Discovery of Novel Allosteric HCV NS5B Inhibitors. 2. Lactam-Containing Thiophene Carboxylates.

Lomibuvir (1) is a non-nucleoside, allosteric inhibitor of the hepatitis C virus NS5B polymerase with demonstrated clinical efficacy. Further development efforts within this class of inhibitor focused on improving the antiviral activity and physicochemical and pharmacokinetic properties. Recently, we reported the development of this series, leading to compound 2, a molecule with comparable potency and an improved physicochemical profile relative to 1. Further exploration of the amino amide-derived side chain led to a series of lactam derivatives, inspired by the X-ray crystal structure of related thiophene carboxylate inhibitors. This series, exemplified by 12f, provided 3-5-fold improvement in potency against HCV replication, as measured by replicon assays. The synthesis, structure-activity relationships, in vitro ADME characterization, and in vivo evaluation of this novel series are discussed.

Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions-an Industry Perspective.

Under the guidance of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), scientists from 20 pharmaceutical companies formed a Victim Drug-Drug Interactions Working Group. This working group has conducted a review of the literature and the practices of each company on the approaches to clearance pathway identification (fCL), estimation of fractional contribution of metabolizing enzyme toward metabolism (fm), along with modeling and simulation-aided strategy in predicting the victim drug-drug interaction (DDI) liability due to modulation of drug metabolizing enzymes. Presented in this perspective are the recommendations from this working group on: 1) strategic and experimental approaches to identify fCL and fm, 2) whether those assessments may be quantitative for certain enzymes (e.g., cytochrome P450, P450, and limited uridine diphosphoglucuronosyltransferase, UGT enzymes) or qualitative (for most of other drug metabolism enzymes), and the impact due to the lack of quantitative information on the latter. Multiple decision trees are presented with stepwise approaches to identify specific enzymes that are involved in the metabolism of a given drug and to aid the prediction and risk assessment of drug as a victim in DDI. Modeling and simulation approaches are also discussed to better predict DDI risk in humans. Variability and parameter sensitivity analysis were emphasized when applying modeling and simulation to capture the differences within the population used and to characterize the parameters that have the most influence on the prediction outcome.

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist.

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.

Discovery of highly potent and efficacious MC4R agonists with spiroindane N-Me-1,2,4-triazole privileged structures for the treatment of obesity.

We report an SAR study of MC4R analogs containing spiroindane heterocyclic privileged structures. Compound 26 with N-Me-1,2,4-triazole moiety possesses exceptional potency at MC4R and potent anti-obesity efficacy in a mouse model. However, the efficacy is not completely mediated through MC4R. Additional SAR studies led to the discovery of compound 32, which is more potent at MC4R. Compound 32 demonstrates MC4R mediated anti-obesity efficacy in rodent models.

Optimization of privileged structures for selective and potent melanocortin subtype-4 receptor ligands.

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties.

Spiroindane based amides as potent and selective MC4R agonists for the treatment of obesity.

We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models.

Discovery of a spiroindane based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor agonist.

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.

Regulation of energy homeostasis by bombesin receptor subtype-3: selective receptor agonists for the treatment of obesity.

Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.

In vitro metabolic activation of lumiracoxib in rat and human liver preparations.

