Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

A novel series of pyrazolylpiperidine N-type calcium channel blockers.

Selective blockers of the N-type calcium channel have proven to be effective in animal models of chronic pain. However, even though intrathecally delivered synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been approved for the treatment of chronic pain in humans, its mode of delivery and narrow therapeutic window have limited its usefulness. Therefore, the identification of orally active, small-molecule N-type calcium channel blockers would represent a significant advancement in the treatment of chronic pain. A novel series of pyrazole-based N-type calcium channel blockers was identified by structural modification of a high-throughput screening hit and further optimized to improve potency and metabolic stability. In vivo efficacy in rat models of inflammatory and neuropathic pain was demonstrated by a representative compound from this series.

Synthesis and biological evaluation of novel pyridazinone-based alpha4 integrin receptor antagonists.

A novel series of pyridazinone-functionalized phenylalanine analogues was prepared and evaluated for inhibition of cellular adhesion mediated by alpha4beta1/VCAM-1 and alpha4beta7/MAdCAM-1 interactions. Concise syntheses were developed and applied for exploration of structure-activity relationships pertaining to the pyridazinone ring as well as the N-acyl phenylalanine scaffold. Potent dual antagonists of alpha4beta1 and alpha4beta7 were generated from an amide subseries; antagonists selective for alpha4beta7 were identified from urea and carbamate-based subseries. The pharmacokinetic properties of selected members of the series have been determined in rats and demonstrate that the use of ester prodrugs and alterations to the amide linkage can lead to improved oral bioavailability in this series. An alpha4beta7-selective member of the carbamate subseries (36c), upon oral administration, demonstrated in vivo efficacy in the mouse DSS colitis model.

Rationale, design, and synthesis of novel phenyl imidazoles as opioid receptor agonists for gastrointestinal disorders.

A small series of novel, imidazoles 4 have been prepared that exhibit very good binding affinities for the delta and mu opioid receptors (ORs), as well as demonstrate potent agonist functional activity at the delta OR. Representative imidazole 4a (K(i) delta = 0.9 nM; K(i) mu = 55 nM; K(i) kappa = 124 nM; EC(50) delta =13-25 nM) was further profiled for OR related in vivo effects. Compound 4a reduced gastrointestinal (GI) propulsive motility in a dose-dependent and naloxone-reversible manner, based on the results of the mouse glass bead expulsion test (3, 5, and 10 mg/kg, ip) and the mouse fecal pellet output test (1 and 3 mg/kg, ip). Compound 4a showed no analgesic activity as measured by the mouse abdominal irritant test (MAIT) when dosed at 100 mg/kg, sc, but did show significant MAIT activity at doses of both 10 microg (40% inhibition) and 100 microg (100% inhibition) when dosed intracerebroventricularly (icv). Taken together, these in vivo results suggest that 4a acts peripherally when dosed systemically, and that these prototypical compounds may prove promising as medicinal leads for GI indications.

Design and optimization of benzimidazole-containing transient receptor potential melastatin 8 (TRPM8) antagonists.

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design, synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the series demonstrated excellent in vivo activity in the TRPM8-selective "wet-dog shakes" (WDS) pharmacodynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types of neuropathic pain.

Small intestinal cannabinoid receptor changes following a single colonic insult with oil of mustard in mice.

