Neuronal N-type calcium channel blockers: a series of 4-piperidinylaniline analogs with analgesic activity.

Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.

Structure-activity relationship at the leucine side chain in a series of N,N-dialkyldipeptidyl-amines as N-type calcium channel blockers.

Exploration of the SAR around the leucine side chain in a series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC is presented. A novel analog is disclosed which possesses improved aqueous solubility, in vivo activity in an audiogenic seizure model, and reversible blockade in electrophysiological assays.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Synthesis and biological activity of substituted bis-(4-hydroxyphenyl)methanes as N-type calcium channel blockers.

Voltage activated calcium channel (VACC) blockers have been demonstrated to have utility in the treatment of stroke and pain. A series of aminomethyl substituted phenol derivatives has been identified with good functional activity and selectivity for N-type VACC's over sodium and potassium channels. The methods of synthesis and preliminary pharmacology are discussed herein.

Structure-activity relationship at the proximal phenyl group in a series of non-peptidyl N-type calcium channel antagonists.

Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.

Structure-activity relationship of N-methyl-N-aralkyl-peptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.

4-Hydroxy-1-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperidine: a novel, potent, and selective NR1/2B NMDA receptor antagonist.

A structure-based search and screen of our compound library identified N-(2-phenoxyethyl)-4-benzylpiperidine (8) as a novel N-methyl-D-aspartate (NMDA) receptor antagonist that has high selectivity for the NR1/2B subunit combination (IC(50) = 0.63 microM). We report on the optimization of this lead compound in terms of potency, side effect liability, and in vivo activity. Potency was assayed by electrical recordings in Xenopus oocytes expressing cloned rat NMDA receptors. Side effect liability was assessed by measuring affinity for alpha(1)-adrenergic receptors and inhibition of neuronal K(+) channels. Central bioavailability was gauged indirectly by determining anticonvulsant activity in a mouse maximal electroshock (MES) assay. Making progressive modifications to 8, a hydroxyl substituent on the phenyl ring para to the oxyethyl tether (10a) resulted in a approximately 25-fold increase in NR1A/2B potency (IC(50) = 0.025 microM). p-Methyl substitution on the benzyl ring (10b) produced a approximately 3-fold increase in MES activity (ED(50) = 0.7 mg/kg iv). Introduction of a second hydroxyl group into the C-4 position on the piperidine ring (10e) resulted in a substantial decrease in affinity for alpha(1) receptors and reduction in inhibition of K(+) channels with only a modest decrease in NR1A/2B and MES potencies. Among the compounds described, 10e (4-hydroxy-N-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperid ine, Co 101244/PD 174494) had the optimum pharmacological profile and was selected for further biological evaluation.

N,N-dialkyl-dipeptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.

Polyamine regulation of N-methyl-D-aspartate receptor channels.

Endogenous polyamines such as spermine and spermidine have multiple effects in the central nervous system and have been suggested to be neurotransmitters or neuromodulators. One effect of the polyamines is to regulate the activity of the N-methyl-D-aspartate receptor (NMDAR) channel subtype of glutamate receptor channels. The effects of polyamines on NMDAR currents are complex, suggesting the presence of one or more polyamine-binding sites on the receptor channel. Electrophysiological studies have shown that polyamines enhance NMDAR currents by increasing channel opening frequency and by increasing the affinity of the receptor for glycine. Polyamines have been shown to reduce NMDAR currents by producing voltage-dependent reduction of single-channel amplitudes and/or by producing an open channel block. Recent molecular biological studies have shown that the polyamine effects on NMDAR channels involve interactions with multiple NMDAR subunits and are characterizing the structural basis for the polyamine regulation of NMDAT receptor channels.

Gabapentin actions on ligand- and voltage-gated responses in cultured rodent neurons.

