Design, Synthesis and In Vitro Characterization of Novel Antimicrobial Agents Based on 6-Chloro-9H-carbazol Derivatives and 1,3,4-Oxadiazole Scaffolds.

In this paper, we aimed to exploit and combine in the same molecule the carbazole and the 1,3,4-oxadiazole pharmacophores, to obtain novel carprofen derivatives, by using two synthesis pathways. For the first route, the following steps have been followed: (i) (RS)-2-(6-chloro-9H-carbazol-2-yl)propanonic acid (carprofen) treatment with methanol, yielding methyl (RS)-2-(6-chloro-9H-carbazol-2-yl)propanoate; (ii) the resulted methylic ester was converted to (RS)-2-(6-chloro-9H-carbazol-2-yl)propane hydrazide (carprofen hydrazide) by treatment with hydrazine hydrate; (iii) reaction of the hydrazide derivative with acyl chlorides led to N-[(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanoil]-N'-R-substituted-benzoylhydrazine formation, which; (iv) in reaction with phosphorus oxychloride gave the (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-(1,3,4-oxadiazol-2-yl)ethane derivatives. In the second synthesis pathway, new 1,3,4-oxadiazole ring compounds were obtained starting from carprofen which was reacted with isoniazid, in the presence of phosphorus oxychloride to form (RS)-1-(6-chloro-9H-carbazol-2-yl)-1-[5-(4-pyridyl)-1,3,4-oxadiazol-2-yl]ethane. The synthesized compounds were characterized by IR, 1H-NMR and 13C-NMR, screened for their drug-like properties and evaluated for in vitro cytotoxicity and antimicrobial activity. The obtained compounds exhibited a good antimicrobial activity, some of the compounds being particularly active on E. coli, while others on C. albicans. The most significant result is represented by their exceptional anti-biofilm activity, particularly against the P. aeruginosa biofilm. The cytotoxicity assay revealed that at concentrations lower than 100 µg/mL, the tested compounds do not induce cytotoxicity and do not alter the mammalian cell cycle. The new synthesized compounds show good drug-like properties. The ADME-Tox profiles indicate a good oral absorption and average permeability through the blood brain barrier. However, further research is needed to reduce the predicted mutagenic potential and the hepatotoxicity.

The conversion of azo-quenchers to fluorophores.

In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.

Affinity purification of the avidin protein family, based on crystal structures of avidin-HABA complexes.

Since the importance of the high affinity between avidin and biotin, Kd = 3 × 10-16 M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4'-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, Kd = 6 × 10-6 M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

Mild disintegration of the green microalgae Chlorella vulgaris using bead milling.

In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25-145 gDW kg(-1)) over a range of agitator speeds (6-12 m s(-1)). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9-10 m s(-1) regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kgDW(-1).

Litiasis pancreática y pancreatitis crónica: importancia del momento del diagnóstico / Pancreatolithiasis and chronic pancreatitis: importance of time of diagnosis

La pancreatitis crónica es una entidad de curso insidioso, que se asocia con brotes de pancreatitis aguda o clínica de dolor recurrente. El manejo depende de la sintomatología del paciente, la posibilidad de manejo con tratamiento conservador y la causas etiológicas asociadas. Así en el caso de las calcificaciones pancreáticas puede ser necesario el abordaje quirúrgico, existiendo la litotripsia extracorpórea como posibilidad menos invasiva en algunos casos

Chronic pancreatitis is associated with exacerbations of acute pancreatitis or recurrent abdominal pain. Management depends on symptoms, results with conservative treatment and etiology. If pancreactic calcificationes are founded on diagnosis, surgical treatment could be necessary, with extracorporeal lithotripsy as less invasive option

Fast mass spectrometry detection of tryptophan-containing peptides and proteins by reduction with pyridine-borane.

In this note, we describe a method devised to detect, by means of mass spectrometry (MS), tryptophan-containing peptides and proteins using pyridine-borane. This reagent selectively reduces tryptophan residues, converting them to 2,3-dihydro-tryptophan, thereby enabling quantitation of tryptophans.

Enrichment of antioxidant compounds from lemon balm (Melissa officinalis) by pressurized liquid extraction and enzyme-assisted extraction.

