Bioisosteric ferrocenyl 1,3-thiazolidine-4-carboxylic acid derivatives: In vitro antiproliferative and antimicrobial evaluations.

To improve the antiproliferative effect of ALC67 (diastereomeric mixture of ethyl 2-phenyl-3-propioloyl-1,3-thiazolidine-4-carboxylate), its structure was modified via (i) bioisosteric substitution of the phenyl ring by the ferrocene unit and (ii) replacing the propiolamide side-chain in ACL67 with other acyl groups having differing electrophilicities. In this way, a small library of methyl N-acyl-2-ferrocenyl-1,3-thiazolidine-4-carboxylates (13 compounds in total) was created and characterized by spectral and crystallographic means. The last N-acylation step was highly diastereoselective toward the cis-diastereomer. In solution, most of the obtained compounds existed as a mixture of two rotamers and displayed a preference for the syn-orientation around the CN bond. A twisted 5T4 envelope conformation was adopted by the derivative containing the N-phenoxyacetyl group in the crystalline state. Two derivatives with chloroacetyl and bromoacetyl groups in the N-3 side chain were cytotoxic to fibroblasts and hepatocellular cancer cells in the low micromolar range (IC50(MRC5) = 9.0 and 11.8 µM, respectively, and IC50(HepG2) = 10.6 and 18.4 µM, respectively) causing an effect similar to the lead compound (IC50(HepG2) = 10.0 µM) and cisplatin (IC50(MRC5) = 4.0 µM and IC50(HepG2) = 7.7 µM). Several derivatives also manifested modest antimicrobial effects against the studied microbial strains (MICs in the range from 0.44 to 4.0 µmol/mL). Our findings demonstrated that the introduction of a ferrocene core facilitated the preparation of optically pure analogs of ALC67 and that the cytotoxicity of compounds may be enhanced by adding proper electrophilic centers to the N-acyl side-chain.

An HPLC-based assay for improved measurement of glutamate decarboxylase inhibition/activation.

L-Glutamic acid decarboxylase (GAD) is an enzyme that ensures the balance between the levels of two neurotransmitters, γ-aminobutyric acid (GABA) and L-glutamic acid (L-Glu), necessary for proper brain functioning. A reduction in the concentrations of GABA and/or GAD activity has been implicated in the symptoms associated with epilepsy, which could be plausibly alleviated by the application of GAD activators. As any unnecessary interference in GAD catalytic activity could be detrimental, it is important to study whether CNS (or other) drug candidates act on GAD or not. The ability to identify and reduce this risk early could significantly improve the process of drug development. Although many methods for measuring GAD activity in various biological samples have been described, only few (such as manometric and radiometric) were adopted as in vitro assays for the screening of potential GAD inhibitors/activators. However, these methods require specialized equipment and/or an expensive radiolabeled substrate, and may have sensitivity and/or reliability issues. Therefore, this study aimed to develop an HPLC-DAD-based assay that would allow a simple and more accurate measurement of GAD inhibition or activation using unpurified mice or rat brain homogenates. This assay is based on the quantification of GABA, formed during the enzymatic reaction, after its derivatization with dansyl chloride. Various parameters were evaluated to optimize the assay procedure (e.g. homogenate volume, incubation time, DMSO content, GAD, GABA, and dansyl-GABA stabilities). This assay was validated for pharmacological screenings using 3-mercaptopropionic acid and gallic acid and GAD obtained from different experimental animals.

New neryl esters from Helichrysum italicum (Roth) G. Don (Asteraceae) essential oil.

Helichrysum italicum (immortelle) is a dwarf aromatic shrub native to the Mediterranean region. The typical subspecies (italicum) produces an essential oil rich in neryl acetate and characteristic ß-diketones, italidiones, highly valued in the perfume industry. As esters are an important group of aroma-active volatiles, herein the composition of the ester fraction of this immortelle chemotype essential oil was studied in detail. Chromatographic separation of Corsican immortelle essential oil enabled the discovery of numerous potentially olfactory-interesting esters of nerol and/or angelic acid, undetectable by direct GC-MS analyses of the unfractioned oil. Four esters of nerol and medium-chain branched fatty acids represent new natural products, while several other esters have a rather restricted occurrence in the Plant Kingdom.

