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Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of M. tuberculosis drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against M. tuberculosis H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis.

A combination of NMR spectroscopy and theoretical methods Density functional theory including dispersion corrections (DFT-D) was used to study the structures of Lumogen and salicylaldazine. In the solid state, Lumogen exists as the dihydroxy tautomer 1a (an azine, C=N-N=C) as was already known from an X-ray determination. In a deuterated dimethyl sulfoxide solution, another tautomer is observed besides 1a; its structure corresponds to the hydroxy-oxo tautomer 1b (a hydrazone, C=N-NH-Csp(2)). In what concerns salicylaldazine, we have observed only the dihydroxy tautomer 2a., (Copyright © 2013 John Wiley & Sons, Ltd.)

Thioridazine (THD) is a commonly prescribed phenotiazine neuroleptic drug, which is extensively biotransformed in the organism producing as main metabolites sulfoxides and a sulfone by sulfur oxidation. Significant differences have been observed in the activity of the THD enantiomers as well as for its main metabolites, and enantioselectivity phenomena have been proved in the metabolic pathway. Here the assignment of the absolute configuration at the sulfur atom of enantiomeric THD-2-sulfoxide (THD-2-SO) has been carried out by circular dichroism (CD) spectroscopy. The stereoisomers were separated by HPLC on Chiralpak AS column, recording the CD spectra for the two collected enantiomeric fractions. The theoretical electronic CD spectrum has been obtained by the TDDFT/B3LYP/6-31G*, as Boltzmann averaging of the contributions calculated for the most stable conformations of the drug. The comparison of the simulated and experimental spectra allowed the absolute configuration at the sulfur atom of the four THD-2-SO stereoisomers to be assigned. The developed method should be useful for a reliable correlation between stereochemistry and activity and/or toxicity.

The purpose of this study was to develop a method for the stereoselective analysis of thioridazine-2-sulfoxide (THD-2-SO) and thioridazine-5-sulfoxide (THD-5-SO) in culture medium and to study the biotransformation of rac-thioridazine (THD) by some endophytic fungi. The simultaneous resolution of THD-2-SO and THD-5-SO diastereoisomers was performed on a CHIRALPAK AS column using a mobile phase of hexane:ethanol:methanol (92:6:2, v/v/v)+0.5% diethylamine; UV detection was carried out at 262 nm. Diethyl ether was used as extractor solvent. The validated method was used to evaluate the biotransformation of THD by 12 endophytic fungi isolated from Tithonia diversifolia, Viguiera arenaria and Viguiera robusta. Among the 12 fungi evaluated, 4 of them deserve prominence for presenting an evidenced stereoselective biotransformation potential: Phomopsis sp. (TD2) presented greater mono-2-sulfoxidation to the form (S)-(SE) (12.1%); Glomerella cingulata (VA1) presented greater mono-5-sulfoxidation to the forms (S)-(SE)+(R)-(FE) (10.5%); Diaporthe phaseolorum (VR4) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(FE) (84.4% and 82.5%, respectively) and Aspergillus fumigatus (VR12) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(SE) (31.5% and 34.4%, respectively).

Thioridazine (TZ) has previously been shown by us to have in vitro and ex vivo activity against antibiotic-susceptible and multidrug-resistant Mycobacterium tuberculosis (MDRTB). Because current therapy of MDRTB is highly problematic even when all five 'first line of defence' drugs are employed, there is a need for effective antituberculosis drugs. New derivatives of TZ were synthesised and their in vitro activity against a reference strain of M. tuberculosis was evaluated with the aid of the BACTEC 460 system. Derivatives that presented significant activity were evaluated by ex vivo studies and were shown to enhance the killing of intracellular M. tuberculosis.

