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.

The discovery of [1-(4-dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethylbutyl)-phen yl]-(3-methyl-but-2-enyl)-amine, an N-type Ca+2 channel blocker with oral activity for analgesia.

Our drug discovery efforts for N-type calcium channel blockers in the 4-piperidinylaniline series led to the discovery of an orally active analgesic agent 26.1-[4-Dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethyl-but yl)-phenyl]-(3-methyl-but-2-enyl)amine (26) showed high affinity to functionally block N-type calcium channels (IC50=0.7 microM in the IMR32 assay) and exhibited high efficacy in the anti-writhing analgesia test with mice (ED50=12 mg/kg by po and 4 mg/kg by iv). In this report, the rationale for the design, synthesis, biological evaluation, and pharmacokinetics of this series of blockers is described.

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.

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.

Multiple parallel synthesis of N,N-dialkyldipeptidylamines as N-type calcium channel blockers.

Selective N-type Voltage Sensitive Calcium Channel (VSCC) blockers have shown utility in several models of stroke and pain. A series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC's has been identified. Multiple parallel synthesis of a focused array of thirty compounds using polymer-supported quenching reagents and preliminary pharmacology are presented. Eighteen compounds were identified with an IC50 below 1 microM in an in vitro functional assay.

Design and synthesis of novel quinoxaline-2,3-dione AMPA/GlyN receptor antagonists: amino acid derivatives.

PNQX (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3, 4-f]quinoxaline-2,3-dione) is a potent AMPA (IC50 = 0.063 microM) and GlyN (IC50 = 0.37 microM) receptor antagonist that was developed in our laboratories. While possessing a desirable in vitro and in vivo activity profile, this compound suffers from low aqueous solubility. In an effort to improve its potency and physical properties, we have designed and synthesized novel ring-opened analogues 4, 6, 9, and 11. Modeling analyses demonstrated that, while the 5-substituent in these analogues was forced to adopt an out-of-plane conformation due to steric contacts with neighboring substituents, the overall structure retained a good fit to a previously described AMPA pharmacophore model. This nonplanar orientation may lessen efficient packing in the solid state, compared to PNQX, leading to increased water solubility. Indeed, several nonplanar analogues containing appropriate functionalities, for example, the sarcosine analogue 9, were found to retain AMPA (IC50 = 0.14 microM) and GlyN (IC50 = 0.47 microM) receptor affinity and possess improved aqueous solubility compared to PNQX. The synthesis and the SAR of these compounds are discussed.

Synthesis and biological evaluation of substituted 4-(OBz)phenylalanine derivatives as novel N-type calcium channel blockers.

Selective N-type Voltage Activated Calcium Channel (VACC) blockers have shown utility in several models of stroke and pain. In the process of searching for small molecules as N-type calcium channel blockers, we have identified a series of N,N-dialkylpeptidylamines (e.g., PD 175069) with potent functional activity at N-type VACC. Further modification of the leucine moiety of PD 175069 with a cyclized ring structure provides a series of novel molecules. Syntheses and pharmacological evaluation of the series are presented.

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.

Acute but not chronic activation of the NMDA-coupled glycine receptor with D-cycloserine facilitates learning and retention.

The memory-enhancing potential of D-cycloserine (cycloserine) a partial agonist at the glycine recognition site on the NMDA receptor, was evaluated in mice using a thirst-motivated linear maze learning task. Immediate acute post-training injections (10, 20 and 80 mg/kg) significantly improved retention relative to vehicle-injected controls. Retention was also facilitated if cycloserine (3 and 10 mg/kg but not 20 or 40 mg/kg) was administered 20 min before the retention test. Acquisition of the habit was accelerated if cycloserine (3 mg/kg) was injected 20 min before the training session. Acute post-training injections failed to facilitate retention if mice were pretreated with cycloserine (3 mg/kg) b.i.d. for 15 days before training on the maze. These results indicate that acute cycloserine administration can enhance consolidation and retrieval of memory but that desensitization may occur with chronic exposure to the drug.

Antinociceptive effect of spinally delivered prostaglandin E receptor antagonists in the formalin test on the rat.

