Design, synthesis, and biological activity of novel semicarbazones as potent Ryanodine receptor1 inhibitors of Alzheimer's disease.

Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 µM, 3 µM and 10 µM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.

Synthesis and characterization of a disubstituted piperazine derivative with T-type channel blocking action and analgesic properties.

BACKGROUND: T-type calcium channels are important contributors to signaling in the primary afferent pain pathway and are thus important targets for the development of analgesics. It has been previously reported that certain piperazine-based compounds such as flunarizine are able to inhibit T-type calcium channels. Thus, we hypothesized that novel piperazine compounds could potentially act as analgesics. RESULTS: Here, we have created a series of 14 compound derivatives around a diphenyl methyl-piperazine core pharmacophore. Testing their effects on transiently expressed Cav3.2 calcium channels revealed one derivative (3-((4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)methyl)-4-(2-methoxyphenyl)-1,2,5-oxadiazole 2-oxide, compound 10e) as a potent blocker. 10e mediate tonic block of these channels with an IC50 of around 4 micromolar. 10e also blocked Cav3.1 and Cav3.3 channels, but only weakly affected high-voltage-activated Cav1.2 and Cav2.2 channels. Intrathecal delivery of 10e mediated relief from formalin and complete Freund's adjuvant induced inflammatory pain that was ablated by genetic knockout of Cav3.2 channels. CONCLUSIONS: Altogether, our data identify a novel T-type calcium channel blocker with tight structure activity relationship (SAR) and relevant in vivo efficacy in inflammatory pain conditions.

Optimization of TRPV6 Calcium Channel Inhibitors Using a 3D Ligand-Based Virtual Screening Method.

Herein, we report the discovery of the first potent and selective inhibitor of TRPV6, a calcium channel overexpressed in breast and prostate cancer, and its use to test the effect of blocking TRPV6-mediated Ca(2+)-influx on cell growth. The inhibitor was discovered through a computational method, xLOS, a 3D-shape and pharmacophore similarity algorithm, a type of ligand-based virtual screening (LBVS) method described briefly here. Starting with a single weakly active seed molecule, two successive rounds of LBVS followed by optimization by chemical synthesis led to a selective molecule with 0.3 µM inhibition of TRPV6. The ability of xLOS to identify different scaffolds early in LBVS was essential to success. The xLOS method may be generally useful to develop tool compounds for poorly characterized targets.

Discovery of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide as a selective T-type calcium channel (Cav3.2) inhibitor.

The T-type calcium channel inhibitor Mibefradil was reported to protect the heart from atrial remodeling, a key process involved in the development of atrial fibrillation and arrhythmias. Mibefradil is not a selective T-type calcium channel inhibitor and also affects the function of different ion channels. Our aim was to develop a selective T-type calcium channel inhibitor to validate the importance of T-type-related pharmacology in atrial fibrillation. Structural optimisation of a previously disclosed hit series focussed on minimising exposure to the central nervous system and improving pharmacokinetic properties, while maintain adequate potency and selectivity. This resulted in the design of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide, a novel, selective, peripherally restricted chemical probe to verify the role of T-type calcium channel inhibition on atrial fibrillation protection.

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.

Synthesis and biological evaluation of 4-piperidinecarboxylate and 4-piperidinecyanide derivatives for T-type calcium channel blockers.

To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 4-piperidinecarboxylate and 4-piperidinecyanide derivatives were prepared and evaluated for in vitro and in vivo activity against α(1G) calcium channel. Among them, several compounds showed good T-type calcium channel inhibitory activity and minimal off-target activity over hERG channel (% inhibition at 10 µM=61.85-71.99, hERG channel IC(50)=1.57 ± 0.14-4.98 ± 0.36 µM). Selected compound 31a was evaluated on SNL model of neuropathic pain and showed inhibitory effect on mechanical allodynia.

A novel membrane-permeant cADPR antagonist modified in the pyrophosphate bridge.

A concise method for the formation of cyclopyrophosphate of cIDPRE as well as sulfur and selenium-substituted pyrophosphate cIDPRE analogues (P(1)(S)-cIDPRE, P(1)(Se)-cIDPRE, P(2)(S)-cIDPRE and P(2)(Se)-cIDPRE) was reported and one of the P(S)-diastereoisomers, P(1)(S)-cIDPRE-1, is a novel membrane-permeant cADPR antagonist.

HTDP-2, a new synthetic compound, inhibits glutamate release through reduction of voltage-dependent Ca²âº influx in rat cerebral cortex nerve terminals.

