A Functional NaV1.7-NaVAb Chimera with a Reconstituted High-Affinity ProTx-II Binding Site.

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

Development of spiroguanidine-derived α7 neuronal nicotinic receptor partial agonists.

We describe the synthesis of quinuclidine-containing spiroguanidines and their utility as α7 neuronal nicotinic acetylcholine receptor (nAChR) partial agonists. The convergent synthetic route developed for this study allowed for rapid SAR investigation and provided access to a structurally diverse set of analogs. A potent and selective α7 nAChR partial agonist, N-(6-methyl-1,3-benzoxazol-2-yl)-3',5'-dihydro-4-azaspiro[bicyclo[2.2.2]octane-2,4'-imidazole]-2'-amine (BMS-910731, 16), was identified. This compound induced immediate early genes c-fos and Arc in a preclinical rodent model of α7 nAChR-derived cellular activation and plasticity. Importantly, the ability to incorporate selectivity for the α7 nACh receptor over the 5-HT3A receptor in this series suggested a significant difference in steric requirements between the two receptors.

Discovery of a novel series of quinolone α7 nicotinic acetylcholine receptor agonists.

High throughput screening led to the identification of a novel series of quinolone α7 nicotinic acetylcholine receptor (nAChR) agonists. Optimization of an HTS hit (1) led to 4-phenyl-1-(quinuclidin-3-ylmethyl)quinolin-2(1H)-one, which was found to be potent and selective. Poor brain penetrance in this series was attributed to transporter-mediated efflux, which was in turn due to high pKa. A novel 4-fluoroquinuclidine significantly lowered the pKa of the quinuclidine moiety, reducing efflux as measured by a Caco-2 assay.

Discovery of Indole- and Indazole-acylsulfonamides as Potent and Selective NaV1.7 Inhibitors for the Treatment of Pain.

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Nav1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund's adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia and models of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The observed efficacy could be correlated with the mouse dorsal root ganglion exposure and NaV1.7 potency associated with 29.

B-973, a novel piperazine positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

The alpha7 (α7) nicotinic acetylcholine receptor is a therapeutic target for cognitive disorders. Here we describe 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide (B-973), a novel piperazine-containing molecule that acts as a positive allosteric modulator of the α7 receptor. We characterize the action of B-973 on the α7 receptor using electrophysiology and radioligand binding. At 0.1mM acetylcholine, 1µM B-973 potentiated peak acetylcholine-induced currents 6-fold relative to maximal acetylcholine (3mM) and slowed channel desensitization, resulting in a 6900-fold increase in charge transfer. The EC50 of B-973 was approximately 0.3µM at acetylcholine concentrations ranging from 0.03 to 3mM. At a concentration of 1µM, B-973 shifted the acetylcholine EC50 of peak currents from 0.30mM in control to 0.007mM. B-973 slowed channel deactivation upon acetylcholine removal (τ=50s) and increased the affinity of the α7 agonist [3H]A-585539. In the absence of exogenously added acetylcholine, application of B-973 at concentrations >1µM induced large methyllycaconitine-sensitive currents, suggesting B-973 can function as an Ago-PAM at high concentrations. B-973 will be a useful probe for investigating the biological consequences of increasing α7 receptor activity through allosteric modulation.

Effects of BMS-902483, an α7 nicotinic acetylcholine receptor partial agonist, on cognition and sensory gating in relation to receptor occupancy in rodents.

The α7 nicotinic acetylcholine receptor is thought to play an important role in human cognition. Here we describe the in vivo effects of BMS-902483, a selective potent α7 nicotinic acetylcholine receptor partial agonist, in relationship to α7 nicotinic acetylcholine receptor occupancy. BMS-902483 has low nanomolar affinity for rat and human α7 nicotinic acetylcholine receptors and elicits currents in cells expressing human or rat α7 nicotinic acetylcholine receptors that are about 60% of the maximal acetylcholine response. BMS-902483 improved 24h novel object recognition memory in mice with a minimal effective dose (MED) of 0.1mg/kg and reversed MK-801-induced deficits in a rat attentional set-shifting model of executive function with an MED of 3mg/kg. Enhancement of novel object recognition was blocked by the silent α7 nicotinic acetylcholine receptor agonist, NS6740, demonstrating that activity of BMS-902483 was mediated by α7 nicotinic acetylcholine receptors. BMS-902483 also reversed ketamine-induced deficits in auditory gating in rats, and enhanced ex vivo hippocampal long-term potentiation examined 24h after dosing in mice. Results from an ex vivo brain homogenate binding assay showed that α7 receptor occupancy ranged from 64% (novel object recognition) to ~90% (set shift and gating) at the MED for behavioral and sensory processing effects of BMS-902483.

