Synthesis, Antimicrobial Activity and in silico Studies on Thymol Esters.

Derivatisation of parent structure in terpenoids often results in enhancement of biological activity of newly obtained compounds. Thymol, a naturally occurring phenol biosynthesized through the terpene pathway, is a well known biocide with strong antimicrobial attributes and diverse therapeutic activities. We have aimed our study on a single modification of phenolic functionality in thymol in order to obtain a small focused library of twenty thymyl esters, ten of which were new compounds. All compounds were involved in in vitro antimicrobial testing. Another important aspect of current study was implementation of in silico calculation of physico-chemical, pharmacokinetic and toxicological properties, which could be helpful by giving an additional guidance in further research.

Naturally occurring N(6)-substituted adenosines (cytokinin ribosides) are in vitro inhibitors of platelet aggregation: an in silico evaluation of their interaction with the P2Y(12) receptor.

A few naturally occurring N(6)-substituted adenosine derivatives (cytokinin ribosides) were investigated as inhibitors of platelet aggregation induced in vitro by collagen and their activity range was demonstrated (IC50: 6.77-141 µM). A docking study suggests that anti-aggregation activity of these compounds could involve an interaction with the P2Y12 receptor binding site.

Evaluation of the radical scavenging activity of some representative isoprenoid and aromatic cytokinin ribosides (N6-substituted adenosines) by in vitro chemical assays.

Cytokinins are naturally occurring adenine derivatives whose physiological role is that of growth regulators in plants and that show also many other activities either in plants and in mammalian cells. In plants, they can be found mainly as free bases ((N6-substituted adenines, CKs), but also as the corresponding N9- ribosides (N6-substituted adenosines, CKRs). In mammalian cells, CKRs are, in general, more active than CKs. In order to evaluate the intrinsic in vitro antioxidant capacity of some significant CKRs, their scavenging activity against synthetic radicals that are at the basis of well-established antioxidant assays (ORAC, TEAC, DPPH) has been evaluated. The results of the in vitro scavenging activity of biologically relevant radicals such as hydroxyl (HO•), superoxide (O2.-) and lipid peroxides (R-OO.) are reported and discussed.

Carvacrol Derivatives as Antifungal Agents: Synthesis, Antimicrobial Activity and in Silico Studies on Carvacryl Esters.

Chemical modifications of natural monoterpenoids to various derivatives have been reported to result in enhancement of biological activities when compared to parent compounds. In this context a well-known biocide and food additive, carvacrol, served as a basic scaffold onto which a phenolic functionality transformation by introducing acyl groups was performed. By using this simple methodology, we obtained a small series of 25 esters. For each of the obtained compounds we have performed structural characterization, in vitro antimicrobial testing and in silico calculation of physico-chemical, pharmacokinetic and toxicological properties. Despite numerous data on the synthesis and bioactivity of carvacryl ester lower homologues, there are scarce data on esters with acid components higher than C9, so that among 25 compounds, 10 were reported for the first time (spectral characterization for 12 are herein the first reported). Our research is also the first comprehensive study of carvacryl esters antifungal and of medium/long chain fatty acid esters antibacterial activities. Interesting result is that all the synthesized esters, regardless the nature of the R residue, have shown activity on fungal strain Aspergilus niger and on yeast Candida albicans comparable to carvacrol. Besides presented experimental data, implementation of in silico calculation of physico-chemical, pharmacokinetic and toxicological properties on the prepared compounds, may be valuable information in further research.

New synthesis of 6[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid and evaluation of the influence of adamantyl group on the DNA binding of a naphthoic retinoid.

6[3-(1-Adamantyl)-4-methoxyphenyl]-2-naphthoic acid (Adapalene®), a synthetic aromatic retinoid specific for RARß and RARγ receptors, has been prepared utilizing a Pd/C-mediated Suzuki coupling between 6-bromo-2-naphthoic acid and 4-methoxyphenyl boronic acid, followed by introduction of an adamantyl group in the position 3 of the formed 6-(4-methoxyphenyl)-2-naphthoic acid. The interaction of 6-(4-methoxyphenyl)-2-naphthoic acid/ethyl ester and the 3-adamantyl analogs with DNA was studied in aqueous solution at physiological conditions by UV-vis spectroscopy. The calculated binding constants K(ligand-DNA) ranged between 1.1×10(4) M(-1) and 1.1×10(5) M(-1), the higher values corresponding to those of the adamantylated compounds. Molecular modeling studies have emphasized that the intercalative binding of adapalene and its derivatives to DNA is mainly stabilized by hydrophobic interactions related to the presence of the adamantyl group.

Measurement of S-methylcysteine and S-methyl-mercapturic acid in human urine by alkyl-chloroformate extractive derivatization and isotope-dilution gas chromatography-mass spectrometry.

