Novel calcium antagonists. Synthesis and structure-activity relationship studies of benzothiazoline derivatives

A series of novel compounds having a benzothiazoline skeleton was studied for their structure-activity relationship (SAR) with respect to Ca2+ antagonistic activity. As test compounds, analogues of 3-acyl-2-arylbenzothiazolines (3) were synthesized. Benzothiazoline derivatives (3) exerted higher Ca2+ antagonistic activity than the corresponding thiazolidine derivatives (2). Effects of substituents R1-R4, the substitution position of the aminoalkoxy group and R2, and the length of the methylene chain on biological activities were examined. Compound 4 [3-acetyl-2-[5-methoxy-2-[4-[N-methyl-N-(3,4,5-trimethoxyphenethyl ) amino]butoxy]phenyl]benzothiazoline hydrochloride] showed a potent Ca2+ antagonistic activity in vitro and dual inhibition on the fast Na+ inward channel and the slow Ca2+ inward channel in Langendorff perfused rabbit hearts. Compound 4 also showed a long-acting hypotensive effect in spontaneously hypertensive rats and prevented acute pulmonary thrombotic death in mice.     

Tissue selectivity of the novel calcium antagonist sesamodil fumarate in isolated smooth muscles and cardiac muscles

The effects of the novel Ca2+ antagonist sesamodil fumarate (JAN), (+)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H- 1,4-benzothiazine hydrogen fumarate (SD-3211), on isolated smooth muscles and cardiac muscles were investigated and compared with those of diltiazem, verapamil, nifedipine and nicardipine. Ca2+ antagonistic activity of SD-3211 (pA2 = 8.42) was more potent than that of diltiazem and verapamil, but less potent than that of nifedipine and nicardipine in isolated pig coronary artery. The inhibition of Ca2(+)-induced contraction by SD-3211 was not reversed by drug washout, whereas that by diltiazem was easily reversed by drug washout. SD-3211 produced a concentration-dependent relaxation (EC50 = 5.7 x 10(-8) mol/l) of KCl-contracted pig coronary artery. The order of potency of the various compounds correlated with their respective Ca2+ antagonistic activities. SD-3211 antagonized KCl-induced contraction, but not that induced by A23187, in the rabbit aorta. On the other hand, negative inotropic and chronotropic effects of SD-3211 on the guinea pig right atria approximated those of diltiazem and verapamil. These results suggest that SD-3211 exerts a potent and long-lasting Ca2+ antagonistic effect on isolated arteries, possessing pharmacological properties diverse from those of known Ca2+ antagonists with respect to tissue selectivity, i.e., it is more vasoselective than diltiazem and verapamil, and more cardioselective than nifedipine and nicardipine.     

Synthesis and Ca2+ antagonistic activity of 2-[2-[(aminoalkyl)oxy]-5-methoxyphenyl]-3,4-dihydro-4-methyl-3-oxo-2H- 1,4-benzothiazines

As an extension of the previous investigation (J. Med. Chem. 1988, 31, 919), we synthesized a series of 2-[2-[(aminoalkyl)oxy]-5-methoxyphenyl]-3,4-dihydro-4-methyl-3-oxo-2H- 1,4-benzothiazines (3) and evaluated their Ca2+ antagonistic activities. Ca2+ antagonistic activity was measured with isolated depolarized guinea pig taenia cecum. On the basis of their potent Ca2+ antagonistic activity, six benzothiazines were selected and further evaluated for their vasocardioselectivity. Among these six compounds, the key compound 15 [3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[3,4- (methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo- 2H-1,4-benzothiazine hydrogen fumarate] was recognized as having the lowest cardioselectivity. Following optical resolution, the absolute configuration of the compound's optically active enantiomer was determined by means of X-ray crystallography of a synthetic precursor (+)-4a. The Ca2+ antagonistic activity of 15 was found to reside primarily in (+)-15 (which was about 7 times more potent than (-)-15). The in vitro study showed that (+)-15 had a low cardioselectivity compared to verapamil and diltiazem. This result suggests that (+)-15 would exhibit less adverse effects due to cardiac inhibition than diltiazem and verapamil in therapeutic use.     

