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Zoliflodacin is a novel spiropyrimidinetrione with activity against bacterial type II topoisomerases that inhibits DNA biosynthesis and results in accumulation of double-strand cleavages in bacteria. We report results from two phase 1 studies that investigated the safety, tolerability, and pharmacokinetics (PK) of zoliflodacin and absorption, distribution, metabolism, and excretion (ADME) after single doses in healthy volunteers. In the single ascending dose study, zoliflodacin was rapidly absorbed, with a time to maximum concentration of drug in serum (Tmax) between 1.5 and 2.3 h. Exposure increased dose proportionally up to 800 mg and less than dose proportionally between 800 and 4,000 mg. Urinary excretion of unchanged zoliflodacin was

The success of bedaquiline as an anti-tubercular agent for the treatment of multidrug-resistant tuberculosis has validated the ATP synthesis pathway and in particular ATP synthase as an attractive target. However, limitations associated with its use in the clinic and the drug–drug interactions with rifampicin have prompted research efforts towards identifying alternative ATP synthesis inhibitors with differentiated mechanisms of action. A biochemical assay was employed to screen AstraZeneca's corporate compound collection to identify the inhibitors of mycobacterial ATP synthesis. The high-throughput screening resulted in the identification of 2,4-diaminoquinazolines as inhibitors of the ATP synthesis pathway. A structure–activity relationship for the quinazolines was established and the knowledge was utilized to morph the quinazoline core into quinoline and pyrazolopyrimidine to expand the scope of chemical diversity. The morphed scaffolds exhibited a 10-fold improvement in enzyme potency and over 100-fold improvement in selectivity against inhibition of mammalian mitochondrial ATP synthesis. These novel compounds were bactericidal and demonstrated growth retardation of Mycobacterium tuberculosis in the acute mouse model of tuberculosis infection.

A noninvasive topical ocular therapy for the treatment of neovascular or “wet” age-related macular degeneration would provide a patient administered alternative to the current standard of care, which requires physician administered intravitreal injections. This manuscript describes a novel strategy for the use of in vivo models of choroidal neovascularization (CNV) as the primary means of developing SAR related to efficacy from topical administration. Ultimately, this effort led to the discovery of acrizanib (LHA510), a small-molecule VEGFR-2 inhibitor with potency and efficacy in rodent CNV models, limited systemic exposure after topical ocular administration, multiple formulation options, and an acceptable rabbit ocular PK profile.

The design, synthesis and structure–activity relationships of a novel series of 3,4-disubstituted pyrrolidine acid analogs as PPAR ligands is outlined. In both the 1,3- and 1,4-oxybenzyl pyrrolidine acid series, the preferred stereochemistry was shown to be the cis-3R,4S isomer, as exemplified by the potent dual PPARα/γ agonists 3k and 4i. The N-4-trifluoromethyl-pyrimidinyl pyrrolidine acid analog 4i was efficacious in lowering fasting glucose and triglyceride levels in diabetic db/db mice.

A novel pyrazolopyridone class of inhibitors was identified from whole cell screening against Mycobacterium tuberculosis (Mtb). The series exhibits excellent bactericidality in vitro, resulting in a 4 log reduction in colony forming units following compound exposure. The significant modulation of minimum inhibitory concentration (MIC) against a Mtb strain overexpressing the Rv3790 gene suggested the target of pyrazolopyridones to be decaprenylphosphoryl-β-D-ribose-2'-epimerase (DprE1). Genetic mapping of resistance mutation coupled with potent enzyme inhibition activity confirmed the molecular target. Detailed biochemical characterization revealed the series to be a noncovalent inhibitor of DprE1. Docking studies at the active site suggest that the series can be further diversified to improve the physicochemical properties without compromising the antimycobacterial activity. The pyrazolopyridone class of inhibitors offers an attractive non-nitro lead series targeting the essential and vulnerable DprE1 enzyme for the discovery of novel antimycobacterial agents to treat both drug susceptible and drug resistant strains of Mtb.

