Dihydro-pyrano[2,3-b]pyridines and tetrahydro-1,8-naphthyridines as CB1 receptor inverse agonists: synthesis, SAR and biological evaluation.

Synthesis and structure-activity relationships of cannabinoid-1 receptor (CB1R) inverse agonists based on dihydro-pyrano[2,3-b] pyridine and tetrahydro-1,8-naphtyridine scaffolds are presented. Rat food intake and pharmacokinetic evaluation of 13g, 13i, 13k and 17a revealed these compounds to be highly efficacious orally active modulators of CB1R.

Discovery of N-[(4R)-6-(4-chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-yl]-5-methyl-1H-pyrazole-3-carboxamide (MK-5596) as a novel cannabinoid-1 receptor (CB1R) inverse agonist for the treatment of obesity.

This paper describes the discovery of N-[(4R)-6-(4-chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-yl]-5-methyl-1H-pyrazole-3-carboxamide (MK-5596, 12c) as a novel cannabinoid-1 receptor (CB1R) inverse agonist for the treatment of obesity. Structure-activity relationship (SAR) studies of lead compound 3, which had off-target hERG (human ether-a-go-go related gene) inhibition activity, led to the identification of several compounds that not only had attenuated hERG inhibition activity but also were subject to glucuronidation in vitro providing the potential for multiple metabolic clearance pathways. Among them, pyrazole 12c was found to be a highly selective CB1R inverse agonist that reduced body weight and food intake in a DIO (diet-induced obese) rat model through a CB1R-mediated mechanism. Although 12c was a substrate of P-glycoprotein (P-gp) transporter, its high in vivo efficacy in rodents, good pharmacokinetic properties in preclinical species, good safety margins, and its potential for a balanced metabolism profile in man allowed for the further evaluation of this compound in the clinic.

Furo[2,3-b]pyridine-based cannabinoid-1 receptor inverse agonists: synthesis and biological evaluation. Part 1.

The synthesis, SAR and binding affinities of cannabinoid-1 receptor (CB1R) inverse agonists based on furo[2,3-b]pyridine scaffolds are described. Food intake, mechanism specific efficacy, pharmacokinetic, and metabolic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.

Pyridopyrimidine based cannabinoid-1 receptor inverse agonists: Synthesis and biological evaluation.

The synthesis, SAR and binding affinities are described for cannabinoid-1 receptor (CB1R) specific inverse agonists based on pyridopyrimidine and heterotricyclic scaffolds. Food intake and pharmacokinetic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.

The triple uptake inhibitor (1R,5S)-(+)-1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0] hexane hydrochloride (DOV 21947) reduces body weight and plasma triglycerides in rodent models of diet-induced obesity.

Selective inhibitors of biogenic amine (e.g., serotonin, norepinephrine, and dopamine) uptake exhibit varying degrees of safety and efficacy as antiobesity agents. Moreover, preclinical findings suggest that the combined inhibition of monoamine neurotransmitter transporters synergistically enhances antiobesity activity. (1R,5S)-(+)-1-(3,4-Dichlorophenyl)-3-azabicyclo-[3.1.0] hexane hydrochloride (DOV 21947) inhibits norepinephrine, 5-hydroxytryptamine, and dopamine uptake, and it reduces body weight in rodent models of diet-induced obesity (DIO). DIO rats treated orally with DOV 21947 for 1 to 24 days showed significantly lower body weights than vehicle-treated DIO rats. The decrease in body weight resulted specifically from a loss of retroperitoneal and mesenteric depots of white adipose tissue. DOV 21947 also reduced daily food intake in DIO rats, but consumption returned to control levels after 11 days of treatment. With the exception of a decrease in triglyceride levels, blood chemistry was unaltered after 24 days of DOV 21947 treatments. DOV 21947 had no effect on motor activity. Although DOV 21947 increased respiratory rate and decreased the tidal volume of normal rats, it did not alter the minute volume. In addition, DOV 21947 did not significantly affect blood pressure, heart rate, electrocardiographic indices or body temperature in telemeterized dogs. However, it caused a sustained, but reversible reduction in the rate of body weight gain for as long as 6 months in normal rats, and for up to 1 year in normal dogs. In summary, DOV 21947 is effective in causing a sustained and selective reduction in fat content and triglyceride levels in animal models of obesity without significantly altering vital organ function.

