Structure-activity relationship study and discovery of indazole 3-carboxamides as calcium-release activated calcium channel blockers

Aberrant activation of mast cells contributes to the development of numerous diseases including cancer, autoimmune disorders, as well as diabetes and its complications. The influx of extracellular calcium via the highly calcium selective calcium-release activated calcium (CRAC) channel controls mast cell functions. Intracellular calcium homeostasis in mast cells can be maintained via the modulation of the CRAC channel, representing a critical point for therapeutic interventions. We describe the structure-activity relationship study (SAR) of indazole-3-carboxamides as potent CRAC channel blockers and their ability to stabilize mast cells. Our SAR results show that the unique regiochemistry of the amide linker is critical for the inhibition of calcium influx, the release of the pro-inflammatory mediators β-hexosaminidase and tumor necrosis factor α by activated mast cells. Thus, the indazole-3-carboxamide 12d actively inhibits calcium influx and stabilizes mast cells with sub-μM IC₅₀. In contrast, its reverse amide isomer 9c is inactive in the calcium influx assay even at 100 μM concentration. This requirement of the specific 3-carboxamide regiochemistry in indazoles is unprecedented in known CRAC channel blockers. The new structural scaffolds described in this report expand the structural diversity of the CRAC channel blockers and may lead to the discovery of novel immune modulators for the treatment of human diseases.     

Effects of calcium channel blockers on phototaxis and motility of Chlamydomonas reinhardtii

The effects of the four calcium channel blockers flunarizine, verapamil, diltiazem and nimodipine on motility and phototaxis of Chlamydomonas reinhardtio have been tested with a fully automated and computerized population system. Flunarizine inhibits motility transiently by causing the detachement of the flagella which, however, are regenerated during some hours. Phototaxis is inhibited to the same extent, but this is simply the result of the decreased motility and, hence, a non-specific effect. Verapamil causes also a detachement of the flagella with following regeneration, but in addition motility and phototaxis are inhibited by this drug to different extents, indicating the involvement of calcium channels in both processes. Diltiazem and nimodipine inhibit phototaxis without impairing motility, indicating that both processes are regulated in different ways. If diltiazem and nimodipine are applied simultaneously, no additive inhibitory effect can be observed. However, the combination of both blockers with verapamil causes and additive inhibitory effect as if verapamil is applied alone. By increasing the external calcium concentration from 10^-4 M to 10^-3 M the optimum of positive phototaxis is shifted to higher fluence rates. This shifting occurs also in the presence of channel blockers, but the strength of the positive reaction is influenced. These results point to the involvement of calcium channels in both phototaxis and motility, but simultaneously demonstrate the different sensitivity of the two processes to these drugs.Abbreviations DIL diltiazem (=benzothiazepine) - FLU flunarizine (=(E)-1-(bis-(4-fluorophenyl(methyl)-4-(3-phenyl-2-propenyl)piperazinex2HCl) - NIM nimodipine (=1,4-dihydropyridine) - VER verapamil (=diphenylalkylamine) CaM, calmodulin - PDE phosphodiesterase - DMSO dimethylsulfoxide     

Synthesis of some new C2 substituted dihydropyrimidines and their electrophysiological evaluation as L-/T-type calcium channel blockers

Drugs targeting different calcium channel subtypes have strong therapeutic potential for future drug development for cardiovascular disorders, neuropsychiatric diseases and cancer. This study aims to design and synthesize a new series of C2 substituted dihydropyrimidines to mimic the structure features of third generation long acting dihydropyridine calcium channel blockers and dihydropyrimidines analogues. The target compounds have been evaluated as blockers for Ca_V1.2 and Ca_V3.2 utilizing the whole-cell patch clamp technique. Among the tested compounds, compound 7a showed moderate calcium channel blockade activity against Ca_V3.2. Moreover, the predicted physicochemical properties and pharmacokinetic profiles of the target compounds recommend that they can be considered as drug-like candidates. The results highlight some significant information for the future design of lead compounds as calcium channel blockers.     

Altered cocaine effects in mice lacking Ca(v)2.3 (alpha(1E)) calcium channel

Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Ca(v)2.3 (alpha(1E)) calcium channel to cocaine effects using Ca(v)2.3 knockout mice (Ca(v)2.3-/-). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Ca(v)2.3+/+), but failed to produce any response in Ca(v)2.3-/- mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Ca(v)2.3+/+ mice; however, it had no significant effect in Ca(v)2.3-/- mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Ca(v)2.3-/- mice. These data indicate that Ca(v)2.3 channel contributes to the locomotor-stimulating effect of cocaine, and the deletion of Ca(v)2.3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist.     

Effects of calcium channel blockers and DCMU on motility and the photophobic response of Gyrodinium dorsum

The effects of the calcium channel blockers, verapamil, diltiazem and lanthanum ions and the Ca²⁺ dependency on motility as well as the photophobic response (stop-response) of Gyrodinium dorsum were studied. At Ca²⁺ concentrations below 10^-3 M, motility was inhibited. La³⁺ inhibits the stop-response, in contrast to verapamil and diltiazem. The only calcium channel blocker that increased the amount of non-motile cells was verapamil. The results indicate that motility are Ca²⁺ dependent and that the stop-responses of G. dorsum could be affected by extracellular Ca²⁺. Effects of the photosythesis inhibitor (DCMU) on the stop-response was also determined. With background light of different wavelength (614, 658 and 686 nm) the stop-response increased. DCMU inhibited this effect of background light. Negative results with the monoclonal antibody Pea-25 directed to phytochrome and the results with DCMU, indicate that the stop-response of G. dorsum is coupled to photosynthesis rather than to a phytochrome-like pigment. Oxygen evolution, but not cell movement, was completely inhibited by 10^-6 M DCMU.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-methylurea - DILT diltiazem - DMSO dimethylsulfoxide - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - VER verapamil