2-[(3aR,4R,5S,7aS)-5-{(1S)-1-[3,5-bis(trifluoromethyl)phenyl]-2-hydroxyethoxy}-4-(2-methylphenyl)octahydro-2H-isoindol-2-yl]-1,3-oxazol-4(5H)-one: a potent human NK1 receptor antagonist with multiple clearance pathways.

Hydroisoindoline 2 has been previously identified as a potent, brain-penetrant NK1 receptor antagonist with a long duration of action and improved profile of CYP3A4 inhibition and induction compared to aprepitant. However, compound 2 is predicted, based on data in preclinical species, to have a human half-life longer than 40 h and likely to have drug-drug-interactions (DDI), as 2 is a victim of CYP3A4 inhibition caused by its exclusive clearance pathway via CYP3A4 oxidation in humans. We now report 2-[(3aR,4R,5S,7aS)-5-{(1S)-1-[3,5-bis(trifluoromethyl)phenyl]-2-hydroxyethoxy}-4-(2-methylphenyl)octahydro-2H-isoindol-2-yl]-1,3-oxazol-4(5H)-one (3) as a next generation NK1 antagonist that possesses an additional clearance pathway through glucuronidation in addition to that via CYP3A4 oxidation. Compound 3 has a much lower propensity for drug-drug interactions and a reduced estimated human half-life consistent with once daily dosing. In preclinical species, compound 3 has demonstrated potency, brain penetration, and a safety profile similar to 2, as well as excellent pharmacokinetics.

In vitro bioactivation of a selective estrogen receptor modulator (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) in liver microsomes: formation of adenine adducts.

As part of our efforts to develop safer selective estrogen receptor modulators (SERMs), compound I {(2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)-phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol} was previously identified as a lead for further development. Subsequent studies showed that compound I is genotoxic in both in vitro Chinese hamster ovary (CHO) cells and in vivo mouse studies. To better understand the possible mechanisms for the observed genetoxicity effects, in vitro incubations of I with liver microsomes of human, monkey, and mouse in the presence of adenine were performed, which led to the detection of five adenine adducts. The formation of these adducts was NADPH-dependent, suggesting the involvement of oxidative bioactivation catalyzed by cytochrome P450 enzymes. The mechanism for the formation of the major adenine adduct (A1) involves the formation of a reactive ring-opened para-quinone intermediate. The formation of four other adenine adducts may involve the formation of a reactive epoxide or ortho-quinone intermediate. Furthermore, incubations of compound I with human hepatocytes showed dose-dependent DNA damages in Comet assays. All of the above suggest that some reactive metabolites of compound I, formed through bioactivation mechanisms, have a potential to interact with DNA molecules in vitro and in vivo. This may be one of the causes of the genotoxicity observed preclinically both in vitro and in vivo. This case study demonstrated an approach using in vitro DNA trapping assays for assessing the genotoxicity potential of drug candidates.

Design of a novel pyrrolidine scaffold utilized in the discovery of potent and selective human ß3 adrenergic receptor agonists.

A novel class of human ß(3)-adrenergic receptor agonists was designed in effort to improve selectivity and metabolic stability versus previous disclosed ß(3)-AR agonists. As observed, many of the ß(3)-AR agonists seem to need the acyclic ethanolamine core for agonist activity. We have synthesized derivatives that constrained this moiety by introduction of a pyrrolidine. This unique modification maintains human ß(3) functional potency with improved selectivity versus ancillary targets and also eliminates the possibility of the same oxidative metabolites formed from cleavage of the N-C bond of the ethanolamine. Compound 39 exhibited excellent functional ß(3) agonist potency across species with good pharmacokinetic properties in rat, dog, and rhesus monkeys. Early de-risking of this novel pyrrolidine core (44) via full AMES study supports further research into various new ß(3)-AR agonists containing the pyrrolidine moiety.

Glutathione S-transferase catalyzed desulfonylation of a sulfonylfuropyridine.

