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The objective of this study was to evaluate the stability of diazoxide in extemporaneously compounded oral suspensions. Oral suspensions of diazoxide 10 mg/mL were prepared from either bulk drug or capsules dispersed in either Oral Mix or Oral Mix Sugar Free. These suspensions were stored at 5°C and 25°C/60%RH in bottles and oral syringes for a total of 90 days. At predetermined time intervals, suspensions were inspected for homogeneity, color or odor change; the pH was measured and the concentration of diazoxide was evaluated by ultraviolet detection using a stability-indicating high pressure liquid chromatography method. All preparations were demonstrated to be chemically stable for at least 90 days., Competing Interests: Since 2013, Pr. Leclair agreed to conduct a total of 12 stability studies using different drugs in Oral Mix and Oral Mix SF, proprietary vehicles of Medisca Pharmaceutique Inc. All these studies were supported by unrestricted grants from Medisca and were subjected to the conditions detailed in the financial disclosure section of this manuscript. Nine of these studies are now completed and the three other studies are ongoing. This situation does not alter our adherence to PLOS One policies on sharing data and materials. After having read the competing interests policy of PLOS One, the authors report no other competing financial or nonfinancial interests.

Background: Hyperinsulinaemic hypoglycaemia (HH) is a group of clinically and genetically heterogeneous disorders characterized by unregulated insulin secretion. Abnormalities in nine different genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A, UCP2 and HNF1A) have been reported in HH, the most common being ABCC8 and KCNJ11. We describe the genetic aetiology and phenotype of Iranian patients with HH., Methods: Retrospective clinical, biochemical and genetic information was collected on 23 patients with biochemically confirmed HH. Mutation analysis was carried out for the ATP-sensitive potassium (K(ATP)) channel genes (ABCC8 and KCNJ11), GLUD1, GCK, HADH and HNF4A., Results: 78% of the patients were identified to have a genetic cause for HH. 48% of patients had mutation in HADH, whilst ABCC8/KCNJ11 mutations were identified in 30% of patients. Among the diazoxide-responsive patients (18/23), mutations were identified in 72%. These include two novel homozygous ABCC8 mutations. Of the five patients with diazoxide-unresponsive HH, three had homozygous ABCC8 mutation, one had heterozygous ABCC8 mutation inherited from an unaffected father and one had homozygous KCNJ11 mutation. 52% of children in our cohort were born to consanguineous parents. Patients with ABCC8/KCNJ11 mutations were noted to be significantly heavier than those with HADH mutation (p = 0.002). Our results revealed neurodevelopmental deficits in 30% and epilepsy in 52% of all patients., Conclusions: To the best of our knowledge, this is the first study of its kind in Iran. We found disease-causing mutations in 78% of HH patients. The predominance of HADH mutation might be due to a high incidence of consanguineous marriage in this population. Further research involving a larger cohort of HH patients is required in Iranian population.

Benzenesulfonylureas and benzenesulfonylthioureas, as well as benzenecarbonylureas and benzenecarbonylthioureas, were prepared and evaluated as myorelaxants on 30mMKCl-precontracted rat aortic rings. The most active compounds were further examined as stimulators of elastin synthesis by vascular smooth muscle cells and as inhibitors of insulin release from pancreaticβ-cells. The drugs were also characterized for their effects on glycaemia in rats. Benzenesulfonylureas and benzenesulfonylthioureas did not display any myorelaxant activity on precontracted rat aortic rings. Such an effect could be attributed to their ionization at physiological pH. By contrast, almost all benzenecarbonylureas and benzenecarbonylthioureas displayed a myorelaxant activity, in particular the benzenecarbonylureas with an oxybenzyl group linked to the ortho position of the phenyl ring. The vasodilatory activity of the most active compounds was reduced when measured in the presence of 80mMKCl or in the presence of 30mM KCl and 10μM glibenclamide. Such results suggested the involvement, at least in part, of KATP channels. Preservation of a vasodilatory activity in rat aortic rings without endothelium indicated that the site of action of such molecules was located on the vascular smooth muscle cells and not on the endothelial cells. Some of the most active compounds also stimulated elastin synthesis by vascular smooth muscle cells. Lastly, most of the active vasorelaxant drugs, except 15k and 15t at high concentrations, did not exhibit marked inhibitory effects on the insulin releasing process and on glycaemia, suggesting a relative tissue selectivity of some of these compounds for the vascular smooth muscle., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The present work aims at identifying new ion channel modulators able to target mitochondrial ATP-sensitive potassium channels (mitoKATP channels). An innovative approach should consist in fixing a cationic and hydrophobic triphenylphosphonium fragment on the structure of known KATP channel openers. Such phosphonium salts are expected to cross the biological membranes and to accumulate into mitochondria. Previous works revealed that the presence of an (R)-1-hydroxy-2-propylamino chain at the 3-position of 4H-1,2,4-benzothiadiazine 1,1-dioxides KATP channel openers increased, in most cases, the selectivity towards the pancreatic-type (SUR1/Kir6.2) KATP channel. In order to target cardiac mitoKATP channels, we decided to introduce a triphenylphosphonium group through an ester link on the SUR1-selective (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide. The new compounds were found to preserve an inhibitory activity on insulin secretion (SUR1-type KATP channel openers) while no clear demonstration of an impact on mitochondria from cardiomyocytes (measurement of oxygen consumption, respiratory parameters and ATP production on H9C2 cells) was observed. However, the most active (inhibition of insulin release) compound 17 was found to penetrate the cardiac cells and to reach mitochondria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