Characterization of a novel and selective cannabinoid CB1 receptor inverse agonist, Imidazole 24b, in rodents.

We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.

Antiobesity efficacy of a novel cannabinoid-1 receptor inverse agonist, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364), in rodents.

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding K(i) of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma C(max) of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30-40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Lead optimization of 5,6-diarylpyridines as CB1 receptor inverse agonists.

Optimization of the biological activity for 5,6-diarylpyridines as CB1 receptor inverse agonists is described. Food intake and pharmacokinetic evaluation of 3f and 15c indicate that these compounds are effective orally active modulators of CB1.

Substituted acyclic sulfonamides as human cannabinoid-1 receptor inverse agonists.

Sulfonamide analogues of the potent CB1R inverse agonist taranabant were prepared and optimized for potency and selectivity for CB1R. They were variably more potent than the corresponding amide analogues. The most potent representative 22 had good pharmacokinetic and brain levels, but was modestly active in blocking CB1R agonist-mediated hypothermia.

Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity.

The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body weight reduction. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compound 48 for development as a clinical candidate for the treatment of obesity.

Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice.

Ghrelin, an acylated peptide secreted from the stomach, acts as a short-term signal of nutrient depletion. Ghrelin is an endogenous ligand for the GH secretagogue receptor 1a, a G protein-coupled receptor expressed in the hypothalamus and pituitary. We used a synthetic oligonucleotide, NOX-B11-2, capable of specific high-affinity binding to bioactive ghrelin to determine whether ghrelin neutralization would alter indices of energy balance in vivo. This novel type of ghrelin-blocking agent, called an RNA Spiegelmer (SPM), is a polyethylene glycol-modified l-RNA oligonucleotide, the nonnatural configuration of which confers in vivo stability. NOX-B11-2 blocked ghrelin mediated activation of GH secretagogue receptor 1a in cell culture (IC50 approximately 5 nm). We explored the effects of acute NOX-B11-2 administration on ghrelin-induced feeding in mice. NOX-B11-2 (66 mg/kg, sc) blocked ghrelin-induced feeding and was without effect on feeding evoked by an orally active nonpeptide ghrelin receptor agonist. We demonstrated that selective ghrelin blockade effectively promoted weight loss in diet-induced obese (DIO) mice. Chronic infusion of NOX-B11-2 (33 mg/kg.d, sc) to DIO mice evoked body weight loss for 13 d and reduced food intake and fat mass relative to control SPM-infused mice. In a 7-d study, DIO mice infused with NOX-B11-2 (33 mg/kg.d, sc) showed body weight loss, compared with animals receiving control SPM. This effect was directly mediated by SPM neutralization of ghrelin because NOX-B11-2 administration to ghrelin-deficient mice resulted in no weight loss. The decreased obesity observed in SPM-treated DIO mice provides validation for ghrelin neutralization as a potential antiobesity therapy.

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.

Synthesis and activity of 4,5-diarylimidazoles as human CB1 receptor inverse agonists.

Structure-activity relationship studies directed toward the optimization of 4,5-diarylimidazole-2-carboxamide analogs as human CB1 receptor inverse agonists resulted in the discovery of the two amide derivatives 24a and b (hCB1 IC50 = 6.1 and 4.0 nM) which also demonstrated efficacy in overnight feeding studies in the rat for reduction in both food intake and overall body weight.

Synthesis and SAR of 5,6-diarylpyridines as human CB1 inverse agonists.

Structure-activity relationship studies for two series of 2-benzyloxy-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)pyridines having either a 3-cyano or 3-carboxamide moiety resulted in the preparation of the 2-(3,4-difluorobenzyloxy)-3-nitrile analog 10d and the 2-(3,4-difluorobenzyloxy)-3-(N-propylcarboxamide) analog 16c, (hCB1 IC(50)=1.3 and 1.7 nM, respectively) as potent and selective hCB1 inverse agonists. Their synthesis and biological activities are described herein.

