Asymmetric synthesis and evaluation of a hydroxyphenylamide voltage-gated sodium channel blocker in human prostate cancer xenografts.

Voltage-gated sodium channels are known to be expressed in neurons and other excitable cells. Recently, voltage-gated sodium channels have been found to be expressed in human prostate cancer cells. α-Hydroxy-α-phenylamides are a new class of small molecules that have demonstrated potent inhibition of voltage-gated sodium channels. The hydroxyamide motif, an isostere of a hydantoin ring, provides an active scaffold from which several potent racemic sodium channel blockers have been derived. With little known about chiral preferences, the development of chiral syntheses to obtain each pure enantiomer for evaluation as sodium channel blockers is important. Using Seebach and Frater's chiral template, cyclocondensation of (R)-3-chloromandelic acid with pivaldehyde furnished both the cis- and trans-2,5-disubsituted dioxolanones. Using this chiral template, we synthesized both enantiomers of 2-(3-chlorophenyl)-2-hydroxynonanamide, and evaluated their ability to functionally inhibit hNa(v) isoforms, human prostate cancer cells and xenograft. Enantiomers of lead demonstrated significant ability to reduce prostate cancer in vivo.

A potent and selective indole N-type calcium channel (Ca(v)2.2) blocker for the treatment of pain.

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Extracellular loop C of NPC1L1 is important for binding to ezetimibe.

Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.

Analgesic effects of a substituted N-triazole oxindole (TROX-1), a state-dependent, voltage-gated calcium channel 2 blocker.

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Substituted biaryl pyrazoles as sodium channel blockers.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. A series of low molecular weight biaryl substituted pyrazole carboxamides were identified with good in-vitro potency and in-vivo efficacy. Compound 26, a Nav1.7 blocker has excellent efficacy in the Chung model of neuropathic pain.

Substituted biaryl oxazoles, imidazoles, and thiazoles as sodium channel blockers.

Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. With a goal to develop potent peripherally active sodium channel blockers, a series of low molecular weight biaryl substituted imidazoles, oxazoles, and thiazole carboxamides were identified with good in vitro and in vivo potency.

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain.

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.

Discovery of isoxazole voltage gated sodium channel blockers for treatment of chronic pain.

A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na(v)1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na(v)1.7 inhibitory activity.

Discovery of a novel class of isoxazoline voltage gated sodium channel blockers.

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.

Madin-Darby canine kidney II cells: a pharmacologically validated system for NPC1L1-mediated cholesterol uptake.

Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.

A high-capacity membrane potential FRET-based assay for the sodium-coupled glucose co-transporter SGLT1.

Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.

3-Amino-1,5-benzodiazepinones: potent, state-dependent sodium channel blockers with anti-epileptic activity.

A series of 3-amino-1,5-benzodiazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of this series displayed subnanomolar, state-dependent sodium channel block, and was orally efficacious in a mouse model of epilepsy.

Imidazopyridines: a novel class of hNav1.7 channel blockers.

A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes.

The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.

Characterization of a new class of potent inhibitors of the voltage-gated sodium channel Nav1.7.

Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. [3H]BNZA binds with high affinity to rat brain synaptosomal membranes (Kd = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (Kd = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (Kd = 1.6 nM) was achieved with [3H]BNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.

Discovery of a novel class of benzazepinone Na(v)1.7 blockers: potential treatments for neuropathic pain.

A series of benzodiazepines and benzazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of the benzazepinone series, compound 47, displayed potent, state-dependent block of hNa(v)1.7, and was orally efficacious in a rat model of neuropathic pain.

Benzazepinone Nav1.7 blockers: potential treatments for neuropathic pain.

A series of benzazepinones were synthesized and evaluated as hNa(v)1.7 sodium channel blockers. Several compounds from this series displayed good oral bioavailability and exposure and were efficacious in a rat model of neuropathic pain.

Synthesis and SAR of 1,2-trans-(1-hydroxy-3-phenylprop-1-yl)cyclopentane carboxamide derivatives, a new class of sodium channel blockers.

Novel cyclopentane-based 3-phenyl-1-hydroxypropyl compounds were evaluated for inhibitory activity against the peripheral nerve sodium channel Na(V)1.7 and off-target activity against the cardiac potassium channel hERG. The stereochemistry of the hydroxyl group and substitution on the phenyl rings with either fluorinated O-alkyl or alkyl groups were found to be critical for conferring potency against Na(V)1.7. A benchmark compound from this series displayed efficacy in rat models of inflammatory and neuropathic pain.

Discovery of potent and use-dependent sodium channel blockers for treatment of chronic pain.

A new series of voltage-gated sodium channel blockers with potential for treatment of chronic pain is reported. Systematic structure-activity relationship studies, starting with compound 1, led to identification of potent analogs that displayed use-dependent block of sodium channels, were efficacious in pain models in vivo, and most importantly, were devoid of activity against the cardiac potassium channel hERG.

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits.

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.