Gabapentin (GBP) is a cyclic gamma-aminobutyric acid (GABA) analog and investigational antiepileptic drug which is effective in the treatment of a variety of human and experimental seizures. GBP's antiepileptic mechanism of action is not known. The present studies tested for effects of GBP on inhibitory (GABA and glycine) and excitatory (N-methyl-D-aspartate (NMDA) and non-NMDA) amino acid neurotransmitter receptors, on repetitive firing of sodium (Na+) action potentials, and on voltage-dependent calcium (Ca2+) channel currents in cultured rodent neurons using intracellular, whole cell, or single channel recording techniques. GBP did not have a significant effect in any experiment when tested at or above concentrations that are therapeutic in humans except for a variable enhancement of NMDA-evoked depolarizations. These results suggest that the antiepileptic activity of GBP is not due to direct effects at receptors for inhibitory or excitatory amino acids or on voltage-dependent Na+ or Ca2+ channels.

The polyamine diaminodecane (DA-10) produces a voltage-dependent flickery block of single NMDA receptor channels.

Receptor binding assays have shown that diaminodecane (DA-10) reduced binding of open channel blockers to the N-methyl-D-aspartate (NMDA) subtype of postsynaptic glutamate receptor through an interaction with the polyamine regulatory site. Because the action of DA-10 was opposite to that of the polyamine agonist spermine and was reversed by polyamine antagonists, DA-10 has been classified as an inverse agonist at the polyamine site. Using whole-cell voltage-clamp and single-channel recordings from cultured rat cortical neurons, we show that at negative holding potentials DA-10 (1-300 microM) reduced NMDA receptor whole cell current (IC50 = 34 microM) and produced a flickery block of NMDA single-channel currents. The flickery block of NMDA single channels was voltage-dependent and not reversed by the polyamine antagonist diethylenetriamine (DET). Potential mechanisms for the flickery block of NMDA single channel currents are discussed.

Spermine and related polyamines produce a voltage-dependent reduction of N-methyl-D-aspartate receptor single-channel conductance.

Several polyamines have been shown to interact with a site on the N-methyl-D-aspartate (NMDA) receptor that regulates the binding of open channel blockers. Spermine (SP) and spermidine (SD), polyamine agonists, enhanced binding of open channel blockers, whereas arcaine (ARC), diethylenetriamine (DET), and putrescine (PUT), polyamine antagonists, reduced the polyamine enhancement of open channel blocker binding. We previously showed that SP had multiple actions on NMDA receptor single-channel currents that underlie its effect on whole-cell NMDA receptor current. At high concentrations, SP produced a voltage-dependent decrease in NMDA receptor single-channel conductance and average open time. In the present study, another polyamine agonist (SD) produced a similar reduction of NMDA receptor single-channel conductance at higher concentrations. The polyamine antagonists (ARC, DET, and PUT), however, produced a voltage-dependent reduction in NMDA receptor whole-cell currents and reductions in single-channel conductance and average open time, even in the absence of polyamine agonists. The rank order of potency for reduction of NMDA receptor single-channel conductance by polyamines was ARC greater than SP greater than SD greater than PUT = DET, a rank order similar to that for the inhibitory actions of polyamines in receptor binding assays and for the effects of the antagonists on NMDA receptor whole-cell currents. The polyamine antagonist DET did not block the reduction of single-channel conductance by the polyamine agonist SP. In fact, the effects of SP and DET on single-channel conductance were additive. DET also showed a variable enhancement of NMDA receptor whole-cell currents in some neurons, suggesting polyamine agonist-like properties. These results are not consistent with the standard pharmacological profile for agonists and antagonists acting at the same site. Potential mechanisms for the effects of the polyamines on single-channel conductance are discussed.

The polyamine spermine has multiple actions on N-methyl-D-aspartate receptor single-channel currents in cultured cortical neurons.

Spermine potentiates the action of N-methyl-D-aspartate (NMDA) at micromolar concentrations but is less effective at millimolar concentrations. In cultured cortical neurons we demonstrate that spermine enhances NMDA receptor currents in a unique manner. At low concentrations (1-10 microM) spermine enhances NMDA receptor current by increasing channel opening frequency, and at higher concentrations (greater than 10 microM) it produces, in addition, a voltage-dependent decrease in channel amplitude and average open time that limits its enhancing action. It is likely that these two actions of spermine, due to differences in concentration and voltage dependence, are mediated by independent sites on the NMDA receptor complex.