Chronic MCH-1 receptor modulation alters appetite, body weight and adiposity in rats.

Central administration of the neuropeptide melanin-concentrating hormone (MCH) stimulates feeding in rodents. We studied the effects of intracerebroventricular (i.c.v.) administration of an MCH-1 receptor agonist (Compound A) and an MCH-1 receptor antagonist (Compound B) on feeding in satiated rats. Compound B (10 microg, i.c.v.) blocked the acute orexigenic effect of Compound A (5 microg, i.c.v.). In an experiment designed to either stimulate or inhibit MCH-1 receptor signaling over an extended period, rats received continuous i.c.v. infusions of vehicle (saline), Compound A (30 microg/day), Compound B (30 or 48 microg/day) or neuropeptide Y (24 microg/day, as positive control) via implantable infusion pumps. Continuous MCH-1 receptor activation recapitulated the obese phenotype of MCH-over-expressor mice, manifest as enhanced feeding (+23%, P<0.001), caloric efficiency and body weight gain (+38%, P<0.005) over the 14-day period relative to controls. Chronic MCH-1 receptor activation also elevated plasma insulin and leptin levels significantly. Conversely, continuous MCH-1 receptor antagonism led to sustained reductions in food intake (-16%, P<0.001), body weight gain (-35%, P<0.01), and body fat gain relative to controls, without an effect on lean mass. Antagonism of the MCH-1 receptor may be an effective approach for the treatment of obesity.

[Platinum compounds in cancer therapy--past, present, and future].

Platinum cytotoxics play an important role globally in the management of solid tumours. Cisplatin sets the standard for efficacy in both regions with careful administration to reduce nephrotoxicity. Carboplatin is associated with neurotoxicity, but has become the leading product in the US due largely to the easier to manage toxicity profile. Both agents have been widely used in both registered and non registered indications and are frequently combined with other cytotoxics. In Japan, cisplatin has been used successfully at low doses in combination with 5-FU based regimens and appears to achieve a synergistic effect, but controlled data are not yet available. More recently oxaliplatin (Europe) and nedaplatin (in Japan) have been introduced, but their clinical roles in therapy have yet to be established. One of the limiting features of the first generation of platinum compounds is that a significant proportion of tumours develop cross resistance to platins due to either changes in uptake or excretion, intracellular detoxification or accelerated DNA repair. The forum discussed the possibility for the development of better new platinum compounds, A new platin agent which had lower toxicity and higher efficacy across a wide range of cancers without the development of resistance would be a significant step forward. If the tolerability profile was suitable, an oral formulation may improve the quality of life for patients but this must not be at the expense of efficacy. Even after the introduction of new target based drugs, platinum cytotoxics are likely to be used to reduce the tumour mass and in some cases can be expected to potentiate the effects of the new agents. In preclinical studies, ZD0473 has been shown to by-pass some major mechanisms of resistance and has the potential to achieve these objectives and is now being evaluated in clinical studies in both Japan and the West.

[Development of molecular targeting drugs for the treatment of cancer-therapeutic potential and issues to be addressed in global development].

A survey of cancer treatment in a sample of hospitals > 100 beds conducted in 1998 compared with experience in the US showed that good progress has been achieved in Japan in the screening and early treatment of gastric cancer, and that the prognosis for breast cancer is better than in the West. Although in the past, the cytotoxic therapies available to physicians in Japan vs the West have been different, recent acceleration of regulatory review will result in a convergence of treatment paradigms and some improvement in acute response in many tumour types. However, world wide there is a need for new improved therapies in all cancers evaluated. Particular needs are in the management of NSCLC, advanced disease and cancers which form micrometastases. The eventual hope is that cancer can be turned from a lethal disease into a chronic disease where patients maintain a good QOL. Apart from anti hormonal therapies, the usual approach has been to kill the cancerous cells. However, the new approaches to intervening in the growth and migration of cancerous cells or the host tissue response by molecular targeting offer the promise of achieving a step change in therapy. Although EGF tyrosine Kinase inhibitors such as ZD 1839 have been shown to cause a conventional tumour response in NSCLC, many of these new approaches are unlikely to show a short term response even if they have the capacity to affect tumour development and increase disease free survival. Some compounds will require combination therapy with a conventional cytotoxic or radiotherapy to show their full benefit. For conventional cytotoxics, the usual approach to development has been to select the maximum tolerated dose and then evaluate the efficacy in advanced disease. However, for the new approaches which will not have such severe dose limiting toxicities, it will be necessary to select a surrogate marker of the intended biological effect to select the optimal biological dose (OBD) and dose regimen in phase I/II studies for further evaluation in phase II or III studies which are designed to show the expected patient benefit. The tumour target, the stage of the disease and the possible need for concomitant therapy will also have to be considered according to the mechanism of action of the product.

