Dibenzazepines and dibenzoxazepines as sodium channel blockers.

We have identified two related series of dibenzazepine and dibenzoxazepine sodium channel blockers, which showed good potency on Nav1.7 in FLIPR-based and electrophysiological functional assays.

N-Aryl azacycles as novel sodium channel blockers.

We have identified a new series of N-aryl azacycles as sodium channel blockers, which showed good potency on Nav1.7 in FLIPR-based and electrophysiological functional assays. Analogs from this series possessed selectivity over hERG, reasonable oral exposure in rat PK studies and are predicted to have limited CNS penetration.

The nociceptin/orphanin FQ receptor partial agonist sunobinop promotes non-REM sleep in rodents and patients with insomnia.

The potent and partial agonist sunobinop activates the nociceptin/orphanin-FQ peptide receptor and promotes non-REM sleep in rodents and patients with insomnia.

Roles of µ-opioid receptors and nociceptin/orphanin FQ peptide receptors in buprenorphine-induced physiological responses in primates.

Buprenorphine is known as a µ-opioid peptide (MOP) receptor agonist, but its antinociception is compromised by the activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine-induced physiological responses in primates (rhesus monkeys). The effects of MOP antagonist (naltrexone), NOP antagonist [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)], and NOP agonists [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one (Ro 64-6198) and 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)] on buprenorphine were studied in three functional assays for measuring analgesia, respiratory depression, and itch in primates. Over the dose range of 0.01 to 0.1 mg/kg, buprenorphine dose-dependently produced antinociception, respiratory depression, and itch/scratching responses, and there was a ceiling effect at higher doses (0.1-1 mg/kg). Naltrexone (0.03 mg/kg) produced similar degrees of rightward shifts of buprenorphine's dose-response curves for all three endpoints. Mean pK(B) values of naltrexone (8.1-8.3) confirmed that MOP receptors mediated mainly buprenorphine-induced antinociception, respiratory depression, and itch/scratching. In contrast, J-113397 (0.1 mg/kg) did not change buprenorphine-induced physiological responses, indicating that there were no functional NOP receptors in buprenorphine-induced effects. More importantly, both NOP agonists, Ro 64-6198 and SCH 221510, enhanced buprenorphine-induced antinociception without respiratory depression and itch/ scratching. The dose-addition analysis revealed that buprenorphine in combination with the NOP agonist synergistically produced antinociceptive effects. These findings provided functional evidence that the activation of NOP receptors did not attenuate buprenorphine-induced antinociception in primates; instead, the coactivation of MOP and NOP receptors produced synergistic antinociception without other side effects. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics.

Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7-selective peptide--protoxin II.

Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N-methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N-methylmorpholine with 2,4,6-collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage-gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized.

Hyperthermia induced by transient receptor potential vanilloid-1 (TRPV1) antagonists in human clinical trials: Insights from mathematical modeling and meta-analysis.

Antagonists of the transient receptor potential vanilloid-1 (TRPV1) channel alter body temperature (Tb) in laboratory animals and humans: most cause hyperthermia; some produce hypothermia; and yet others have no effect. TRPV1 can be activated by capsaicin (CAP), protons (low pH), and heat. First-generation (polymodal) TRPV1 antagonists potently block all three TRPV1 activation modes. Second-generation (mode-selective) TRPV1 antagonists potently block channel activation by CAP, but exert different effects (e.g., potentiation, no effect, or low-potency inhibition) in the proton mode, heat mode, or both. Based on our earlier studies in rats, only one mode of TRPV1 activation - by protons - is involved in thermoregulatory responses to TRPV1 antagonists. In rats, compounds that potently block, potentiate, or have no effect on proton activation cause hyperthermia, hypothermia, or no effect on Tb, respectively. A Tb response occurs when a TRPV1 antagonist blocks (in case of hyperthermia) or potentiates (hypothermia) the tonic TRPV1 activation by protons somewhere in the trunk, perhaps in muscles, and - via the acido-antithermogenic and acido-antivasoconstrictor reflexes - modulates thermogenesis and skin vasoconstriction. In this work, we used a mathematical model to analyze Tb data from human clinical trials of TRPV1 antagonists. The analysis suggests that, in humans, the hyperthermic effect depends on the antagonist's potency to block TRPV1 activation not only by protons, but also by heat, while the CAP activation mode is uninvolved. Whereas in rats TRPV1 drives thermoeffectors by mediating pH signals from the trunk, but not Tb signals, our analysis suggests that TRPV1 mediates both pH and thermal signals driving thermoregulation in humans. Hence, in humans (but not in rats), TRPV1 is likely to serve as a thermosensor of the thermoregulation system. We also conducted a meta-analysis of Tb data from human trials and found that polymodal TRPV1 antagonists (ABT-102, AZD1386, and V116517) increase Tb, whereas the mode-selective blocker NEO6860 does not. Several strategies of harnessing the thermoregulatory effects of TRPV1 antagonists in humans are discussed.