Thioridazine (THD) is a phenothiazine neuroleptic drug used for the treatment of psychiatric disorders. After oral administration THD is extensively biotransformed to thioridazine 2-sulfone (THD 2-SO(2)), thioridazine 5-sulfoxide (THD 5-SO) and thioridazine 2-sulfoxide (THD 2-SO). THD 2-SO and THD 5-SO have two chiral centres and therefore exist as two diastereoisomeric pairs. The degradation and epimerization of THD 2-SO in human plasma, buffer and methanolic solutions were studied using an enantioselective HPLC method. The samples were prepared by liquid-liquid extraction with diethyl ether and the chiral resolution of the enantiomers was carried out on a Chiralpak AD column using a mobile phase consisting of hexane:ethanol:2-propanol (90:7:3, v/v/v) containing 0.2% diethylamine. The method was validated and used to study the degradation and epimerization under different conditions of incubation. Our results showed that both enantiomers were stable at varying temperatures, pH and ionic strengths; however, solubility problems were observed, mainly at pH 8.5. The influence of light on stability was studied using methanolic solutions and degradation and epimerization of the THD 2-SO enantiomers were observed under UV light of 366 and 254nm, respectively.

We present a method for the stereoselective analysis of thioridazine 5-sulfoxide (THD 5-SO) in human plasma based on liquid-liquid extraction with diethyl ether and chiral resolution of the stereoisomers by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin and sulfated beta-cyclodextrin as chiral selectors. The method showed recovery rates of 85.5% for both THD 5-SO (slow-eluting, SE) enantiomers. The coefficients of variation observed in the precision studies, as well as the accuracy values, were below 10%. After validation, the method was used to study the stability and configurational changes of this THD metabolite. Our results showed that both enantiomers of THD 5-SO (SE) were stable under conditions of variation of temperatures (38 degrees C, 4 degrees C and -20 degrees C), pH (5.0, 7.0 and 8.5) and ionic strengths (0.2, 0.5 and 1.0 mol/L). The influence of light on the stability of the THD 5-SO (SE) stereoisomers was also studied using standard solutions prepared in methanol and an inversion in configuration was observed under UV light (254 and 366 nm).

We present two methods for the enantioselective analysis of thioridazine (THD) and thioridazine 2-sulfone (THD 2-SO(2)) in human plasma based on liquid-liquid extraction with diethyl ether and chiral resolution of the enantiomers on Chiralpak AD and Chiralcel OD-H columns, respectively. After validation, the methods were used to study the degradation and racemization of both drug and metabolite. Our results showed that both enantiomers of THD and THD 2-SO(2) were stable at varying temperatures, pH, and ionic strengths; however, solubility problems for THD and THD 2-SO(2) enantiomers were observed, mainly at pH 8.5. The influence of light on the stability of the THD and THD 2-SO(2) enantiomers was also studied. Degradation of the THD enantiomers was observed under UV light (254 and 366 nm) while THD 2-SO(2) enantiomers were stable at these wavelengths and also when exposed to visible light., (Copyright 2003 Wiley-Liss, Inc.)

This study was aimed at investigation of the pharmacokinetics of thioridazine and its metabolites after a single and repeated administrations. Male Wistar rats received thioridazine as a single dose (10 mg/kg i.p.) or they were treated chronically with the neuroleptic (10 mg/kg i.p., twice a day for two weeks). Plasma and brain concentrations of thioridazine and its metabolites (N-desmethylthioridazine, mesoridazine, sulforidazine, and the ring sulfoxide) were determined using the HPLC method. The obtained data showed that sulfoxidation in position 2 of the thiomethyl substituent and in the thiazine ring are main metabolic pathways of thioridazine, and showed that, in contrast to humans, in the rat N-desmethylthioridazine is formed in appreciable amount. The biotransformation of thioridazine was rather fast yielding plasma peak concentrations of metabolites lower than that of the parent compound. The maximum concentrations of thioridazine and its metabolites in the brain appeared later than in plasma. The peak concentrations and AUC values of thioridazine and its metabolites were higher in the brain than in plasma and this corresponded well with their longer half-lives in the brain as compared to plasma. The drug was not taken up by the brain as efficiently as other phenothiazines. Chronic treatment with thioridazine produced significant increases (with the exception of thioridazine ring sulfoxide) in the plasma concentrations of the parent compound and its metabolites which was accompanied with the prolongation of their plasma half-lives. The observed plasma levels of thioridazine were within 'therapeutic range' while the concentrations of its metabolites were relatively lower as compared to those observed in psychiatric patients. The increased plasma concentrations of thioridazine and its metabolites observed in plasma after chronic treatment were not followed by parallel changes in the brain.