S-methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O-6-methyl-guanine to revert the normal GC base pairing. To assess the value of SMC and of S-methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC-MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra- and inter-individual variability. Extractive alkylation with butylchloroformate-n-butanol-pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC-MS with S-[CD(3) ]-SMC and -SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium-polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile-age women followed for one month above the minimum detectable level of approx. 500 µg/L while SMC concentrations were in the 0.02-0.7 µg/mL range (n = 61) with large inter-day and inter-individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90-810 µg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes.

Pharmacogenomics and analogues of the antitumour agent N6-isopentenyladenosine.

N(6)-isopentenyladenosine (i(6)A), a member of the cytokinin family of plant hormones, has potent in vitro antitumour activity in different types of human epithelial cancer cell lines. Gene expression profile analysis of i(6)A-treated cells revealed induction of genes (e.g., PPP1R15A, DNAJB9, DDIT3, and HBP1) involved in the negative regulation of cell cycle progression and reportedly up-regulated during cell cycle arrest in stress conditions. Of 6 i(6)A analogues synthesized, only the 1 with a saturated double bond of the isopentenyl side chain had in vitro antitumour activity, although weaker than that of i(6)A, suggesting that i(6)A biological activity is highly linked to its structure. In vivo analysis of i(6)A and the active analogue revealed no significant inhibition of cancer cell growth in mice by either reagent. Thus, although i(6)A may inhibit cell proliferation by regulating the cell cycle, further studies are needed to identify active analogues potentially useful in vivo.

Enhanced activity or resistance of adenosine derivatives towards adenosine deaminase-catalyzed deamination: Influence of ribose modifications.

The effect of the presence of the 1'-C-methyl group and 2',3'-O-substitution in the adenosine structure on ADA activity has been investigated by modeling studies. Results show that the 2'- and 3'-O- substituents are harbored in a quite large cavity of intermediate polarity, whereas the 1'-C-substituent clashes against Ala180 distorting the architecture of the catalytic centre. Globally, the study emphasizes the ability of ADA to transform a large set of 2',3'-O-substituted adenosine analogues as well as the opportunity to design 1'-C-substituted adenosine derivatives resistant to ADA-catalyzed deamination.

Activity of adenosine deaminase and adenylate deaminase on adenosine and 2', 3(')-isopropylidene adenosine: role of the protecting group at different pH values.

The deamination rate of 2',3'-isopropylidene adenosine catalyzed by adenosine deaminase (ADA) from calf intestine and adenylate deaminase (AMPDA) from Aspergillus species has been evaluated and compared with that of the enzymatic reactions of adenosine, to elucidate the influence of the protecting group on enzyme activity.

Synthesis, Antimicrobial Activity and in silico Studies on Eugenol Eters.

The results presented herein represent our continued study based on the modification of phenolic functionality in molecules originated from natural sources by acylation. A small focused library of nineteen eugenyl esters, with four of which are new compounds, is reported. All compounds were subjected to in vitro antimicrobial testing. In silico studies were carried out calculating physico-chemical, pharmacokinetic and toxicological properties, providing more data as additional guidance for further research.

Peroxynitrite and nitrosoperoxycarbonate, a tightly connected oxidizing-nitrating couple in the reactive nitrogen-oxygen species family: new perspectives for protection from radical-promoted injury by flavonoids.

Peroxynitrite is the product of the reaction of nitric oxide with superoxide radical and is implicated in the pathogenesis of a wide variety of human diseases, being responsible for in-vivo oxidation/nitration events. Nitrosoperoxycarbonate anion, formed by the interaction of peroxynitrite with CO(2)/bicarbonate at physiological concentrations, provides a new interpretation of oxidative/nitrative processes formerly attributed to peroxynitrite. The aim of this review is to summarize the chemistry and biology of peroxynitrite and radical species related to nitrosoperoxycarbonate anion, as well as the information available regarding the molecular mechanisms that determine and regulate radical-promoted injury by the two tightly connected species at physiological concentrations. Interception of carbonate and nitro radicals produced by interaction of peroxynitrite with CO(2)/bicarbonate, as in-vivo prevention of pathological events, creates new perspectives for the evaluation of safe scavengers of oxidative/nitrative stress at the physiological level. In this respect, natural products such as flavonoids hold a preeminent position among the vast array of compounds endowed with such properties.

2',3'-isopropylidene group, a molecular scaffold to study the activity of adenosine and adenylate deaminase on adenosine analogues modified in the ribose moiety.

2 ',3 '-Isopropylidene group can be used as a molecular scaffold for the introduction of modifications at 5 ' and 1 ' positions of adenosine and these modified nucleosides are used to evaluate the biocatalytic activity of adenosine and adenylate deaminase.