The discovery of novel therapeutics for the treatment of tuberculosis involves routine testing in a mouse model over four weeks of daily dosing with test compounds. In this model, daily oral administration of rifampin (10 mg/kg) showed significantly lower plasma exposure on day 5 compared to day 1. The absence of PXR-mediated induction of mouse Cyp3a isoforms was confirmed in the present study by incubating liver microsomes prepared from control and rifampin treated mice with probe substrates of CYP3A. To test whether the reduction in exposure was due to Pgp-mediated efflux, verapamil, a known Pgp inhibitor, was dosed to the rifampin pre-treated mice which led to an increase in exposure to that obtained after a single dose of rifampin, suggesting the role of Pgp induction in reducing exposure to rifampin. To further confirm Pgp induction in rifampin treated mice, digoxin, a known substrate of Pgp, was administered to the rifampin pre-treated mice, and a significant drop in the digoxin exposure was observed compared to the control group. Collectively, our results show that repeated administration of rifampin in mice leads to a reduction in oral exposure due to induction of Pgp-mediated efflux of rifampin, and not via induction of CYP3A isoforms.

The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD / HDAC5 signaling.

A novel 2,6-naphthyridine was identified by high throughput screen (HTS) as a dual protein kinase C/D (PKC/PKD) inhibitor. PKD inhibition in the heart was proposed as a potential antihypertrophic mechanism with application as a heart failure therapy. As PKC was previously identified as the immediate upstream activator of PKD, PKD vs PKC selectivity was essential to understand the effect of PKD inhibition in models of cardiac hypertrophy and heart failure. The present study describes the modification of the HTS hit to a series of prototype pan-PKD inhibitors with routine 1000-fold PKD vs PKC selectivity. Example compounds inhibited PKD activity in vitro, in cells, and in vivo following oral administration. Their effects on heart morphology and function are discussed herein.

The synthesis and biological evaluation of potent and selective PKD inhibitors are described herein. The compounds described in the present study selectively inhibit PKD among other putative HDAC kinases. The PKD inhibitors of the present study blunt phosphorylation and subsequent nuclear export of HDAC4/5 in response to diverse agonists. These compounds further establish the central role of PKD as an HDAC4/5 kinase and enhance the current understanding of cardiac myocyte signal transduction. The in vivo efficacy of a representative example compound on heart morphology is reported herein.

A novel class of azetidinone acid-derived dual PPARalpha/gamma agonists has been synthesized for the treatment of diabetes and dyslipidemia. The preferred stereochemistry in this series for binding and functional agonist activity against both PPARalpha and PPARgamma receptors was shown to be 3S,4S. Synthesis, in vitro and in vivo activities of compounds in this series are described. A high-yielding method for N-arylation of azetidinone esters is also described.

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99, The emergence of resistant Plasmodium strains fuels the search for new antimalarials. Here, the authors present a new class of potent antimalarial compounds, the triaminopyrimidines, that display low toxicity and long half-life in animal models.

Reactive metabolites (RMs) have been implicated as causal factors in many drug-associated idiosyncratic toxicities. This study aims at identification and mitigation of an RM liability associated with aminoimidazole and amino(aza)benzimidazole structural motifs from an antimalarial project. Nineteen compounds with different structural modifications were studied in rat and human liver microsomes using glutathione (GSH) and N-acetyl cysteine (NAC) as trapping agents for RM. Metabolite profiling of aminoimidazole compounds in initial studies revealed the presence of dihydrodiol metabolites suggestive of reactive epoxide precursors, confirmed by the identification of a dihydrohydroxy GSH conjugate in GSH supplemented incubations. Substitution of methyl group at a potential site of metabolism blocked the epoxidation; however, formation of an imine-methide RM was suspected. Masking the site of metabolism via benzimidazole and 4/7-azabenzimidazole resulted in the possible formation of quinone-imine intermediates as a product of bioactivation. Further, substitutions with electron withdrawing groups and steric crowding did not address this liability. Mitigation of bioactivation was achieved with 5/6-azabenzimidazole and with CF3 substitution at the 6-position of the 7-azabenzimidazole ring. Moreover, compounds devoid of imidazole -NH2 do not undergo bioactivation. This study, therefore, establishes aminoimidazole and amino(aza)benzimidazoles as potential toxicophores and describes ways to mitigate this bioactivation liability by chemical modification.

