Playing with opening and closing of heterocycles: using the cusmano-ruccia reaction to develop a novel class of oxadiazolothiazinones, active as calcium channel modulators and P-glycoprotein inhibitors.

As a result of the ring-into-ring conversion of nitrosoimidazole derivatives, we obtained a molecular scaffold that, when properly decorated, is able to decrease inotropy by blocking L-type calcium channels. Previously, we used this scaffold to develop a quantitative structure-activity relationship (QSAR) model, and we used the most potent oxadiazolothiazinone as a template for ligand-based virtual screening. Here, we enlarge the diversity of chemical decorations, present the synthesis and in vitro data for 11 new derivatives, and develop a new 3D-QSAR model with recent in silico techniques. We observed a key role played by the oxadiazolone moiety: given the presence of positively charged calcium ions in the transmembrane channel protein, we hypothesize the formation of a ternary complex between the oxadiazolothiazinone, the Ca2+ ion and the protein. We have supported this hypothesis by means of pharmacophore generation and through the docking of the pharmacophore into a homology model of the protein. We also studied with docking experiments the interaction with a homology model of P-glycoprotein, which is inhibited by this series of molecules, and provided further evidence toward the relevance of this scaffold in biological interactions.

Absolute configuration and biological profile of two thiazinooxadiazol-3-ones with L-type calcium channel activity: a study of the structural effects.

In the framework of our interest in racemic thiazinooxadiazol-3-ones we determined the absolute configuration and the biological activity as L-type calcium channel blockers of two compounds that differ in the length of the acetal chain, which could affect the pharmacological profile. We observed an interesting inversion of the stereoselectivity, with the activity residing on the R-form for a short chain compound (n = 1) and on the S-form for a long chain one (n = 12). The length of the linear acetal chain appears to be able to invert the stereoselectivity of such a class of compounds, and in silico simulations suggested that this different behaviour might be explained by different hydrophilic and hydrophobic interactions with the binding site.

Inhibition of MDR1 activity and induction of apoptosis by analogues of nifedipine and diltiazem: an in vitro analysis.

We report herein the reversal of multidrug resistance-1 (MDR1) in A2780/DX3 cells by the two nifedipine-like compounds 1 and 2 that are part of a library of 1,4-dihydropyridines (1,4-DHPs) calcium-channel modulators bearing in C-4 a different substituted imidazo[2,1-b]thiazole system. By methylthiazol tetrazolium (MTT) assay, cytofluorimetry, and fluorescence microscopy we evaluated their ability to reverse MDR in our cell system. Moreover, together with compound 3 (the diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one) we analyzed their ability to potentiate the triggering of apoptosis after exposure to doxorubicin, through the nuclear morphological analysis after 4',6-diamidino-2-phenylindole (DAPI), the fluorescein isothiocyanate (FITC)-Annexin-V/propidium iodide (PI) staining and the caspase activity determination. Our results demonstrate that compounds 1 and 2, at concentrations showing a very low (5%) or absent inhibition of cell proliferation, in combination with doxorubicin enhance its antiproliferative activity (from 30% to 54% IC(50) reduction) in A2780/DX3 cells through an increase of doxorubicin intracellular accumulation. These compounds together with compound 3, which has already been demonstrated to act as a potent inhibitor of MDR1 function, were also able to significantly potentiate the activation of the apoptosis machinery triggered by the exposure to doxorubicin. In conclusion, our results identify two new molecules structurally related to the calcium-channel blocker nifedipine, but characterized by a very low LTCC blockers activity, able to potentiate the antiproliferative and apoptotic activities of doxorubicin through an increase of its intracellular concentration likely caused by the inhibition of MDR1 function.

Imidazo[2,1-b]thiazole system: a scaffold endowing dihydropyridines with selective cardiodepressant activity.

The synthesis, characterization, and functional in vitro assays in cardiac tissues and smooth muscle (vascular and nonvascular) of a number of 4-imidazo[2,1- b]thiazole-1,4-dihydropyridines are reported. The binding properties for the novel compounds have been investigated and the interaction with the binding site common to other aryl-dihydropyridines has been demonstrated. Interestingly, the novel 4-aryl-dihydropyridines are L-type calcium channel blockers with a peculiar pharmacological behavior. Indeed, the imidazo[2,1- b]thiazole system is found to confer to the dihydropyridine scaffold an inotropic and/or chronotropic cardiovascular activity with a high selectivity toward the nonvascular tissue. Finally, molecular modeling studies were undertaken for the most representative compounds with the aim of describing the binding properties of the new ligands at molecular level and to rationalize the found structure-activity relationship data. Due to the observed pharmacological behavior of our compounds, they might be promising agents for the treatment of specific cardiovascular pathologies such as cardiac hypertrophy and ischemia.