8-Substituted analogues of 3-(3-cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine: highly potent and selective PDE4 inhibitors.

3-(3-Cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine V11294 (1) has been identified as a lead structure, which selectively inhibits human lung PDE4 (436 nM) and is also active in a number of in vitro and in vivo models of inflammation. Here we describe the synthesis and pharmacology of a series of highly potent 8-[(benzyloxy)methyl]-substituted analogues, with potencies in the range 10-300 nM. In addition, several compounds showed interesting PDE4 subtype specificities, for example, the 3-thienyl derivative 5v, which showed 6-10 nM potency at PDE4B, D3, and D5 and a 20- to 200-fold selectivity over A and D2, respectively.

Novel, potent ORL-1 receptor agonist peptides containing alpha-Helix-promoting conformational constraints.

The ORL-1 receptor has recently been cloned and is implicated in a wide variety of physiological and pathophysiological processes. Toward the goal of elucidating important features of the receptor-bound conformation of the endogenous ligand, nociceptin (NC), several conformationally constrained analogues were prepared. Either alpha-aminoisobutyric acid (Aib) or N-methylalanine (MeAla) were inserted as replacement(s) for Ala7, Ala11, or Ala15 in the native NC sequence (FGGFTGARKSARKLANQ). In vitro assays measuring human ORL-1 receptor affinity (competition binding against [3H] NC), functional potency ([35S]GTP gamma S), and efficacy (as compared to NC) were performed for each new peptide. The receptor affinities of the Aib-containing peptides generally matched NC, showing K(i)'s in the range of 0.1-0.5 nM. By comparison, the receptor affinities of the MeAla-containing peptides were significantly diminished. Peptide 14 (FGGFTG[Aib]RKS[Aib]RKLANQ-NH2), which contains two constrained alanine residues (positions 7 and 11) and a C-terminal amide modification, was found to be a very potent agonist with K(i) = 0.05 nM and EC50 = 0.08 nM in the human ORL-1 assays. The data support a hypothesis that the receptor-bound form of NC might adopt an amphipathic helix in the "address" segment of the sequence.

An efficient parallel synthesis of capsazepine and capsazepine analogs.

Capsazepine (CPZ, 1) is a well-known vanilloid receptor (VR1) antagonist that has been cited widely used in the literature. However the current synthetic methods used for the total synthesis of CPZ are lengthy, involve multiple purification steps, and produce low yields. Here we describe a new and highly efficient synthesis of benzazepine 3, a synthetic precursor of CPZ, in only two steps and 59% overall yield from a commercially available tetralone 2 via a Schmidt reaction as a key step. Moreover, we apply parallel synthesis techniques to prepare CPZ and CPZ analogs. Our approach enables the possibility of preparing larger, and more diverse libraries of CPZ analogs.

Solid-phase and solution-phase parallel synthesis of tetrahydro-isoquinolines via Pictet-Spengler reaction.

An efficient parallel synthesis of 6,7-dimethoxytetrahydroisoquinolines is reported. The key reaction step is 3,4-dimethoxyphenethylimines reacting with acid chlorides to form an N-acyliminium ion intermediate, which undergoes Pictet-Spengler condensation to give the desired products in >80% yield. Both solution-phase and solid-phase synthesis of 6,7-dimethoxytetrahydroisoquinolines are described.

Parallel synthesis of a biased library of thiazolidinones as novel sodium channel antagonists.

A biased chemical library containing 91 differentially substituted thiazolidinones was prepared in an effort to improve the pharmacology of a known anticonvulsant agent V102862. The collection was prepared in a single step multi-component condensation reaction that produced good yields and very high crude purity (75%-85%). Seven compounds, identified within the library were shown to be more potent than V102862, our parent reference compound, in an electrophysiological assay measuring sodium channel antagonism. The most potent compound, 3-(2-piperidinylethyl)-2-(3-(3-trifluoromethylphenoxy)phenyl)thiazolidinone, has a Ki of 90 nM.