On the basis of the results obtained in previous series of AMPA potentiators belonging to 3,4-dihydro-2H-benzo- and 3,4-dihydro-2H-pyrido-1,2,4-thiadiazine 1,1-dioxides, the present work focuses on the design of original isosteric 3,4-dihydro-2H-thieno-1,2,4-thiadiazine 1,1-dioxides. Owing to the sulfur position, three series of compounds were developed and their activity as AMPA potentiators was characterized. In each of the developed series, potent compounds were discovered. After screening the selected active compounds on a safety in vivo test, 6-chloro-4-ethyl-3,4-dihydro-2H-thieno[2,3-e]-1,2,4-thiadiazine 1,1-dioxide (24) appeared as the most promising compound and was further evaluated. Its effects on long-term potentiation in vivo and on AMPA-mediated noradrenaline release were measured to predict its potential cognitive enhancing properties. Finally, an object recognition test performed in mice revealed that 24 was able to significantly enhance cognition, after oral administration, at doses as low as 0.3 mg/kg. This study validates the interest of the isosteric replacement of the benzene or pyridine nuclei by the thiophene nucleus in the ring-fused thiadiazine dioxides class of AMPA potentiators.

This paper shows a new finding about the decrease of relaxative response to loperamide in prostate of spontaneously hypertensive rats (SHR) as compare to normal rats (WKY). Authors demonstrated the reduction of ATP-sensitive potassium channels is responsible for this change using immunoblotting analysis and the decrease of action induced by diazoxide. This view is not mentioned before and is the first one reporting this result.

The synthesis of different series of 4- and 6-substituted R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans is described. All of these new benzopyran derivatives were bearing, at the 4-position, a phenylthiourea moiety substituted on the phenyl ring by a meta or a para-electron-withdrawing group such as Cl or CN. The study aimed at exploring the influence of the nature of the substituent at the 6-position in order to develop new benzopyran-type K(ATP) channel activators exhibiting an improved selectivity towards the insulin secreting cells. The original compounds were examined in vitro on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (vasorelaxant effect) and their activity was compared to that of the reference K(ATP) channel activators (±)-cromakalim, (±)-pinacidil, diazoxide and to previously synthesized cromakalim analogues. Structure-activity relationships indicated that the inhibitory effect on the insulin secreting cells was related to the lipophilicity of the molecules and to the size of the substituent located at the 6-position. A marked inhibitory activity on the insulin secretory process was obtained with molecules bearing a bulky tert-butyloxycarbonylamino group at the 6-position (20-23). The latter compounds were found to have the same efficacy on the pancreatic endocrine tissue than some previously described molecules. Lastly, radioisotopic experiments further identified R/S-N-4-chlorophenyl-N'-(6-tert-butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (23) as a K(ATP) channel opener., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The synthesis of 5-chloro-, 6-chloro-, and 8-chloro-substituted 3-alkylamino/cycloalkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides is described. Their inhibitory effect on the insulin releasing process and their vasorelaxant activity was compared to that of previously reported 7-chloro-3-alkylamino/cycloalkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides. "5-Chloro" compounds were found to be essentially inactive on both the insulin-secreting and the smooth muscle cells. By contrast, "8-chloro" and "6-chloro" compounds were found to be active on insulin-secreting cells, with the "6-chloro" derivatives emerging as the most potent drugs. Moreover, the "6-chloro" analogues exhibited less myorelaxant activity than their "7-chloro" counterparts. 8-Chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (25b) and 6-chloro-3-cyclobutylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (19e) were further identified as K(ATP) channel openers by radioisotopic measurements conducted on insulin-secreting cells. Likewise, current recordings on HEK293 cells expressing human SUR1/Kir6.2 channels confirmed the highly potent activity of 19e (EC(50) = 80 nM) on such types of K(ATP) channels. The present work indicates that 6-chloro-3-alkylamino/cycloalkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides appear to be more attractive than their previously described 7-chloro-substituted analogues as original drugs activating the SUR1/Kir6.2 K(ATP) channels.