MRL-1, a cannabinoid receptor-1 inverse agonist, was a member of a lead candidate series for the treatment of obesity. In rats, MRL-1 is eliminated mainly via metabolism, followed by excretion of the metabolites into bile. The major metabolite M1, a glutathione conjugate of MRL-1, was isolated and characterized by liquid chromatography/mass spectrometry and NMR spectroscopic methods. The data suggest that the t-butylsulfonyl group at C-2 of furopyridine was displaced by the glutathionyl group. In vitro experiments using rat and monkey liver microsomes in the presence of reduced glutathione (GSH) showed that the formation of M1 was independent of NADPH and molecular oxygen, suggesting that this reaction was not mediated by an oxidative reaction and a glutathione S-transferase (GST) was likely involved in catalyzing this reaction. Furthermore, a rat hepatic GST was capable of catalyzing the conversion of MRL-1 to M1 in the presence of GSH. When a close analog of MRL-1, a p-chlorobenzenesulfonyl furopyridine derivative (MRL-2), was incubated with rat liver microsomes in the presence of GSH, p-chlorobenzene sulfinic acid (M2) was also identified as a product in addition to the expected M1. Based on these data, a mechanism is proposed involving direct nucleophilic addition of GSH to sulfonylfuropyridine, resulting in an unstable adduct that spontaneously decomposes to form M1 and M2.

Metabolism and renal elimination of gaboxadol in humans: role of UDP-glucuronosyltransferases and transporters.

PURPOSE: Gaboxadol, a selective extrasynaptic agonist of the delta-containing gamma-aminobutyric acid type A (GABAA) receptor, is excreted in humans into the urine as parent drug and glucuronide conjugate. The goal of this study was to identify the UDP-Glucuronosyltransferase (UGT) enzymes and the transporters involved in the metabolism and active renal secretion of gaboxadol and its metabolite in humans.Methods. The structure of the glucuronide conjugate of gaboxadol in human urine was identified by LC/MS/MS. Human recombinant UGT isoforms were used to identify the enzymes responsible for the glucuronidation of gaboxadol. Transport of gaboxadol and its glucuronide was evaluated using cell lines and membrane vesicles expressing human organic anion transporters hOAT1 and hOAT3, organic cation transporter hOCT2, and the multidrug resistance proteins MRP2 and MRP4.Results. Our study indicated that the gaboxadol-O-glucuronide was the major metabolite excreted in human urine. UGT1A9, and to a lesser extent UGT1A6, UGT1A7 and UGT1A8, catalyzed the O-glucuronidation of gaboxadol in vitro. Gaboxadol was transported by hOAT1, but not by hOCT2, hOAT3, MRP2, and MRP4. Gaboxadol-O-glucuronide was transported by MRP4, but not MRP2.Conlusion. Gaboxadol could be taken up into the kidney by hOAT1 followed by glucuronidation and efflux of the conjugate into urine via MRP4.

Conformational analysis and receptor docking of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (taranabant, MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist.

X-ray crystallographic, NMR spectroscopic, and computational studies of taranabant afforded similar low-energy conformers with a significant degree of rigidity along the C11-N13-C14-C16-C17 backbone but with more flexibility around bonds C8-C11 and C8-O7. Mutagenesis and docking studies suggested that taranabant and rimonabant shared the same general binding area of CB1R but with significant differences in detailed interactions. Similar to rimonabant, taranabant interacted with a cluster of aromatic residues (F(3.36)200, W(5.43)279, W(6.48)356, and Y(5.39)275) through the two phenyl rings and with F(2.57)170 and L(7.42)387 through the CF 3-Pyr ring. The notable distinction between taranabant and rimonabant was that taranabant was hydrogen-bonded with S(7.39)383 but not with K(3.28)192, while rimonabant was hydrogen-bonded with K(3.28)192 but not with S(7.39)383. The strong hydrogen bonding between the amide NH of taranabant and hydroxyl of S(7.39)383 was key to the superior affinity of taranabant to CB1R.

