phzO, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84

Certain strains of root-colonizing fluorescent Pseudomonas spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent Pseudomonas spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of Pseudomonas aureofaciens 30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA). P. aureofaciens 30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described.     

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 μm) using water–acetonitrile–methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 μg/ml. The limit of quantitation was 0.08 μg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b]pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane—dichloromethane (7:3) and analysed on a C₁₈ column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 μg/ml for III and 0.05 μg/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.     

Exercise training is more effective than resveratrol supplementation on alleviation of inflammation in peritoneal macrophages of high fat diet mice

[Purpose]

This study investigated the effect of exercise training and resveratrol supplementation with low fat diet on proinflammatory profiles by Lipopolysaccharide (LPS)-stimulation in peritoneal macrophage of high fat diet mice.

[Methods]

To accomplish the purpose of this study, C57BL/6 male mice were fed high fat diet (45% fat diet) for 8 weeks. Then these mice were divided into 3 groups; HLC (high fat diet and low fat diet for 8 weeks as the control, n=10), HLR (high fat diet and low fat diet for 8 weeks with resveratrol supplementation, n=10). HLE (high fat diet and low fat diet for 8 weeks with moderate exercise training, n=10). Resveratrol (10 mg/kg) was administrated once a day, 5 days/week for 8 weeks. Exercise training was performed for 8 weeks on a treadmill running for 30-60 min/day at 10-22 m/min, 0% grade, 5 days/week. After exercise training, all the peritoneal macrophage was collected and LPS (0, 0.5, 1.0 μg/ml) were used to stimulate the cells. Then peritoneal macrophage TNF-α, IL-6, MCP-1, IL12p70, IFN-γ, IL-10 were measured by BD cytometric bead array mouse inflammation kit.

[Results]

As a result, body weight and total cholesterol were significantly reduced in HLE compared with HLC (p<.05). Also, TNF-α and MCP-1 were decreased in HLE compared with HLC (p<.05) by LPS-stimulation (0, 0.5, 1.0 μg/ml) and IL-6, IL-12p70 and IFN-r were decreased in HLE compared with HLC (p<.05) by LPS-stimulation (1.0 μg/ml). But resveratrol supplementation did not affect the result.

[Conclusion]

These findings suggest that exercise training has beneficial effects on body weight, total cholesterol, peritoneal macrophage and proinflammatory cytokine in high fat diet mice.     

Synthesis of 5-(4-hydroxy-3-methylphenyl)-5-(substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N¹-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.     

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 mM) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125×4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 μl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 μg/ml and was capable of detecting a minimum concentration of 0.029 μg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.     

Screening of natural phenazine producers for electroactivity in bioelectrochemical systems

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1 μg/ml mediating an anodic current of 15.1 μA/cm². On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5 μA/cm². Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.     

Mapping the regioisomeric distribution of fatty acids in triacylglycerols by hybrid mass spectrometry

This study describes the use of hybrid mass spectrometry for the mapping, identification, and semi-quantitation of triacylglycerol regioisomers in fats and oils. The identification was performed based on the accurate mass and fragmentation pattern obtained by data-dependent fragmentation. Quantitation was based on the high-resolution ion chromatograms, and relative proportion of sn-1(3)/sn-2 regioisomers was calculated based on generalized fragmentation models and the relative intensities observed in the product ion spectra. The key performance features of the developed method are inter-batch mass accuracy < 1 ppm (n = 10); lower limit of detection (triggering threshold) 0.1 μg/ml (equivalent to 0.2 weight % in oil); lower limit of quantitation 0.2 μg/ml (equivalent to 0.4 weight % in oil); peak area precision 6.5% at 2 μg/ml concentration and 15% at 0.2 μM concentration; inter-batch precision of fragment intensities < 1% (n = 10) independent of the investigated concentration; and averaged accuracy using the generic calibration 3.8% in the 1–10 μg/ml range and varies between 1–23% depending on analytes. Inter-esterified fat, beef tallow, pork lard, and butter fat samples were used to show how well regioisomeric distribution of palmitic acid can be captured by this method.     

Synthesis of 5-(4-hydroxy-3-methylphenyl) -5- (substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N1-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1- pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyri- dylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.     