L-cis diltiazem attenuates intracellular Ca(2+) overload by metabolic inhibition in guinea pig myocytes

We have previously demonstrated that treatment with L-cis diltiazem reduced cardiac infarct size in vivo. To examine the effect of L-cis diltiazem on Ca(2+) overload induced by ischemia/reperfusion, we used a model for Ca(2+) overload produced by metabolic inhibition in isolated guinea pig myocytes. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was quantified by fura-2 fluorescence microscopy and Ca(2+) overload was induced by inclusion of 1 microM of carbonyl cyanide m-chrolophenylhydrazone (CCCP) for 40 min treatment followed by washout for 30 min. This treatment caused a large [Ca(2+)](i) elevation as well as a sustained contracture of the cardiomyocytes. The increase was suppressed by 10 microM of 2-[2-[4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea methanesulphonate (KB-R7943), a specific inhibitor of the Na(+)/Ca(2+) exchanger, but not by nitrendipine (10 microM). L-cis Diltiazem (10 microM) attenuated the [Ca(2+)](i) increase, suggesting that L-cis diltiazem elicits a cardioprotective effect via attenuation of the [Ca(2+)](i) increase induced by metabolic inhibition and energy repletion.     

Influence of platelet activating factor antagonists on the protamine sulphate stimulated release of histamine from peritoneal mast cells of rats in vitro.

The phospholipase (PL)A2 inhibitor p-bromophenacyl bromide (p-BPB), the antagonists of the platelet activating factor (PAF) 3-(4-(2-chlorophenyl)-9-methyl-6H-thieno(3,2-f)-(1,2,4)triazolo(4, 3-a)(1,4)diazepine-2-yl)-1-(4-morpholinyl)-1-propanone (a thieno-triazolo-diazepine, WEB 2086) and terpene-ginkgolide B and the antihistamine with PAF antagonistic qualities 4,9-dihydro-4-(1-methyl-4-piperidinylidene)-10H-benzo[2,5]cyclo hep ta[1,2-b]thiophen-10-one (ketotifen) inhibit in the order p-BPB greater than terpene-ginkgolide B greater than ketotifen greater than WEB 2086 with decreasing activity the protamine sulphate activated histamine release from peritoneal rat mast cells (pRMC) in vitro. All compounds also inhibit the PAF induced aggregation of human platelets. The order of inhibition of the histamine release by these compounds does not agree with the order of their inhibitory activity on PAF induced aggregation of human platelets, indicating that the inhibition of the mast cell degranulation caused by PAF antagonists does not occur via PAF receptors. A generally membrane stabilizing quality of terpene-ginkgolide B and WEB 2086 may be ruled out as the cause of degranulation inhibition because none of the compounds suppresses the cytotoxic, triton X-100 induced release of histamine from pRMC. The mechanism of mast cell degranulation inhibition by PAF antagonists is unclear. An inhibition of PLA2 and/or inhibition of the Ca(2+)-activation are suggested.     

Activation of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by analogues of sulmazole.

