Syntheses of C2'-Fluorinated Analogs of Solamin.

The details of the total syntheses of C2'-fluorinated analogs of solamin, an antitumor annonaceous acetogenin, are described. Fluorine was enantioselectively introduced at the C2'-position by organocatalytic α-fluorination of the aldehyde according to a previously reported method. C2'-fluorinated solamin and its C2'-diastereomer were synthesized by the Sonogashira coupling of a tetrahydrofuran fragment and fluorine-containing γ-lactone fragments.

Identification and Characterization of Menadione and Benzethonium Chloride as Potential Treatments of Pierce's Disease of Grapevines.

Xylella fastidiosa infects a wide range of plant hosts and causes Pierce's disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naphthoquinone, vitamin K) from the phytochemical library and benzethonium chloride (a topical disinfectant) from the chemical library both showed significant bactericidal activity against X. fastidiosa. Both menadione and benzethonium chloride foliar spray (15 and 5 mM, respectively) and soil drench (5 and 25 mM, respectively) treatments were equally effective in reducing PD symptoms by 54 to 59% and revealed that the effects of both chemical treatments became systemic. However, menadione was phytotoxic when applied as a foliar spray at effective concentrations, causing significant loss of photosynthetic capacity.

Glutaraldehyde collagen cross-linking stabilizes resin-dentin interfaces and reduces bond degradation.

This study evaluated the stability of resin-dentin interfaces treated with glutaraldehyde-containing agents, and assessed collagen degradation in dentin matrices treated with Gluma. Microtensile bond strength (µTBS) was evaluated 24 h and 6 months after treatment with three desensitizers (Gluma Desensitizer, Gluma Desensitizer Power Gel, and MicroPrime G) and two etch-and-rinse adhesives (Comfort Bond & Desensitizer and iBond TE). Demineralized beams of human dentin were treated with water or Gluma, and the degradation of collagen in these beams was assessed by quantification of the dry mass loss and of the amount of hydroxyproline released from hydrolyzed specimens after 1 or 4 wk. All groups demonstrated significant reduction in µTBS after 6 months, except for Gluma Desensitizer and iBond TE groups, which showed decreases of 7.2% and 10.8%, respectively. The most prevalent failure mode was 'mixed'. Significantly less hydroxyproline was released from Gluma-treated beams than from control beams after 4 wk. Beams treated with Gluma yielded significantly less dry mass loss than did beams in the control group. Collagen cross-linking with glutaraldehyde-containing agents may assist in the stabilization of resin-dentin bonds by reducing the amount of collagen solubilized from dental matrices in the hybrid layer. In turn, this may contribute to the preservation of adhesive interfaces.

Short Route to the Total Synthesis of Natural Muricadienin and Investigation of Its Cytotoxic Properties.

An original synthesis of the acetogenin muricadienin, the bioprecursor of solamin, has been developed. The key step in the five-step 41% overall yield synthesis is the catalytic cross-cyclomagnesiation reaction of functionally substituted 1,2-dienes with EtMgBr in the presence of Cp2TiCl2 and magnesium metal. It has been demonstrated for the first time that muricadienin exhibits a moderate in vitro inhibitory activity against topoisomerases I and IIα, key cell cycle enzymes. Using flow cytometry, muricadienin was shown to have high cytotoxicity toward the HEK293 kidney cancer cells (IC50 0.39 µM).

[Formulation of benzethonium chloride into gels]. / Formulácia benzetóniumchloridu do gélov.

This study is focused on the preparation of gels with antimicrobial effects. A quaternary ammonium salt, benzethonium chloride, in a concentration of 0.01-0.5% (w/w) was employed as the drug. The humectant employed was propylene glycol in concentrations of 5% and 10% (w/w). Two types of polymers, chitosan and hydroxyethyl cellulose, in the same concentrations of 2.5% (w/w), were used for gel preparation. Finally the flow properties, rheological parameters and pH values of the gels were evaluated. Based on the obtained results, the samples of the gels prepared on the basis of chitosan and hydroxyethyl cellulose, which have the following optimum composition shown below, were found: 2,5% (w/w) CHIT + 0,5% (w/w) BZCl + 10% (w/w) PG; 2,5% (w/w) HEC + 0,5% (w/w) BZCl + 5% (w/w) PG.

Solvent-free production of thermoplastic lignocellulose from wood pulp by reactive extrusion.

A novel acylation approach suited to rapid bulk thermoplasticization of lignocellulose without solvents was previously demonstrated by the authors in benchtop batch studies. The method relies upon a benzethonium chloride/sulfuric acid functionalizing agent at low concentrations to act as a wetting agent for the wood pulp, similar to an ionic liquid, yet binds to the lignocellulose ester as a flow aid in the final thermoplastic. The present investigation evaluates the approach in a residence time-limited (45-90 s) continuous twin-screw reactor, where intensive mixing and heat were found to yield high acylation. The modified lignocellulose exhibited desired thermoplasticity by being melt moldable without the need for plasticizers and maintained much of the excellent stiffness of cellulose, demonstrating a maximum flexural modulus of 5.4 GPa and tensile modulus of 1.8 GPa. The influence of extrusion conditions on thermoplasticity was examined by a Design of Experiments (DOE) analysis.