From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-beta-D-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of similar to 100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

Several bioartificial liver devices have been developed as temporary therapy for patients suffering from fulminant hepatic failure. Some of these devices contain porcine hepatocytes entrapped in collagen matrices. In order to improve the function of these BAL devices, there exists a need to optimize metabolic function of cultured hepatocytes. The goal of these investigations was to evaluate the effect of altering culture conditions on rifampin-mediated induction of CYP3A isoforms in cultured porcine hepatocytes. Midazolam metabolism was compared in porcine hepatocytes cultured in a monolayer configuration on collagen gels, in a sandwich configuration between collagen gels and a Matrigel overlay, and in spheroidal cultures. The effect of culture conditions was evaluated, by measuring CYP3A-mediated metabolism of midazolam and by immunoblotting to detect CYP3A proteins, in control cultures and in rifampin-treated cultures. Results obtained by normalizing the metabolism rate data to cell numbers (based on DNA content) present at the end of the culture experiment, showed that there was no difference between the different culture conditions tested. Our results suggest that culturing porcine hepatocytes as spheroids or in a sandwich configuration between collagen and Matrigel, offers no advantage in terms of CYP3A-mediated metabolic function on a per cell basis compared to culturing on collagen gels.

The cytochrome P450s are hemoproteins that play critical roles in the bioactivation and detoxication of a wide variety of xenobiotic substances. The enzymes are encoded by a superfamily of genes. The term “cytochrome P450” originates from the observation that the reduced state of the proteins form complexes with carbon monoxide that exhibit absorbance maxima at 450 nm (Omura and Sato, 1964). The P450 enzymes are now referred to as heme-thiolate proteins. P450s have been characterized in many species of organisms, including bacteria, fungi, plants, fish, and mammalian systems (Nebert and McKinnon, 1994). In mammalian cells, P450s are localized predominantly in the smooth endoplasmic reticulum of the cell and are entirely distinct from the cytochrome proteins that comprise the mitochondrial electron-transport function. Currently, .700 P450s have been characterized, inclusive of the many different species of organisms that have been studied (Nelson et al., 1996). Based primarily on sequence similarities, a standardized nomenclature has been adopted that categorizes the individual P450s into respective families and subfamilies. P450 proteins exhibiting .40% similarity in protein sequence are classified within the same family; proteins exhibiting .55% sequence similarity are grouped into the same subfamily (Nebert and McKinnon, 1994; Nelson et al., 1996). Although the catalog is updated regularly, the current P450 nomenclature and classification scheme was most recently reviewed in 1996 (Nelson et al., 1996). The purpose of this current review is to very briefly highlight some of the more important mammalian P450s with respect to their expression, inducibility and inhibition character, and substrate preferences, especially as they relate to human toxicology. Due to this limited scope, it is impossible to thoroughly reference all the important and relevant literature pertaining to this topic. The reader is referred to other recent reviews for more detailed information (Dogra et al., 1998; Meyer and Zanger, 1997; Pelkonen and Raunio, 1997; Rendic and Di-Carlo, 1997). Information from these and other references has been used as source material to compile the contents of Table 1, summarizing some of the salient features of several relevant P450s.

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-β-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