The mitochondrial benzodiazepine receptor: Evidence for association with the voltage-dependent anion channel (VDAC)

Specific, high-affinity receptors for numerous drugs have recently been localized to mitochondrial membrane proteins. This review discusses the association of the mitochondrial receptor for benzodiazepines (mBzR) with the voltage-dependent anion channel (VDAC), indicating a possible auxiliary role for VDAC as a putative drug binding protein. The proposed subunit composition of the purified mBzR complex isolated from rat kidney mitochondria includes VDAC, which functions as a recognition site for benzodiazepines (e.g., flunitrazepam), the adenine nucleotide carrier (ADC), and an 18 kDa outer membrane protein identified by covalent labelling with the mBzR antagonists isoquinoline carboxamides (e.g., PK 14105).Abbreviations and chemical names: Ro5-4864: 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-one; Ro15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-]-[1,4]benzodiazepine-3-carboxylate; AHN-086: (1-(2-isothiocyanatoethyl-7-chloro-1,3-dihydro-5-(4-chlorophenyl)-2H-1,4-benzodiazepin-2-one hydrochloride;) PK11195: 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-isoquinoline-3-carboxamide; PK14105: 1-(2-fluoro-5-nitrophenyl)-3-isoquinoline-carboxylic acid.     

Determination of lamotrigine in human serum by liquid chromatography

A rapid high-performance liquid chromatographic method was developed using a short silica column (30 mm×4.6 mm) with an aqueous methanol mobile phase consisting of methanol–water–NH₄H₂PO₄ (94:5.96:0.04) adjusted to a final apparent pH of 5.0 and pumped at a flow-rate of 1 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm, and serum samples were prepared for HPLC analysis by extraction into dichloromethane after basification. Lamotrigine was eluted at 0.96 min. Within-day variation of the method was 4.46% at 0.75 μg/ml and 2.37% at 6.0 μg/ml, and day-to-day variation was 9.10% at 0.75 μg/ml and 7.28% at 6.0 μg/ml.     

High-performance liquid chromatographic assay for cefepime in serum

A simple, rapid, specific and sensitive high-performance liquid chromatographic method was developed for the determination of cefepime 1-[[6R, 7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-2-carboxy-8-oxo-5-thia-1-azabicyclo-[4.2.0] oct-2-en-3-yl]methyl]-1-methylpyrrolidinium hydroxide, inner salt, 7²-(Z)-(O-methyloxime) in human serum. Separation was achieved on a reversed-phase Ultrasphere XL-ODS column (75×4.6 mm I.D.). The mobile phase was 7% acetonitrile in 20 mM ammonium acetate (pH 4). Cefepime eluted in the range of 1.8–2.2 min. Detection was by UV absorbance at 254 nm. The lower limit of quantitation of cefepime in plasma was 0.5 μg/ml. The average absolute recovery was 106.2±2.1%. The linear range was from 0.1 to 50 μg/ml, with a correlation coefficient greater than 0.999. The within-day C.V.s for human samples were 4.9 and 2.3% for 1 and 50 μg/ml, respectively. The between-day C.V.s for human serum samples were 14.5, 7.4 and 6.7 for 1, 25 and 50 μg/ml, respectively. Cefepime was found to be unstable in serum at room temperature. For delayed assay, samples must be stored at −80°C.     

Determination of gabapentin in plasma by high-performance liquid chromatography

A rapid and simple method for determination of the novel antiepileptic compound gabapentin [1-(aminomethyl)cyclohexaneacetic acid] in plasma is described. Blank human plasma was spiked with gabapentin (1.0–10.0 μg/ml) and internal standard [1-(aminomethyl)-cycloheptaneacetic acid; 5.0 μg/ml]. Individual samples were treated with 2 M perchloric acid, centrifuged and then derivatised with o-phthalaldehyde-3-mercaptopropionic acid. Separation was achieved on a Beckman Ultrasphere 5 μm reversed-phase column with mobile phase consisting of 0.33 M acetate buffer (pH 3.7; containing 100 mg/l EDTA)-methanol-acetonitrile (40:30:30, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 330 and 440 nm, respectively. The calibration curve for gabapentin in plasma was linear (r=0.9997) over the concentration range 1.0–10.0 μg/ml. Recovery was seen to be 90%. The inter- and intra-assay variations for three different gabapentin concentrations were 10% throughout. The lower limit of quantitation was found to be 0.5 μg/ml. Chromatography was unaffacted by a range of commonly employed antiepileptic drugs or selected amino acids.     