Recent clinical reports have suggested that the cyclooxygenase-2 inhibitor, lumiracoxib (Prexige), may cause a rare but serious hepatotoxicity in patients. In view of the close structural resemblance between lumiracoxib and diclofenac, a widely used nonsteroidal anti-inflammatory drug whose use also has been associated with rare cases of liver injury, it is possible that the toxicity of the two agents may share a common mechanism. Because it is believed that chemically reactive metabolites may play a role as mediators of diclofenac-mediated hepatotoxicity, the present in vitro study was carried out to test the hypothesis that lumiracoxib also undergoes metabolic activation when incubated with liver microsomal preparations and hepatocytes from rats and humans. By means of liquid chromatography tandem mass spectrometry and nuclear magnetic resonance spectrometry techniques, two previously unknown N-acetylcysteine (NAC) conjugates were identified, namely, 3'-NAC-4'-hydroxy lumiracoxib (M1) and 4'-hydroxy-6'-NAC-desfluoro lumiracoxib (M2), the structures of which reveal the intermediacy of an electrophilic quinone imine species. Based on the results of studies with immunoinhibitory antibodies, it was demonstrated that the formation of M1 and M2 in human liver microsomes was catalyzed by cytochrome P450 (P450) 2C9. These findings demonstrate that lumiracoxib is subject to P450-mediated bioactivation in both rat and human liver preparations, leading to the formation of a reactive intermediate analogous to species generated during the metabolism of diclofenac.

Characterization of two cyclic metabolites of sitagliptin.

Two novel metabolites of the dipeptidyl peptidase inhibitor sitagliptin (MK-0431, (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)-butan-2-amine), were identified after purification from dog urine. The metabolites (referred to as M2 and M5) were characterized by hydrogen/deuterium exchange tandem mass spectrometry and NMR spectroscopy nuclear Overhauser effect experiments as the cis and trans stereoisomers formed by cyclization of the primary amino group with the alpha carbon of the piperazine ring, following oxidative desaturation.

Disposition of the dipeptidyl peptidase 4 inhibitor sitagliptin in rats and dogs.

The pharmacokinetics, metabolism, and excretion of sitagliptin [MK-0431; (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine], a potent dipeptidyl peptidase 4 inhibitor, were evaluated in male Sprague-Dawley rats and beagle dogs. The plasma clearance and volume of distribution of sitagliptin were higher in rats (40-48 ml/min/kg, 7-9 l/kg) than in dogs ( approximately 9 ml/min/kg, approximately 3 l/kg), and its half-life was shorter in rats, approximately 2 h compared with approximately 4 h in dogs. Sitagliptin was absorbed rapidly after oral administration of a solution of the phosphate salt. The absolute oral bioavailability was high, and the pharmacokinetics were fairly dose-proportional. After administration of [(14)C]sitagliptin, parent drug was the major radioactive component in rat and dog plasma, urine, bile, and feces. Sitagliptin was eliminated primarily by renal excretion of parent drug; biliary excretion was an important pathway in rats, whereas metabolism was minimal in both species in vitro and in vivo. Approximately 10 to 16% of the radiolabeled dose was recovered in the rat and dog excreta as phase I and II metabolites, which were formed by N-sulfation, N-carbamoyl glucuronidation, hydroxylation of the triazolopiperazine ring, and oxidative desaturation of the piperazine ring followed by cyclization via the primary amine. The renal clearance of unbound drug in rats, 32 to 39 ml/min/kg, far exceeded the glomerular filtration rate, indicative of active renal elimination of parent drug.

Evidence for the bioactivation of zomepirac and tolmetin by an oxidative pathway: identification of glutathione adducts in vitro in human liver microsomes and in vivo in rats.

Although zomepirac (ZP) and tolmetin (TM) induce anaphylactic reactions and form reactive acyl glucuronides, a direct link between the two events remains obscure. We report herein that, in addition to acyl glucuronidation, both drugs are subject to oxidative bioactivation. Following incubations of ZP with human liver microsomes fortified with NADPH and glutathione (GSH), a metabolite with an MH+ ion at m/z 597 was detected by LC/MS/MS. On the basis of collision-induced dissociation and NMR evidence, the structure of this metabolite was determined to be 5-[4'-chlorobenzoyl]-1,4-dimethyl-3-glutathionylpyrrole-2-acetic acid (ZP-SG), suggesting that the pyrrole moiety of ZP had undergone oxidation to an epoxide intermediate, followed by addition of GSH and loss of the elements of H2O to yield the observed conjugate. The oxidative bioactivation of ZP most likely is catalyzed by cytochrome P450 (P450) 3A4, since the formation of ZP-SG was reduced to approximately 10% of control values following pretreatment of human liver microsomes with ketoconazole or with an inhibitory anti-P450 3A4 IgG. A similar GSH adduct, namely 5-[4'-methylbenzoyl]-1-methyl-3-glutathionylpyrrole-2-acetic acid (TM-SG), was identified when TM was incubated with human liver microsomal preparations. The relevance of these in vitro findings to the in vivo situation was established through the detection of the same thiol adducts in rats treated with ZP and TM, respectively. Taken together, these data suggest that, in addition to the formation of acyl glucuronides, oxidative metabolism of ZP and TM affords reactive species that may haptenize proteins and thereby contribute to the drug-mediated anaphylactic reactions.