In this work enzyme-assisted extraction (EAE) and pressurized liquid extraction (PLE) are applied for extraction of natural compounds from lemon balm (Melissa officinalis). Cellulase, endo-ß-1,4 xylanase and pectinase were studied in order to degrade cell wall of lemon balm leaves and to release phenolic compounds. On the other hand, in order to compare the performance obtained with EAE, PLE using water and ethanol was employed maintaining 150°C as extraction temperature. The obtained extracts were characterized in terms of antioxidant capacity by using DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and trolox equivalent antioxidant capacity (TEAC) in vitro assays, whereas the Folin-Ciocalteu procedure was employed to estimate the total phenols content. On the other hand, extracts were chemically characterized by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that EAE enhanced the total phenolic content and the antioxidant capacity compared to a non-enzymatic control. PLE extracts presented higher amount of phenols and antioxidant capacity than enzyme-assisted extracts, reaching the highest values on water extracts (193.18mggallicacid/gextract and EC50=6.81µg/mL). Among the bioactive phenolic compounds identified in lemon balm, rosmarinic acid was the main component, although other important compounds were also identified, such as caffeic acid derivatives (salvianolic acids, lithospermic acid) and rosmarinic acid derivatives (rosmarinic acid hexoside, sagerinic acid, sulfated rosmarinic acid). The present study confirms that EAE and PLE can be considered alternative methods for the extraction of natural compounds with biological activity from natural sources.

Chemical composition of bioactive pressurized extracts of Romanian aromatic plants.

In this contribution, pressurized liquid extraction (PLE) has been employed to isolate bioactive compounds from three native Romanian plants, oregano (Origanum vulgare), tarragon (Artemisia dracunculus) and wild thyme (Thymus serpyllum). Different PLE conditions have been tested including extraction with water, ethanol and their mixtures in a wide range of extraction temperatures (50-200°C), and the antioxidant capacity of the extracts was measured using different assays (DPPH radical scavenging, TEAC assay and Folin-Ciocalteau assay to measure total phenols). Moreover, a complete chemical characterization by using LC-MS/MS was carried out to be able to correlate the bioactivity with the particular chemical composition of each extract and plant. The use of PLE with water as a solvent at the highest temperature tested (200°C) always provided the highest extraction yields for the three studied plants, being maximum for oregano (>60%). Besides, oregano's pressurized water extracts at lower temperatures (50°C) presented the highest content on total phenols (184.9 mg gallic acid/g extract) and the best antioxidant activities (EC(50) 6.98 µg/ml). In general, oregano extracts were the most active, followed by wild thyme extracts. The antioxidant capacity measured by DPPH assay was highly correlated with the amount of total phenols. Moreover, the use of a LC-MS/MS method allowed the identification of 30 different phenolic compounds in the different extracts, including phenolic acids, flavones, flavanones and flavonols, which have an important influence on the total antioxidant capacity of the different extracts.

Novel derivatives of 6-mercaptopurine: synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines.

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

[3H]Allicin: preparation and applications.

Allicin (diallylthiosulfinate), the active substance of garlic, has been shown to possess a variety of biological activities. Mechanistic and pharmacokinetic studies of allicin and its derivatives raise the need for a labeled compound. However, labeling of this volatile and unstable liquid requires delicate handling. Here, we describe a simple method for the preparation of (3)H-labeled allicin. This was achieved by applying synthetic [(3)H]alliin ([2,3-(3)H]allylcysteine sulfoxide) to a column containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [(3)H]allicin was done by differential adsorbtion of the reaction components on a neutral polystyrene resin, Porapak Q. Thiol-containing compounds are known to be the main target of allicin. In this work we demonstrated that [(3)H]allicin can be used for the synthesis of labeled [(3)H]allylmercapto derivatives of SH peptides and proteins. Thus, we prepared [(3)H]S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we showed that incubation of alliinase with [(3)H]allicin led to modification of 1.4 cysteine residues per subunit of the enzyme.

Carbonic anhydrase inhibitors. Inhibition of tumor-associated isozyme IX by halogenosulfanilamide and halogenophenylaminobenzolamide derivatives.

Two series of halogenated sulfonamides have been prepared. The first consists of mono/dihalogenated sulfanilamides, whereas the second one consists of the mono/dihalogenated aminobenzolamides, incorporating equal or different halogens (F, Cl, Br, and I). These sulfonamides have been synthesized from the corresponding anilines by acetylation (protection of the amino group), chlorosulfonylation, followed either by amidation, or reaction with 5-amino-1,3,4-thiadiazole-2-sulfonamide (and eventually deacetylation). All these compounds, together with the six clinically used sulfonamide inhibitors (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, and brinzolamide) were investigated as inhibitors of the transmembrane, tumor-associated isozyme carbonic anhydrase (CA) IX. Inhibition data against the classical, physiologically relevant isozymes I, II, and IV were also obtained. CA IX shows an inhibition profile which is generally completely different from those of isozymes I, II, and IV, with potent inhibitors (inhibition constants in the range of 12-40 nM) among both simple aromatic (such as 3-fluoro-5-chloro-4-aminobenzenesulfonamide) as well as heterocyclic compounds (such as acetazolamide, methazolamide, 5-amino-1,3,4-thiadiazole-2-sulfonamide, aminobenzolamide, and dihalogenated aminobenzolamides). This first detailed CA IX inhibition study revealed many interesting leads, suggesting the possibility to design even more potent and eventually CA IX-selective inhibitors, with putative applications as antitumor agents.