Concentrations of total (R) + (S) and of the enantiomers (R) and (S) of thioridazine and metabolites were measured in 21 patients who were receiving 100 mg thioridazine for 14 days and who were comedicated with moclobemide (450 mg/day). Two patients were poor metabolizers of dextromethorphan and one was a poor metabolizer of mephenytoin. Cytochrome P450IID6 (CYP2D6) is involved in the formation of thioridazine 2-sulfoxide (2-SO) from thioridazine and also probably partially in the formation of thioridazine 5-sulfoxide (5-SO), but not in the formation of thioridazine 2-sulfone (2-SO2) from thioridazine 2-SO. Significant correlations between the mephenytoin enantiomeric ratio and concentrations of thioridazine and metabolites suggest that cytochrome P450IIC19 could contribute to the biotransformation of thioridazine into yet-unknown metabolites, other than thioridazine 2-SO, thioridazine 2-SO2, or thioridazine 5-SO. An enantioselectivity and a large interindividual variability in the metabolism of thioridazine have been shown: measured (R)/(S) ratios of thioridazine, thioridazine 2-SO fast eluting (FE), thioridazine 2-SO slow eluting (SE), thioridazine 2-SO (FE+SE), thioridazine 2-SO2, thioridazine 5-SO(FE), and thioridazine 5-SO(SE) were (mean +/- SD) 3.48 +/- 0 .93 (range, 2.30 to 5.80), 0.45 +/- 0.22 (range, 0.21 to 1.20), 2.27 +/- 8.1 (range, 6.1 to 40.1), 4.64 +/- 0.68 (range, 2.85 to 5.70), 3.26 +/- 0.58 (range, 2.30 to 4.30), 0.049 +/- 0.019 (range, (0.021 to 0.087), and 67.2 +/- 66.2 (range, 16.8 to 248), respectively. CYP2D6 is apparently involved in the formation of (S)-thioridazine 2-SO(FE), (R)-thioridazine 2-SO(SE), and also probably (S)-thioridazine 5-SO(FE) and (R)-thioridazine 5-SO(SE).

Thioridazine is a commonly prescribed phenothiazine drug administered as a racemate and it is believed that its antipsychotic effect is mainly associated with (R)-thioridazine. A method based on high-performance liquid chromatography has been developed for the determination of the enantiomers of thioridazine and thioridazine 2-sulfone (THD 2-SO2 or sulforidazine) and of the enantiomers of the diastereoisomeric pairs of thioridazine 2-sulfoxide (THD 2-SO or mesoridazine) and thioridazine 5-sulfoxide (THD 5-SO) in the plasma of thioridazine-treated patients. The method involves sequential achiral and chiral HPLC. The limits of quantitation for total (R) + (S) concentrations were found to be 15 ng/ml for thioridazine and 5 ng/ml for its metabolites. The limits for the determination of the (R)/(S) ratios were found to be 60 ng/ml for racemic THD and 10 ng/ml for racemic THD 2-SO, THD 2-SO2, THD 5-SO (FE) and THD 5-SO (SE). The method has been used to determine the concentrations of the enantiomers of thioridazine and of its metabolites in the plasma of a patient treated with 100 mg of racemic thioridazine hydrochloride per os per day for 14 days. The results show a high enantioselectivity in the metabolism of this drug: the (R)/(S) ratios for THD, THD 2-SO (FE), THD 2-SO (SE), THD 2-SO2, THD 5-SO (FE) and THD 5-SO (SE) were found to be 3.90, 1.22, 6.10, 4.10, 0.09 and 28.0, respectively.

