Discovery of novel phenylpyridone derivatives as potent and selective MCH1R antagonists.

The design, synthesis and structure-activity relationships of a novel class of N-phenylpyridone MCH1R antagonists are described. The core part of the N-phenylpyridone structure was newly designed and the side chain moieties that were attached to the core part were extensively explored. As a result of optimization of the N-phenylpyridone leads, we successfully developed the orally available, and brain-penetrable MCH1R selective antagonist 7c, exhibiting excellent anti-obese effect in diet-induced obese (DIO) mice.

Melanin-concentrating hormone 1-receptor antagonist suppresses body weight gain correlated with high receptor occupancy levels in diet-induced obesity mice.

Melanin-concentrating hormone (MCH), which is a neuropeptide expressed in the hypothalamus of the brain, is involved in regulating feeding behavior and energy homeostasis via the MCH(1) receptor in rodents. It is widely considered that MCH(1) receptor antagonists are worthy of development for medical treatment of obesity. Here we report on the development of an ex vivo receptor occupancy assay using a new radiolabeled MCH(1) receptor antagonist, [(35)S]-compound D. An MCH(1) receptor antagonist inhibited the binding of [(35)S]-compound D to brain slices in a dose-dependent manner. The result showed a good correlation between the receptor occupancy levels and plasma or brain levels of the MCH(1) receptor antagonist, suggesting that the ex vivo receptor binding assay using this radioligand is practical. Quantitative analysis in diet-induced obese mice showed that the efficacy of body weight reduction correlated with the receptor occupancy levels at 24h. Furthermore, more than 90% occupancy levels of MCH(1) receptor antagonists during 24h post-dosing are required for potent efficacy on body weight reduction. The present occupancy assay could be a useful pharmacodynamic marker to quantitatively estimate anti-obese efficacy, and would accelerate the development of MCH(1) receptor antagonists for treatment of obesity.

Inverse agonist histamine H3 receptor PET tracers labelled with carbon-11 or fluorine-18.

Two histamine H3 receptor (H3R) inverse agonist PET tracers have been synthesized and characterized in preclinical studies. Each tracer has high affinity for the histamine H3 receptor, has suitable lipophilicity, and neither is a substrate for the P-glycoprotein efflux pump. A common phenolic precursor was used to synthesize each tracer with high specific activity and radiochemical purity by an alkylation reaction using either [(11)C]MeI or [(18)F]FCD(2)Br. Autoradiographic studies in rhesus monkey and human brain slices showed that each tracer had a widespread distribution with high binding densities in frontal cortex, globus pallidus and striatum, and lower uptake in cerebellum. The specificity of this expression pattern was demonstrated by the blockade of the autoradiographic signal by either the H3R agonist R-alpha-methylhistamine or a histamine H3R inverse agonist. In vivo PET imaging studies in rhesus monkey showed rapid uptake of each tracer into the brain with the same distribution seen in the autoradiographic studies. Each tracer could be blocked by pretreatment with a histamine H3R inverse agonist giving a good specific signal. Comparison of the in vitro metabolism of each compound showed slower metabolism in human liver microsomes than in rhesus monkey liver microsomes, with each compound having a similar clearance rate in humans. The in vivo metabolism of 1b in rhesus monkey showed that at 60 min, approximately 35% of the circulating counts were due to the parent. These tracers are very promising candidates as clinical PET tracers to both study the histamine H3R system and measure receptor occupancy of H3R therapeutic compounds.

Discovery of novel diarylketoxime derivatives as selective and orally active melanin-concentrating hormone 1 receptor antagonists.

Optimization of the lead 2a led to the identification of a novel diarylketoxime class of melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. Our focus was directed toward improvement of hERG activity and metabolic stability. The representative derivative 4b showed potent and dose-dependent body weight reduction in diet-induced obese (DIO) C57BL/6J mice after oral administration. The synthesis and structure-activity relationships of the novel diarylketoxime MCH-1R antagonists are described.

Discovery of imidazo[1,2-a]pyridines as potent MCH1R antagonists.

A series of imidazo[1,2-a]pyridine derivatives was identified and evaluated for MCH1R binding and antagonistic activity. Introduction of a methyl substituent at the 3-position of imidazo[1,2-a]pyridine provided compounds with a significant improvement in MCH1R affinity. Representative compounds in this series exhibited good potency and brain exposure in rats.

Synthesis, structure-activity relationships, and biological profiles of a dihydrobenzoxathiin class of histamine H(3) receptor inverse agonists.

