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 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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Synthesis and SAR studies of a novel series of T-type calcium channel blockers

For the novel, potent, and selective T-type Ca2+ channel blockers, a series of sulfonamido-containing 3,4-dihydroquinazoline derivatives were prepared and evaluated for their blocking actions on T- and N-type Ca2+ channels. Among them, 9c (KYS05064, IC50 = 0.96 +/- 0.22 microM) was found to be as potent as Mibefradil and also showed the highest selectivity for T-type Ca2+ channel with no effect on N-type Ca2+ 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。     

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.     

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.     

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.     

Comparison of Effects of Vanadium Absorbed by Coprinus comatus with Those of Inorganic Vanadium on Bone in Streptozotocin-Diabetic Rats

The purpose of this study was to compare the effect of vanadium absorbed by Coprinus comatus (VACC) with inorganic vanadium (vanadium nitrate, IV) in preventing diabetes-related osteopenia in streptozotocin-diabetic rats. Sixty Wistar female rats used were divided into four groups: (1) normal rats (control), (2) diabetic rats, (3) diabetic rats treated with VACC, and (4) diabetic rats treated with vanadium nitrate. A standardized type 1-like diabetes model was induced by injection of streptozotocin. After the rats were treated orally with VACC and IV respectively, plasma glucose, body weights, micro-CT, biomechanical testing, and histomorphometry were examined. In addition, bone samples were obtained to evaluate the content of mineral substances in bones. Treatments were performed over a 12-week period. Both VACC and IV have a positive effect on plasma glucose and body weights of STZ-induced diabetic rats. However, treatment with IV only caused a 39.6?% decrease in glucose levels and a 14.6?% increase in body weights, whereas VACC decreased plasma glucose and increased body weights by up to 52.2 and 24.5?%, respectively. At the same time, VACC significantly improved trabecular microstructure and mechanical strength, while IV did not exhibit desirable such effects. Also, bone Ca and bone P were not significantly increased by IV. These results indicated that both VACC and IV have hypoglycemic activity on diabetic rats, while IV did not improve bone properties. In conclusion, this study suggests that VACC improves diabetes-related bone dysfunction, primarily by improving the diabetic states.     

Electrophysiological characterization of volume-activated chloride currents in mouse cholangiocyte cell line

Recent electrophysiological and radioisotope efflux studies have demonstrated various Cl(-) channels in cholangiocytes including volume-activated Cl(-) channels (VACC). Because VACCs play prominent roles in many vital cellular functions and physiology in cholangiocytes, we have examined their electrophysiological characteristics in mouse cholangiocytes to provide an important framework for studying in the future. The present study is to characterize VACCs expressed in the mouse bile duct cell (MBDC) line, conditionally immortalized by SV40 virus. Conventional whole cell patch-clamp techniques were used to study the electrophysiological characteristics of VACC in MBDC. When the MBDCs were exposed to hypotonic solution, they exhibited an outwardly rectified current, which was significantly inhibited by replacing chloride in the bath solution with gluconate or glutamate and by administration of classic chloride channel inhibitors 5-nitro-2-(3-phenylpropylamino)-benzoate, glybenclamide, DIDS, and tamoxifen. These inhibitory effects were reversible with washing them out from the bath solution. Moreover, the ion selectivity of the volume-activated channel to different anions indicates that it is more permeable to SCN(-) > I(-) >/= Cl(-) > F(-) >/= acetate >/= glutamate >/= gluconate. These electrophysiological characteristics demonstrate that the volume-activated current observed is a VACC. In addition, the VACC in MBDC has electrophysiological characteristics similar to those of the VACC in human cholangiocarcinoma cell line. The present study is the first to characterize the VACC in mouse cholangiocyte and will provide an important framework for further studies to examine and understand the role of the VACC in biliary secretion and ion-transport physiology.     

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.