BMS-933043, a Selective α7 nAChR Partial Agonist for the Treatment of Cognitive Deficits Associated with Schizophrenia.

The therapeutic treatment of negative symptoms and cognitive dysfunction associated with schizophrenia is a significant unmet medical need. Preclinical literature indicates that α7 neuronal nicotinic acetylcholine (nACh) receptor agonists may provide an effective approach to treating cognitive dysfunction in schizophrenia. We report herein the discovery and evaluation of 1c (BMS-933043), a novel and potent α7 nACh receptor partial agonist with high selectivity against other nicotinic acetylcholine receptor subtypes (>100-fold) and the 5-HT3A receptor (>300-fold). In vivo activity was demonstrated in a preclinical model of cognitive impairment, mouse novel object recognition. BMS-933043 has completed Phase I clinical trials.

Development of 4-Heteroarylamino-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octanes] as α7 Nicotinic Receptor Agonists.

We describe the synthesis of quinuclidine-containing spiroimidates and their utility as α7 nicotinic acetylcholine receptor (nAChR) partial agonists. A convergent synthetic route allowed for rapid SAR investigation and provided a diverse set of fused 6,5-heteroaryl analogs. Two potent and selective α7 nAChR partial agonists, (1'S,3'R,4'S)-N-(7-bromopyrrolo[2,1-f][1,2,4]triazin-4-yl)-4H-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octan]-2-amine (20) and (1'S,3'R,4'S)-N-(7-chloropyrrolo[2,1-f][1,2,4]triazin-4-yl)-4H-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octan]-2-amine (21), were identified. Both agonists improved cognition in a preclinical rodent model of learning and memory. Additionally, 5-HT3A receptor SAR suggested the presence of a steric site that when engaged led to significant loss of affinity at that receptor.

Development of New Benzenesulfonamides As Potent and Selective Nav1.7 Inhibitors for the Treatment of Pain.

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans.

Design and Synthesis of a New Series of 4-Heteroarylamino-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octanes as α7 Nicotinic Receptor Agonists. 1. Development of Pharmacophore and Early Structure-Activity Relationship.

The design and synthesis of a series of quinuclidine-containing spirooxazolidines ("spiroimidates") and their utility as α7 nicotinic acetylcholine receptor partial agonists are described. Selected members of the series demonstrated excellent selectivity for α7 over the highly homologous 5-HT3A receptor. Modification of the N-spiroimidate heterocycle substituent led to (1S,2R,4S)-N-isoquinolin-3-yl)-4'H-4-azaspiro[bicyclo[2.2.2]octane-2,5'oxazol]-2'-amine (BMS-902483), a potent α7 partial agonist, which improved cognition in preclinical rodent models.

Synthesis and structure-activity relationships of 3-aryloxindoles: a new class of calcium-dependent, large conductance potassium (maxi-K) channel openers with neuroprotective properties.

