-infected patients, and new inhibitor molecules continue to appear on the horizon. Herein, toe relate the remarkable progress of these drug candidates to recent evidence that suggests HCV might depend on NS3.4A protease to subvert multiple innate cellular defense mechanisms.

The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log(10) was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log(10) reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.

The hepatitis C virus (HCV) NS3.4A protease, which is essential for viral replication, is considered one of the most attractive targets for developing novel anti-HCV therapies. However, discovery of potent and selective small-molecule inhibitors of HCV NS3.4A protease as oral drug candidates has been hampered by the shallow substrate-binding groove of the protease. Serine trap warheads have been used to covalently anchor inhibitor scaffolds and to increase their affinity to the protease. This review will examine the evolution of covalent inhibitors of the HCV NS3.4A protease from early aldehyde molecules to alpha-ketoamide inhibitors. Kinetic and structural studies of alpha-ketoacid and alpha-ketoamide inhibitors revealed an unusual mechanism of binding in the catalytic site. Optimization of alpha-ketoamide scaffolds by scientists at Vertex and Eli Lilly led to the discovery of VX-950, a novel, potent, selective inhibitor of HCV NS3.4A protease. VX-950 possesses excellent antiviral activity in both HCV replicon cells and human fetal hepatocytes infected with HCV-positive patient sera. In addition, VX-950 exhibits a favorable pharmacokinetic profile in several animal species and demonstrates potent inhibition of the HCV NS3.4A protease activity in a mouse model. In a recent phase 1b clinical trial, VX-950 was able to rapidly reduce the plasma viral load of patients chronically infected with genotype 1 HCV by a mean approximately 3 log(10) in 2 days. The median viral load reduction was 4.4 log(10) for the best dose group after 14 days of dosing. The pre-clinical profile and early clinical data of VX-950 will be discussed in this review.

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.

We recently described the identification of an optimized alpha-ketoamide warhead for our series of HCV NS3.4A inhibitors. We report herein a series of HCV protease inhibitors incorporating 3-alkyl-substituted prolines in P(2). These compounds show exceptional enzymatic and cellular potency given their relatively small size. The marked enhancement of activity of these 3-substituted proline derivatives relative to previously reported 4-hydroxyproline derivatives constitutes additional evidence for the importance of the S(2) binding pocket as the defining pharmacophore for inhibition of the NS3.4A enzyme.

The alpha-ketoamide warhead (e.g., 15) was found to be a practical replacement for aliphatic aldehydes in a series of HCV NS3.4A protease inhibitors. Structure-activity relationships and prime side optimization are discussed.

Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.

A non-nucleoside class of compounds that inhibits the replication of hepatitis B virus (HBV) in cell culture has been discovered. A series of substituted analogues of phenylpropenamide 6 has been prepared and evaluated in the HepAD38 cellular assay. Structure-activity relationships of this series are discussed.

The hepatitis C virus (HCV) epidemic represents a significant unmet medical need. Current treatment is arduous and less than 50% effective. Indeed, interferon-alpha treatment is so unpleasant that asymptomatic patients will often stop treatment. Heartened by the success of HIV protease inhibitors, researchers have considered inhibition of the HCV NS3 serine protease an attractive mode of intervention. This account discusses the structure and function of NS3 4A and the prospects of its success as a therapeutic target. The review includes a detailed discussion of the role of the NS4A co-factor, requirements for inhibition and some recent examples of peptidomimetic and small-molecule inhibitors.

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

Two new series of antitumor agents, 4-aminomethylthioxanthenones (6-50) and 5-aminomethylbenzothiopyranoindazoles (51-61), are described and compared. Nearly all members of both series display excellent in vivo activity versus murine pancreatic adenocarcinoma 03 (Panc03) although there is little to distinguish the two series from each other. In both series there is no discernible relationship between structure and in vivo efficacy. Selected analogues were evaluated in vitro; all were observed to have moderate to strong DNA binding via intercalation. However, varying degrees of in vitro P388 cytotoxicity and topoisomerase II inhibition were seen. In general, those molecules which exhibited strong topoisomerase II inhibition were significantly more cytotoxic than those which did not. In both series, those derivatives (48-50, 60, and 61) having a phenolic hydroxy substitution exhibited the most potent P388 cytotoxicity and topoisomerase II inhibition.