The antinociceptive effect of spinally administered prostaglandin E2 receptor antagonists, SC-51089 and SC-51234A, which are selective for EP1 receptors, was examined in rats using the formalin test. Intrathecal injection of SC-51089 (30-300 micrograms) or SC-51234A (30-300 micrograms) resulted in a significant, dose-dependent, suppression of the second phase (10-60 min), but not the first phase (0-9 min), flinching behavior evoked by formalin injection into the paw. ED25 values and 95% confidence intervals for the second phase were 120 (70-200) micrograms for SC-51089 and 80 (50-140) micrograms for SC-51234A. These data demonstrate that specific nociceptive behaviors are attenuated by spinal prostaglandin E2 receptor antagonists and suggest that prostaglandin E2 is involved in facilitated processing at the spinal level.

Evidence for chemical differentiation of delta opioid receptor subtypes by the sulfhydryl reagent N-ethylmaleimide.

In this study, the delta receptor-selective nonequilibrium affinity ligands, 5'-NTII and DALCE, and the nonspecific sulfhydryl reagent NEM were evaluated over a range of concentrations and treatment conditions for their ability to selectively alter the binding properties of delta 1- or delta 2-preferring opioid radioligands in brain homogenate. Treatment of tissue preparations with DALCE (0-10,000 nM) or NTII (0-10,000 nM) resulted in an equivalent concentration-dependent loss of binding capacity for the delta 1 agonist 3H-DPDPE and the mu/delta 2 agonist 3H-DSLET. In contrast, treatment of tissue with NEM (0-8000 microM) resulted in greater loss of 3H-DPDPE binding. Scatchard analysis of the binding of 3H-DPDPE, 3H-DSLET, and 3H-NTI in 3 mM NEM-treated rat brain P2 preparation revealed an equivalent decrease in affinity for the agonist ligands, but a significantly greater decrease in Bmax for 3H-DPDPE compared with control tissue values. Comparison of the K(i) values for a series of delta-selective compounds against 3H-DSLET binding in control vs 3 mM NEM treated P2 fraction showed differential effects of NEM on affinity within the series that were consistent with a selective depletion of delta 1 sites. Overall, these results indicate that NEM treatment selectively reduced delta 1 receptor binding, resulting in a preparation that is enriched in delta 2 sites.

N-substituted dibenzoxazepines as analgesic PGE2 antagonists.

8-Chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide (1, SC-19220) has been previously reported by us and others to be a PGE2 antagonist selective for the EP1 receptor subtype with antinociceptive activities. Analogs of SC-19220, in which the acetyl moiety has been replaced with pyridylpropionyl groups and their homologs, have been synthesized as illustrated by compounds 13 and 29. These and other members of this series have been shown to be efficacious analgesics and PGE2 antagonists of the EP1 subtype. This report discusses the structure activity relationships within this series.

[3H]naltrindole: a potent and selective ligand for labeling delta-opioid receptors.

Naltrindole (NTI) is a selective and potent delta-opioid antagonist which preferentially antagonizes a subset of selective delta-opioid agonists. The purpose of this study was to evaluate whether [3H]NTI, the first radiolabeled delta-opioid antagonist, could selectively label delta-opioid receptors in a synaptosomal preparation. Increasing temperature and protein concentration (0.1-1.6 mg protein) increased the specific binding of [3H]NTI. Monovalent and divalent cations (0.01-100 mM) had minimal effects on the binding properties of [3H]NTI, in contrast to their effects on binding of the delta agonists [3H]DPDPE and [3H]DSLET. Subfractionation of rat brain homogenates revealed that [3H]NTI and [3H]DSLET primarily labeled binding sites in synaptosomal and microsomal fractions, whereas [3H]DPDPE labelled half as many sites in synaptosomal fraction. The Bmax determined for [3H]NTI in crude synaptosomal fraction was 95 +/- 12 fmol/mg. The dissociation constant (Kd) was determined from three different methods to be 0.08 +/- 0.02 nM (Scatchard analysis), 0.07 +/- 0.02 nM (competition study) and 0.03 +/- 0.005 nM (kinetic analysis). [3H]NTI binding was not significantly inhibited by mu- or kappa-opioid ligands or by nonopioid compounds. These results demonstrate that [3H]NTI is a potent and selective radioligand for delta-opioid receptors in rat brain preparations.

Quantitative light microscopic localization of [3H]naltrindole binding sites in the rat brain.

The binding of radiolabeled naltrindole ([3H]NTI), a selective delta-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent Kd of 0.25 +/- 0.08 nM (Bmax of 597.5 fmol/mg protein). Autoradiographic localization of [3H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1-3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.

Antinociception after intracerebroventricular administration of naltrindole in the mouse.

