Role of the Benzodioxole Group in the Interactions between the Natural Alkaloids Chelerythrine and Coptisine and the Human Telomeric G-Quadruplex DNA. A Multiapproach Investigation.

The binding properties toward the human telomeric G-quadruplex of the two natural alkaloids coptisine and chelerythrine were studied using spectroscopic techniques, molecular modeling, and X-ray diffraction analysis. The results were compared with reported data for the parent compounds berberine and sanguinarine. Spectroscopic studies showed modest, but different rearrangements of the DNA-ligand complexes, which can be explained considering particular stereochemical features for these alkaloids, in spite of the similarity of their skeletons. In fact, the presence of a dioxolo moiety rather than the two methoxy functions improves the efficiency of coptisine and sanguinarine in comparison to berberine and chelerythrine, and the overall stability trend is sanguinarine > chelerythrine ≈ coptisine > berberine. Accordingly, the X-ray diffraction analysis confirmed the involvement of the benzodioxolo groups in the coptisine/DNA binding by means of π···π, O···π, and CH···O interactions. Similar information is provided by modeling studies, which, additionally, evidenced reasons for the quadruplex vs double-helix selectivity shown by these alkaloids. Thus, the analyses shed light on the key role of the benzodioxolo moieties in strengthening the interaction with the G4-folded human telomeric sequence and indicated the superior G4 stabilizing properties of the benzophenanthridine scaffold with respect to the protoberberine one and conversely the better G4 vs dsDNA selectivity profile of coptisine over the other alkaloids.

Interactions of selected gold(III) complexes with DNA G quadruplexes.

The interactions of three representative gold(III) complexes with human telomeric DNA sequences were analysed using a variety of biophysical methods, including DNA melting, circular dichroism, SPR and ESI MS; remarkable interactions were highlighted for all tested complexes, although they were associated to significantly different binding profiles. The most interesting compound was Auoxo6, a dinuclear gold(III) complex, which beyond manifesting a conspicuous binding affinity for the G-quadruplex conformation, turned out to be very effective in inducing a non-canonical secondary structure. These findings may clear the way for novel biological and pharmacological applications of this class of metal compounds.

FILO (field interaction ligand optimization): a simplex strategy for searching the optimal ligand interaction field in drug design.

A method (FILO, Field Interaction Ligand Optimization) for obtaining the optimal molecular interaction field was developed on the basis of the Simplex optimization procedure applied to a matrix of interaction energies obtained by performing a GRID computation on a suitable data set. The FILO procedure was tested on a set of nine HIV-1 protease inhibitors with known crystal structures. The results of FILO consist of the optimal molecular interaction field of a putative new ligand with optimal binding affinity. The final FILO model yields R2 and R2(CV) values of 0.993 and 0.936, respectively, and finds eight negative and four positive interaction nodes for the OH probe taken as an example. The eight H bonding interactions pointed out by FILO identified well the binding site AA-residues Gly A27, Asp A29, water 501, Gly B48 and Asp A25 of HIV-1 protease.

Benzodiazepine receptor ligands. III. Synthesis and biological evaluation of 2- and/or 3-substituted pyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides.

A new series of 2- and/or 3-substituted pyrazolo [5,1-c][benzotriazine 5-oxides and their 8-chloro derivatives were synthesized, and their benzodiazepine receptor (BZR) affinities were evaluated in vitro in comparison to lead compound 3-ethoxycarbonyl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide (29) [1,2]. None of the new compounds showed significant affinity for BZR. On the basis of a pharmacophore/receptor model suggested for lead compound 29, some hypotheses to explain the inactivity of new derivatives are discussed.

Experimental design and multivariate calibration in the development, set-up and validation of a differential pulse polarographic and UV spectrophotometric method for the simultaneous plasmatic determination of the therapeutic metronidazole-pefloxacin combination.

Partial least squares regression (PLS1 and PLS2) and GOLPE variable selection procedures were used for the treatment of differential pulse polarographic and UV spectrophotometric data obtained from the analysis of the therapeutic combination of metronidazole and pefloxacin. The analytical method used for the determination was set up using experimental design strategies (Doehlert's design, full factorial design, fractional face center cube design, etc.) and by involving the simultaneous optimization of several responses (desirability function). Method validation was also performed, determining accuracy, precision, linearity and range, detection and quantification limits and robustness. The quantitative prediction abilities in determining metronidazole and pefloxacin plasma levels of the PLS1 and PLS2 models were tested on spiked plasma samples and good results were obtained (metronidazole, 97.5%, RSD = 4.8%, n = 3; pefloxacin, 100.6%, RSD = 3.6%, n = 3). The use of multivariate calibration was particularly useful for spectrophotometric quantification because of the highly overlapping spectra of the binary mixture.

Experimental design strategies in the optimization and robustness testing of adsorptive stripping voltammetric conditions for kynurenic acid determination.

Experimental design was used for the optimization and robustness testing of an adsorptive stripping voltammetric procedure for kynurenic acid determination. The optimization of the peak height response proceeded through a screening phase (D-optimal design strategy) followed by a response surface study (Doehlert design) applied to the variables pH, pulse amplitude and stirring rate. An interaction between pH and stirring rate was pointed out. The optimized method was validated and the variation of factors that was expected to occur in practice was simulated in a robustness test. A composite fractional matrix for the evaluation of method robustness was used and pH emerged as the only critical factor. The linear range found applying the optimized conditions was 2.5 x 10(-9) to 2.5 x 10(-7) M and the calculated limit of detection was 1.72 x 10(-9) M.