AIM: The present study was aimed at investigating the effect of trans-6-(4-chlorobutyl)-5-hydroxy-4-(phenylthio)-1-tosyl-5,6-dihydropyridine-2(1H)-one (HTDP-2), a novel synthetic compound, on the release of endogenous glutamate in rat cerebrocortical nerve terminals (synaptosomes) and exploring the possible mechanism. METHODS: The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an on-line enzyme-coupled fluorimetric assay. We also used a membrane potential-sensitive dye to assay nerve terminal excitability and depolarization, and a Ca²âº indicator, Fura-2-acetoxymethyl ester, to monitor cytosolic Ca²âº concentrations ([Ca²âº](c)). RESULTS: HTDP-2 inhibited the release of glutamate evoked by 4-AP in a concentration-dependent manner. Inhibition of glutamate release by HTDP-2 was prevented by the chelating intraterminal Ca²âº ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-ß-benzyloxyaspartate. HTDP-2 did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in [Ca²âº](c). Furthermore, the inhibitory effect of HTDP-2 on the evoked glutamate release was abolished by the N-, and P/Q-type Ca²âº channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na⁺/Ca²âº exchanger blocker CGP37157. CONCLUSION: Based on these results, we suggest that, in rat cerebrocortical nerve terminals, HTDP-2 decreases voltage-dependent Ca²âº channel activity and, in so doing, inhibits the evoked glutamate release.

Synthesis and biological evaluation of oxazole derivatives as T-type calcium channel blockers.

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pain. In this study, as a part of our ongoing efforts to develop potent T-type calcium channel blockers, we designed oxazole derivatives substituted with arylpiperazinylalkylamines. The oxazoles were synthesized in a convenient convergent synthetic method, and biologically evaluated against alpha(1G) (Ca(V)3.1) T-type calcium channel. Among total 41 oxazole compounds synthesized, the most active one was the compound 10-35 with an IC(50) value of 0.65 microM, which is comparable with that of mibefradil.

Identification and characterization of a dual-acting antinematodal agent against the pinewood nematode, Bursaphelenchus xylophilus.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca(2+) channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.

A fluorescence-based high-throughput screening assay for the identification of T-type calcium channel blockers.

T-type voltage-gated Ca(2+) channels have been implicated in contributing to a broad variety of human disorders, including pain, epilepsy, sleep disturbances, cardiac arrhythmias, and certain types of cancer. However, potent and selective T-type Ca(2+) channel modulators are not yet available for clinical use. This may in part be due to their unique biophysical properties that have delayed the development of high-throughput screening (HTS) assays for identifying blockers. One notable challenge is that at the normal resting membrane potential (V(m)) of cell lines commonly utilized for drug screening purposes, T-type Ca(2+) channels are largely inactivated and thus cannot be supported by typical formats of functional HTS assays to both evoke and quantify the Ca(2+) channel signal. Here we describe a simple method that can successfully support a fluorescence-based functional assay for compounds that modulate T-type Ca(2+)channels. The assay functions by exploiting the pore-forming properties of gramicidin to control the cellular V(m) in advance of T-type Ca(2+) channel activation. Using selected ionic conditions in the presence of gramicidin, T-type Ca(2+) channels are converted from the unavailable, inactivated state to the available, resting state, where they can be subsequently activated by application of extracellular K(+). The fidelity of the assay has been pharmacologically characterized with sample T-type Ca(2+) channel blockers whose potency has been determined by conventional manual patch-clamp techniques. This method has the potential for applications in high-throughput fluorometric imaging plate reader (FLIPR(R), Molecular Devices, Sunnyvale, CA) formats with cell lines expressing either recombinant or endogenous T-type Ca(2+) channels.

Discovery of 1,4-substituted piperidines as potent and selective inhibitors of T-type calcium channels.

The discovery of a novel series of potent and selective T-type calcium channel antagonists is reported. Initial optimization of high-throughput screening leads afforded a 1,4-substituted piperidine amide 6 with good potency and limited selectivity over hERG and L-type channels and other off-target activities. Further SAR on reducing the basicity of the piperidine and introducing polarity led to the discovery of 3-axial fluoropiperidine 30 with a significantly improved selectivity profile. Compound 30 showed good oral bioavailability and brain penetration across species. In a rat genetic model of absence epilepsy, compound 30 demonstrated a robust reduction in the number and duration of seizures at 33 nM plasma concentration, with no cardiovascular effects at up to 5.6 microM. Compound 30 also showed good efficacy in rodent models of essential tremor and Parkinson's disease. Compound 30 thus demonstrates a wide margin between CNS and peripheral effects and is a useful tool for probing the effects of T-type calcium channel inhibition.

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.

Novel T-type calcium channel blockers: dioxoquinazoline carboxamide derivatives.