A series of novel dihydrobenzoxathiin derivatives was synthesized and evaluated as potent human histamine H(3) receptor inverse agonists. After systematic modification of lead 1a, the potent and selective histamine H(3) inverse agonist 1-(3-{4-[(2S,3S)-8-methoxy-3-methyl-4,4-dioxido-2,3-dihydro-1,4-benzoxathiin-2-yl]phenoxy}propyl)pyrrolidine (5k) was identified. Compound 5k showed good pharmacokinetic profiles and brain penetrability in laboratory animals. After 3mg/kg oral administration of 5k, significant elevation of brain histamine levels was observed in rats where the brain H(3) receptor was fully occupied.

Identification of 2-aminobenzimidazoles as potent melanin-concentrating hormone 1-receptor (MCH1R) antagonists.

A series of 2-aminobenzimidazole-based MCH1R antagonists was identified by core replacement of the aminoquinoline lead 1. Subsequent modification of the 2- and 5-positions led to improvement in potency and intrinsic clearance. Compound 25 exhibited good plasma and brain exposure, and attenuated MCH induced food intake at 30mg/kg PO in rats.

Hemodynamic and cardiac neurotransmitter-releasing effects in conscious dogs of attention- and wake-promoting agents: a comparison of d-amphetamine, atomoxetine, modafinil, and a novel quinazolinone H3 inverse agonist.

Conscious coronary sinus-cannulated dogs were used to assess the hemodynamic effects and local cardiac norepinephrine (NE) and histamine (HA) release of 4 mechanistically diverse agents either clinically approved or representing a potential novel mechanism for the promotion of wakefulness or attention. Dosing regimens were based on reported or concurrently determined wake-promoting activities in canine models. The central nervous system stimulant, d-amphetamine [0.1 mg x kg(-1) x 10 min intravenous (IV)], significantly elevated mean arterial pressure (+30%) and increased coronary sinus and peripheral venous NE concentrations, indicative of cardiac neurotransmitter release. The selective NE reuptake inhibitor atomoxetine (2.0 mg x kg(-1) x 10 min(-1) IV) and modafinil (30.0 mg x kg(-1) x 10 min(-1) IV) also significantly elevated mean arterial pressure (+15% and +30%, respectively), but with no effect on coronary sinus or peripheral NE concentration, suggesting central mechanisms underlying the hemodynamic effects. The preclinical demonstrations of pressor effects with d-amphetamine, atomoxetine, and modafinil are consistent with clinically reported hemodynamic effects with these agents. The quinazolinone HA receptor subtype H3 inverse agonist 5r (0.3 mg x kg(-1) x 10 min(-1) IV) displayed no effect on hemodynamics or on coronary sinus or peripheral NE and HA concentrations. These data suggest the potential for therapeutic effect with the latter mechanism in the absence of peripheral cardiac neurotransmitter release or obvious changes in cardiovascular function.

Synthesis and evaluation of structurally constrained quinazolinone derivatives as potent and selective histamine H3 receptor inverse agonists.

A series of structurally constrained derivatives of the potent H 3 inverse agonist 1 was designed, synthesized, and evaluated as histamine H 3 receptor inverse agonists. As a result, the N-cyclobutylpiperidin-4-yloxy group as in 2f was identified as an optimal surrogate structure for the flexible 1-pyrrolidinopropoxy group of 1. Subsequent optimization of the quinazolinone core of 2f revealed that substitution at the 5-position of the quinazolinone ring influences potency. Representative derivatives 5a and 5s showed improved potency in a histamine release assay in rats and a receptor occupancy assay in mice.

Development of novel 2-[4-(aminoalkoxy)phenyl]-4(3H)-quinazolinone derivatives as potent and selective histamine H3 receptor inverse agonists.

Novel 2-[4-(aminoalkoxy)phenyl]-4(3H)-quinazolinone derivatives were identified as potent human H(3) receptor inverse agonists. After systematic modification of lead 5a, the potent and selective analog 5r was identified. Elimination of hERG K(+) channel and human alpha(1A)-adrenoceptor activities is the main focus of the present study.

Synthesis and evaluation of a spiro-isobenzofuranone class of histamine H3 receptor inverse agonists.

Spiro-isobenzofuranones 1a and 1b were discovered as potent, selective, and brain-penetrable non-imidazole H3 receptor inverse agonists. Our corporate sample collection was screened to identify 2a as a lead. Recognizing the right-hand portion of 2a as an essential pharmacophore, an extensive screen of the left-hand piperidine portion was carried out to yield the potent spiro-derivatives 2t-x. Spiro-isobenzofuranone 2x, the most potent among the derivatives, was converted to the corresponding amide 1a, which possessed dramatically improved H3 activity (IC(50)=0.72 nM; more than 20-fold improvement over 2x). Further elaboration led to the identification of 1b, a 5-methoxy derivative with an IC(50) of 0.54 nM. Our studies demonstrated that derivatives 1a and 1b to be potent, selective, and brain-penetrable H3 inverse agonists.