1. The effect of sulmazole and several structurally related analogues on cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel gating and on [3H]-ryanodine binding to isolated SR membrane vesicles has been investigated. 2. The optical isomers, (+)- and (-)-sulmazole, increased the open probability (Po) of single Ca(2+)-release channels incorporated into phospholipid bilayers held under voltage clamp by increasing the frequency and duration of open events. The respective EC50s were 423 microM and 465 microM at 10 microM activating cytosolic Ca2+ and the Hill coefficients for activation were approximately two, suggesting that at least two molecules of either enantiomer are required to bind for channel activation. 3. Similarly the related enantiomers, (+)- and (-)-isomazole, which differ from sulmazole in the position of the pyridine nitrogen (4.5b for sulmazole; 4.5c for isomazole), were approximately as potent as each other and as potent as the isomers of sulmazole with EC50s of approximately 445 microM. 4. In contrast, EMD 46512 and EMD 41000, which are sulmazole and isomazole analogues respectively, each with the methylsulphinyl oxygen removed, increased single-channel Po with EC50s of 42 microM and 40 microM. The open and closed lifetime distributions were similar to those of the less potent analogues and the Hill coefficients were the same, suggesting that these compounds act at the sulmazole site on the Ca(2+)-release channel. 5. All of the compounds tested were able to increase the Po of channels in the absence of activating Ca2+ but were less potent than in the presence of Ca2+. The drugs were effective only when added to the cytosolic face of the channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of stereoselectivity in the inotropic and phosphodiesterase inhibitory effects of saterinone enantiomers

The enantiomers of the positive inotropic and a1-adrenoceptor blocking agent saterinone (+/-)-1,2-dihydro-5-[4-[2-hydroxy-3- [4-(2-methoxyphenyl)-1-piperazinyl]propoxy] phenyl]-6-methyl-2-oxo-3-pyridine-carbonitrile, BDF 8634) have been investigated with in vitro and in vivo models in laboratory animals. In the guinea pig papillary muscle, saterinone enantiomers had equipotent inotropic activity and were also as potent as racemic saterinone; the (R)-enantiomer, however, exhibited a greater efficacy than the related compounds. Saterinone and its enantiomers were equipotent in the inhibition of phosphodiesterase PDE III activity in the guinea pig myocardium. The equipotent inotropic effects were also observed after parenteral and enteral administration in cats. In receptor binding studies, (S)-saterinone was 10-fold more potent than (R)-saterinone by inhibiting [3H]-prazosin binding to specific alpha 1-adrenoceptor sites in rat brain cortex membranes. However, in the isolated thoracic aorta of the rabbit, (S)-saterinone was only 3-fold more potent than (R)-saterinone at preventing the pressor effects of phenylephrine. When the enantiomers were tested in vivo against the pressor effects of phenylephrine in the pithed rat, (S)-saterinone was only 2-fold more potent than (R)-saterinone in its alpha 1-adrenoceptor blocking potency. Thus the enantiomers of saterinone do not display enantio-selectivity in their inotropic and PDE III inhibitory effects in vitro, nor in their cardiotonic effects in vivo. There is a slight enantio-selectivity at alpha 1-adrenoceptors in receptor binding studies, but this is reduced to biologically irrelevant magnitude in functional studies in vitro and in vivo.     

Iodoaminopotentidine and related compounds: a new class of ligands with high affinity and selectivity for the histamine H2 receptor.

The synthesis and biological evaluation of a new class of histamine H2 antagonists with N-cyano-N'-[omega-[3-(1-piperidinylmethyl)phenoxy] alkyl]guanidine partial structure are described as part of an extensive research program to find model compounds for the development of new radioligands with high H2 affinity and specific activity. High receptor affinity is achieved by an additional (substituted) aromatic ring, which is connected with the third guanidine N by a carbon chain spacer and an amine, carboxamide, ester, or sulfonamide link ("polar group"). In functional studies for H2 antagonistic activity and other pharmacological actions [e.g. H1 antihistaminic, antimuscarinic, antiadrenergic (alpha 1, beta 1), 5-HT2 blocking activity] in the isolated guinea pig atrium and ileum and rat aorta and tail artery, the compounds proved to be highly potent and selective histamine H2 receptor antagonists. The H2 antagonistic activity is mainly depending on the length of both the N'-alkyl chain (chain A) and the N"-spacer (chain B). Compounds with a C3 chain A and a C2 chain B are most potent in the preferred group of substances, i.e., the carboxamide series. A wide variety of substituents at the aromatic ring is tolerated, among them iodine, amino, and azido groups. These compounds are up to 32 times more potent than cimetidine in the isolated guinea pig right atrium. The replacement of the carboxamide by an ester group (44c) is well tolerated, while replacement of the cyanoguanidine by an urea group results in nearly 100-fold decrease in activity (46c,e). The iodinated benzamides are among the most potent H2 antagonists known so far. The [125I]-labeled form of 31f ([125I]iodoaminopotentidine, [125I]-N-[2-(4-amino-3-iodobenzamido) ethyl]-N'-cyano-N"-[3-[3-(1-piperidinylmethyl) phenoxy]propyl]guanidine) and its photolabile analogue 31h ([125I]iodoazidopotentidine, [125I]-N-[2-(4-azido-3- iodobenzamido)ethyl]-N'-cyano-N"-[3-[3-(1-piperidinyl-methyl)pheno xy] propyl]guanidine) proved to be useful probes for reversible and irreversible labeling of the histamine H2 receptor. Radioligand binding studies in guinea pig cerebral membranes revealed considerably higher H2 receptor affinity for 31f (pKi = 9.15), 31h (pKi = 8.58), and some analogues than functional experiments (guinea pig atrium), presumably reflecting an easier access to the H2 receptors in membranes.     