The ring sulfoxidation of thioridazine (THD), a widely used neuroleptic agent, yields two diastereoisomeric pairs, fast- and slow-eluting (FE and SE) thioridazine 5-sulfoxide (THD 5-SO). Until now, studies in which concentrations of these metabolites were measured in THD-treated patients have revealed no significant differences in their concentrations. Preliminary experiments in our laboratory had shown that sunlight and, to a lesser extent, dim daylight led to racemization and probably also to photolysis of the diastereoisomeric pairs as measured by high-performance liquid chromatography. Similar results were also obtained with direct UV light (UV lamp). In appropriate light-protected conditions, THD, northioridazine, mesoridazine, sulforidazine, and FE and SE THD 5-SO were measured in 11 patients treated with various doses of THD for at least 1 week. Significantly higher concentrations of the FE stereoisomeric pair were found. The concentration ratios THD 5-SO (FE)/THD 5-SO (SE) ranged from 0.89 to 1.75 in plasma and from 1.15 to 2.05 in urine. Because it is known that the ring sulfoxide contributes to the cardiotoxicity of the drug even more potently than the parent compound does, these results justify further studies to determine whether there is stereoselectivity in the cardiotoxicity of THD 5-SO.

(+/-)-Thioridazine and (+/-)-desmethylthioridazine have been oxidized to produce a number of chiral sulphoxide and amine oxide compounds. Diastereoisomeric isomers were separated by thin-layer and high performance liquid chromatography. Thioridazine-5-sulphoxide, N-desmethylthioridazine-5-sulphoxide and thioridazine-N-oxide diastereoisomers were found to be thioridazine metabolites following dosing in rats or after in-vitro incubation with rat liver homogenate.

Chlorpromazine and thioridazine are widely used antipsychotic agents that are extensively metabolized. Parent compounds and metabolites have diverse pharmacologic activities, and differences in patterns of metabolism may explain differences in therapeutic and side effects from individual to individual. Radioreceptor assays were used to determine the neuroleptic, antimuscarinic, and anti-alpha-noradrenergic potency of chlorpromazine, thioridazine, and their metabolites. The results indicate that these metabolites show a wide range of potencies. The spectrum of activity of a metabolite may be quite different from that of its parent compound. The clinical relevance of these findings to individual differences in drug response is discussed. The combined use of radioreceptor assays and chemical assays in future clinical research is proposed.

Thioridazine, thioridazine side chain sulfoxide, side chain sulfone and ring sulfoxide were each given to two dogs in incremental doses. Profound changes in cardiac output and blood pressure were seen only in the dogs receiving the ring sulfoxide. Although all four drugs increased the QRS and Q-Tc interval, ventricular arrhythmias and "torsades de pointes" were seen only in dogs receiving the ring sulfoxide. Total plasma concentrations of thioridazine ring sulfoxide were in the same range as those seen in some patients on thioridazine therapy although the free concentrations were higher. Thioridazine ring sulfoxide appears to be the most toxic metabolite of thioridazine.

A selective oxidation of thioridazine to give exclusively its ring sulfoxides and a separation of the resulting products as diastereoisomeric pairs of enantiomers (DL, LD and DD, LL) are reported. These pairs were characterized by TLC, high-performance liquid chromatographic, IR, UV, 1H-NMR, 13C-NMR, GC-MS, and elemental analyses, and by reduction to thioridazine by lithium aluminum hydride. Structural data for the separated diastereoisomeric pairs or their nitric acid salts were obtained from NMR and IR studies. Gram quantities of each of the two diastereoisomeric pairs of enantiomers were isolated in better than 99% purity.