A series of 3-aryloxindole derivatives were synthesized and evaluated as activators of the cloned maxi-K channel mSlo expressed in Xenopus laevis oocytes using electrophysiological methods. The most promising maxi-K openers to emerge from this study were (+/-)-3-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-3-hydroxy-6-(trifluoromethyl)-2H-indol-2-one ((+/-)-8c) and its 3-des-hydroxy analogue (+/-)-11b. The individual enantiomers of (+/-)-8c were synthesized, and the maxi-K channel-opening properties were shown to depend on the absolute configuration of the single stereogenic center with the efficacy of (-)-8c superior to that of both (+)-8c and the racemic mixture when evaluated at a concentration of 20 microM. Racemic 11b exhibited greater efficacy than either the racemic 8c or the more active enantiomer in the electrophysiological evaluation. In vitro metabolic stability studies conducted with (+/-)-8c and (+/-)-11b in rat liver S9 microsomal fractions revealed significant oxidative degradation with two hydroxylated metabolites observed by liquid chromatography/mass spectrometry for each compound in addition to the production of 8c from 11b. The pharmacokinetic properties of (+/-)-8c and (+/-)-11b were determined in rats as a prelude to evaluation in a rat model of stroke that involved permanent occlusion of the middle cerebral artery (MCAO model). In the MCAO model, conducted in the spontaneously hypertensive rat, the more polar 3-hydroxy derivative (+/-)-8c did not demonstrate a significant reduction in cortical infarct volume when administered intravenously at doses ranging from 0.1 to 10 mg/kg as a single bolus 2 h after middle cerebral artery occlusion when compared to vehicle-treated controls. In contrast, intravenous administration of (+/-)-11b at a dose of 0.03 mg/kg was found to reduce the measured cortical infarct volume by approximately 18% when compared to vehicle-treated control animals. Intraperitoneal administration of (+/-)-11b at a dose of 10 mg/kg 2 h following artery occlusion was shown to reduce infarct volume by 26% when compared to vehicle-treated controls. To further probe the effects of compounds (+/-)-8c and (+/-)-11b on neurotransmitter release in vitro, both compounds were examined for their ability to reduce electrically stimulated [3H]-glutamate release from rat hippocampal slices that had been preloaded with [3H]-glutamate. Only (+/-)-11b was able to demonstrate a significant inhibition [3H]-glutamate release in this assay at a concentration of 20 microM, providing concordance with the profile of these compounds in the MCAO model. Although (+/-)-11b showed some promise as a potential developmental candidate for the treatment of the sequelae of stroke based on its efficacy in the rat MCAO model, the pharmacokinetic profile of this compound was considered to be less than optimal and was not pursued in favor of derivatives with enhanced metabolic stability.

Pharmacological and behavioral characterization of the novel CRF1 antagonist BMS-763534.

BMS-763534 is a potent (CRF(1) IC(50) = 0.4 nM) and selective (>1000-fold selectivity vs. all other sites tested) CRF(1) receptor antagonist (pA2 = 9.47 vs. CRF(1)-mediated cAMP production in Y79 cells). BMS-763534 accelerated the dissociation of (125)I-o-CRF from rat frontal cortex membrane CRF(1) receptors consistent with a negative allosteric modulation of CRF binding. BMS-763534 produced dose-dependent increases in CRF(1) receptor occupancy and anxiolytic efficacy; lowest effective anxiolytic dose = 0.56 mg/kg, PO, which was associated with 71 ± 5% CRF(1) receptor occupancy of frontoparietal CRF(1) receptors. Sedative/ataxic effects of BMS-763534 were only observed at high dose multiples (54-179×) relative to the lowest dose required for anxiolytic efficacy. At doses of 5- to 18-fold higher than the lowest efficacious dose in the anxiety assay, BMS-763534 shared subjective effects with the benzodiazepine chlordiazepoxide. Interestingly BMS-790318, the O-demethylated metabolite of BMS-763534, showed weak affinity for the TBOB site of the GABA(A) receptor (67% inhibition at 10 µM) and augmented GABA evoked currents (EC(50) = 1.6 µM). Thus, the unanticipated signal in the drug discrimination assay may have resulted from an interaction of the metabolite BMS-790318 with the TBOB site on the GABA(A) channel where it appears to behave as an allosteric potentiator of GABA evoked currents.

The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channels.

3-Aryl-3-fluorooxindoles can be efficiently synthesized in two steps by the addition of an aryl Grignard to an isatin, followed by treatment with DAST. Oxindole 1 (BMS-204352; MaxiPost) can be isolated using chiral HPLC or prepared by employing chiral resolution. Cloned maxi-K channels are opened by 1, which demonstrates a brain/plasma ratio >9 in rats.