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.     

A novel series of pyrazolylpiperidine N-type calcium channel blockers

Selective blockers of the N-type calcium channel have proven to be effective in animal models of chronic pain. However, even though intrathecally delivered synthetic ω-conotoxin MVIIA from Conus magnus (ziconotide [Prialt®]) has been approved for the treatment of chronic pain in humans, its mode of delivery and narrow therapeutic window have limited its usefulness. Therefore, the identification of orally active, small-molecule N-type calcium channel blockers would represent a significant advancement in the treatment of chronic pain. A novel series of pyrazole-based N-type calcium channel blockers was identified by structural modification of a high-throughput screening hit and further optimized to improve potency and metabolic stability. In vivo efficacy in rat models of inflammatory and neuropathic pain was demonstrated by a representative compound from this series.     

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.     

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.     

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.     

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.     

Discovery and evaluation of selective N-type calcium channel blockers: 6-unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives

A structure-activity relationship study of 6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-cinnamyl ester was found to be an effective and selective N-type calcium channel blocker with oral analgesic potential.     

Structure-activity study of L-cysteine-based N-type calcium channel blockers: optimization of N- and C-terminal substituents

Synthesis and structure-activity relationship (SAR) studies of L-cysteine-based N-type calcium channel blockers are described. In the course of exploring SAR of the N- and C-terminal substituents, the L-cysteine derivative was found to be a potent N-type calcium channel blocker with an IC(50) value of 0.14 microM on IMR-32 assay. Compound showed 12-fold selectivity for N-type over L-type calcium channels on AtT-20 assay.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

Synthesis and biological evaluation of 1,4-diazepane derivatives as T-type calcium channel blockers

We have synthesized and biologically evaluated 1,4-diazepane derivatives as T-type calcium channel blockers. In this study, we discovered compound 4s, a potential T-type calcium channel blocker with good selectivity over hERG and N-type calcium channels. In addition, it exhibited favorable pharmacokinetic characteristics for further investigation of T-type calcium channel related diseases.     

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.     

Modular,efficient synthesis of asymmetrically substituted piperazine scaffolds as potent calcium channel blockers

A novel approach to the synthesis of substituted piperazines and their investigation as N-type calcium channel blockers is presented. A common scaffold exhibiting high activity as N-type blockers is N-substituted piperazine. Using recently developed titanium and zirconium catalysts, we describe the efficient and modular synthesis of 2,5-asymmetrically disubstituted piperazines from simple amines and alkynes. The method requires only three isolation/purification protocols and no protection/deprotection steps for the diastereoselective synthesis of 2,5-dialkylated piperazines in moderate to high yield. Screening of the synthesized piperazines for N-type channel blocking activity and selectivity shows the highest activity for a compound with a benzhydryl group on the nitrogen (position 1) and an unprotected alcohol-functionalized side chain.     

Effects of the blockade of high voltage-activated calcium channels on in vitro pineal melatonin synthesis

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.