Incorporation of [14C]shikimate into phenazines and their further metabolism by Pseudomonas phenazinium

1. During growth of Pseudomonas phenazinium on l-threonine medium, phenazine pigment formation commenced early and 1,6-dihydroxyphenazine 5,10-dioxide (iodinin) was the major component. Growth on l-[U-¹⁴C]threonine showed that when growth was complete about 25% of the label had been incorporated into phenazines and 30% into cell substance. 2. The addition of d-[2,3,4,5(n)-¹⁴C]shikimate to cultures at different phases of growth showed that the greatest efficiency of incorporation (about 70%) occurred in the mid- to late-exponential phase. Phenazines accounting for most of the ¹⁴C supplied were iodinin and 9-hydroxyphenazine-1-carboxylate plus 2,9-dihydroxyphenazine-1-carboxylate. Radioactivity incorporated into cell substance was about one-third of the amount found in phenazines. 3. Kinetic studies showed that radioactivity from a pulse of [¹⁴C]-shikimate was incorporated into phenazines immediately, without a discernible lag, and into all detectable phenazines simultaneously rather than sequentially. 4. Radioactive phenazines isolated from culture media were fed to growing cultures and their metabolism was studied. The results supported a scheme for the biosynthesis of iodinin and 1,8-dihydroxyphenazine 10-monoxide by a branched pathway. 5. It is proposed that phenazine-1,6-dicarboxylate is the common precursor of all naturally occurring phenazines.     

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column eluted with a phosphate buffer—acetonitrile—methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195–200 μg/ml, the propionic acid metabolite 0.195–50.0 μg/ml, the p-hydroxy metabolite 0.781 to 50.0 μg/ml, and the monocarbamate metabolite 0.098–50.0 μg/ml.     

Validation of an analytical procedure for the determination of the fluoroquinolone ofloxacin in chicken tissues

A novel analytical procedure was developed for the determination of the fluoroquinolone ofloxacin in chicken kidney, liver, muscle and fat plus skin tissues. The procedure involved a preliminary extraction with 0.15 M HCl followed by solid-phase extraction clean-up. The purification step was performed using a polymeric sorbent coated cartridge. Ofloxacin was analyzed by reversed-phase HPLC using UV detection at 295 nm. The mobile phase used was water–acetonitrile–triethylamine (83:14:0.45, v/v, pH 2.30). The use of triethylamine and the acidic pH modulated the retention of ofloxacin and avoided chemical tailing. The amine modifier and acetonitrile content of the mobile phase were optimized to provide the best selectivity. A flow-rate of 1 ml/min was used and ofloxacin eluted at 5.1 min. HPLC analysis of the tissue samples was performed in 12 min. The procedure was validated according to the International Conference on Harmonisation guidelines across the concentration ranges (100 μg/kg–1.7 mg/kg for kidney and liver tissues and 50 μg/kg–1.0 mg/kg for muscle and fat plus skin tissues). The linearity, the intra- and inter-day accuracies and precisions were determined. The limits of quantification were 50 μg/kg for muscle and fat plus skin tissues and 100 μg/kg for liver and kidney tissues. The procedure was specific and the accuracy values were lower than 20% at the limit of quantitation of spiked samples. The recovery values ranged from 80 to 100%. The limits of detection were established at 60 μg/kg for liver and kidney tissues and at 25 μg/kg for muscle and fat plus skin tissues. Finally, ofloxacin was found to be stable in acidic conditions. The developed procedure is simple, sensitive, accurate and adapted to routine sample analyses such as those carried out for residue depletion studies.     

Synthesis of novel 2-pyrazoline analogues with potent anti-inflammatory effect mediated by inhibition of phospholipase A2: Crystallographic,in silico docking and QSAR analysis

Oxidative-stress induces inflammatory diseases. Further, infections caused by drug-resistant microbial strains are on the rise. This necessitates the discovery of novel small-molecules for intervention therapy. A series of 3-(2,3-dichlorophenyl)-1-(aryl)prop-2-en-1-ones are synthesized as intermediates via Claisen-Schmidt reaction approach. Subsequently, these intermediates were transformed into 2-pyrazolines by their reaction with phenylhydrazine hydrochlorides in methanol and few drops of acetic acid under reflux conditions. Synthesized compounds were characterized by spectroscopic, crystallographic and elemental analyses studies and then, were evaluated for their in vitro antimicrobial and anti-inflammatory activities. Amongst the series, 3-(4-chlorophenyl)-5-(2,3-dichlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (5e), 5-(2,3-dichlorophenyl)-3-(4-fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (5c) and 5-(2,3-dichlorophenyl)-3-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (5h) showed significant inhibition of phospholipase A2 with IC₅₀ values of 10.2, 11.1 and 11.9 µM, respectively. Protein structure modelling and docking studies indicated that the compounds showed binding to a highly conserved calcium-binding pocket on the enzyme. Further, compounds (5e), 1-(3-chlorophenyl)-5-(2,3-dichlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (5b), and 1-(3-chlorophenyl)-3-(4-chlorophenyl)-5-(2,3-dichlorophenyl)-4,5-dihydro-1H-pyrazole (5f) showed excellent antimicrobial activities against various bacterial and fungal strains. In conclusion, this study is a successful attempt at the synthesis and characterization of chalcone derivatives that can target phospholipase A2, an enzyme that is a prominent player in the physiological inflammatory cascade. Thus, these compounds show promise for development as next-generation nonsteroidal anti-inflammatory drugs.     