Studies examining the relationship between the chemical structure of protoxin II and its activity on voltage gated sodium channels.

The aqueous solution structure of protoxin II (ProTx II) indicated that the toxin comprises a well-defined inhibitor cystine knot (ICK) backbone region and a flexible C-terminal tail region, similar to previously described NaSpTx III tarantula toxins. In the present study we sought to explore the structure-activity relationship of the two regions of the ProTx II molecule. As a first step, chimeric toxins of ProTx II and PaTx I were synthesized and their biological activities on Nav1.7 and Nav1.2 channels were investigated. Other tail region modifications to this chimera explored the effects of tail length and tertiary structure on sodium channel activity. In addition, the activity of various C-terminal modifications of the native ProTx II was assayed and resulted in the identification of protoxin II-NHCH3, a molecule with greater potency against Nav1.7 channels (IC50=42 pM) than the original ProTx II.

Structure-activity relationship studies and discovery of a potent transient receptor potential vanilloid (TRPV1) antagonist 4-[3-chloro-5-[(1S)-1,2-dihydroxyethyl]-2-pyridyl]-N-[5-(trifluoromethyl)-2-pyridyl]-3,6-dihydro-2H-pyridine-1-carboxamide (V116517) as a clinical candidate for pain management.

A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazinecarboxamide series. The compounds were evaluated for their ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human TRPV1. The most potent of these TRPV1 antagonists were further characterized in pharmacokinetic, efficacy, and body temperature studies. On the basis of its pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was selected for further evaluation in human clinical trials.

Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain.

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.

Design and synthesis of novel small molecule N/OFQ receptor antagonists.

Small molecule N/OFQ receptor antagonists were designed and synthesized to further investigate the therapeutic potential of N/OFQ receptor modulators. The resulting octahydrobenzimidazol-2-ones 14 and 23 show excellent antagonistic activity towards both N/OFQ and mu receptors with high affinity to the human N/OFQ receptor.

Synthesis and evaluation of pyridazinylpiperazines as vanilloid receptor 1 antagonists.

A structurally biased chemical library of pyridazinylpiperazine analogs was prepared in an effort to improve the pharmaceutical and pharmacological profile of the lead compound N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC). The library was evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH5.5-induced (pH) FLIPR assays in a human VR1-expressing HEK293 cell line. The most potent VR1 antagonists were found to have IC(50) values in the range of 9-200nM with improved pharmaceutical and pharmacological profiles versus the lead BCTC. These compounds represent possible second-generation BCTC analogs.

Design and synthesis of 4-phenyl piperidine compounds targeting the mu receptor.

Small molecule mu agonists based on the 4-phenyl piperidine scaffold were designed and synthesized to further investigate the therapeutic potential of loperamide analogs. The resulting compounds show excellent agonistic activity towards the human mu receptor with interesting SAR trends within the series.

Human B1 and B2 bradykinin receptors and their agonists target caveolae-related lipid rafts to different degrees in HEK293 cells.

To address the targeting of G protein-coupled receptors to caveolae-related lipid rafts (CLR), we studied the human B2 (B2R) and B1 (B1R) bradykinin receptor subtypes in HEK293 cells. CLR were enriched on the basis of their unique buoyant density and composition of cholesterol, caveolin-1, and flotillin-1 but not clathrin. CLR contained B2R and B1R as determined by both receptor immunoblotting and the increase in specific activity of receptor agonist binding to cells at both 4 and 37 degrees C when binding was followed by CLR enrichment. B2R was highly enriched in this fraction, whereas B1R was not enriched. Furthermore, acid washing of cells prior to cell disruption minimally affected the CLR-associated B2R agonist binding, whereas it dissociated a major portion of the CLR-associated B1R agonist binding. In addition, when agonist binding at 4 degrees C was followed by an increase in the temperature to 37 degrees C, B2R agonist binding in CLR transiently increased, and this increase was dependent on the C-terminal domain. On the other hand, B1R agonist binding remained unchanged and was independent of the C-terminal domain. Our results show that B2R is constitutively targeted to CLR in HEK293 cells and appears to shuttle the agonist through these domains, whereas B1R may be there by default.