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium channel blockers. A small molecule library of dioxoquinazoline carboxamide derivatives containing 155 compounds was designed, synthesized, and biologically evaluated for T-type calcium channel blocking activity. Among those compounds synthesized, the compound 1n shows the most potent T-type calcium current blocking activity with an IC(50) value of 1.52 microM, which is comparable to that of mibefradil.

3D pharmacophore based virtual screening of T-type calcium channel blockers.

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYSTtrade mark program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC(50) value of 0.10 microM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC(50) values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.

Discovery, structure-activity relationship study, and oral analgesic efficacy of cyproheptadine derivatives possessing N-type calcium channel inhibitory activity.

Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization, we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium channel, histamine (H1), and serotonin (5-HT2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat formalin-induced pain model.

Novel potent and selective calcium-release-activated calcium (CRAC) channel inhibitors. Part 2: Synthesis and inhibitory activity of aryl-3-trifluoromethylpyrazoles.

To identify potent and selective calcium-release-activated calcium (CRAC) channel inhibitors, we examined the structure-activity relationships of the pyrazole and thiophene moieties in compound 4. Compound 25b was found to exhibit highly potent and selective inhibitory activity for CRAC channels and further modifications of the pyrazole and benzoyl moieties of compound 25b produced compound 29. These compounds were potent inhibitors of IL-2 production in vitro and also acted as inhibitors in pharmacological models of diseases resulting from T-lymphocyte activation, after oral administration.

Novel potent and selective calcium-release-activated calcium (CRAC) channel inhibitors. Part 1: synthesis and inhibitory activity of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamides.

We synthesized and evaluated a series of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamides to identify potent inhibitors of calcium-release-activated calcium (CRAC) channels with greater selectivity than voltage-operated calcium (VOC) channels. These efforts resulted in identification of compounds 22 and 24. The former exhibits highly potent and selective CRAC channel inhibitory activity, and the latter inhibited phytohemagglutinin-induced interleukin-2 production by Jurkat T lymphocytes and concanavalin A-induced hepatitis in mice.

[Semi-synthesis of derivatives with C-3' and C-4' trans-configuration from (+)-praeruptorin A].

AIM: In order to compare the calcium antagonist activity between the derivatives of (+)-praeruptorin A with C-3' and C-4' cis-configuration and trans-configuration, and to look for new active compounds, some derivatives with C-3', C-4' trans-configuration of (+)-praeruptorin A were semi-synthsized. METHODS: (+)-Praeruptorin A was isolated from the root of Peucedanum praeruptorum. Basic hydrolysis of (+)-praeruptorin A was carried out. From the alkaline hydrolysis product (2), eight new products (5-12) with C-3', C-4' trans-configuration were semi-synthsized whose C-3' was linked to angeloyloxy and C-4' was linked to various acyloxy, using respective acids as acylating agents, DCC as a dehydrant, DMAP as catalyst. From the alkaline hydrolysis product (4), five new products (13-17) with C-3', C-4' trans-configuration were obtained whose C-3', C-4' is linked to various same acyloxys, using respective acids as acylating agents, DCC as dehydrant, DMAP as catalyst. Also from the alkaline hydrolysis product (4), using respective acyl chlorides as acylating agents, anhydrous dichloromethane containing minor pyridine as a solvent, the improved Schotten-Baumann reactions were carried out, two new products (18, 20) with C-3', C-4' trans-configuration were obtained whose only C-3' linked to acyloxy and two other new products (19, 21) with C-3', C-4' trans-configuration were obtained whose C-3' and C-4' linked two acyloxys. The structures of all the products were elucidated by spectral analyses including IR, 1HNMR and EIMS. The calcium antagonist activity of all of the products were tested by inhibition of the systole of rat artery ring. RESULTS: Seventeen compounds with C-3', C-4' trans-configuration were semi-synthesized from (+)-praeruptorin A for the first time and their calcium antagonist activity were evaluated. CONCLUSION: All of the derivatives were new compounds. Bioactivity assay indicated that some new compounds with C-3', C-4' trans-configuration showed obvious calcium antagonist activity, but they are not as strong as (+)-praeruptorin A. The activity of some products was shown to be similar to that of the derivatives with C-3', C-4' cis-configuration for the first time.

[Studies on structure modification of (+)-praeruptorin A].

AIM: In order to look for new active compounds, the structure of (+)-praeruptorin A is modified. METHODS: (+)-Praeruptorin A was isolated from the root of Peucedanum praeruptorum, basic hydrolysis of (+)-praeruptorin A and acyled reactions of hydrolysis product of (+)-praeruptorin A were carried out. RESULTS: Eighteen compounds were semi-synthesized from (+)-praeruptorin A. CONCLUSION: Fourteen compounds (5-18) among them are new compounds. Preliminary bioactivity assay indicated that the new compounds show calcium antagonist activity, but they are not as strong as (+)-praeruptorin A.