Magnetic "fishing" assay to screen small-molecule mixtures for modulators of protein-protein interactions.

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 µM), with the latter two values being in close agreement with literature values.

Drug repurposing approach to target FtsZ cell division protein from Salmonella Typhi.

Drug repurposing is an efficient alternative approach to counter the increasing drug-resistant pathogens to treat infectious diseases. FtsZ is an essential bacterial cytokinesis protein involved in the formation of cell-division complex and targeting FtsZ using FDA approved drugs is a promising strategy to identify and develop a new antibacterial drug. Using in silico pharmacophore-based screening of drug bank, molecular docking and molecular dynamics simulations, we identified six drugs inhibiting the function of stFtsZ from Salmonella Typhi. The selected drugs target stFtsZ at the hydrophobic cleft formed between the C-terminal domain and helix α7 with binding energy better than -8 kcal/mol. Out of these six drugs, benzethonium chloride showed promising results at 8 µM concentration where it inhibits stFtsZ GTPase activity by 80% and prevents polymerization. Benzethonium chloride also possesses an excellent antibacterial activity against the bacterial culture of Salmonella Typhi (ATCC 19430), Staphylococcus aureus (ATCC 43300) and Escherichia coli (ATCC 25922) with the MIC values of 8 µg/mL, 1 µg/mL and 12 µg/mL, respectively. Based on our current study, the scaffold of benzethonium chloride can be used for the development of broad-spectrum antibacterial agents against drug-resistant pathogens.

Implication of foam sclerosant inactivation by human whole blood in a laboratory setting.

Background During sclerotherapy, it has been recommended to confirm intravenous placement of the needle by aspirating blood into the sclerosant syringe. This may inactivate some, or all of the sclerosant. Aims To quantify the volume of human blood needed to completely inactivate 1 ml of sodium tetradecyl sulphate, and comparing fresh blood and blood that has been stored in an ethylenediaminetetraacetic acid tube. Methods A series of manual titrations were carried out following a procedure developed at STD Pharmaceutical Products Ltd (Hereford, UK) and listed in the British Pharmacopeia. Three percent of sodium tetradecyl sulphate stock solutions were made with increasing volumes of blood and titrated against benzethonium chloride to determine the active concentration (% w/v) of sodium tetradecyl sulphate remaining in the solution. Results A calculated approximation showed 0.3 ml of blood is required to fully inactivate 1 ml of 3% sodium tetradecyl sulphate when made into a foam. A comparison was made between the use of fresh blood and blood stored in ethylenediaminetetraacetic acid tubes. Blood stored in ethylenediaminetetraacetic acid tubes showed more inactivation of sodium tetradecyl sulphate, but this was not significant at the P ≤ 0.05 level. Conclusion The data from our study have shown that a minimum of 0.3 ml of fresh blood is required to inactivate 1 ml of 3% sodium tetradecyl sulphate as a foam and it is not significantly affected by storing blood in an ethylenediaminetetraacetic acid tube. Our methodology suggests that during foam sclerotherapy treatment, blood should not be aspirated into the syringe to confirm position, and that ultrasound guidance is more appropriate for needle placement.

Displacement chromatography of proteins on hydrophobic charge induction adsorbent column.

Displacement chromatography of protein mixtures is proposed on hydrophobic charge induction chromatography (HCIC). We have used an HCIC medium, MEP-Hypercel as the stationary phase and a quaternary ammonium salt, benzethonium chloride, as the displacer. It was found that the multiple interactions between proteins/displacer and the HCIC sorbent, i.e. hydrophobic interaction and charge repulsion, enabled a greater flexibility for the design of displacement processes and ease of column regeneration by adjustment of pH. The capacity factors of proteins and displacers were used to predict their performances in column displacement, and the experimental results agreed well with the prediction. An isotachic displacement train of lysozyme and alpha-chymotrypsinogen A was formed with benzethonium chloride as the displacer at pH 5.0 with good yields and purities of the two proteins. Column regeneration was efficiently achieved by charge repulsion between the displacer and the adsorbent at lower pH values (pH 3 and 4). The results indicate that the displacement chromatography on HCIC is a good alternative to traditional hydrophobic displacement chromatography.

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation.

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.

Ormosil nanocomposite materials as modifiers for acrylic coating systems.