Ralitoline (CI-946) and CI-953 block sustained repetitive sodium action potentials in cultured mouse spinal cord neurons and displace batrachotoxinin A 20-alpha-benzoate binding in vitro.

Ralitoline and CI-953 are anticonvulsant compounds active in both maximal electroshock and kindling models of seizures with rodents. CI-953 (IC50 = 5 microM) and ralitoline (IC50 = 2 microM) both blocked sustained repetitive firing of sodium action potentials with effects on firing activity triggered by spontaneous excitatory postsynaptic potentials at higher concentrations. No effects on iontophoretic GABA and glutamate responses were noted. Both compounds inhibited the binding of tritiated batrachotoxinin A 20-alpha-benzoate ([3H]BTX-b) to rat brain synaptosomes with apparent Kd values of 29 microM (CI-953) and 25 microM (ralitoline). Our results suggest that effects on voltage-dependent sodium channels may underlie the anticonvulsant action of these compounds.

Comparison of phenytoin with noncompetitive N-methyl-D-aspartate antagonists in a model of focal brain ischemia in rat.

Recent in vitro and in vivo experiments have suggested that excitatory amino acid antagonists, particularly those active at the N-methyl-D-aspartate receptor subtype, are effective in ameliorating ischemic injury due to their antiexcitotoxic activity. However, these drugs are also potent and effective in vivo anticonvulsants. The present experiments compared the noncompetitive N-methyl-D-aspartate antagonists phencyclidine and MK-801 with the anticonvulsant phenytoin in a model of focal brain ischemia. Fisher F-344 rats were subjected to tandem occlusion of the middle cerebral and ipsilateral common carotid arteries under halothane anesthesia. Compounds were administered intravenously 30 minutes and 24 hours after arterial occlusion; infarct size was assessed at 48 hours after occlusion. Phencyclidine had no effect on infarct volume at 1 mg/kg, significantly reduced (by 36%) infarct volume at 3 mg/kg, and produced a nonsignificant 26% decrease at 10 mg/kg. The more potent and selective noncompetitive antagonist MK-801 reduced (by 32%) infarct volume significantly at 0.1 mg/kg, produced a nonsignificant 23% decrease at 0.3 mg/kg, and had no effect at 0.5 mg/kg. Phenytoin, which is not a glutamate antagonist, reduced the infarct volume by 45% at 28 mg/kg. A single dose of phenytoin (28 mg/kg) administered 30 minutes after occlusion was neuroprotective, but delaying drug administration for more than 2 hours was ineffective. These data suggest that blockade of the N-methyl-D-aspartate receptor is effective in reducing the infarct size after focal cerebral ischemia. The neuroprotective activity of phenytoin suggests that this may be related to the common anticonvulsant action.

Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD 810, CI 912), a novel anticonvulsant.

Zonisamide is a novel anticonvulsant that prevents seizures in laboratory animals and in man. Zonisamide (3 micrograms/ml and above) blocked the sustained firing of action potentials induced by depolarizing steps of current injected across the membrane of intracellularly recorded spinal cord neurons. Responses to GABA and glutamate were not altered by zonisamide, and spontaneous synaptically evoked activity was not reduced until higher concentrations of zonisamide (10 micrograms/ml) were applied.

Effects of diazepam, pentobarbital, phenytoin and pentylenetetrazol on hippocampal paired-pulse inhibition in vivo.

The effects of diazepam, pentobarbital, phenytoin and pentylenetetrazol on paired-pulse inhibition were assessed in the intact rat hippocampus. Diazepam (4 mg/kg i.p.) and pentobarbital (15 mg/kg i.p.) increased inhibition over control levels, while phenytoin (15 mg/kg i.p.) and pentylenetetrazol (20 mg/kg i.p.) decreased inhibition from control levels. The present results with diazepam, pentobarbital and pentylenetetrazol confirm previous reports. However, the present results indicate that augmentation of GABA-ergic inhibition is not involved in the anticonvulsant action of phenytoin.