Intracerebroventricular (i.c.v.) injection of the delta-opioid receptor antagonist naltrindole hydrochloride (2.2-22.2 nmol) in mice produced a dose-dependent increase in tail flick and hot plate latencies with respective ED50 and 95% confidence limits of 10.6 (8.3-13.9) and 16.4 (9.2-62.3) nmol. This increase in response latencies was antagonized by 1 mg/kg s.c. naloxone or by i.c.v. coadministration of 1.4 nmol ICI-174,864, a selective peptidergic delta-receptor antagonist. Pretreatment 24 h earlier with the irreversible mu-receptor antagonist beta-funaltrexamine (6 nmol i.c.v.) or 1 h earlier with the selective kappa-receptor antagonist nor-binaltorphimine (100 nmol i.c.v.) did not attenuate the antinociceptive effects of naltrindole. These data indicate that high doses of naltrindole may have agonist activity at supraspinal delta-opioid receptors in the mouse.

Selective antagonism by naltrindole of the antinociceptive effects of the delta opioid agonist cyclic[D-penicillamine2-D-penicillamine5]enkephalin in the rat.

Spinal delta opioid receptors have been proposed to mediate antinociception in the rat on the basis of 1) the efficacy of a small number of agonists; 2) the lack of effect of mu-selective antagonists; and 3) the lack of cross-tolerance with mu-selective agonists. However, direct evidence to support or refute this postulate has not been obtained in the rat due to a lack of suitable delta-selective antagonists. The present study characterized the ability of Naltrindole (NTI, 17-cyclopropylmethyl-6,7-dehydro-4,5 alpha-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan), a recently discovered delta-selective antagonist, to antagonize the antinocieption produced by intrathecal (i.t.) administration of the prototypic delta-selective agonist cyclic[D-penicillamine2-D-penicillamine5]enkephalin (DPDPE) or the mu-selective agonists morphine and [D-Ala2,MePhe4,Gly-ol5] enkephalin (DAMGO) in the rat. Intrathecal coadministration of NTI with DPDPE significantly antagonized the increase in tail-flick latency (TFL) and hot-plate latency (HPL) produced by DPDPE. In the absence of NTI, the ED50 values and 95% CL of DPDPE in the tail-flick and hot-plate tests were 2.8 (1.1-4.7) and 19.5 (13.3-33.7) micrograms, respectively. In the presence of 10 micrograms of NTI, the ED50 value of DPDPE in the tail-flick test was unchanged and was increased by 2-fold in the hot-plate test to 35.9 (26.2-60.1) micrograms. In the presence of 30 micrograms of NTI, the ED50 value of DPDPE in the tail-flick test was increased by 5-fold to 14.5 (8.5-24.9) micrograms and its antinociceptive effect in the hot-plate test was antagonized completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis, configuration, and evaluation of two conformationally restrained analogues of phencyclidine.

Brown oxidation of cis-bicyclo[3.1.0]hexan-3-ol afforded bicyclo[3.1.0]hexan-3-one in 98% yield. Treatment of this ketone with either phenyllithium or phenylamagnesium bromide in ether at room temperature followed by solvolysis of the resulting alcohol in a mixture of trifluoroacetic acid, sodium azide, and chloroform gave a mixture of cis- and trans-3-azido-3-phenylbicyclo[3.1.0]hexanes. LAH reduction of this crude mixture of azides afforded a 1:3.5 mixture of cis- and trans-3-phenyl-3-bicyclo[3.1.0]hexylamine, respectively, in 51% overall yield from the alcohol. Separation of the mixture of amines by column chromatography followed by cyclization of each by heating at 60 degrees C in DMF solution with 1 equiv of 1,5-dibromopentane furnished the two conformationally restrained analogues of phencyclidine (PCP), cis- and trans-3-phenyl-3-piperidinylbicyclo[3.1.0]hexane (1 and 2, respectively), in high yield. Configurations were assigned on the basis of an X-ray crystallographic analysis of the cis isomer (1). Bond lengths and angles are similar to those found in PCP and its derivatives. Binding to PCP receptors and sigma sites as well as behavioral effects of 1 and 2 in rats was determined relative to PCP. In displacement of specifically bound [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine) from PCP receptors, 1 and 2 were nearly equipotent and about one-seventh as potent as PCP. These compounds were about one-fifth as potent as PCP in displacing [3H]-(+)-SKF 10,047 from its binding site. Calculation of the ED50 values of 1 and 2 for stereotyped behavior and ataxia indicated that they were about equipotent, and 2-3-fold less active than PCP.