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A "heart-cut" two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC-LC-MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-1,1-dioxide ((±)-1), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)-1 and its two hepatic metabolites. In the "heart-cut" two-dimension achiral-chiral configuration, (±)-1 and (±)-1-d4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)-1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A stopped-flow HPLC method was developed to evaluate configurational and chemical stability of pharmaceutical compounds employing immobilized artificial membranes (IAM) column to simulate conditions that pharmaceutical compounds will meet in vivo. The method was applied to recent developed chiral 5-arylbenzothiadiazine derivatives possessing high positive allosteric modulatory (PAM) activity on AMPA receptor. In particular the stopped-flow HPLC method developed used a chiral column to separate single enantiomer of the compounds that are forced into an IAM column where configurational and chemical stability was evaluated in simulated gastrointestinal fluids (pH 1.2 and 6.8 at 37.5 °C) to simulate in vivo conditions. The results were compared to those obtained by dynamic and off-column methods to evaluate the effects of stationary phases on kinetic constant of enantiomerization and hydrolysis. The results suggested that the phospholipids environment of IAM stationary phases, which mimes biological membrane, greatly influence the hydrolysis process increasing the chemical stability of tested compounds while no influence on enantiomerization kinetic was observed. Therefore it is possible to suppose that 5-arylbenzothiadiazine derivatives should not hydrolysed in vivo while they should rapidly racemized in aqueous solvents. The method could represents a rapid and value tool to predict chemical and configurational stability of new chemical entities to decrease the number of animal studies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, configurational and chemical stability of (R,R),(S,S),(R,S),(S,R)-3,6-dimethyl-2,3,5,6-tetrahydro[1,2,4]thiadiazino[6,5,4-hi]indole 1,1-dioxide (1) were investigated by dynamic and stopped-flow HPLC methods. Single epimeric mixtures (R,R),(R,S)-1 and (S,S),(S,R)-1 were obtained combining synthetic and chromatographic strategies. Separation of (R,R)-1 and (R,S)-1 was achieved by chiral chromatography and absolute configuration of eluted epimers has been assigned basing on molecular modelling calculations. Epimerization and hydrolysis of (R,R),(R,S)-1 have been studied by classical off-column, dynamic HPLC and stopped-flow HPLC methods. The influence of different parameters, such as temperature, pH and dielectric constant was evaluated. The data obtained indicate that (R,R),(R,S)-1 undergoes to a rapid epimerization in aqueous solvent and hydrolysis in acidic conditions. Moreover, epimerization and hydrolysis were investigated in presence of an artificial membrane and in physiological buffers (pH 2.2 and 7.0 at 37.5°C) to simulate in vivo conditions., (Copyright © 2010 Elsevier B.V. All rights reserved.)

An on-column stopped-flow bidimensional recycling HPLC procedure was developed to obtain an enantiomeric enrichment starting from a racemic mixture. The method developed was applied to two chiral compounds of pharmaceutical interest, (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide (1) and (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-)IDRA21, (2)), since the pharmacological activity of the two benzothiadiazine derivatives investigated has been ascribed to only one enantiomer. Starting from a racemic mixture it was possible to obtain about 95% of pure enantiomer. The procedure was applied both in reverse-phase mode and in normal-phase mode. The scaled up and automatization of the novel analytical HPLC procedure represents a powerful tool to obtain pure enantiomer starting from racemic compounds without cumbersome stereoselective synthesis or expensive enantiopurification processes.

A novel stopped-flow multidimensional HPLC (sf-MD-HPLC) procedure has been developed to investigate simultaneously the effect of the pH on the enantiostability and hydrolysis of (+/-)-7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide [(+/-)IDRA21]. It was possible to determinate the rate constants and free energy barriers of enantiomerization and hydrolysis rate constants of (+/-)IDRA21, by using two chiral stationary phases (CSPs) and one achiral C18 column. A classical batchwise kinetic method was used to calculate rate constants of hydrolysis at the same temperature and in the same buffers used in sf-MD-HPLC. The good agreement of the results obtained validate the sf-MD-HPLC procedure. Furthermore, hydrolysis rate constants of (+/-)IDRA21 were calculated in a series of buffers over a pH range of 1.20-10.60 at 37 degrees C in order to evaluate the influence of the pH on hydrolysis.

An on-column stopped-flow multidimensional HPLC (sfMDHPLC) procedure using two chiral stationary phases (CSPs) and one achiral C18 column was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide. Moreover, a stopped-flow HPLC (sfHPLC) method previously developed was applied to the determination of kinetic parameters of enantiomerization of the above compound in the presence of a CSP. The individual enantiomers of the studied compound were isolated in parallel by preparative HPLC and the rate constants and free energy barriers of enantiomerization were determined in different solvents (off-column method). The data obtained by sfMDHPLC, sfHPLC and off-column methods were compared. The (S) enantiomer of the studied compound (S18986) was prepared by asymmetric synthesis and subsequently purified by preparative HPLC, followed by the determination of rate constants and free energy barriers of enantiomerization in different buffer solutions at pH 2-9.3.

Taurine has been shown to exert many biological effects. Among them, the ability of this amino acid to induce a reduction of arterial blood pressure, when administered intracerebroventricularly (i.c.v.) to mammals, has been established since a long time ago. To gain further information on the structure-activity requirements for taurine's hypotensive effect, the synthesis of 2-methyltaurine (2-aminopropanesulphonic acid) and the asymmetric synthesis of its enantiomers were performed; the enantiomeric purity of (R)- and (S)-2-methyltaurine was assayed by differential scanning calorimetry (DSC) and HPLC. The findings here reported show that the hypotensive effect elicited by the i.c.v. administration of racemate is mainly due to the (S)-enantiomer. The specificity of this effect was proved by the ability of the taurine antagonist TAG (6-aminomethyl-4H-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) to prevent it.

A cyclodextrin-modified micellar capillary electrophoretic method was developed for the chiral separation of 2-methyltaurine. The racemic mixture was separated after derivatization with dansyl chloride. The enantiomeric separation of the dansyl derivative was done using electrolyte solutions containing beta-cyclodextrin (beta-CD) or different ratios of beta- and gamma-CD. The mixture of beta- and gamma-CD optimally resolved the compound studied.

In this study we tested the potential taurine-antagonistic properties of three rigid analogs of taurine, 3-amino- (1), 3-hydrazino- (2) and 3-aminomethyl-1,2,4-benzothiadiazine-1,1-dioxide (3), which were prepared in our laboratory, using TAG (6-aminomethyl-3-methyl-1,2,4-benzothiadiazine-1,1- dioxide) (4), the only antagonist of taurine so far available, as reference compound in "in vivo" and "in vitro" experiments. Some physicochemical properties of (1), (2) and (3) were studied and the synthesis of TAG (4) was improved with a new preparative method. A dose-effect study performed by injecting intracerebroventricularly (1), (2) and (3) showed that these compounds have none of exciting effects exerted by the high doses of TAG (4). (1) and (3) as well as TAG (4), were found to antagonize the controlateral turning induced by the intracerebro injection of taurine and to potentiate the sedative effect of diazepam. We failed to find specific binding for taurine in different brain synaptic membrane preparations using 3H-taurine as radioligand and taurine, (1) and (3) as binding displacer. (1), (3) and TAG (4) however were found to antagonize the inhibitory effect of taurine on 3H-diazepam binding. These results seem to indicative that at least (1) and (3), which were more extensively studied than (2) because of their better solubility, are taurine antagonists with an apparent better selectivity than TAG (4).