The present work aimed at exploring a series of diversely 4-arylthiourea-substituted R/S-3,4-dihydro-2,2-dimethyl-6-halo-2H-1-benzopyrans structurally related to (+/-)-cromakalim. These new compounds were examined in vitro as putative potassium channel openers (PCOs) on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (relaxation of aorta ring) and their activity was compared to that of the reference K(ATP) channel activators (+/-)-cromakalim, (+/-)-pinacidil, diazoxide and of previously reported cromakalim analogues. Structure-activity relationships indicated that the most pronounced inhibitory activity on the insulin secretory process was obtained with molecules bearing a strong meta- or para-electron-withdrawing group (CN or NO(2)) on the phenyl ring of the arylthiourea moiety at the 4-position of the benzopyran nucleus (compounds 12-23). Among those, R/S-6-chloro-4-(4-cyanophenylaminothiocarbonylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran (16) was found to be the most potent benzopyran-type inhibitor of insulin release ever described. Most of these original benzopyran derivatives show increased selectivity for pancreatic versus vascular tissue. Radioisotopic investigations indicated that these new compounds activated pancreatic K(ATP) channels.

4H-1,2,4-Benzothiadiazine-1,1-dioxides with various substituents in positions 3, 5, and 7 were synthesized and tested as K(ATP) channel agonists in artificial cell systems (CHO cells transfected with SUR1/Kir6.2, and HEK 293 transfected with SUR2B/Kir6.1) as model systems for insulin-secreting pancreatic beta-cells and for smooth muscle cells, respectively. The effects of agonists were tested in intact cells using DiBAC4(3) [bis-(1,3-dibarbituric acid)trimethine oxanol] as a membrane potential dye, and the results compared with their binding affinity for the SUR2B-type K(ATP) channels using the radioligand [(3)H]P1075. Compounds with cycloalkyl and (cycloalkyl)methyl side chains in position 3 had higher affinities towards the SUR2B/Kir6.1 receptor compared with the parent compound diazoxide (1 a). Compounds with bulky, nonpolar residues in position 3 exhibited remarkable selectivity for SUR2B-type K(ATP) channels. The compound substituted with a bulky (1-adamantyl)methyl residue exhibited micromolar affinity and activity on SUR2B-type K(ATP) channels without being able to activate the SUR1-type K(ATP) channels.

Taking into account structure-activity relationships obtained with our previous series, new diversely substituted 1,2,4-pyridothiadiazine 1,1-dioxides were designed to obtain novel AMPA potentiators. The aim of this work was focused on the improvement of lipophilicity, which is well known as a critical parameter to obtain in vivo active central nervous system agents. For this purpose, two positions on the pyridine ring were privileged to insert selected groups. Among the synthesized compounds emerged 7-chloro-4-ethyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide (12d), which was evaluated in two memory tests in Wistar rats and showed cognition enhancing effects after intraperitoneal injection at doses as low as 0.3mg/kg.

The present work was aimed at exploring a series of R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(phenylaminothiocarbonylamino)-2H-1-benzopyrans structurally related to (+/-)-cromakalim and differently substituted at the 4- and 6-positions. The biological effects of these putative activators of ATP-sensitive potassium channels (K(ATP)) were characterized in vitro on the pancreatic endocrine tissue (inhibition of insulin release) and on the vascular smooth muscle tissue (relaxation of aorta rings). The biological activity of these new dimethylchroman derivatives was further compared to that of (+/-)-cromakalim, (+/-)-pinacidil, diazoxide and BPDZ 73. Structure-activity relationships indicated that an improved potency for the pancreatic tissue was obtained by introducing a meta- or a para-electron-withdrawing group such as a chlorine atom on the C-4 phenyl ring, independently of the nature of the halogen atom at the 6-position of the benzopyran nucleus. Most original dimethylchroman thioureas were more potent than their 'urea' homologues and even more potent than diazoxide at inhibiting insulin release. Moreover, and unlike (+/-)-cromakalim or (+/-)-pinacidil, such compounds appeared to be highly selective towards the pancreatic tissue. Radioisotopic and fluorimetric investigations indicated that the new drugs activated pancreatic K(ATP) channels. Lastly, conformational studies suggested that the urea/thiourea dimethylchromans can be regarded as hybrid compounds between cromakalim and pinacidil.

Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K(ATP) channel openers.

2-(4-Methoxyphenoxy)-5-nitro-N-(4-sulfamoylphenyl)benzamide and close analogues inhibit glucose stimulated insulin release through activation of Kir6.2/SUR1 K(ATP) channels of beta cells.

This communication describes the discovery and synthesis of a series of 3-trifluoromethyl-4-nitro-5-arylpyrazoles as potent K(ATP) channel agonists. The most potent compound reported is ca. 100-fold more potent than diazoxide and exhibits selectivity for the SUR1 K(ATP) channel subtype. The 4-nitro substitutent on the pyrazole ring was required for activity, and limited SAR suggests that the de-protonated pyrazole maybe the active species.

3-(Alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides were synthesized, and their activity on rat-insulin-secreting cells and rat aorta rings was compared to that of the K(ATP) channel activators diazoxide and pinacidil. Structure-activity relationships indicated that an improved potency and selectivity for the pancreatic tissue was obtained by introducing a fluorine atom in the 7-position and a short linear (preferably ethyl) or cyclic (preferably cyclobutyl) hydrocarbon chain on the nitrogen atom in the 3-position. By contrast, strong myorelaxant activity was gained by the introduction of a halogen atom different from the fluorine atom in the 7-position and a bulky branched alkylamino chain in the 3-position. Thus, 3-(ethylamino)-7-fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide (11) expressed a marked inhibitory activity on pancreatic B-cells (IC(50) = 1 microM) associated with a weak vasorelaxant effect (ED(50) > 300 microM), whereas 7-chloro-3-(1,1-dimethylpropyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide (27), which was only slightly active on insulin-secreting cells (IC(50) > 10 microM), was found to be very potent on vascular smooth muscle cells (ED(50) = 0.29 microM). Radioisotopic and electrophysiological investigations performed with 7-chlorinated, 7-iodinated, and 7-fluorinated 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides confirmed that the drugs activated K(ATP) channels. The present data revealed that subtle structural modifications of 3-(alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides can generate original compounds activating K(ATP) channels and exhibiting different in vitro tissue selectivity profiles.

Original quinolinone derivatives structurally related to diazoxide were synthesized and their effects on insulin secretion from rat pancreatic islets and the contractile activity of rat aortic rings determined. A concentration-dependent decrease of insulin release was induced by 6-chloro-2-methylquinolin-4(1H)-one (HEI 713). The average IC(50) values were 16.9+/-0.8 microM for HEI 713 and 18.4+/-2.2 microM for diazoxide. HEI 713 increased the rate of (86)Rb outflow from perifused pancreatic islets. This effect persisted in the absence of external Ca(2+) but was inhibited by glibenclamide, a K(ATP) channel blocker. Inside-out patch-clamp experiments revealed that HEI 713 increased K(ATP) channel openings. HEI 713 decreased (45)Ca outflow, insulin output and cytosolic free Ca(2+) concentration in pancreatic islets and islet cells incubated in the presence of 16.7 or 20 mM glucose and extracellular Ca(2+). The drug did not affect the K(+)(50 mM)-induced increase in (45)Ca outflow. In aortic rings, the vasorelaxant effects of HEI 713, less potent than diazoxide, were sensitive to glibenclamide and to the extracellular K(+) concentration. The drug elicited a glibenclamide-sensitive increase in (86)Rb outflow from perifused rat aortic rings. Our data describe an original compound which inhibits insulin release with a similar potency to diazoxide but which has fewer vasorelaxant effects. Our results suggest that, in both aortic rings and islet tissue, the biological effects of HEI 713 mainly result from activation of K(ATP) channels ultimately leading to a decrease in Ca(2+) inflow.