[Early detection and prevention of cancers in various therapeutic areas. Discussion].

As illustrated by prostate cancer screening provides an opportunity for early intervention and treatment. However the screening test needs to detect patients with cancer with a low rate of false positives and at a stage which can be treated. Recently the concept of treating patients at high risk of developing breast cancer or suffering a recurrence has been highlighted by the western studies with Nolvadex (tamoxifen). Thus roundtable discussion (held in Tokyo) discussed the different strategies in Japan compared to US & Europe for screening & early intervention/prevention of cancer for breast, prostate, bladder, liver, lung, gynaecological & GI cancers. The range of strategies for cancer screening, how it is funded, whether it is appropriately targeted and whether there is any evidence for a beneficial effect on morbidity or mortality & future prospects for improved sensitivity through new methodology or markers were discussed. Although the relative rates of cancer vary between Japan & the West, the same factors seem to influence cancer development & the data on intervention were seen to be valid. The changing patterns of cancer in Japan suggest a clear opportunity for reducing, the incidence of cancer through lifestyle modification. For some cancers, e.g. cervical & bladder where there is a clear link between abnormal cytology & development cancer true prevention is already practiced. In other cases, preventive treatment is limited by the efficacy of available therapies. As far as drug treatment is concerned, funding of healthcare in Japan does not recognise the concept of prevention although there is, in practice, no barrier to the use of interventions where there is a clear link between biochemical/histological markers & development of cancer.

Protein kinase C beta II specifically binds to and is activated by F-actin.

The two most closely related isoenzymes of protein kinase C (PKC), PKC betaI and betaII, are distinct but highly homologous isoenzymes derived via alternative splicing of the same gene product. In this study, PKC betaII, but not PKC betaI, translocated to the actin cytoskeleton upon stimulation of cells with phorbol esters. In cells, antibodies to PKC betaII, but not to PKC betaI, co-immunoprecipitated actin. Using an actin-binding co-sedimentation assay, we show in vitro that PKC betaII, but not PKC betaI, binds to actin specifically. This binding was inhibited by peptides based on sequences unique to PKC betaII; thus defining an actin-binding site in PKC betaII that is not present in PKC betaI. The binding of PKC betaII to actin was not inhibited by kinase inhibitors of PKC (sphingosine and staurosporine), suggesting that prior activation and/or substrate phosphorylation are not required for the interaction of PKC betaII with actin. On the other hand, the interaction of PKC betaII with actin resulted in marked enhancement of autophosphorylation of PKC betaII and in an alteration in substrate specificity. These studies serve to define a novel functional domain in the carboxyl-terminal region of PKC beta, which is involved in directing isoenzyme-specific protein-protein interactions, and consequently, isoenzyme-specific functions in vivo.

Selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis. 1. [4-[2-[(2-Hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetates.

The ester methyl [4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetate (8) has been identified as the most interesting member of a series of selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis in the rat. In vivo it acts mainly via the related acid 10. Potency was generally markedly reduced by placing substituents on the phenyl ring of the phenoxypropanolamine unit of 8; only the 2-fluoro analogue 16 had comparable potency to 8. Other structure-activity relationships are discussed. Further testing of 8 (ICI 198157) has shown that in the rat it stimulates the beta 3-adrenergic receptor in brown adipose tissue at doses lower than those at which it affects beta 1 and beta 2 adrenergic receptors in other tissues. It increases metabolic rate, as judged by an increase in oxygen consumption, and in the genetically obese Zucker rat it causes a reduced rate of weight gain. This class of compound may be useful in the treatment of obesity in man.