Interaction of cis-(6-benzhydrylpiperidin-3-yl)benzylamine analogues with monoamine transporters: structure-activity relationship study of structurally constrained 3,6-disubstituted piperidine analogues of (2,2-diphenylethyl)-[1-(4-fluorobenzyl)piperidin-4-ylmethyl]amine

To explore structure-activity relationships (SAR) of a novel conformationally constrained lead cis-3,6-disubstituted piperidine derivative derived from (2,2-diphenylethyl)-[1-(4-fluorobenzyl)piperidine-4-ylmethyl]amine (I), a series of compounds was synthesized by derivatizing the exocyclic N-atom at the 3-position of the lead. This study led to the formation of substituted phenyl and heterocyclic derivatives. All novel compounds were tested for their affinity at the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [3H]WIN 35 428, [3H]citalopram, and [3H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [3H]DA. The SAR results demonstrated that the nature of substitutions on the phenyl ring is important in activity at the DAT with the presence of an electron-withdrawing group having the maximum effect on potency. Replacement of the phenyl ring in the benzyl group by heterocyclic moieties resulted in the development of compounds with moderate activity for the DAT. Two most potent racemic compounds were separated by a diastereoisomeric separation procedure, and differential affinities were observed for the enantiomers. Absolute configuration of the enantiomers was obtained unambiguously by X-ray crystal structural study. One of the enantiomers, compound S,S-(-)-19a, exhibited the highest potency for the DAT (IC50 = 11.3 nM) among all the compounds tested and was as potent as GBR 12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine). However, the compound (-)-19a was more selective than GBR 12909 in binding to the DAT compared with binding to the SERT and NET. The present results establish the newly developed 3,6-disubstituted piperidine derivatives as a novel template for high-affinity inhibitors of DAT. Structurally these molecules are more constrained compared to our earlier flexible piperidine molecules and, thus, should provide more insights about their bioactive conformations.     

Synthesis and aromatase inhibitory activity of novel 1-(4-aminophenyl)-3-azabicyclo[3.1.0]hexane- and -[3.1.1]heptane-2,4- diones

The synthesis of 3-(cyclohexymethyl)-1-(4-aminophenyl)-3-azabicyclo[3.1.0]hexane-2, 4-dione (1h), with its optical enantiomers, and a series of novel achiral 1-(4-aminophenyl)-3-azabicyclo[3.1.1]haptane-2,4-diones (2a-i,k) is described. These compounds were tested in vitro for inhibition of human placental aromatase, a cytochrome-P450-dependent enzyme responsible for the conversion of androgens to estrogens. All of them displayed enzyme-inhibiting activity, and 3-cyclohexyl derivative 2g and 3-cyclohexylmethyl derivative 1h both proved more potent (greater than 140-fold) than the clinically effective agent aminoglutethimide [3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione, AG]. As with AG and its derivatives, the 1R-(+)-enantiomer of 1h was responsible for the enzyme inhibitory activity. These novel compounds are of interest as potential drugs for endocrine therapy of hormone-dependent tumors, e.g. breast cancer.     