7-Chloro-3-pyridyl(alkyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxides and 3-alkylamino-7-chloro-4H-1,2,4-benzothiadiazine 1,1-dioxides containing one or more heteroatoms on the side chain in the 3 position have been synthesized in an attempt to discover new potent KATP-channel openers. The compounds were tested as putative pancreatic B-cells KATP channel openers by measuring their inhibitory activity on the insulin releasing process. The influence on the biological activity of the nature of the side chain in the 3 position is discussed.

A series of 3-alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides structurally related to diazoxide and pinacidil were synthesized and tested as possible K(ATP) channel openers on isolated pancreatic endocrine tissue as well as on isolated vascular, intestinal, and uterine smooth muscle. In contrast to previously described 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1, 1-dioxides, most of the new compounds were found to be poorly active on B-cells but exhibited clear vasorelaxant properties. 3-(3, 3-Dimethyl-2-butylamino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxide (4d) and 7-chloro-3-(3, 3-dimethyl-2-butylamino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxide (5d), two compounds bearing the alkyl side chain of pinacidil, were found to be the most active representatives of their respective series on rat aorta rings. 3-Cycloalkylalkylamino- and 3-aralkylamino-7-chloro-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides also expressed myorelaxant activity on electrically stimulated guinea pig ileum and on oxytocin-induced contractions of the rat uterus. Further biological investigations ((86)Rb efflux measurements, vasodilator potency on 30 and 80 mM KCl-induced contractions in the absence and presence of glibenclamide) revealed that compounds 4d and 5d, but not compound 5f, expressed the pharmacological profile of classical K(ATP) channel openers. In conclusion, by changing the position of the nitrogen atom in the pyridine ring, we now have obtained a family of drugs expressing an opposite tissue selectivity. Taken as a whole, the present findings also suggest that 3-alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides such as 4c, 4d, 5c, and 5d may be considered as new examples of K(ATP) channel openers expressing a pharmacological profile similar to that of pinacidil and diazoxide.

A series of 4H-1,2,4-pyridothiadiazine 1,1-dioxides and 2, 3-dihydro-4H-1,2,4-pyridothiadiazine 1,1-dioxides bearing various alkyl and aryl substituents on the 2-, 3-, and 4-positions was synthesized and tested as possible positive allosteric modulators of the (R/S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. Many compounds were found to be more potent than the reference compounds diazoxide and aniracetam as potentiators of the AMPA current in rat cortex mRNA-injected Xenopus oocytes. The most active compound, 4-ethyl-2,3-dihydro-4H-pyrido[3,2-e]-1,2, 4-thiadiazine 1,1-dioxide (31b), revealed an in vitro activity on Xenopus oocytes not far from that of cyclothiazide, the most potent allosteric modulator of AMPA receptors reported to date. Moreover, 31b, but not cyclothiazide, was found to potentiate the duration and the amplitude of the excitatory postsynaptic field potentials induced by electric stimulation in rat hippocampal slices. Such an effect could indicate, for 31b, but not for cyclothiazide, a possible interaction with postsynaptic AMPA receptor binding sites located on hippocampal CA1 neurons. Structure-activity relationships indicated that the structural requirements responsible for a biological activity on AMPA receptors are different from those responsible for an inhibitory activity on the insulin releasing process (putative ATP-sensitive K+-channel openers). For instance, 31b and other related dihydropyridothiadiazines were found to be ineffective as inhibitors of insulin release from rat pancreatic B-cells, in contrast to diazoxide and known pyridothiadiazines reported as ATP-sensitive K+-channel openers. Conversely, the pyridothiadiazines active on B-cells were found to be ineffective as potentiators of the AMPA currents in Xenopus oocytes. Thus, 31b appeared to be more specific than diazoxide as an AMPA receptor modulator. This compound may be considered as a new pharmacological tool, different from diazoxide and cyclothiazide, for studying AMPA receptors. Moreover, 31b can also constitute a new therapeutic agent for the treatment of cognitive disorders.

The diazoxide derivative IDRA 21 and other positive modulators of (AMPA)-type glutamate receptors are considered potential memory-enhancing agents. However, AMPA receptor activation contributes to CA1 hippocampal neuron damage from global ischemia in rodents, raising the possibility that 7-chloro-3-methyl-3-4-dihydro-2H-1,2,4 benzothiadiazine S,S-dioxide (IDRA 21) or drugs with similar actions may worsen ischemic neuronal injury. Here we demonstrate that glutamate plus IDRA 21 kills cultured rat hippocampal neurons by AMPA receptor activation, and, in vivo, 12 and 24 mg/kg of IDRA 21 given orally increases CA1 neuron loss produced by 10 minutes of global ischemia. Treating patients with drugs that potentiate AMPA receptor activation will have to consider these potential effects, particularly when coexistent with conditions in which excessive activation of AMPA receptors may occur (eg, stroke, seizures).