Antimalarial activity of phenazines from lapachol, beta-lapachone and its derivatives against Plasmodium falciparum in vitro and Plasmodium berghei in vivo

The antimalarial activity of benzo[a]phenazines synthesized from 1,2-naphthoquinone, lapachol, beta-lapachone and several derivatives have been tested against Plasmodium falciparum in vitro using isolates of parasites with various susceptibilities to chloroquine and/or mefloquine. Parasite growth in the presence of the test drugs was measured by incorporation of [(3)H]-hipoxanthine in comparison to controls with no drugs, always testing in parallel chloroquine, a standard antimalarial. Among seven benzophenazines tested, four had significant in vitro activities; important, the parasites resistant to chloroquine were more susceptible to the active phenazines in vitro. The doses of phenazines causing 50% inhibition of parasite growth varied from 1.67 to 9.44 microM. The two most active ones were also tested in vivo against Plasmodium berghei in mice, in parallel with lapachol and beta-lapachone. The 3-sulfonic acid-beta-lapachone-derived phenazine was the most active causing up to 98% inhibition of parasitaemia in long term treatment (7 doses) subcutaneously, whereas the phenazine from 3-bromo-beta-lapachone was inactive. Thus, these simple phenazines, containing polar (-Br,-I) and ionizable (-SO(3)H, -OH) groups, easily synthesized from cheap, natural or synthetic precursors (lapachol and beta-lapachone), at rather low cost, provide prototypes for development of new antimalarials aiming the chloroquine resistant parasites.     

POMA analyses as new efficient bioinformatics’ platform to predict and optimise bioactivity of synthesized 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues

A series of 43, 3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide/carbothioamide analogues (D01–D43) were analysed using Petra, Osiris, Molinspiration and ALOGPS (POMA) to identify pharmacophore, toxicity prediction, lipophilicity and bioactivity. All the compounds were evaluated for anti-HIV activity. 3-(4-Chlorophenyl)-N-(4-fluorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide (D07) was found to be the most active with IC₅₀ > 4.83 μM and CC₅₀ 4.83 μM. 3-(4-Fluorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carbothioamide (D41) was found to be the most active compound against bacterial strains with MIC of 4 μg/ml, comparable to the standard drug ciprofloxacin while 3-(4-methoxyphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide (D38) was found to be the most active compound against fungal strains with MIC 2–4 μg/ml, however less active than standard fluconazole. Toxicities prediction by Osiris were well supported and experimentally verified with exception of some compounds. In anticonvulsant screening, 3-(4-fluorophenyl)-N-(4-chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide (D09) showed maximum activity showing 100% (4/4, 0.25–0.5 h) and 75% (3/4, 1.0 h) protection against minimal clonic seizure test without any toxicity.     

Stereospecific determination of mefloquine in biological fluids by high-performance liquid chromatography

A sensitive stereoselective HPLC method was developed for determination of mefloquine (MFQ) enantiomers in plasma, urine and whole blood. The assay involved liquid-liquid extraction of MFQ from biological fluids with a mixture of hexane and isopropanol in the presence of sodium hydroxide and derivatization of the residue by (+)-(S)-naphthylethylisocyanate (NEIC) as chiral derivatizing reagent. Separation of the resulting diastereomers was performed on a silica normal-phase column using chloroform-hexane-methanol (25:74:1) as the mobile phase with a flow-rate of 1 ml/min. Using 200 μl of plasma or whole blood, the limit of determination was 0.2 μg/ml with UV detection for both enantiomers. The limit of determination in 500 μl of urine was 0.08 μg/ml with UV detection.