Synthesis and structure-activity relationships of 1-substituted 4-(1,2-diphenylethyl)piperazine derivatives having narcotic agonist and antagonist activity

Racemates and enantiomers of 1-substituted 4-[2-(3-hydroxyphenyl)-1-phenylethyl]piperazine derivatives (3-18) were synthesized, and their analgesic and other pharmacological activities and structure-activity relationships were investigated. The S-(+) enantiomers of 2a, 5, 7, 9, 10, and 15-18 had a stronger analgesic activity than their R-(-) enantiomers; analgesic activity of the strongest one [(S)-(+)-10] was 105 times as potent as that of morphine. The S-(+) enantiomers of these compounds had the opposite configuration to that of morphine with respect to its (C-9) asymmetric center but the same configuration to that of the tyrosine residue of Met5-enkephalin. The R-(-) enantiomers of 16 and 18 showed narcotic antagonist activity, but the S-(+) enantiomers did not. (R)-(-)-18 had analgesic and narcotic antagonist activities comparable to pentazocine but showed no significant physical dependence liability. From these results, it is suggested that these compounds show an uncommon enantioselectivity in comparison with morphine and its surrogates, and belong to a new series of compounds having a potent analgesic activity.     

Inhibition of turkey gizzard myosin light chain. Kinase activity by dihydropyridine calcium antagonists

Drugs which block the influx of calcium (Ca2+) across plasma membranes may additionally have direct effects upon smooth muscle contractile proteins. In a system of purified proteins comprised of calmodulin, turkey gizzard myosin light chains, and turkey gizzard myosin light chain kinase, the inhibition of myosin light chain phosphorylation by the dihydropyridine Ca2+ antagonists felodipine [3-ethyl-5-methyl-1-1,4-dihydro-2,6-dimethyl-4-(2,3-dichlorophenyl)-3, 5-pyridine dicarboxylate] and nitrendipine [3-ethyl-5-methyl-1-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate] was studied. In the presence of excess myosin light chain kinase, 50% inhibition of myosin light chain phosphorylation occurred at felodipine and nitrendipine concentrations of 9.8 +/- 1.1 X 10(-6) M and 5.6 +/- 0.6 X 10(-5) M respectively. Inhibition of light chain kinase activity could not be overcome by increasing the free Ca2+ concentration from 0.05 to 5.0 mM. Felodipine was unable to inhibit the activity of myosin light chain kinase rendered Ca2+/calmodulin-independent by limited tryptic digestion. Using molecular sieve chromatography, nitrendipine was found to bind to calmodulin with an apparent dissociation constant (Kapp) of 5.2 +/- 0.3 X 10(-5) M, and this binding was Ca2+ dependent. These data suggest that dihydropyridines inhibit the phosphorylation of smooth muscle myosin light chains in vitro by binding to Ca2+/calmodulin and inhibiting the activation of myosin light chain kinase.     

Interphenylene 7-oxabicyclo[2.2.1]heptane thromboxane A2 antagonists. Semicarbazone omega-chains