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties.

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.

Carbonic anhydrase activators: design of high affinity isozymes I, II, and IV activators, incorporating tri-/tetrasubstituted-pyridinium-azole moieties.

A series of tight binding carbonic anhydrase (CA) activators was obtained by reaction of amino-azoles (3-amino-pyrazole, 2-amino-imidazole, and 5-amino-tetrazole) with tri- or tetrasubstituted pyrylium salts. Many of the new pyridinium salts incorporating azole moieties reported here proved to be efficient in vitro activators of three CA isozymes, CA I, II, and IV. Very good activity was detected against hCA I and bCA IV (h = human; b = bovine isozymes), for which some of the new compounds showed affinities in the low nanomolar range, whereas against hCA II, their affinities were in the range of 95-150 nM. Substitution patterns of the pyridinium ring leading to best activity included 4-phenyl-2,6-dialkyl moieties or 2,4,6-tri- and 2,3,4,6-tetraalkyl groups. Ex vivo experiments showed some of the new activators to strongly enhance CA activity after incubation with human erythrocytes. Furthermore, due to their cationic nature, some of these compounds (the imidazole and pyrazole derivatives) are membrane-impermeant, discriminating thus between cytosolic and membrane-bound CA isozymes. The present paper is the first report of membrane-impermeant CA activators. The pyridinium tetrazole derivatives on the other hand do penetrate through biological membranes. Such CA activators might lead to the development of drugs/diagnostic tools for the management of CA deficiency syndromes as well as for the pharmacological enhancement of synaptic efficacy, spatial learning, and memory. This may constitute a new approach for the treatment of Alzheimer disease and other conditions in need of achieving memory therapy.

The effects of allicin and enalapril in fructose-induced hyperinsulinemic hyperlipidemic hypertensive rats.

The effects of a synthetic preparation of an active constituent of garlic, allicin, were studied on blood pressure (BP), triglycerides, and insulin levels in Sprague-Dawley rats in which high fructose feeding elicited hyperinsulinemia, hypertension, and hypertriglyceridemia. Results were compared with those of the antihypertensive drug enalapril. Three groups of male Sprague-Dawley rats were fed a fructose-enriched diet for 5 weeks. During the last 2 weeks 10 animals received only fructose, 10 received allicin, and 10 received enalapril. Blood pressure, insulin level, and triglyceride levels were measured at the beginning of the experiment and after 3 and 5 weeks on the fructose diet, fructose/allicin diet, or fructose/enalapril diet. Allicin lowered BP from the maximal level (after 3 weeks of fructose) of 153.4 +/- 8 mm Hg to 139.7 +/- 12 mm Hg after 2 weeks on allicin; insulin from 11.7 +/- 3.7 ng/mL on fructose diet to 6.92 +/- 3.3 ng/mL on allicin; and triglycerides from 132.8 +/- 18 mg/dL on fructose to 59.6 +/- 27 mg/dL on allicin. The similar effect of allicin and enalapril on BP, insulin, and triglycerides reinforces the trend toward combining the nonpharmacologic approach with drug therapy.

S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.

Natural antibodies against alliinase in human serum and polyclonal antibodies elicited in rabbit share the same immunogenic determinants.

Human serum contains natural antibodies against alliinase, a protein abundantly found in garlic (Allium sativum) cloves. In order to study the epitope(s) of this protein recognized by anti-alliinase antibodies, we used a random hexapeptide library displayed on filamentous M13 phage. Analysis of the phagotopes selected on rabbit anti-alliinase antibodies revealed that the motif-GKXVXX- was common for all peptides. The most frequent phage displaying -GKHVAV- sequence has a 50% identity with the original alliinase sequence (amino acid residues 156-161). The position of this epitope is only nine amino acids apart from the oligosaccharide chain attached to the N146. The rabbit anti-alliinase immunoglobulin G (IgG), which bound the phages displaying this phagotope, also bound the corresponding peptide derived from the alliinase sequence. Affinity-purified natural antibodies against alliinase, present in normal human serum (which can specifically recognize the native and denaturated protein) also bound the selected phagotope. Thus, our results indicate that specific natural anti-dietary protein antibodies presented in human serum can have the same. or overlapping. epitopes with the IgG evoked during the active (experimental) immunization in animals.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 microM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 microM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.