Organically modified silica (ormosil) particles were prepared using hydrolytic sol-gel methods and the miniemulsion polymerisation approach. Methyl-, ethyl-, and phenylsilsesquioxane nanoparticles with diameters in the range 50-180 nm were obtained using the modified Stöber method with an aqueous sodium silicate solution used as a seed for further growth of the particles. Methyl- and phenylsilsesquioxane particles were prepared by a sol-gel method in the presence of benzethonium chloride used as a surfactant. The miniemulsion polymerisation approach has been extended to the production of methacrylate-modified silica nanoparticles with vinylpyridine or lauryl methacrylate used as comonomers both in the presence and in the absence of a surfactant. Styryl-modified silica particles with diameters in the range 300-800 nm were obtained by the Stöber method using pre-formed SiO2 nanoparticles as the seeds for silsesquioxane nanoparticle formation. The effect of incorporation of the particles produced using different synthetic methods on hardness and water resistance of acrylic coating systems was studied here.

[Properties of benzethonium chloride in micellar solutions and the effect of added sodium chloride]. / Vlastnosti benzethonium chloridu v micelárních roztocích a vliv prídavku chloridu sodného.

Aqueous solutions of the antimicrobially effective quaternary ammonium salt benzethonium chloride (hyamine 1622) were studied using UV spectrophotometry and partially conductometry. The spectra of micellar solutions of benzethonium chloride revealed a concentration-dependent bathochromic and hyperchromic shift of a weak UV absorption band in the region 250-300 nm. This served to elaborate the spectrophotometric determination of the critical micellar concentration (CMC) of benzethonium chloride and the concentration of free benzethonium cations in micellar solutions without an addition of NaCl and with a constant addition of NaCl 0.003, 0.1 and 0.15 mol/l. Premicellar associations were not observed and in NaCl-free solutions CMC 0.0028 mol/l was spectrophotometrically determined. An addition of NaCl resulted in an increased hyperchromic effect and strengthening of micellization, manifested by a more than ten-times decrease in the CMC as well as the concentration of free benzethonium cations in micellar solutions. The courses of the determined concentrations of free benzethonium cations in the solutions both without and with the presence of NaCl were quite similar; their maximal values were always just a little higher than the corresponding CMC and with a further growth of the total concentration of benzethonium chloride there was, on the other hand, a marked decrease in the concentration of its free cations in micellar solution. Possible effects of a decreased concentration of free benzethonium cations due to an added electrolyte on antimicrobial activity and formation of ionic pairs are discussed.

Headspace gas chromatography based methodology for the analysis of aromatic substituted quaternary ammonium salts.

The analysis of quaternary ammonium salts (QAS) using GC is often performed by "in injector" pyrolysis to create volatile degradation products for quantification purposes. Besides the risk of severe system contamination, the application of this approach on aqueous samples is problematic. In this work, the sample is treated in a vial with 2,2-dimethoxypropane (DMP) under acidic catalysis. In addition to the removal of water and sample enrichment, the QAS are decomposed. As HS transfers only volatile compounds to the GC system, contamination is avoided. It was found that depending on the presence of benzyl, phenyl or methyl groups on the quaternary nitrogen; benzyl chloride, N,N-dimethylaniline or chloromethane are formed respectively in the sealed vial. All these can be used as an analytical target. A calibration curve for benzyl chloride could be derived from the pure compound. Chloromethane was generated from pure benzyldimethyldecylammonium chloride (BEDIDE), a pure QAS with benzyl and methyl groups, to construct a secondary calibration curve using a back analysis approach. It has been proven that by quantifying the formed analytical targets, the mass balance for the QAS under investigation was close to 100%. The presented procedure allows the quantification of any aromatic substituted QAS without the need for a matching reference, which is a major advantage over existing CE and LC methods The proposed methodology was validated for mouth sprays containing benzethonium chloride (BZTCl) or benzoxonium chloride (BZOCl) and for denatonium benzoate (DB) in ethylene glycol (EG) based cooling liquids. Results showed that the approach provided excellent linearity (R2≥0.999) and limits of detection around 0.01µg/vial for benzyl chloride. It was found that the reaction product of DMP and glycerol which was also present in the mouthspray and some cooling liquids, caused chromatographic interference with benzyl chloride. Treating those samples in the vial with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) after the enrichment step removes the interference and leaves a possible pathway for the simultaneous determination of glycerol in those samples.

Effect of desensitizers on the bond strength of a self-etching adhesive system to caries-affected dentin on the gingival wall.