Diltiazem analogues: the last ten years on structure activity relationships.

Cardiovascular diseases as hypertension, angina and/or supraventricular arrhythmias are among the most important death causes in the world. For the treatment of heart pathologies, calcium channel entry blockers are very important drugs, owing to their therapeutic versatility. Although few calcium antagonists described until today are structurally related to diltiazem and to the benzothiazepine class, the still high pharmaceutical interest on diltiazem analogues justifies this review. Diltiazem and its first analogues developed in the early '70s became popular in the '80s, and were pharmacologically characterized for a long time. It is in the '90s that several research groups carried out structural variations identifying novel scaffolds for diltiazem-related compounds, with significant calcium antagonist behaviour. Recently, a series of thiazino-oxadiazolone derivatives were identified as potent and selective antagonists for calcium influx into cardiac cells, and they were subsequently used to search for novel chemotypes by means of virtual screening techniques. The resulting hits could open interesting perspectives for the development of drugs to treat cardiovascular diseases. In the present review, an updated collection of diltiazem analogues is reported over the last ten years. The chemical structure and the structure activity relationships will be given, with additional mention to the potential therapeutic applications.

Novel quinolizidinyl derivatives as antiarrhythmic agents.

Eighteen analogues of lidocaine, mexiletine, and procainamide were synthesized, replacing their aminoalkyl chains with the rigid and cumbersome quinolizidine nucleus. The target compounds were tested for antiarrhythmic, inotropic, and chronotropic effects on isolated guinea pig (gp) heart tissues and to assess calcium antagonist activity. Most compounds exhibited from moderate to high antiarrhythmic activity, and compounds 7, 9, and 19 were more active and potent than quinidine and lidocaine, while producing only modest inotropic, chronotropic, and vasorelaxant effects. These compounds were studied on spontaneously beating Langendorff-perfused gp heart. While quinidine and amiodarone produced a dose-dependent prolongation of all the ECG intervals, compounds 7, 9, and 19, even at concentrations 10-20 times higher than EC50 for the antiarrhythmic activity, only moderately prolonged the PR and QT intervals, leaving unchanged the QRS complex. Ether 7 deserves further investigations due to its interesting cardiovascular profile.

WB4101-related compounds: new, subtype-selective alpha1-adrenoreceptor antagonists (or inverse agonists?).

Our previous structure-affinity relationship study had considered the enantiomers of the naphthodioxane, tetrahydronaphthodioxane, and 2-methoxy-1-naphthoxy analogues (compounds 1, 3, and 2, respectively) of 2-(2,6-dimethoxyphenoxyethylaminomethyl)-1,4-benzodioxane, the well-known alpha1-adrenoceptor (alpha1-AR) antagonist WB4101, showing that such modifications significantly modulate the affinity and selectivity profile for alpha1-AR subtypes and 5-HT1A receptor. Here, we extend investigations to antagonist activity enclosing new enantiomeric pairs, namely those of the methoxytetrahydronaphthoxy and methoxybiphenyloxy WB4101 analogues (4 and 5-7, respectively) and of a double-modified WB4101 derivative (8) resulting from hybridization between 2 and 3. We found that (S)-2 is a very potent (pA2 10.68) and moderately selective alpha1D-AR antagonist and the hybrid (S)-8 is a potent (pA2 7.98) and highly selective alpha1A-AR antagonist. Both of these compounds and (S)-WB4101 seem to act as inverse agonists in a vascular model. The results, which generally validate the logic we followed in designing these eight compounds, are acceptably rationalized by comparative SAR analysis of binding and functional affinities.

Calcium channel antagonists discovered by a multidisciplinary approach.

A multidisciplinary project has led to the discovery of novel, structurally diverse, L-type calcium entry blockers (CEBs). The absolute configuration of a recently reported CEB has been determined by vibrational circular dichroism spectroscopy, to assign the stereospecificity of the ligand-channel interaction. Thereafter, a virtual screening procedure was performed with the aim of identifying novel chemotypes for CEBs, starting from a database of purchasable compounds; 340,000 molecules were screened in silico in order to prioritize structures of interest for bioscreening. As a result, 20 compounds were tested in vitro, and functional and binding assays revealed several hits with promising behavior as CEBs.

Effects of probiotic bacteria on gastrointestinal motility in guinea-pig isolated tissue.