Two diastereoisomeric pairs of thioridazine 5-sulfoxide (T5SO)(ring sulfoxide) were detected in serum and urine from rats and patients following chronic thioridazine administration. The isolation and identification of each pair of isomers was by thin layer chromatography, high pressure liquid chromatography, and mass spectrometry. Equal concentrations of T5SO pairs were present in serum and urine specimens from both rats and man. Therefore, the biotransformation and renal clearance of the isomers does not appear to be stereoselective in either species. An evaluation of chromatographic systems used in previous metabolic studies of thioridazine, and reasons for the lack of prior recognition of thioridazine 5-sulfoxide isomers is also presented.

The use of combined UV-fluorescence detection for the evaluation of incompletely resolved compounds and trace components in the presence of large quantities of major components is described, analysis for thioridazine and some of its oxidation products by high-pressure liquid chromatography being chosen as a practical example. Mesoridazine and the ring oxide of thioridazine have been determined quantitatively with relative standard deviations (n = 6) of 2.0 and 3.6%, respectively, at concentrations below 0.1 mug per injection. Resolution of the two components is difficult and, in this instance, unnecessary. By a similar approach, it was possible to determine the highly fluoresecent sulforidazine at a concentration of 0.4% of the thioridazine with 6.2 mug of thioridazine injected. A relative standard deviation of 5% was attainable at this concentration. Fluorescence detection limits for mesoridazine and sulforidazine at a signal-to-noise ratio of 4:1 are between 5 and 10 ng per injection; this corresponds to about 0.1% of the active substance for the above example.

The concentrations of thioridazine and its main metabolites in erythrocytes from 61 thioridazine-treated patients were determined by gas-liquid chromatography. The mean and range of the erythrocyte concentrations, expressed as percentage of the corresponding plasma concentrations, were: thioridazine, 5.1% (2.0-10.6); side-chain sulfoxide, 5.6% (1.6-10.4); side-chain sulfone, 3.3% (1.1-6.8); ring sulfoxide 2.7% (0.8-4.9). The erythrocyte and plasma concentrations were significantly correlated. The erythrocyte/plasma concentration ratios, all the erythrocyte concentrations, but none of the plasma concentrations except ring sulfoxide were significantly positively correlated to the dose of thioridazine. The erythrocyte/plasma concentration ratio was not correlated to age. In vitro experiments indicated no clinically relevant erythrocyte-mediated oxidation of thioridazine.

The binding of thioridazine, northioridazine, mesoridazine and sulforidazine to bovine serum albumin is studied by means of equilibrium dialysis. For these four substances the percent binding in function of the albumin concentration is determined, as well as the apparent binding constant in the concentration range studied. The importance of the hydrophobic character and the chemical structure for the albumin binding is discussed.

The potential cardiotoxicity of a racemic mixture of thioridazine-5-sulfoxide, an oxidative metabolite of thioridazine, was studied in the isolated, perfused rat heart. Thioridazine-5-sulfoxide (ring sulfoxide) was prepared by a new method in which a racemic mixture of the two diastereoisomeric forms of the compound was formed. Hearts from male Sprague-Dawley rats were perfused using a modified Langendorff preparation. Quinidine (31 microM) served as a positive control for the measurements of the electrocardiogram (ECG) and the heart rate. Thioridazine (13 microM) perfusion resulted in a variable heart rate and elevated S-T segment. Perfusate concentrations of the ring sulfoxide as low as 12 microM increased P-R and Q-T intervals, produced delays in A-V and ventricular conduction, premature ventricular contractions and ultimately A-V block. These findings suggest that thioridazine cardiotoxicity may be due in part to the actions of thioridazine ring sulfoxide.