Deamination of 2',3'-O-isopropylideneadenosine-5'- carboxylic acid catalyzed by adenosine deaminase (ADA) and adenylate deaminase (AMPDA): influence of substrate ionization on the activity of the enzymes.

Adenosine deaminase (ADA) and adenylate deaminase (AMPDA) catalyze the deamination of 2 ',3 '-O-isopropylideneadenosine-5'-carboxylic acid to the corresponding inosine derivative and dependence of the rate of enzymatic reaction on the ionization degree of the substrate has been studied at different pH values.

Effect of organic co-solvents in the evaluation of the hydroxyl radical scavenging activity by the 2-deoxyribose degradation assay: The paradigmatic case of α-lipoic acid.

The 2-deoxyribose degradation assay (2-DR assay) is an in vitro method broadly used for evaluating the scavenging activity against the hydroxyl radical (HO). One of the major drawbacks of the assay, however, is that only water soluble compounds can be tested for their radical-scavenging activity. Lipoic acid (LA) is an excellent scavenger of HO but it exhibits a low solubility in the aqueous milieu of the 2-DR assay and a high tendency to polymerize under a variety of conditions. We used LA as a paradigmatic substrate to evaluate the effect of several organic co-solvents in increasing its solubility. Most of these solvents, however, demonstrated to be potent scavengers of HO making their use in the 2-DR assay improper. On the other hand, acetonitrile showed a remarkably low reactivity toward HO (rate constant ~8.7×106M-1s-1) which allowed us to use it as a co-solvent in the preparation of stock solutions of LA ~5mM. We therefore evaluated the radical-scavenging activity of LA by the 2-DR assay in a relatively large range of concentrations, 1-200µM. We found that the rate constant for LA+HO is diffusion-controlled (~1×1010M-1s-1 in water at 25°C) and uninfluenced by the presence of small quantities of acetonitrile. Therefore, the use of acetonitrile in the 2-DR assay does not interfere with the test and may increase the solubility of the radical scavengers.

Evaluation of the antioxidant activity and capacity of some natural N6-substituted adenine derivatives (cytokinins) by fluorimetric and spectrophotometric assays.

Four natural N(6)-substituted adenine derivatives (cytokinins) were evaluated for the first time in vitro for they antioxidant capacity by using fluorimetric and spectrophotometric assays, i.e., the oxygen radical absorbance capacity (ORAC), trolox equivalence antioxidant capacity (TEAC) and the 2-deoxyribose degradation (2-DRA) assays. The results from the TEAC assay show that only N(6)-(4-hydroxybenzyl)adenine (p-topolin) shows an electron transfer capacity due to the presence of a phenolic moiety in the N(6)-position. The results from the ORAC test show that the antioxidant activity of N(6)-furfuryladenine (kinetin, K) is the highest up to a concentration of 1 µM, whereas at concentrations higher than 1 µM p-topolin is the most efficient antioxidant. Analysis of the kinetic data suggests that, compared to the other cytokinins, more sites of the molecular structure of p-topolin are available for the quenching of peroxyl radicals. The hydroxyl radical scavenger ability, as measured by the 2-DRA assay, showed that all tested cytokinins react in this test and that N(6)-(Δ(2)-isopentenyl)adenine is slightly more potent, probably because of the allylic methylene group present in the N(6)-isopentenyl moiety. Our data suggest that a part of the biological activity of the evaluated cytokinins is likely to be related to an intrinsic antioxidant capacity.

A minimally invasive technique for the evaluation of the regulatory steps of the two major pathways of bile acid synthesis.

BACKGROUND: Bile acid synthesis accounts for more than 95% of total cholesterol catabolism per day. We have developed a minimally invasive technique in humans that quantifies the rates of plasma appearance of 7alpha- and 27-hydroxycholesterol, representing the first steps of the "classical" and "alternative" pathways of bile acid synthesis, respectively. METHODS: For this purpose, during the intravenous infusion of synthetic deuterated isotopomers of 7alpha-hydroxycholesterol and 27-hydroxycholesterol plasma samples are collected and analysed by a GC-MS based method that allows to quantify the exogenous/natural isotopomer ratio of the two sterols. From this data, the rates of plasma appearance of 7alpha- and 27-hydroxycholesterol are calculated. RESULTS: In a group of healthy individuals steady state kinetics are obtained during a 2 h period yielding mean values of 2.0+/-0.8 and 3.7+/-0.6 mg/h for 7alpha- and 27-hydroxycholesterol, respectively. The data are consistent with findings using older techniques that require studies over several days. CONCLUSION: Considering that at steady state of the exogenous/natural isotopomer ratio the plasma appearance of the two regulatory hydroxysterols are related to the rate of bile acid synthesis via the "classical" and the "alternative" pathways, respectively, the proposed method could be used to evaluate the immediate effects of different diets and drugs and other determinants on cholesterol catabolism.