AIM: To evaluate the intestinal motility changes evoked by 8 bacterial strains belonging to Bifidobacterium, Lactobacillus and Streptococcus genera within the probiotic preparation VSL#3. METHODS: Ileum and proximal colon segments isolated from guinea-pigs were used as a study model. Entire cells and cell fractions (cell debris, cell wall fraction, cytoplasmatic fraction, proteinaceous and non-proteinaceous cytoplasmatic components) of VSL#3 strains and, as controls, Escherichia coli, Salmonella aboni and Bacillus licheniformis were tested in this in vitro model. RESULTS: Among the bacterial cell fractions tested, only the cytoplasmatic fraction modified intestinal motility. Lactobacillus strains stimulated the contraction of ileum segment, whereas all probiotic strains tested induced proximal colon relaxation response. The non-proteinaceous cytoplasmatic components were responsible for the colon relaxation. CONCLUSION: The results obtained in this study suggest that the proximal colon relaxation activity showed by the probiotic bacteria could be one of the possible mechanisms of action by which probiotics exert their positive effects in regulating intestinal motility.

Understanding Oxadiazolothiazinone Biological Properties: Negative Inotropic Activity versus Cytochrome P450-Mediated Metabolism.

We present a series of oxadiazolothiazinones, selective inotropic agents on isolated cardiac tissues, devoid of chronotropy and vasorelaxant activity. Functional and binding data for the precursor of the series (compound 1) let us hypothesize LTCC blocking activity and the existence of a recognition site specific for this scaffold. We synthesized and tested 22 new derivatives: introducing a para-methoxyphenyl at C-8 led to compound 12 (EC50 = 0.022 µM), twice as potent as its para-bromo analogue (1). For 10 analogues, we extended the characterization of the biological properties by including the assessment of metabolic stability in human liver microsomes and cytochrome P450 inhibition potential. We observed that the methoxy group led to active compounds with low metabolic stability and high CYP inhibition, whereas the protective effect of bromine resulted in enhanced metabolic stability and reduced CYP inhibition. Thus, we identified two para-bromo benzothiazino-analogues as candidates for further studies.

A new class of selective myocardial calcium channel modulators. 2. Role of the acetal chain in oxadiazol-3-one derivatives.

In the framework of the continuing interest of this research group in the use of 8-aryl-8-hydroxy-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3-ones (1) as calcium entry blockers, a number of acetals were synthesized and assayed "in vitro". All of them are structurally related to diltiazem and pyrrolobenzothiazines. The effect on the biological profile was measured by functional assays for a wide variety of acetal residues: saturated linear and branched chains, short and long unsaturated E and/or Z chains as well as benzyl and methylcyclohexyl residues. From selective assays on the most active derivative (5b) (EC(50) = 0.04 microM), which is 20 times more active than diltiazem (EC(50) = 0.79 microM), a muscarinic or adenosinic mechanism of action was excluded. A 3D QSAR model was obtained and validated with homologous literature data, and a virtual receptor scheme was derived for the unknown binding site. The following pharmacophoric features favorably affect the potency: one positively charged center, three lipophilic groups, and two hydrogen-bonding acceptor groups.

Alpha1- and alpha2-adrenoreceptor antagonist profiles of 1- and 2-[omega-(4-arylpiperazin-1-yl)alkyl]-1,2,3-benzotriazoles.

A series of pharmacologically interesting 1- and 2-[omega-(4-arylpiperazin-1-yl)alkyl]-1,2,3-benzotriazoles, compounds 1-27, were synthesized (Scheme) and subjected to various biological studies to identify structure-activity relationships (SAR). The new compounds were found to exhibit good non-selective binding affinity towards the alpha1-adrenoreceptor (Table 1). In several cases, high functional antagonism was observed towards the alpha1A-, alpha1B-, and alpha1D-adrenoreceptor subtypes (Table 2). The selectivity for these three subtypes was comparable with or superior to that displayed by the standard drug prazosin. The most-common selectivity rank order was alpha1D > alpha1B > alpha1A, followed by alpha1B > alpha1D > alpha1A. In functional experiments, antagonism towards the alpha2-adrenoreceptor was generally low; however, a few compounds were endowed with significant antagonist properties (pA2 values of up to 7.87).

Synthesis and L-type calcium channel blocking activity of new chiral oxadiazolothiazinones.