A self-etching dentin adhesive was evaluated for its ability to bond to caries-affected and sound dentin after applying three desensitizers to the gingival walls. Sixty extracted human molars, with approximal dentin caries, were cut horizontally on the long axis of the tooth through caries-affected gingival walls. Carious dentin was removed with SiC paper by means of a caries detector to expose caries-affected dentin. The molars were randomly assigned to four groups: control and three experimental groups-Micro Prime, Glauma Desentizer and Cervitec. Desensitizers were applied to the dentinal surfaces according to manufacturers' instructions. A resin composite was bonded to both the caries-affected and sound dentin of each tooth using a bonding system and plastic rings. The restoration was debonded by shear bond strength. The application of Micro Prime and Gluma Desensitizer to caries-affected dentin did not show any effect on bond strength testing. However, Cervitec caused a decrease in bond strength to caries-affected dentin. The effect of desensitizers on the bond strength of the self-etch bonding agent to caries-affected dentin changed according to the chemical composition of the materials. Desensitizer application on sound dentin is recommended with self-etch bonding systems.

Co-delivery of chemosensitizing siRNA and an anticancer agent via multiple monocomplexation-induced hydrophobic association.

Synergistic combination of gene targeting and chemotherapy by co-delivering siRNA and anticancer drugs has widely been investigated to develop siRNA-based therapeutics for cancer treatment. Despite clinical potential of this approach, big challenges still remain such as delivery efficiency or stability/biocompatibility of the siRNA delivery system. Here we report a simple and biocompatible co-delivering formulation based on a unique complexation method, i.e., multiple monocomplexation-induced hydrophobic association between Bcl-2 targeting siRNA and a monocationic anticancer agent (benzethonium chloride, BZT). A colloidal formulation of the hydrophobically associated multiple monocomplex (HMplex) composed of siRNA, BZT and Pluronic F-68 was spontaneously constructed by physical mixing of the ternary constituents. In vitro and in vivo studies revealed that the ternary HMplex with a low charge ratio (N/P=4) possesses a tightly complexed stable nanostructure with Pluronic surface and small colloidal size less than 10nm, which allowed for 1) suitable protection of siRNA in serum-rich physiological environment, 2) efficient intracellular transfection into the cytoplasm, and 3) successful peritumoral co-delivery into the tumor tissue with dense interstitial matrix. Compared to non-targeting HMplexes between scrambled siRNA and BZT, Bcl-2 targeting HMplexes enhanced significantly both mRNA down-regulation by siRNA and apoptosis induction by BZT, and thus greatly suppressed the tumor volume when administered to highly aggressive and resistant human breast cancer xenografts (MDA-MB-231) in mice. These results elucidate that the co-complexed siRNA and BZT were liberated by intracellular decomplexation to trigger a synergistically combined therapeutic action. The successful siRNA/chemodrug co-delivery in vivo via peritumoral route and the greatly promoted therapeutic efficacy thereby represent the clinical potential of HMplexes for adjuvant locoregional cancer treatment by gene-targeted combination therapy.

Polyurethane elastomer with PEO-PTMO-PEO soft segment for sustained release of drugs.

Blood-compatible segmented polyurethanes and polyurethaneureas were evaluated as drug delivery matrices using crystal violet and benzethonium chloride as model drugs. These polymers were synthesized from ABA-type triblock copolyether as a prepolymer, where A stands for poly(ethylene oxide) and B for poly(tetramethylene oxide). Microphase separation was observed in segmented polyurethaneureas, including drug-doped films. Crystal violet dissolved more in the hard segment domain than in the soft segment matrix, whereas benzethonium chloride was easily dissolved in the soft segment matrix. The drug release behaviours from these films were analysed by the exponent relation Mt/M infinity = ktn, where k and n are constants and Mt/M infinity is the fraction of drug released until time, t. The constant k grew with increasing poly(ethylene oxide) content in the prepolymer, i.e. increased swelling. The constant n was found to be close to 0.5 in many samples, which suggests the release of drug from these polymers is explained by the Fickian diffusion model. However, the mechanism became non-Fickian with increased swelling of the devices.

Synthesis of cis-solamin using a permanganate-mediated oxidative cyclization.

[reaction: see text] cis-Solamin (1) and its diastereoisomer 14 have been synthesized in 13 steps using the diastereoselective permanganate-promoted oxidative cyclization of 1,5-dienes to create the tetrahydrofuran diol core. Notably, no protecting groups are required during the stages of fragment assembly.

Cellulose materials modified by antiseptics and their antimicrobial properties.

Adsorption properties of dressing cellulose materials with respect to surfactant antiseptics were studied. These antiseptics are a complex of the copolymer of N-vinylpyrrolidone and crotonic acid with dimethylbenzylalkylammonium chloride (a synthetic polymer with a wide spectrum of antimicrobial effect) and its low molecular weight analogue (dimethylbenzylalkylammonium chloride). It was established that cellulose materials reversibly adsorb mentioned surfactant antiseptics depending on their concentration in the initial solutions. Maximum release of surfactant antiseptics is achieved at solutions at pH = 7.0. Microbiological tests of cellulose materials modified by antiseptics have shown that they exhibit antimicrobial activity. These results can be used in medical practice in clinics for imparting antimicrobial properties to dressing materials.