A series of chiral interphenylene 7-oxabicyclo[2.2.1]heptane semicarbazones 19-26 were prepared and evaluated for their in vitro thromboxane (TxA2) antagonistic activity and in vivo duration of action. The potency of 19-26 was found to highly dependent on the substitution pattern of the interphenylene ring and decreased in the order ortho greater than meta much greater than para. SQ 35,091 (25), [1S-(1 alpha,2 alpha,3 alpha,4 alpha)]-2-[[3-[[[(phenylamino) carbonyl]hydrazono]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl] benzenepropanoic acid, was identified as a potent and long-acting TxA2 antagonist. In human platelet rich plasma SQ 35,091 inhibited arachidonic acid (800 microM) and U-46,619 (10 microM) induced aggregation with I50 values of 3 and 12 nM, respectively. In contrast, no inhibition of ADP (20 microM) induced aggregation was observed at greater than 1000 microM. Receptor binding studies with [3H]-SQ 29,548 showed SQ 35,091 was a competitive antagonist with a Kd value of 1.0 +/- 0.1 nM in human platelet membranes. In vivo SQ 35,091 (0.2 mg/kg po) showed extended protection (T50 = 16 h) from U-46,619 (2 mg/kg iv) induced death in mice. These compounds have for the first time demonstrated that a metabolically stable interphenylene alpha-sidechain can be introduced into a prostanoid-like series of TxA2 antagonists with the maintainance of potent antagonistic activity.     

Stereospecific regulation of [3H]inositol monophosphate accumulation by calcium channel drugs from all three main chemical classes

Depolarization of [3H]inositol-prelabelled rat cortical slices through the elevation of extracellular K+ levels leads to increased accumulation of [3H]inositol phosphates. In the presence of 18 mM K+, Ca2+ channel activators selectively stimulated the formation of [3H]inositol monophosphate ([3H]IP1) whereas Ca2+ channel blockers were inhibitory. Blockade of the Na+ channel by 1 microM tetrodotoxin had no effect but chelation of extracellular Ca2+ abolished the response. The enantiomers of the benzoxadiazol 1,4-dihydropyridine 202-791 showed opposite stereospecific regulation of [3H]IP1 formation: (+)-(S)-202-791 stimulated (252%; ED50: 88 nM), whereas (-)-(R)-202-791 inhibited (65% inhibition, ED50: 602 nM). The (-) enantiomer of Bay K 8644 was a potent [3H]IP1 stimulator (258%; ED50: 82 nM). While (+)-Bay K 8644 was inactive in the presence of 18 mM K+, it completely inhibited the (-)-Bay K 8644-induced stimulation with a Ki of 103 nM. Representatives of the other two main classes of Ca2+ channel blockers (phenylalkylamines and benzothiazepines) inhibited K+ depolarization-induced and (-)-Bay K 8644 enhanced [3H]IP1 formation in a dose-dependent, stereospecific manner. The results show that Ca2+ channel blockers are efficient modulators of depolarization-induced and Ca2+ channel activator-induced [3H]inositol monophosphate formation in brain, and demonstrate the functional coupling of three distinct drug receptor sites on neuronal Ca2+ channels.     

Effect of R56865 on cardiac sarcoplasmic reticulum function and its role as an antagonist of digoxin at the sarcoplasmic reticulum calcium release channel.

1. The effect of R56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl]-N-methyl-2- benzothiazolamine) on cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel function was investigated. The effect of R56865 on [3H]-ryanodine and [3H]-digoxin binding to SR vesicles and its effect on the ATP-stimulated 45Ca2+ uptake into SR vesicles was also studied. 2. R56865 (0.5-50 microM) had no effect on single-channel open probability (Po) when added to native cardiac SR Ca(2+)-release channels, incorporated into planar phospholipid bilayers, that had previously been activated by 10 microM Ca2+. The single-channel conductance (93 pS) and the Ca2+/Tris+ permeability ratio (12.5) were also unaffected by R56865. 3. R56865 failed to affect the rapid Ca(2+)-induced efflux of 45Ca2+ from cardiac SR vesicles. The initial efflux rate at an extravesicular [Ca2+] of 0.1 microM was 176 +/- 33 nmol 45Ca2+ mg-1 protein s-1 (n = 5). Addition of 0.5-50 microM R56865 to the efflux solution did not affect the initial efflux rate or the total amount of 45Ca2+ released from the vesicles. 4. The specific binding of [3H]-ryanodine to SR vesicles can be viewed as a marker for SR Ca(2+)-release channel activation. R56865 (0.05-50 microM) did not change the amount of specific [3H]-ryanodine bound at 10 microM activating Ca2+. Taken together these data (points 2, 3 and 4) suggest that R56865 does not affect the Ca2+ activation of the cardiac SR Ca(2+)-release channel. 5. R56865 (0.5-50 microM) decreased the ATP-stimulated uptake of 45Ca2+ into cardiac SR vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