Oxadiazolo[3,4-c][1,4]thiazin-3-ones are cardiovascular agents that block L-type calcium channels. Previous data of cardiac and vasorelaxant activity on guinea-pig for several derivatives indicated the two positions ortho to the thiazine's sulphur as crucial for modulating the activity; but these positions are likely susceptible to metabolic biotransformations, as indicated by in silico predictions. We designed new derivatives, and obtained three negative inotropic agents with EC50 in the low nanomolar range, more potent than all the precursors published so far. In particular, benzocondensation at the thiazine ring led to 3a (EC50 = 0.013 µM) and 3b (EC50 = 0.006 µM). Besides negative inotropy, we also observed relaxant activity on nonvascular muscle in the micromolar range. We resolved the new derivatives by chiral chromatography, and determined their absolute configuration by comparing experimental and calculated chiroptical properties (VCD, ECD and ORD): they hold the same absolute configuration-optical rotation relationship, (S)-(+)/(R)-(-). Both cardiac and nonvascular activity are majorly or mostly retained in the R-form for all the compounds, but for the nonvascular activity we observed a strong stereoselectivity for 3a, with the R-form in the nanomolar range (IC50 = 0.020 µM) and 259-fold more potent than the S-one.

Cardiovascular characterization of [1,4]thiazino[3,4-c][1,2,4]oxadiazol-1-one derivatives: selective myocardial calcium channel modulators.

As an extension of previous investigations (Tetrahedron 1999, 55, 5433-5440; J. Heterocycl. Chem. 2000, 37, 875-878), a series of 21 [1,4]thiazino[3,4-c][1,2,4]oxadiazolones, which has already been synthesized (except for compounds 5a, 5b, 6), was evaluated as calcium entry blockers by functional studies, namely, in isolated guinea-pig left and right atria and K(+)-depolarized aortic strips. With the aim of investigating the effect of a condensed benzene ring on the molecular structure and the influence of substituents on the 8-phenyl ring of 4a, ab initio computations (RHF/3-21G) were performed on compounds 3, 4a-d, 4f, and 4k. The results obtained show that many of the compounds studied are potent and selective negative inotropic agents; in particular, compounds 4e and 4f are about 3- and 2-fold more potent than diltiazem, respectively.

Castanea sativa Mill. extract contracts gallbladder and relaxes sphincter of Oddi in guinea pig: a natural approach to biliary tract motility disorders.

Impaired gallbladder motility is a contributing factor to gallstone formation. Since many drugs delaying intestinal motility inhibit gallbladder emptying, the aim of the present study was to evaluate the effect on gallbladder and sphincter of Oddi motility of a Natural Chestnut Wood Extract (NEC) that reduces intestinal motility. In order to evaluate the effect of the extract in normal- and high-risk gallstone conditions, the investigation was performed using tissues from animals fed normal and lithogenic diet. Fifty guinea pigs were administered either control or lithogenic diet. The spontaneous motility of the gallbladder and sphincter of Oddi were recorded on isolated gallbladder tissues; thereafter, the effect of NEC on motility was tested and compared with carbachol (CCh), potassium chloride (KCl), noradrenaline (NA), and A71623. Compared to controls, the lithogenic diet induced an irregular and disordered motor pattern in both the gallbladder and sphincter of Oddi. NEC increased gallbladder and decreased sphincter of Oddi spontaneous motility independently of cholinergic, adrenergic, and CCK-1 receptor-mediated pathways both in controls and in lithogenic diet-fed animals, although the effect was lower in the latter group. The effect was reversible and mediated by calcium channels. The natural extract of chestnut increasing gallbladder contraction and inducing the relaxation of the sphincter of Oddi can be of benefit in pathological conditions associated with increased transit time at risk of gallstones.

Ligand based approach to L-type calcium channel by imidazo[2,1-b]thiazole-1,4-dihydropyridines: from heart activity to brain affinity.

The synthesis, characterization, and functional in vitro assay in cardiac and smooth muscle (vascular and nonvascular) of a series of 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines are reported. To define the calcium blocker nature of the imidazo[2,1-b]thiazole-1,4-DHPs and their selectivity on Cav1.2 and Cav1.3 isoforms, we performed binding studies on guinea pig atrial and ventricular membranes on intact cells expressing the cloned Cav1.2a subunit and on rat brain cortex. To get major insights into the reasons for the affinity for Cav1.2 and/or Cav1.3, molecular modeling studies were also undertaken. Some physicochemical and pharmacokinetic properties of selected compounds were calculated and compared. All the biological data collected and reported herein allowed us to rationalize the structure-activity relationship of the 4-imidazo[2,1-b]thiazole-1,4-DHPs and to identify which of these enhanced the activity at the central level.