Conformational studies of 3-amino-1-alkyl-cyclopentane carboxamide CCR2 antagonists leading to new spirocyclic antagonists.

In an effort to shed light on the active binding conformation of our 3-amino-1-alkyl-cyclopentane carboxamide CCR2 antagonists, we prepared several conformationally constrained analogs resulting from backbone cyclization. Evaluation of CCR2 binding affinities for these analogs gave insight into the optimal relative positions of the piperidine and benzylamide moieties while simultaneously leading to the discovery of a new, potent lead type based upon a spirocyclic acetal scaffold.

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 1'-hydroxymidazolam glucuronidation in human liver microsomes.

Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1'-hydroxymidazolam, which is equipotent to midazolam. 1'-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1'-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1'-hydroxymidazolam in human liver microsomes enriched with uridine 5'-diphosphoglucuronic acid (UDPGA). The structure of the N-glucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1'-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n = 1.2-1.5). The apparent S(50) values for the formation of O- and N-glucuronides were 43 and 18 microM, respectively, and the corresponding apparent V(max) values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1'-hydroxymidazolam in human liver microsomes.

Rational design of a novel, potent, and orally bioavailable cyclohexylamine DPP-4 inhibitor by application of molecular modeling and X-ray crystallography of sitagliptin.

Molecular modeling was used to design a rigid analog of sitagliptin 1. The X-ray crystal structure of sitagliptin bound to DPP-4 suggested that the central beta-amino butyl amide moiety could be replaced with a cyclohexylamine group. This was confirmed by structural analysis and the resulting analog 2a was synthesized and found to be a potent DPP-4 inhibitor (IC(50)=21 nM) with excellent in vivo activity and pharmacokinetic profile.

Highly constrained bicyclic VLA-4 antagonists.

VLA-4 is implicated in several inflammatory and autoimmune disease states. A series of cyclic beta-amino acids (beta-aa) was studied as VLA-4 antagonists. Binding affinity was highly dependent on the dihedral angle (phi) between the amino and the carboxyl termini of the beta-aa. Compound 5 m where the beta-aa is embedded in a bicycle possesses the most preferred phi (120 degrees). It is a potent and bioavailable VLA-4 antagonist (VCAM-Ig alpha4beta1 IC50 = 54 nM, rat po F = 49%).

Addressing metabolic activation as an integral component of drug design.

Formation of reactive intermediates by metabolism of xenobiotics represents a potential liability in drug discovery and development. Although it is difficult, if not impossible, to predict toxicities of drug candidates accurately, it is prudent to try to minimize bioactivation liabilities as early as possible in the stage of drug discovery and lead optimization. Measurement of covalent binding to liver microsomal proteins in the presence and the absence of NADPH, as well as the use of trapping agents such as glutathione or cyanide ions to provide structural information on reactive intermediates, have been used routinely to screen drug candidates. These in vitro experiments are often supplemented with in vivo covalent binding data in rats. The resulting data are not only used to eliminate potentially risky compounds, but, more importantly, they provide invaluable information to direct the Medicinal Chemistry group efforts to design analogs with less propensity to undergo bioactivation. Select case histories are presented in which this approach was successfully applied at Merck.

Species differences in metabolism and pharmacokinetics of a sphingosine-1-phosphate receptor agonist in rats and dogs: formation of a unique glutathione adduct in the rat.

The pharmacokinetics and metabolism of 1-(4-((4-phenyl-5-trifluoromethyl-2-thienyl)methoxy)benzyl)azetidine-3-carboxylic acid (MRL-A), a selective agonist for the sphingosine-1-phosphate 1 (S1P1) receptor, were investigated in rats and dogs. In both species, more than 50% of the dose was excreted in bile. Specific to the rat, and observed in bile, were a taurine conjugate of MRL-A and a glucuronide conjugate of an azetidine lactam metabolite. In dogs, a smaller portion of the dose (54% of administered dose) was excreted intact in bile, and the major metabolites detected were an azetidine N-oxide of MRL-A and an acylglucuronide of an N-dealkylation product. This latter metabolite was also observed in rat bile. Stereoselective formation of the N-oxide isomer was observed in dogs, whereas the rat produced comparable amounts of both isomers. The formation of a unique glutathione adduct was observed in rat bile, which was proposed to occur via N-dealkylation, followed by reduction of the putative aldehyde product to form the alcohol, and dehydration of the alcohol to generate a reactive quinone methide intermediate. Incubation of a synthetic standard of this alcohol in rat microsomes fortified with reduced glutathione or rat hepatocytes resulted in formation of this unique glutathione adduct.