Improved determination of malonaldehyde by high-performance liquid chromatography with UV detection as 2,3-diaminonaphthalene derivative.

A rapid, specific and simple procedure is proposed for the determination of free malonaldehyde (MA) contained in fish tissue. The method is the optimization of the reaction of MA with 2,3-diaminonaphthalene to afford a naphtodiazepinium ion that present a UV absorption at 311nm, useful for MA determination by HPLC with UV detection. The reaction proceeds in the presence of 25% acetonitrile at 37°C in 20min at pH 2 using 2,4-pentanedione as internal standard. The method has been applied to homogenized samples of canned mackerel fillets that were treated with 2,3-diaminonaphthalene in an acidic aqueous:acetonitrile mixture. The produced naphtodiazepinium ion was extracted in acetonitrile by a salting-out homogeneous liquid-liquid extraction. A standard calibration was carried out in the range 0.625-10nmol/g. The reliability of the procedure is demonstrated by linearity (r(2)=0.998), limit of detection (0.16nmol/g), limit of quantification (0.22nmol/g), repeatibility (RSD 5.57%), and intermediate precision (RSD 8.92%).

Detoxification of 4-hydroxynonenal (HNE) in keratinocytes: characterization of conjugated metabolites by liquid chromatography/electrospray ionization tandem mass spectrometry.

Keratinocytes are potential targets of lipid peroxidation products (alpha,beta-unsaturated aldehydes) generated in the skin following UV exposure, among which the most abundant and toxic product is 4-hydroxy-trans-2,3-nonenal (HNE). The aim of this study was to investigate the ability of keratinocytes (NCTC2544 cell lines) to detoxify HNE, through characterization of metabolites, until now never demonstrated, using a combined analytical approach (liquid chromatography (LC) and liquid chromatography/mass spectrometry (LC/MS)). Incubation of cells with HNE (up to 200 micro M) was performed in order to evaluate the ability of the cells to detoxify this toxic aldehyde, and indicated that the cell viability was maintained under these conditions. LC analysis of the extracellular media from keratinocytes incubated with 100 micro M HNE shows a time-dependent decrease of HNE, disappearance from the medium within 2 h and concomitant formation of two unconjugated (phase I) metabolites, 4-hydroxy-2-nonenoic acid (HNA) and 1,4-dihydroxy-2-nonene (DHN), which were both identified and quantified by LC and accounted for 48.8 +/- 4.6% of the HNE dose. Four additional metabolites were identified in the extracellular medium by reversed-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection as Michael adducts (phase II metabolites), arising by the addition of the nucleophilic sulfur of glutathione (GSH) to the electrophilic C-3 of HNE, followed by oxidation-reduction enzymatic processes. The GSH-HNE conjugates were (a) S-(4-hydroxynonanal-3-yl)glutathione, (b) S-(1,4-dihydroxy-nonane-3-yl)glutathione, (c) S-(4-oxononanal-3-yl)glutathione and (d) S-(4-oxo-nonan-1-ol-3-yl)glutathione, and accounted for 52.3 +/- 5.8% of the HNE dose (35 nmol mg(-1) protein), as estimated indirectly by measuring the extent of cellular GSH consumption (18.7 +/- 1.8 nmol mg(-1) protein). The time course of HNE biotransformation was then determined by monitoring the formation of metabolites inside and outside the cell at different times after HNE addition (5-120 min). A time-dependent and almost linear formation inside the cell was observed for all the metabolites (plateau after 15 min of incubation), followed by a rapid decay and a concomitant increase in the extracellular medium (plateau of formation after 60 min). This confirms that HNE diffuses into the cell where is totally metabolized through phase I and phase II reactions to unreactive products, which are then exported outside the cell. This is the first demonstration that skin epidermal cells are able to detoxify the cytotoxic products of lipid peroxidation.

Clemmensen reduction of diosgenin and kryptogenin: synthesis of [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol.

A new experimental protocol has been established for the Clemmensen reduction of diosgenin and kryptogenin with the aim to prepare deuterated isotopomers of (25R)-26-hydroxycholesterol. Uncontrolled deuteration has been achieved from diosgenin, whereas [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol (1) can be synthesized from kryptogenin.

Synthesis of (20S)-[7,7,21,21-2H4]-3beta-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, a useful intermediate for the preparation of deuterated isotopomers of sterols.

(20S)-[7,7,21,21-2H(4)]-3beta-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3beta-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7beta-hydroxy derivative in a highly stereoselective manner.