The disposition and urinary elimination of thioridazine 5-sulfoxide (ring sulfoxide) following subacute administration of thioridazine was investigated in control and phenobarbital (Pb) induced rats. The ring sulfoxide exists as two stereoisomeric pairs, identified by high performance liquid chromatography as fast eluting (RSF) and slow eluting (RSS) ring sulfoxide. No major differences were detected in the distribution in serum or tissue of the ring sulfoxide between control and induced animals. The ring sulfoxide accumulated in all tissues studied. RSF tissue concentrations were significantly greater than those of RSS in both groups. However, stereoselective formation and elimination of the two ring sulfoxides was not noted in either group. Pb inducible cytochrome P-450 drug metabolizing enzymes do not appear to play a major role in the biotransformation of thioridazine to the ring sulfoxide.

The binding of thioridazine and some of its psychoactive metabolites to human serum and to human albumin was studied using equilibrium dialysis. A very important binding capacity was found for each of the products tested, but significant differences between binding to human serum and binding to human albumin were observed.

The disposition and elimination of 2 stereoisomeric forms of thioridazine 5-sulfoxide (ring sulfoxide) were followed for 72 hr in male Sprague-Dawley rats following the administration of a single 40 mg/kg intraperitoneal dosage. The distribution half-lives for the fast (RSF) and slow (RSS) eluting ring sulfoxides in serum were 4.40 and 3.23 min, respectively. Elimination half-lives were 1.79 and 2.06 h. Both RSF and RSS were distributed throughout the tissue in equal amounts based on HPLC analysis of the tissues. Both isomers were found in lung tissue at concentrations 10 times that for other organs. Elimination of the ring sulfoxide was complete by 72 hours except in the brain, lung and kidney. The brain, heart, liver and kidney exhibited periodicity, or recycling of both RSF and RSS during the elimination phase. Release of RSF and RSS from myocardial tissue was slower than that for all other tissues. Excretion of unchanged thioridazine 5-sulfoxide occurred via renal, and to a lesser extent, biliary mechanisms. The biliary excretion pattern suggested an enterohepatic circulation of both isomers. These results indicate a lack of stereoselectivity in the disposition and clearance of the two ring sulfoxide isomers in the animal model; results that mirror those seen in man.

The cardiotoxic potential of the phenothiazine neuroleptic thioridazine and five of its metabolites, including two stereoisomeric forms of thioridazine 5-sulfoxide (ring sulfoxide), was characterized in the isolated perfused rat heart. Hearts from male Sprague-Dawley rats were perfused using a modified Langendorff technique. After a 30-min control period, hearts were perfused for 30 min with 15 or 30 microM thioridazine, racemic and isomeric forms of thioridazine 5-sulfoxide, or thioridazine 2-sulfoxide. Thioridazine disulfoxide, thioridazine 2-sulfone, and desmethylthioridazine 2-sulfoxide were perfused at 15 microM. Thioridazine (15 microM) severely reduced contractile tension and the rate of tension development (dT/dt), transiently increased coronary flow rate but prolonged conductance through the AV node. No arrhythmias were seen. At 30 microM, ventricular premature contractions and irregular rhythms occurred, progressing to asystole. Thioridazine 5-sulfoxide was arrhythmogenic at 15 and 30 microM. Atrial premature contractions and paroxysmal atrial tachycardia progressed to second degree AV block. Contractile tension and dT/dt declined although not as quickly when compared to thioridazine. Each isomeric form of thioridazine 5-sulfoxide was also cardiotoxic at 15 and 30 microM. There were minor differences in the onset and degree of toxicity but the overall results did not suggest a stereoselective effect. Lactic dehydrogenase and aspartate aminotransferase concentrations were unchanged after thioridazine 5-sulfoxide perfusion indicating no direct tissue damage. Thioridazine 5-sulfoxide induced arrhythmias could not be prevented or reversed by treatment with adrenergic agonists. They were reversible upon washout, however. The other metabolites were not arrhythmogenic at 15 microM. Racemic thioridazine 5-sulfoxide appears to be qualitatively and quantitatively more arrhythmogenic than thioridazine, an action that does not appear to be stereoselective.