Sweet chestnut (Castanea sativa Mill.) bark extract: cardiovascular activity and myocyte protection against oxidative damage.

This work was aimed at evaluating the cardioprotective effects of Castanea sativa Mill. (CSM) bark extract characterized in its phenolic composition by HPLC-DAD-MS analysis. The study was performed using primary cultures of neonatal rat cardiomyocytes to investigate the antioxidant and cytoprotective effects of CSM bark extract and isolated guinea pig left and right atria, left papillary muscle, and aorta to evaluate its direct effect on cholinergic and adrenergic response. In cultured cardiomyocytes the CSM bark extract reduced intracellular reactive oxygen species formation and improved cell viability following oxidative stress in dose-dependent manner. Moreover, the extract decreased the contraction induced by noradrenaline (1 µ M) in guinea pig aortic strips and induced transient negative chronotropic and positive inotropic effects without involvement of cholinergic or adrenergic receptors in the guinea pig atria. Our results indicate that CSM bark extract exhibits antioxidant activity and might induce cardioprotective effect.

Curcuma longa extract exerts a myorelaxant effect on the ileum and colon in a mouse experimental colitis model, independent of the anti-inflammatory effect.

BACKGROUND: Curcuma has long been used as an anti-inflammatory agent in inflammatory bowel disease. Since gastrointestinal motility is impaired in inflammatory states, the aim of this work was to evaluate if Curcuma Longa had any effect on intestinal motility. METHODS: The biological activity of Curcuma extract was evaluated against Carbachol induced contraction in isolated mice intestine. Acute and chronic colitis were induced in Balb/c mice by Dextran Sulphate Sodium administration (5% and 2.5% respectively) and either Curcuma extract (200 mg/kg/day) or placebo was thereafter administered for 7 and 21 days respectively. Spontaneous contractions and the response to Carbachol and Atropine of ileum and colon were studied after colitis induction and Curcuma administration. RESULTS: Curcuma extract reduced the spontaneous contractions in the ileum and colon; the maximal response to Carbachol was inhibited in a non-competitive and reversible manner. Similar results were obtained in ileum and colon from Curcuma fed mice. DSS administration decreased the motility, mainly in the colon and Curcuma almost restored both the spontaneous contractions and the response to Carbachol after 14 days assumption, compared to standard diet, but a prolonged assumption of Curcuma decreased the spontaneous and Carbachol-induced contractions. CONCLUSIONS: Curcuma extract has a direct and indirect myorelaxant effect on mouse ileum and colon, independent of the anti-inflammatory effect. The indirect effect is reversible and non-competitive with the cholinergic agent. These results suggest the use of curcuma extract as a spasmolytic agent.

Cystic fibrosis: a new target for 4-Imidazo[2,1-b]thiazole-1,4-dihydropyridines.

The pharmacology of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel has attracted significant interest in recent years with the aim to search for rational new therapies for diseases caused by CFTR malfunction. Mutations that abolish the function of CFTR cause the life-threatening genetic disease cystic fibrosis (CF). The most common cause of CF is the deletion of phenylalanine 508 (ΔF508) in the CFTR chloride channel. Felodipine, nifedipine, and other antihypertensive 1,4-dihydropyridines (1,4-DHPs) that block L-type Ca(2+) channels are also effective potentiators of CFTR gating, able to correct the defective activity of ΔF508 and other CFTR mutants ( Mol. Pharmacol. 2005 , 68 , 1736 ). For this purpose, we evaluated the ability of the previously and newly synthesized 4-imidazo[2,1-b]thiazoles-1,4-dihydropyridines without vascular activity and inotropic and/or chronotropic cardiac effects ( J. Med. Chem. 2008 , 51 , 1592 ) to enhance the activity of ΔF508-CFTR. Our studies indicate compounds 17, 18, 20, 21, 38, and 39 as 1,4-DHPs with an interesting profile of activity.

Preliminary finding on a new calcium channel entry blocker chemotype: 5,6-diamino-4-hydroxy-2-mercaptopyrimidine derivatives.

We report the preliminary in vitro characterization of a series of pyrimidines as a new chemotype that modulates cardiovascular parameters and relaxes ileum smooth muscle according to classical calcium entry blockers. Tested compounds showed an interesting negative inotropic selectivity. In patch-clamp experiments they block L- over T-type calcium currents. Two requisites seem essential for the activity: lipophilic substituents in positions 2 and 5 of the pyrimidine ring and the acetamidic function in position 6.