Novel antiherpetic 3-quinolinecarboxamides were discovered as part of a drug discovery program at Sterling Winthrop Inc. A major goal of this research was to identify novel non-nucleoside agents possessing activity against acyclovir resistant herpes simplex virus. From screening compound libraries in an HSV-2 plaque reduction assay, 1-ethyl-1,4-dihydro-4-oxo-7-(4-pyridinyl)-3-quinolinecarboxamide (1) emerged as an attractive lead structure. By modifying the quinoline ring at the 1-, 2-, 3-, 4-, and 7-positions, analogues were identified that have up to 5-fold increased in vitro potency relative to acyclovir. In a single dose mouse model of infection the 1-(4-FC6H4) analogue 17, one of the most potent derivatives in vitro, displayed comparable oral antiherpetic efficacy to acyclovir at 1/16 the dose; in a multiple dose regimen, however, it was 2-fold less potent. Mechanism of action studies indicate that these new compounds interact with a different, as yet undefined, molecular target than acyclovir.

Supported by the antiherpetic properties of 3-quinolinecarboxamides and the importance of the planar intramolecular H-bonded beta-keto amide pharmacophore, a series of novel conformationally rigid analogues that contain a heterocyclic bridge between the 3- and 4-positions of the quinoline ring have been evaluated. Two isoxazolo-fused derivatives 17 and 23 displayed good in vitro antiherpetic potency that was similar to that of 1, the 3-quinolinecarboxamide that served as the comparison structure for this study. The pyrazolo, pyrrolo, and pyrimido derivatives showed considerably less or no activity. In vitro activity did not translate to in vivo efficacy. For 17, the lack of in vivo activity is likely a consequence of insufficient plasma drug levels (both Cmax and duration) in mice relative to the MIC versus HSV-2.

The palladium-catalyzed coupling of 3- and 4-(trialkylstannyl)pyridines with 7-bromo or 7-chloro 1-substituted 1,4-dihydro-4-oxo-3-quinolinecarboxylates has provided access to the corresponding 1-substituted 1,4-dihydro-4-oxo-7-pyridinyl-3-quinolinecarboxylic acids. The antibacterial activity of these derivatives was studied with the finding that the optimal 1- and 7-position substituents for Gram positive activity are cyclopropyl and 4-(2,6-dimethylpyridinyl), respectively. We find that for the fluorine-substituted derivatives studied, the position of the fluorine on the quinolone nucleus or the number of fluorine atoms does not seem to be important for good Gram positive activity. For 1-cyclopropyl 7-(2,6-dimethyl-4-pyridinyl) derivatives, the 6-fluoro 4a, 8-fluoro 10d, 6,8-difluoro 10b, and 5,6,8-trifluoro 8, all provided equal antibacterial activity against Staphylococcus aureus ATCC 29213. There is also a correlation between the substitution on the 7-(4-pyridinyl) group and the Gram positive activity, particularly for S. aureus, clearly indicating that the 2,6-dimethylpyridinyl group is optimal. The MIC50 value for the most potent agents in this study against S. aureus ATCC 29213 is 0.008 microgram/mL. By comparison, ciprofloxacin and aminopyrrolidine 28 gave values of 0.25 and 0.015 microgram/mL, respectively, against this organism.

A series of novel 3-quinolinecarboxamides that are structurally similar to the quinolone class of antibacterial agents possess excellent antiherpetic properties. By modifying the quinoline ring at the 1-, 2-, 3-, and 7-positions, analogues were identified that have up to 5-fold increased HSV-2 plaque-reduction potency relative to acyclovir. In a single-dose mouse model of infection, one of the most potent derivatives in vitro, 1-(4-fluorophenyl)-1,4-dihydro-4-oxo-7-(4-pyridinyl)-3-quinolinecarbo xamide (97), displayed comparable oral antiherpetic efficacy to acyclovir at 1/16 the dose; in a multiple-dose regimen, however, 97 was 2-fold less potent. In mice dosed orally with 97, sustained plasma drug levels were evident that may account for the high efficacy observed. The molecular mechanism of action of these agents is not known; however, based on in vitro studies with acyclovir resistant mutants, it is likely that the mechanism differs from that of acyclovir. In vitro plaque-reduction potency was not generally predictive of oral efficacy in mice. An X-ray crystal structure of 97 corroborated the assignment of structure and provided useful insights as to the effect of conformation on plaque-reduction potency.

A novel conformationally restricted 1-cyclopropylquinolone (1) that incorporates structural features of both ofloxacin and ciprofloxacin has been prepared. Compound 1 was found to be a DNA gyrase inhibitor having potency similar to ofloxacin but less than ciprofloxacin. The cellular inhibitory and in vivo antibacterial potencies of 1 were found to be less than those of the two reference agents.