Experimental design in the development of voltammetric method for the assay of omeprazole.

A multivariate strategy was used to optimize an adsorptive stripping voltammetric method for the determination of the antiulcer drug omeprazole. A 3/4 matrix was used for the variable screening while a central composite design was chosen in the subsequent step to evaluate the response surfaces. Simultaneous optimization of the response peak height (hp) and peak half width w1/2), the latter being a peak shape measure, was achieved. The factors accumulation time, pulse amplitude, scan rate and stirring rate were all found to be statistically significant for the response hp, while for the response w1/2 only the stirring rate was found to be significant. The optimized method shows a good linearity between peak height and analyte concentration in the concentration range from 8.33 x 10(-9) M to 1.42 x 10(-7) M with a LOD of 6.5 x 10(-9) M. The mean recovery of omeprazole in capsules was 101.9% with a SD of 2.04 (RSD = 200).

Design and optimization of the variables in the adsorptive stripping voltammetric determination of rufloxacin in tablets, human plasma and urine.

An adsorptive stripping voltammetric method with a hanging mercury drop electrode was developed for the determination of the fluoroquinolone rufloxacin in tablets, human plasma and urine. Measurements were obtained in differential pulse mode and a multivariate strategy was used to optimize the variables involved. Besides the independent effects of the variables, a strong interaction between scan rate and pulse duration has been found. Rufloxacin was analysed at concentrations between 1.7 x 10(-8) and 1.9 x 10(-7) M with a detection limit of 9.2 x 10(-9) M. Diluted tablet solutions and urine samples were analysed directly, while plasma samples needed an extraction procedure before voltammetric analysis. An improved HPLC procedure was used as comparative method.

High-performance liquid chromatographic determination of benzalkonium and naphazoline or tetrahydrozoline in nasal and ophthalmic solutions.

A high-performance liquid chromatographic method is described for the determination of benzalkonium chloride homologues and naphazoline nitrate or tetrahydrozoline hydrochloride in ophthalmic and nasal solutions. The technique involves a one-step dilution (1:5) for sample preparation and direct injection onto a 5 mm RP C-8 column. The mobile phase is acetonitrile-diluted acetic acid (80:20, v/v) with 6 mM tetramethylammonium bromide. Detection is carried out at 260 nm with a diode array. The method is rapid, specific, reproducible and is especially useful for quality control procedures.

Characterization of Sanguinaria canadensis L. fluid extract by FAB mass spectrometry.

Positive-ion fast atom bombardment mass spectrometry was used for the rapid characterization of commercial Sanguinaria canadensis L. fluid extracts. Quaternary and non-quaternary benzophenanthridine alkaloids afford persistent peaks due to [M]+ and [M+H]+ ionic species, respectively, and their relative abundances are in good agreement with previously reported per cent analytical data. The procedure allowed sanguinarine, chelerythrine, chelirubine, sanguilutine, protopine, allocryptopine and the isomers sanguirubine and/or chelilutine to be effectively detected by means of persistent and intense peaks in all the samples examined.

Determination of flubendazole in pharmaceutical dosage forms by differential pulse polarography and UV spectroscopy.

A precise and accurate differential pulse polarographic method was developed for the determination of flubendazole in dosage forms without any prior extraction procedure of interference from the other stated ingredients. A UV spectroscopic procedure was also described and used as reference method. Analyses were generally performed at the 4 micrograms ml-1 flubendazole level. Flubendazole or its dosage forms were dissolved in 70% perchloric acid and diluted with a pH 2.6 sodium phosphate-citric acid buffer as polarographic supporting electrolyte or spectrophotometric solvent. The peak potential occurred at about -0.9 V (vs. Ag/AgCl reference electrode), depending on the pH of the assayed solution. The irreversible electrochemical reduction involved the transfer of two electrons. The UV absorption spectrum showed a sharp maximum at 237 nm with a specific extinction coefficient of 886. No advantage was found in the use of first and second-order derivative spectrophotometry.

Determination of isosorbide-5-mononitrate in tablets by differential pulse polarography.

A differential pulse polarographic method requiring little sample separation was developed for the determination of isosorbide-5-mononitrate in tablet dosage form with the standard addition technique and without interference from common excipients. Britton-Welford buffer (pH 3.0) was used as the supporting electrolyte, the single peak occurring at -0.36 V vs. a reference Ag/AgCl electrode. The irreversible, diffusion controlled, two-electron reduction process at the dropping mercury electrode permits a precise and accurate determination of the active ingredient in the 0.4-20 microgram/ml concentration range.

Determination of benzalkonium chloride in contact lens solutions by positive-ion fast atom bombardment mass spectrometry.

Under positive-ion fast atom bombardment (FAB) mass spectrometric conditions, benzalkonium chloride (BAK) afforded intense peaks at m/z 304 and 332, corresponding to the intact cations [M--Cl]+ of C12 and C14 homologues, respectively. The use of benzethonium chloride as an internal standard and thioglycerol as a FAB matrix allowed the direct and specific determination of the BAK content (0.004-0.020%) in commercial hard contact lens solutions through the individual assay of the two alkyl homologues. A linear relationship between the homologue concentration and the peak-area ratio was observed over the concentration range 3-180 micrograms ml-1.