Short synthesis of octosyl nucleosides.

[reaction: see text] Commercial 1,2:5,6-di-O-isopropylidene-alpha-d-allofuranose was converted to a protected bicyclic octosyl acid thioglycoside donor by a 10-step sequence that features an intramolecular ester enolate alkylation. Glycosylation of N-benzoyladenine and methyl uridine-5-carboxylate followed by deprotection gave the respective nucleosides "octosyl adenine" and octosyl acid A.

Synthesis of functionalized 1,8-naphthyridinones and their evaluation as novel, orally active CB1 receptor inverse agonists.

Synthesis, SAR, and binding affinities are described for a new class of 1,8-naphthyridinone CB1 receptor specific inverse agonists. Food intake, knockout mouse, and pharmacokinetic evaluation of 14 indicate that this compound is an effective orally active modulator of CB1.

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.

Zafirlukast metabolism by cytochrome P450 3A4 produces an electrophilic alpha,beta-unsaturated iminium species that results in the selective mechanism-based inactivation of the enzyme.

Zafirlukast is a leukotriene antagonist indicated for the treatment of mild to moderate asthma, but the drug has been associated with occasional idiosyncratic hepatotoxicity. Structurally, zafirlukast is similar to 3-methylindole because it contains an N-methylindole moiety that has a 3-alkyl substituent on the indole ring. The results presented here describe the metabolic activation of zafirlukast via a similar mechanism to that described for 3-methylindole. NADP(H)-dependent biotransformation of zafirlukast by hepatic microsomes from rats and humans afforded a reactive metabolite, which was detected as its GSH adduct. Mass spectrometry and NMR data indicated that the GSH adduct was formed by the addition of GSH to the methylene carbon between the indole- and methoxy-substituted phenyl rings of zafirlukast. The formation of this reactive metabolite in human liver microsomes was shown to be exclusively catalyzed by CYP3A enzymes. Evidence for in vivo metabolic activation of zafirlukast was obtained when the same GSH adduct was detected in bile of rats given an iv or oral dose of the drug. On the basis of results with model peroxidases and of the structures of product alcohols from incubations containing H2(18)O, it appeared that zafirlukast underwent dehydrogenation by two sequential one-electron oxidations. In addition, zafirlukast proved to be a mechanism-based inhibitor of CYP3A4 activity in human liver microsomes and in microsomes containing cDNA-expressed CYP3A4. The enzyme inhibitory property of zafirlukast was selective for this enzyme among all of the P450 enzymes that were tested in human liver microsomes. The inactivation was characterized by a K(I) of 13.4 microM and k(inact) of 0.026 min(-1). In summary, zafirlukast dehydrogenation to an electrophilic alpha,beta-unsaturated iminium intermediate may be associated with idiosyncratic hepatotoxicity and/or cause drug-drug interactions through inactivation of CYP3A4.

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.

Dipeptidyl peptidase IV inhibitors derived from beta-aminoacylpiperidines bearing a fused thiazole, oxazole, isoxazole, or pyrazole.

A series of beta-aminoacylpiperidines bearing various fused five-membered heterocyclic rings was synthesized as dipeptidyl peptidase IV inhibitors. Potent and relatively selective inhibition could be obtained, depending on choice of heterocycle, regioisomerism, and substitution. In particular, one analog (74, DPP-IV IC50=26 nM) exhibited good oral bioavailability and acceptable half-life in the rat, albeit with rather high clearance.