1-Amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid as a Tic mimetic: application in the synthesis of potent human melanocortin-4 receptor selective agonists.

The discovery of 1-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid analogs as potent human melanocortin-4 selective agonists is described.

Discovery and activity of (1R,4S,6R)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl]-2-methyl-2-azabicyclo[2.2.2]octane-6-carboxamide (3, RY764), a potent and selective melanocortin subtype-4 receptor agonist.

A novel isoquinuclidine containing selective melanocortin subtype-4 receptor small molecule agonist, 3 (RY764), is reported. Its in vivo characterization revealed mechanism-based food intake reduction and erectile activity augmentation in rodents.

2-Piperazinecarboxamides as potent and selective melanocortin subtype-4 receptor agonists.

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. The 5- and 6-alkylated piperazine compounds exhibit low bioactivation potential as measured by covalent binding in microsome preparations.

In vitro bioactivation of dihydrobenzoxathiin selective estrogen receptor modulators by cytochrome P450 3A4 in human liver microsomes: formation of reactive iminium and quinone type metabolites.

Estrogens and selective estrogen receptor modulators (SERMs) are prescribed widely in the clinic to alleviate symptoms in postmenopausal women, and they are metabolized to reactive intermediates, which may elicit adverse effects. As part of our efforts to develop safer SERMs, in vitro covalent protein binding of (2S,3R)-(+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) was evaluated. Radioactivity from [3H]I became covalently bound to proteins in a fashion that was both time- and NADPH-dependent in human liver microsomes and reached a value of 1106 pmol equiv/mg protein following a 45 min incubation. At least three pathways are involved in the bioactivation of I, namely, oxidative cleavage of the dihydrobenzoxathiin moiety to give a hydroquinone/para-benzoquinone redox couple, hydroxylation at position 5 or 7 of the benzoxathiin moiety leading to an o-quinone intermediate, and metabolism of the piperidine ring to give an iminium ion. The latter reactive intermediate was identified as its bis-cyano adduct when human liver microsomal incubations were performed in the presence of sodium cyanide. Structural modification of I, including a replacement of the piperidine with a pyrrolidine group, led to (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (II), which did not form a reactive iminium ion. Following the incubation of II with human liver microsomes, covalent binding to proteins was reduced (461 pmol equiv/mg protein), the residual level of binding apparently due to the formation of a rearranged biphenyl quinone type metabolite. Studies with inhibitory antibodies and chemical inhibitors showed that P450 3A4 was the primary enzyme responsible for oxidative bioactivation of I and II in human liver microsomes. These studies thus demonstrated that gaining an understanding of bioactivation mechanisms may be exploited in terms of guiding structural modifications of drug candidates to minimize covalent protein binding and, hopefully, to lower the potential for drug-mediated adverse effects.

Metabolic activation of a 1,3-disubstituted piperazine derivative: evidence for a novel ring contraction to an imidazoline.

