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.     

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Ca_v2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.     

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.     

Discovery and SAR of novel tetrahydropyrrolo[3,4-c]pyrazoles as inhibitors of the N-type calcium channel

A novel series of substituted tetrahydropyrrolo[3,4-c]pyrazoles were investigated as blockers of the N-type calcium channel (Ca_v2.2 channels), a chronic pain target.     

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety,and oral analgesic efficacy in rat pain models

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.     

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.     

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.     

High-throughput screening for N-type calcium channel blockers using a scintillation proximity assay

N-type calcium channels located on presynaptic nerve terminals regulate neurotransmitter release, including that from the spinal terminations of primary afferent nociceptors. Accordingly, N-type calcium channel blockers may have clinical utility as analgesic drugs. A selective N-type calcium channel inhibitor, ziconotide (Prialt), is a neuroactive peptide recently marketed as a novel nonopioid treatment for severe chronic pain. To develop a small-molecule N-type calcium channel blocker, the authors developed a 96-well plate high-throughput screening scintillation proximity assay (SPA) for N-type calcium channel blockers using [125I]-labeled omega-conotoxin GVIA as a channel-specific ligand. Assay reagents were handled using Caliper's Allegro automation system, and bound ligands were detected using a PerkinElmer TopCount. Using this assay, more than 150,000 compounds were screened at 10 microM and approximately 340 compounds were identified as hits, exhibiting at least 40% inhibition of [125I]GVIA binding. This is the 1st demonstration of the use of [125I]-labeled peptides with SPA beads to provide a binding assay for the evaluation of ligand binding to calcium channels. This assay could be a useful tool for drug discovery.     

Structure-activity study and analgesic efficacy of amino acid derivatives as N-type calcium channel blockers

The synthesis and structure-activity relationship (SAR) study of a novel series of N-type calcium channel blockers are described. L-Cysteine derivative 2a was found to be a potent and selective N-type calcium channel blocker with IC(50) 0.63 microM on IMR-32 assay. Compound 2a showed analgesic efficacy in the rat formalin-induced pain model by intrathecal and oral administration.     

Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels

N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain.     

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.     

Synthesis and SAR of 4-aminocyclopentapyrrolidines as N-type Ca²⁺ channel blockers with analgesic activity

A novel 4-aminocyclopentapyrrolidine series of N-type Ca(2+) channel blockers have been discovered. Enantioselective synthesis of the 4-aminocyclopentapyrrolidines was enabled using N-tert-butyl sulfinamide chemistry. SAR studies demonstrate selectivity over L-type Ca(2+) channels. N-type Ca(2+) channel blockade was confirmed using electrophysiological recording techniques. Compound 25 is an N-type Ca(2+) channel blocker that produces antinociception in inflammatory and nociceptive pain models without exhibiting cardiovascular or motor liabilities.     

Scaffold-based design and synthesis of potent N-type calcium channel blockers

The therapeutic agents flunarizine and lomerizine exhibit inhibitory activities against a variety of ion channels and neurotransmitter receptors. We have optimized their scaffolds to obtain more selective N-type calcium channel blockers. During this optimization, we discovered NP118809 and NP078585, two potent N-type calcium channel blockers which have good selectivity over L-type calcium channels. Upon intraperitoneal administration both compounds exhibit analgesic activity in a rodent model of inflammatory pain. NP118809 further exhibits a number of favorable preclinical characteristics as they relate to overall pharmacokinetics and minimal off-target activity including the hERG potassium channel.     

Design and biological evaluation of non-peptide analogues of omega-conotoxin MVIIA

Omega-conotoxin MVIIA, a highly potent antagonist of the N-type voltage sensitive calcium channel, has shown utility in several models of pain and ischemia. We report a series of three alkylphenyl ether based analogues which mimic three key amino acids of the toxin. Two of the compounds have been found to exhibit IC50 values of 2.7 and 3.3 microM at the human N-type voltage sensitive calcium channel.     

Synthesis and SAR of novel 2-arylthiazolidinones as selective analgesic N-type calcium channel blockers

A series of new N-type (Ca(v)2.2) calcium channel blockers derived from the 'hit' structures 2-(3-bromo-4-fluorophenyl)-3-(2-pyridin-2-ylethyl)thiazolidin-4-one 9 and its 2-[4-(4-bromophenyl)pyridin-3-yl]-3-isobutyl analogue 10 is described. Extensive SAR studies using a range of synthetic approaches resulted in novel, patented compounds with IC50 values of up to 0.2 microM in an in vitro IMR32 assay, and selectivities for N/L of up to 30-fold. The new compounds described have potential in treatment of neuropathic pain.     

N型钙通道与疼痛

N型电压依赖性钙通道(VDCCs)在疼痛的传递与调控中具有重要作用。它们密集分布于脊髓背角伤害感受性神经元突触前末梢,参与主要疼痛介质如谷氨酸和P物质等释放的调节。通过阻断上述通道,选择性N型VDCCs阻断剂表现出强效镇痛作用,N型VDCCs Cav2．2亚基基因敲除小鼠也表现为痛阈提高。N型VDCCs还分布于自主神经系统和中枢神经系统突触部位,现有的N型VDCCs阻断剂用于疼痛治疗时出现的各种副作用与这些部位的突触抑制有关。最近发现,背根节伤害感受性神经元上存在一种特异的N型VDCCs亚型,这为疼痛治疗提供了一个非常有意义的新靶标。     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Structure-activity relationship study of 1,4-dihydropyridine derivatives blocking N-type calcium channels

Cilnidipine is a 1,4-dihydropyridine derived L/N-type calcium channel dual blocker possessing neuroprotective and analgesic effects which are related to its N-type calcium channel inhibitory activity. In order to find specific N-type calcium channel blockers with the least effects on cardiovascular system, we performed structure-activity relationship study on APJ2708, which is a derivative of cilnidipine, and found a promising N-type calcium channel blocker 21b possessing analgesic effect in vivo with a 1600-fold lower activity against L-type calcium channels than that of cilnidipine.     

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

N-type calcium channels (Ca_v2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca_v2.2 blockers with good in vitro and in vivo potency.     

Cell-specific alternative splicing increases calcium channel current density in the pain pathway

N-type calcium channels are critical for pain transduction. Inhibitors of these channels are powerful analgesics, but clinical use of current N-type blockers remains limited by undesirable actions in other regions of the nervous system. We now demonstrate that a unique splice isoform of the N-type channel is restricted exclusively to dorsal root ganglia. By a combination of functional and molecular analyses at the single-cell level, we show that the DRG-specific exon, e37a, is preferentially present in Ca(V)2.2 mRNAs expressed in neurons that contain nociceptive markers, VR1 and Na(V)1.8. Cell-specific inclusion of exon 37a correlates closely with significantly larger N-type currents in nociceptive neurons. This unique splice isoform of the N-type channel could represent a novel target for pain management.