Synthesis, structure-activity relationships, and biological profiles of a quinazolinone class of histamine H3 receptor inverse agonists.

A new series of quinazolinone derivatives was synthesized and evaluated as nonimidazole H 3 receptor inverse agonists. 2-Methyl-3-(4-[[3-(1-pyrrolidinyl)propyl]oxy]phenyl)-5-(trifluoromethyl)-4(3 H)-quinazolinone ( 1) was identified as a promising derivative for further evaluation following optimization of key parameters. Compound 1 has potent H 3 inverse agonist activity and excellent selectivity over other histamine receptor subtypes and a panel of 115 unrelated diverse binding sites. Compound 1 also shows satisfactory pharmacokinetic profiles and brain penetrability in laboratory animals. Two hours after oral administration of 30 mg/kg of 1 to SD rats, significant elevation of brain histamine levels was observed where the brain H 3 receptor was highly occupied (>90%). On the basis of species differences in P-glycoprotein (P-gp) susceptibility of 1 between human and rodent P-gps, the observed rodent brain permeability of 1 is significantly limited by P-gp mediated efflux in rodents, whereas the extent of P-gp mediated efflux in humans should be very small or negligible. The potential of 1 to be an efficacious drug was demonstrated by its excellent brain penetrability and receptor occupancy in P-gp-deficient CF-1 mice.

Metabolism of rofecoxib in vitro using human liver subcellular fractions.

The metabolism of rofecoxib, a potent and selective inhibitor of cyclooxygenase-2, was examined in vitro using human liver subcellular fractions. The biotransformation of rofecoxib was highly dependent on the subcellular fraction and the redox system used. In liver microsomal incubations, NADPH-dependent oxidation of rofecoxib to 5-hydroxyrofecoxib predominated, whereas NADPH-dependent reduction of rofecoxib to the 3,4-dihydrohydroxy acid metabolites predominated in cytosolic incubations. In incubations with S9 fractions, metabolites resulting from both oxidative and reductive pathways were observed. In contrast to microsomes, the oxidation of rofecoxib to 5-hydroxyrofecoxib by S9 fractions followed two pathways, one NADPH-dependent and one NAD+-dependent (non-cytochrome P450), with the latter accounting for about 40% of total activity. The 5-hydroxyrofecoxib thus formed was found to undergo NADPH-dependent reduction ("back reduction") to rofecoxib in incubations with liver cytosolic fractions. In incubations with dialyzed liver cytosol, net hydration of rofecoxib to form 3,4-dihydro-5-hydroxyrofecoxib was observed, whereas the 3,4-dihydrohydroxy acid derivatives were formed when NADPH was present. Although 3,4-dihydro-5-hydroxyrofecoxib could be reduced to the 3,4-dihydrohydroxy acid by cytosol in the presence of NADPH, the former species does not appear to serve as an intermediate in the overall reductive pathway of rofecoxib metabolism. In incubations of greater than 2 h with S9 fractions, net reductive metabolism predominated over oxidative metabolism. These in vitro results are consistent with previous findings on the metabolism of rofecoxib in vivo in human and provide a valuable insight into mechanistic aspects of the complex metabolism of this drug.

Structure-activity relationship in O-glucuronidation of indolocarbazole analogs.

The glucuronidation of 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(beta-D-glucopyranosyl)5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (compound 1), a potent inhibitor of DNA topoisomerase I, and its related indolocarbazole compounds was studied using human liver microsomes. Compound 1 and its structurally related compounds with the NHCHO moiety at the N-6 position were glucuronidated even if the positions of the phenolic hydroxy moiety were different in these molecules. Compounds that have the NHCH(CH(2)OH)(2) moiety at the N-6 position, however, were not glucuronidated. The three-dimensional structure of these substrates was determined by the semiempirical molecular-orbitals calculation method. Computer-modeling studies, however, revealed that the O-glucuronidation of indolocarbazole analogs depended on the molecular size of the substrates. Compounds larger than 14.5 A in diameter perpendicular to the phenolic hydroxy moiety were not glucuronidated. The chemical reactivity of the hydroxy moiety, evaluated by the atom electron density and the electrostatic potential charges, was very similar in these substrates. These results suggest that a molecular length less than 14.5 A may be required for a substrate to interact with the active site of UDP-glucuronosyltransferase (UGT). To further characterize the glucuronidation of indolocarbazole analogs, compound 1 was used as a representative compound to assess expressed human UGTs. The glucuronidation of compound 1 was catalyzed by recombinant UGT1A9 and UGT1A10 among UGT isoforms tested.