MB243 (a 1,3-disubstituted piperazine) is a new, potent, and selective melanocortin receptor subtype-4 agonist with potential application in the treatment of obesity and/or erectile dysfunction. MB243 was observed to covalently bind extensively to liver microsomal proteins from rats and humans. In the presence of glutathione, two thioether adducts were detected in liver microsomal incubations by radiochromatography and LC/MS/MS analysis. These adducts were also formed when bile duct-cannulated rats were dosed with MB243. The two adducts were isolated, and their structures were determined by accurate mass MS/MS and NMR analyses. The proposed structures resulted from a novel contraction of the piperazine ring to yield a substituted imidazoline. A mechanism is proposed, which involves an initial six electron oxidation of the piperazine ring to form a reactive intermediate, which is trapped by glutathione. Hydrolysis of the glutamic acid residue followed by internal aminolysis by the cysteine amino group resulted in opening of the piperazine ring, which is followed by ring closure to an imidazoline. The resulting cysteinyl-glycine conjugate underwent subsequent hydrolysis of the glycine residue. Understanding of the mechanism of bioactivation led to the design of MB243 analogues that exhibited reduced covalent protein binding.

Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomes.

The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogs (1S)-1-(trans-4-([(4-trifluoromethoxyphenyl)sulfonyl]amino)cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride and (1S)-1-(trans-4-([(2,4-difluorophenyl)sulfonyl]amino)cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritium-labeled compounds to rat liver microsomal protein was time- and NADPH-dependent and was attenuated by the addition of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chemically reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping experiments with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidation and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant CYP3A1 and CYP3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16alpha-carbonitrile and dexamethasone) enhanced the extent of bioactivation which, in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5- and 9-fold increase, respectively). Herein, we describe studies that demonstrate that the fluoropyrrolidine ring is prone to metabolic activation and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation.

Discovery of (2S)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl]-4-methyl-2-piperazinecarboxamide (MB243), a potent and selective melanocortin subtype-4 receptor agonist.

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.

Conversion of the 2,2,6,6-tetramethylpiperidine moiety to a 2,2-dimethylpyrrolidine by cytochrome P450: evidence for a mechanism involving nitroxide radicals and heme iron.

Earlier we described a novel cytochrome P450 (CYP) catalyzed metabolism of the 2,2,6,6-tetramethylpiperidine (2,2,6,6-TMPi) moiety in human liver microsomes to a ring-contracted 2,2-dimethylpyrrolidine (2,2-DMPy) [Yin, W., et al. (2003) Drug Metab. Dispos. 31, 215-223]. In the current report, evidence is provided for the involvement of 2,2,6,6-TMPi hydroxylamines and their one-electron oxidation products, the nitroxide radicals, as intermediates in this pathway. Nitroxide radicals could be converted to their corresponding 2,2-DMPy metabolites by "inactivated CYP3A4", as well as by a number of other heme proteins and hemin, suggesting that this is a heme-catalyzed process. The conversion of nitroxide radicals to the 2,2-DMPy products by CYP3A4 or hemin was accompanied by the generation of acetone in incubations, providing evidence that the three-carbon unit from 2,2,6,6-TMPi was lost as acetone. With one model 2,2,6,6-TMPi nitroxide radical, evidence for an alternate pathway, which resulted in the formation of an intermediate that incorporated two oxygen atoms from water of the incubation medium before collapsing to the 2,2-DMPy product, was also obtained. To account for both pathways, a mechanism involving interaction of the nitroxide radicals with heme iron (Fe(III)), followed by a homolytic scission of the N-O bond and transfer of the nitroxide oxygen to heme iron to form a perferryl-oxygen complex, is proposed. The nitrogen-centered 2,2,6,6-TMPi radical thus formed then precipitates the contraction of the piperidine ring via C2-C3 bond cleavage, and the resulting product further oxidizes to an exocyclic iminium ion (by the perferryl-oxygen complex); the latter may undergo capture by water from the incubation medium and eliminate the three-carbon unit via N-dealkylation. It remains to be determined whether this novel interaction of nitroxide radicals with heme iron has any relevance in regard to the known biological properties of these stable radical species.