Standard incubation procedures for carrying out microsomal assays involve the use of less than 1% w/v organic solvents to minimize the potential inhibitory effects of organic solvents on metabolic activity. This presents a practical limitation for poorly soluble xenobiotics, which cannot be incubated at concentrations high enough to obtain a V(max), and therefore subsequent values for K(m) and Cl(int) cannot be calculated. Our goal was to study the application of a variety of pharmaceutical excipients to aid the solubilization of compounds in vitro in glucuronidation incubations, without affecting the reaction kinetics. In vitro glucuronidation incubations were carried out in human liver microsomes with 4-methylumbelliferone (4-MU) and the kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations without any excipients. In addition, IC(75) values were calculated for each excipient. We observed that HPBCD (Hydroxypropyl-β-cyclodextrin) may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting the Cl(int) of 4-MU. Although NMP (N-methyl-2-pyrrolidone) and DMA (N,N-dimethylacetamide); showed low IC(75) values approximately 0.1% w/v each, neither excipients altered the Cl(int) of 4-MUG (4-methylumbelliferyl-β-D-glucuronide) formation. Our studies point toward possible applications of pharmaceutical excipients to carry out in vitro glucuronidation of substrates with poor aqueous solubility, in order to estimate Cl(int) and subsequently scaled organ clearance values.

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) alpha agonist, with an EC(50) of 10 nM for human PPARalpha and approximately 410-fold selectivity vs human PPARgamma in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPARdelta. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystal structures of the early lead compound 12 and compound 2 in complex with PPARalpha ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPARalpha in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.

1-[4-Aminomethyl-4-(3-chlorophenyl)-cyclohexyl]-tetrahydro-pyrimidin- 2-one, 1, was developed as an inhibitor of dipeptidyl peptidase-4 enzyme. Biotransformation studies with 1 revealed the presence of an N-carbamoyl glucuronide metabolite (M1) in rat bile and urine. N-Carbamoyl glucuronides are rarely observed, and little is understood regarding the mechanism of N-carbamoyl glucuronidation. The objectives of the current investigation were to elucidate the structure of the novel N-carbamoyl glucuronide, to investigate the mechanism of N-carbamoyl glucuronide formation in vitro using stable labeled CO(2), UDP glucuronosyltransferase (UGT) reaction phenotyping, and to assess whether M1 was formed to the same extent in vitro across species-mouse, rat, hamster, dog, monkey, and human. Structure elucidation was performed on a mass spectrometer with accurate mass measurement and MS(n) capabilities. (13)C-labeled carbon dioxide was used for identification of the mechanism of N-carbamoyl glucuronidation. Mechanistic studies with (13)C-labeled CO(2) in rat liver microsomes revealed that CO(2) from the bicarbonate buffer (in equilibrium with exogenous CO(2)) may be responsible for the formation of M1. M1 was formed in vitro in liver microsomes from multiple species, mainly rat and hamster, followed by similar formation in dog, monkey, mouse, and human. M1 could be detected in UGT1A1, UGT1A3, and UGT2B7 Supersomes in a CO(2)-rich environment. In conclusion, our study demonstrates that formation of M1 was observed in microsomal incubations across various species and strongly suggests incorporation of CO(2) from the bicarbonate buffer, in equilibrium with exogenous CO(2), into the carbamoyl moiety of the formed N-carbamoyl glucuronide.

The design, synthesis and structure–activity relationships of a novel series of N-phenyl-substituted pyrrole, 1,2-pyrazole and 1,2,3-triazole acid analogs as PPAR ligands are outlined. The triazole acid analogs 3f and 4f were identified as potent dual PPARα/γ agonists both in binding and functional assays in vitro. The 3-oxybenzyl triazole acetic acid analog 3f showed excellent glucose and triglyceride lowering in diabetic db/db mice.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) activator, has both glucose- and lipid-lowering effects in animal models and in patients with diabetes. This study describes the in vivo and in vitro comparative metabolism of [(14)C]muraglitazar in rats, dogs, monkeys, and humans by quantitative and qualitative metabolite profiling. Metabolite identification and quantification methods used in these studies included liquid chromatography/mass spectrometry (LC/MS), LC/tandem MS, LC/radiodetection, LC/UV, and a newly described mass defect filtering technique in conjunction with high resolution MS. After oral administration of [(14)C]muraglitazar, absorption was rapid in all species, reaching a concentration peak for parent and total radioactivity in plasma within 1 h. The most abundant component in plasma at all times in all species was the parent drug, and no metabolite was present in greater than 2.5% of the muraglitazar concentrations at 1 h postdose in rats, dogs, and humans. All metabolites observed in human plasma were also present in rats, dogs, or monkeys. Urinary excretion of radioactivity was low (