New 1,4-dihydropyridines endowed with NO-donor and calcium channel agonist properties

A new series of calcium channel agonists structurally related to Bay K8644, containing NO donor furoxans and the related furazans unable to release NO, is described. The racemic mixtures were studied for their action on L-type Ca(2+) channels expressed in cultured rat insulinoma RINm5F cells. All the products proved to be potent calcium channel agonists. All the racemic mixtures, with the only exception of the carbamoyl derivatives 9, 12 endowed with scanty solubility, were separated by chiral chromatography into the corresponding enantiomers; the (+) enantiomers were found to be potent agonists while the (-) ones were feeble antagonists. The racemic mixtures were also assessed for their positive inotropic activity on electrically stimulated rat papillary muscle and for their ability to increase Ca(2+) entry into the vascular smooth muscle of rat aorta strips. The cyanofuroxan 8 proved to be an interesting product with dual Ca(2+)-dependent positive inotropic and NO-dependent vasodilating activity.     

Agonist-stimulated Ca(2+) transport in mesenteric vascular smooth muscle cells of vitamin D(3)-induced calcium overload rats

Altered intracellular Ca(2+) homeostasis and accumulated Ca(2+) deposition in arterial walls contribute to the natural arterial aging and aging-related vascular pathologies. To gain further insight into internal relationship between these two factors, a vitamin D(3)-induced vascular Ca(2+) overload rat model was employed. Mesenteric vascular smooth muscle cells (VSMCs) were isolated from both vitamin D(3) and Wistar control rats and were maintained in primary culture for 24 h. Cytosolic and nuclear Ca(2+) ([Ca(2+)](i), [Ca(2+)](n)) in VSMCs were compared between vitamin D(3) and Wistar groups using laser scanning confocal microscopy and Ca(2+)-sensitive-dye Fluo-3. Cytosolic and nuclear Ca(2+) were evaluated under both resting and agonist-stimulated conditions including the voltage-dependent Ca(2+) channel openers BayK8644 and KCl, the inositol-1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) release channel activator IP(3), the ryanodine-sensitive Ca(2+) release channel activator trichloromethane, the sarcoplasmic reticulum Ca(2+) -ATPase inhibitor cyclopiazonic acid, and angiotensin II. Although the levels of [Ca(2+)](n) and [Ca(2+)](i) were comparable between vitamin D(3) and Wistar groups under the resting condition, the increase of [Ca(2+)](n) and [Ca(2+)](i) elicited by various agonists was significantly enhanced in VSMCs from the vitamin D(3) group compared with those from the Wistar group, suggesting abnormality of membrane Ca(2+) gating and intracellular Ca(2+) release under Ca(2+) overload condition. In conclusion, our study indicated that vitamin D(3)-induced vascular Ca(2+) overload may directly interrupt cytosolic and nuclear Ca(2+) homeostasis.     

Multiple actions of glaucine on cyclic nucleotide phosphodiesterases, alpha 1-adrenoceptor and benzothiazepine binding site at the calcium channel.