Cannabinoids are known to be clinically beneficial for control of appetite disorders and nausea/vomiting, with emerging data that they can impact other GI disorders, such as inflammation. Post-inflammatory irritable bowel syndrome (PI-IBS) is a condition of perturbed intestinal function that occurs subsequent to earlier periods of intestinal inflammation. Cannabinoid 1 receptor (CB1R) and CB2R alterations in GI inflammation have been demonstrated in both animal models and clinically, but their continuing role in the post-inflammatory period has only been implicated to date. Therefore, to provide direct evidence for CBR involvement in altered GI functions in the absence of overt inflammation, we used a model of enhanced upper GI transit that persists for up to 4 weeks after a single insult by intracolonic 0.5% oil of mustard (OM) in mice. In mice administered OM, CB1R immunostaining in the myenteric plexus was reduced at day 7, when colonic inflammation is subsiding, and then increased at 28 days, compared to tissue from age-matched vehicle-treated mice. In the lamina propria CB2R immunostaining density was also increased at day 28. In mice tested 28 day after OM, either a CB1R-selective agonist, ACEA (1 and 3 mg/kg, s.c.) or a CB2R-selective agonist, JWH-133 (3 and 10 mg/kg, s.c.) reduced the enhanced small intestinal transit in a dose-related manner. Doses of ACEA and JWH-133 (1 mg/kg), alone or combined, reduced small intestinal transit of OM-treated mice to a greater extent than control mice. Thus, in this post-colonic inflammation model, both CBR subtypes are up-regulated and there is increased efficacy of both CB1R and CB2R agonists. We conclude that CBR remodeling occurs not only during GI inflammation but continues during the recovery phase. Thus, either CB1R- or CB2-selective agonists could be efficacious for modulating GI motility in individuals experiencing diarrhea-predominant PI-IBS.

Agonists of cannabinoid receptor 1 and 2 inhibit experimental colitis induced by oil of mustard and by dextran sulfate sodium.

Oil of mustard (OM) is a potent neuronal activator that is known to elicit visceral hyperalgesia when given intracolonically, but the full extent to which OM is also proinflammatory in the gastrointestinal tract is not known. We have previously shown that male CD-1 mice given a single administration of 0.5% OM develop a severe colitis that is maximum at day 3 and that gradually lessens until essentially absent by day 14. OM-induced neuronal stimulation is reported to be reduced by cannabinoid agonists, and cannabinoid receptor 1 (CB1R)-/- mice have exacerbated experimental colitis. Therefore, we examined the role of cannabinoids in this OM-induced 3-day model of colitis in CD-1 mice and in a 7-day dextran sulfate sodium (DSS) colitis model in BALB/c mice. In OM colitis, the CB1R-selective agonist ACEA and the CB2R-selective agonist JWH-133 reduced (P < 0.05) colon weight gain (means +/- SE; 82 +/- 13% and 47 +/- 15% inhibition, respectively), colon shrinkage (98 +/- 24% and 42 +/- 12%, respectively), colon inflammatory damage score (49 +/- 11% and 40 +/- 12%, respectively), and diarrhea (58 +/- 12% and 43 +/- 11%, respectively). Histological damage was similarly reduced by these treatments. Likewise, CBR agonists attenuated DSS colitis, albeit at higher doses; ACEA at 10 mg/kg, twice daily, inhibited (P < 0.05) macroscopic and microscopic scores (46 +/- 9% and 63 +/- 7%, respectively); whereas 20 mg/kg, twice daily, of JWH-133 was required to diminish (P < 0.05) macroscopic and microscopic scores (29 +/- 7% and 43 +/- 5%, respectively). CB1R and CB2R immunostaining of colon sections revealed that CB1R in enteric neurons was more intense in colitic vs. control mice; however, CB1R was also increased in the endothelial layer in OM colitis only. CB2R immunostaining was more marked in infiltrated immune cells in OM colitis. These findings validate the OM colitis model with respect to the DSS model and provide strong support to the emerging idea that cannabinoid receptor activation mediates protective mechanisms in experimental colitis. The demonstration of CB1R agonist effects in colitis support the neurogenic nature of the OM-induced colitis model and reinforce the importance of neuronal activation in intestinal inflammation.

Aza-bicyclic amino acid carboxamides as alpha4beta1/alpha4beta7 integrin receptor antagonists.

A series of N-carboxy, N-alkyl, and N-carboxamido azabicyclo[2.2.2]octane carboxamides were prepared and assayed for inhibition of alpha4beta1-VCAM-1 and alpha4beta7-MAdCAM-1 interactions. Potency and alpha4beta1/alpha4beta7 selectivity were sensitive to the substituent R1-R3 in the structures 6, 7, and 8. Several compounds demonstrated low nanomolar balanced alpha4beta1/alpha4beta7 in vitro activity. Two compounds were selected for in vivo leukocytosis studies and demonstrated increases in circulating lymphocytes up to 250% over control.