Human microsomal epoxide hydrolase (EPHX1) is active in the metabolism of many potentially carcinogenic or otherwise genotoxic epoxides, such as those derived from the oxidation of polyaromatic hydrocarbons. EPHX1 is polymorphic and encodes allelic variation at least two amino acid positions, Y113H and H139R. In a number of recent molecular epidemiological investigations, EPHX1 polymorphism has been suggested as a susceptibility factor for several human diseases. To better evaluate the functional contribution of EPHX1 genetic polymorphism, we characterized the enzymatic properties associated with each of the respective variant proteins. Enzymatic profiles were evaluated with cis-stilbene oxide (cSO) and benzo[a]pyrene-4,5-epoxide (BaPO), two prototypical substrates for the hydrolase. In one series of experiments, activities of recombinant EPHX1 proteins were analyzed subsequent to their expression using the pFastbac® baculovirus vector in Spodoptera frugiperda-9 (Sf9) insect cells, and purification by column chromatography. In parallel studies, EPHX1 activities were evaluated with human liver microsomes derived from individuals of known EPHX1 genotype. Using the purified protein preparations, rates of cSO and BaPO hydrolysis for the reference protein, Y113/H139, were approximately 2-fold greater than those measured with the other EPHX1 allelic variants. However, when activities were analyzed using human liver microsomal fractions, no major differences were evident in the reaction rates generated among preparations representing the different EPHX1 alleles. Collectively, these results suggest that the structural differences encoded by the Y113H and H139R variant alleles exert only modest impact on EPHX1-specific enzymatic activities in vivo.

Liver X receptors (LXRs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. LXRs activate transcription of a spectrum of genes that regulate reverse cholesterol transport, including the ATP binding cassette transporter A1 (ABCA1), and raise HDL cholesterol (HDL-C) levels. However, LXR agonists also induce genes that stimulate lipogenesis, including the sterol response element binding protein (SREBP1-c) and fatty acid synthetase (FAS). The induction of these genes in the liver cause increased hepatic triglyceride synthesis, hypertriglyceridemia, and hepatic steatosis. As LXR response elements have been identified in these promoters, it is not clear if these two processes can be separated. Herein, we demonstrate that plasma HDL-C elevation and intestinal ABCA1 induction can occur with relatively little induction of FAS and SREBP1-c in mouse liver via a selective LXR modulator GW3965. This is in contrast to the strong induction of hepatic lipogenic genes by the well-characterized LXR agonist T0901317 (T317). Consistent with the in vivo results, GW3965 is a very weak LXR activator compared with T317 in human hepatoma cells. GW3965-liganded LXR recruits selected coactivators less effectively than T317 and may explain in part the tissue selective gene induction. This demonstration that tissue and gene selective modulation is possible with selective LXR modulators has positive implications for the development of this class of antiatherosclerotic agents.

Microsomal epoxide hydrolase is a critical biotransformation enzyme that catalyzes the conversion of a broad array of xenobiotic epoxide substrates to more polar diol metabolites. The gene has been shown previously to exhibit polymorphism, including variation in the coding region leading to amino acid substitutions at positions 113 (Y/H) and 139 (H/R). To better evaluate the phenotype associated with the structural region genetic polymorphisms associated with mEH, we performed enzymatic analyses using purified mEH proteins that were expressed using a baculovirus system, or with microsomal preparations obtained from liver tissues that were derived from individuals with homozygous mEH allelic status. Benzo[a]pyrene-4, 5-oxide and cis-stilbene oxide were employed as substrates for the enzymatic determinations. Results obtained with the purified enzymes suggested that the reaction velocity catalyzed by the wild type (Y113/H139) protein was approximately two-fold greater than the corresponding velocities for the variant forms of the enzyme. However, when reaction rates were analyzed using human liver microsomal preparations, the maximal velocities generated among the variant mEH proteins were not statistically different. Collectively, these results indicate that the structural differences coded by the mEH genetic variants may have only modest impact on the enzyme's specific activity in vivo.