1. In the present study, the properties of glaucine (an aporphine structurally related to papaverine) were compared with those of papaverine, diltiazem, nifedipine and prazosin. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KCl, and a determination of the affinity of glaucine at calcium channel binding sites of alpha-adrenoceptors, by use of [3H]-(+)-cis-diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The effects of glaucine on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined. 2. Contraction evoked by noradrenaline (1 microM) or depolarizing solution (60 mM KCl) were inhibited in a concentration-dependent manner by all the compounds tested. As expected, prazosin showed a greater selectivity of action on NA-induced contraction, whereas nifedipine and diltiazem appeared more potent on KCl-induced contraction. Glaucine had a greater potency on the contraction elicited by noradrenaline whereas papaverine acted non specifically. 3. In Ca(2+)-free solution, prazosin (0.1 microM) and glaucine (0.1 mM) inhibited the contraction evoked by NA; diltiazem (0.1 mM) diminished this contraction whereas nifedipine (1 microM) had no effect. Preincubation of tissues with glaucine, diltiazem, nifedipine and prazosin did not modify the contractile response induced by caffeine. In contrast, papaverine (0.1 mM) significantly inhibited the contractions evoked by NA or caffeine in Ca(2+)-free medium. 4. Glaucine and papaverine show affinity at the [3H]-prazosin binding site and at the benzothiazepine binding site of the Ca(2+)-channel receptor complex, but have no effect at the dihydropyridine binding site in rat cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

M3-subtype muscarinic receptor that controls intracellular calcium release and inositol phosphate accumulation in gastric parietal cells.

The muscarinic receptor subtype which triggers acid secretion was investigated in isolated rabbit gastric parietal cells. Cytosolic free Ca2+ concentration ([Ca2+]i), measured with the fluorescent indicator FURA-2, increased rapidly after full agonist (carbachol) stimulation (6-8 sec), then returned to an intermediate sustained value. Other M2-agonists, oxotremorine and arecoline, produced a partial [Ca2+]i increase, whereas M1-agonists, pilocarpine and [4-m-chlorophenylcarbamoyloxyl]-2-butynyl-trimethylammonium, were without any significant effect. [Ca2+]i rise was inhibited by selective muscarinic antagonists: atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than quinuclidinylbenzilate (QNB) greater than pirenzepine greater than 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one, this sequence being characteristic of the involvement of an M3-subtype. This inhibition was shown to be stereoselective; dexetimide and (-)QNB were more potent than levetimide and (+)QNB. The IC50 values for inhibition of [Ca2+]i increase by muscarinic antagonists were in good agreement with those obtained for inhibition of phospholipase C activation. In conclusion, the muscarinic receptor that controls acid secretion appears to be of the M3-subtype and the biochemical events coupled to the activation of this receptor system are also controlled through the same subtype.     

Lipophilic amino alcohols with calcium channel blocking activity.

A series of novel lipophilic amino alcohols, analogs of the anticholinergic drug vesamicol, were evaluated for Ca2+ channel blocking activity. The effects of these drugs on depolarization-induced intracellular free Ca2+ concentration ([Ca2+]i) transients were examined in single NG108-15 cells and dorsal root ganglion (DRG) neurons in culture. [Ca2+] was recorded with the Ca2+ indicator Indo-1 and a dual emission microfluorimeter. Structure-activity studies indicated that features required for Ca2+ channel blocking activity were distinct from those required for anticholinergic activity. In particular, the Ca2+ channel blocking activity was insensitive to the configuration at the chiral center, whereas the anticholinergic activity was clearly enantioselective. One of the most active compounds, 3-(3-bromophenyl)-2-hydroxy-1-[1-(4-phenylpiperidinyl)]propane (2b), was characterized in more detail. This compound inhibited the dihydropyridine-sensitive Ca2+ channel response in NG108-15 cells, evoked by depolarization with 50 mM K+, with an IC50 of 5 microM. Field potential stimulation of DRG neurons elicited [Ca2+]i transients mediated by at least three Ca2+ channel subtypes; compound 2b inhibited the entire Ca2+ channel response with an IC50 of 1 microM. A key element required for Ca2+ channel blocking activity was the presence of an electron withdrawing substituent on the pendant phenyl ring. Modification of the amino alcohol structure may lead to more potent compounds with broad spectrum Ca2+ channel blocking activity. These structures provide a new chemical starting point for the development of Ca2+ antagonists.