4-N-Substituted and -unsubstituted 3-alkyl- and 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides were synthesized and tested vs diazoxide and selected 3-alykl- and 3-(alkylamino)-7-chloro-4H-1,2,4-benzothiadiazine 1,1-dioxides as potassium channel openers on pancreatic and vascular tissues. Several 4-N-unsubstituted 3-(alkylamino)pyridothiadiazines and some 3-(alkylamino)-7-chlorobenzothiadiazines were found to be more potent than diazoxide for the inhibition of the insulin-releasing process. Moreover, the 3-(alkylamino)pyridothiadiazines appeared to be more selective for the pancreatic than for the vascular tissue. By means of the pharmacological results obtained on pancreatic B-cells, structure--activity relationships were deduced and a pharmacophoric model for the interaction of these drugs with their receptor site associated to the pancreatic K(ATP) channel was proposed. According to their selectivity for the B-cell (endocrine tissue) vs the vascular (smooth muscle tissue) ionic channel, selected 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4,-thiadiazine 1,1-dioxides may serve as pharmacological tools in studying the K(ATP) channels ("pancreatic-like" K(ATP) channels) in other tissues.

A series of substituted-1,2,4-benzothiadiazin-1,1-dioxide derivatives was designed and synthesized as potassium channel modulators. Various sulfonylurea moieties were introduced on positions 3 and 7 of the heterocycle without, or by means of, methylene and phenyl spacers. On rat aortic rings, several compounds displayed vasodilating activities, especially compound 24, which was more active than cromakalim and diazoxide at low doses (0.1 microM) and more active than diazoxide between 1 and 10 microM.

A conformational analysis has been performed with SYBYL starting from the energy optimized (Tripos force field) X-ray conformation of (R,S)-pinacidil. The geometry of four selected low energy conformers has been reoptimized using the AM1 semiempirical Molecular Orbital method (MOPAC 5.0), and the total energy of the optimized conformers has been compared. In spite of an apparent structural dissimilarity, a good analogy has been found between the calculated isopotential map of diazoxide and that of at least one selected low energy conformer of pinacidil. It has been suggested that, unlike cromakalim, the low but observable activity of pinacidil on pancreatic B-cells could be explained by adoption for this compound of a conformation having a diazoxide-like stereoelectronical imprint which could assume a similar interaction on the same biological receptor.

Among the non-NMDA (non-N-methyl-D-aspartic acid) glutamate receptors, the AMPA (alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid) selective receptors are characterized by a fast occurring desensitization. We and others have searched for specific modifiers of the rapid desensitization of AMPA responses in hippocampal slices using the patch-clamp technique. Aniracetam (1-(4-methoxybenzoyl)-2-pyrrolidinone) and diazoxide (7-chloro-3-methyl-2H-1,2,4-benzo-thiadiazine 1,1-dioxide) (1 mM) increased glutamate-activated currents recorded from voltage-clamped CA1 pyramidal neurons in presence of 5 microM MK-801 (dizocilpine; 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine) by 2.5 fold. Cyclothiazide (3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-2H-1,2,4-benzoth ia diazine-7-sulfonamide 1,1-dioxide) (100 microM), a chemical congener of diazoxide, completely removed the desensitization of the AMPA response measured with fast application in excised outside-out patches. At this concentration cyclothiazide produced an 18 fold enhancement of the glutamate current. Eighteen diazoxide analogues (2H-1,2,4-benzothiadizines: IDRA 2-19) were then tested but none of them was as effective as diazoxide. Three analogues of cyclothiazide (3,4-dihydro-2H-1,2,4-benzothiadiazines: IDRA 20-22) were also tested and none of them were as potent as the parent compound. However, IDRA 21 produced a response 3 times larger than diazoxide. Moreover, while cyclothiazide and diazoxide potentiated kainate responses for all the doses that decreased AMPA receptor desensitization, IDRA 21, similarly to aniracetam, inhibited AMPA receptor desensitization preferentially. These results suggest that similarly to NMDA receptors the structure of AMPA receptors may include a center that regulates desensitization.