Linking the antimicrobial and anti-inflammatory effects of immortelle essential oil with its chemical composition - The interplay between the major and minor constituents.

Immortelle (Helichrysum italicum, Asteraceae) essential oil has been widely used in alternative medicine to accelerate wound healing, as well as in cosmetic products to stimulate skin regeneration and to reduce the appearance of wrinkles. It is also considered a natural and safe culinary spice that could also be applied in the food industry as a preservative in the future. The therapeutic efficacy of this oil changes with the natural variability of the composition. Herein we tested and mutually compared the antimicrobial and anti-inflammatory activities of four commercial immortelle oils differing in the relative amounts of marker compounds, i.e. neryl esters, α-pinene, γ- and ar-curcumenes, and ß-diketones. The anti-inflammatory effect of selected chromatographic fractions, enriched in the aforementioned constituents, was evaluated by studying toxicity toward rat peritoneal macrophages, their nitric oxide production, myeloperoxidase, and arginase activities. Subsequently, the compositional and activity data were subjected to a multivariate statistical treatment to reveal the possible correlation(s) between the percentage of essential-oil constituents and the observed activities. The obtained results imply that immortelle oil efficiency as an antimicrobial and/or anti-inflammatory agent is most plausibly a result of a synergistic action between its constituents, and/or, rather unexpectedly, the presence of some minor constituents.

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C21 -C35 ), fatty acid benzyl esters (FABEs; C20 -C32 ), and 2-alkanones (C23 -C35 ) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.

Genomics-Based Insights Into the Biosynthesis and Unusually High Accumulation of Free Fatty Acids by Streptomyces sp. NP10.

Streptomyces sp. NP10 was previously shown to synthesize large amounts of free fatty acids (FFAs). In this work, we report the first insights into the biosynthesis of these fatty acids (FAs) gained after genome sequencing and identification of the genes involved. Analysis of the Streptomyces sp. NP10 draft genome revealed that it is closely related to several strains of Streptomyces griseus. Comparative analyses of secondary metabolite biosynthetic gene clusters, as well as those presumably involved in FA biosynthesis, allowed identification of an unusual cluster C12-2, which could be identified in only one other S. griseus-related streptomycete. To prove the involvement of identified cluster in FFA biosynthesis, one of its three ketosynthase genes was insertionally inactivated to generate mutant strain mNP10. Accumulation of FFAs in mNP10 was almost completely abolished, reaching less than 0.01% compared to the wild-type strain. Cloning and transfer of the C12-2 cluster to the mNP10 mutant partially restored FFA production, albeit to a low level. The discovery of this rare FFA biosynthesis cluster opens possibilities for detailed characterization of the roles of individual genes and their products in the biosynthesis of FFAs in NP10.

Prenylated ß-diketones, two new additions to the family of biologically active Hypericum perforatum L. (Hypericaceae) secondary metabolites.

Two novel ß-diketones, 2,6,9-trimethyl-8-decene-3,5-dione (A) and 3,7,10-trimethyl-9-undecene-4,6-dione (B), were identified from the renowned medicinal plant Hypericum perforatum L. The structures of ß-diketones A and B were corroborated by syntheses (4 steps starting from methyl acetoacetate, overall yields 30% and 23%, respectively). In solution, these ß-diketones predominantly exist as two rapidly interconverting ß-keto-enol tautomers. The structures of A and B show some common fragments with the molecules of hyperforin and adhyperforin, respectively, the acknowledged multi-target secondary metabolites from St. John's wort. It is therefore not surprising that A displayed a noteworthy biological activity profile as well (including brine shrimp toxicity, antinociceptive, antidepressant and acetylcholinesterase inhibitory activity). ß-Diketone A manifested the most outstanding potency as an acetylcholinesterase inhibitor with IC50 value of 1.51 µM pointing again to the ß-keto-enol moiety as a promising lead structure for the development of drugs that could lessen symptoms of Alzheimer's disease (such as dementia, depression and pain).

Toxic essential oils. Part V: Behaviour modulating and toxic properties of thujones and thujone-containing essential oils of Salvia officinalis L., Artemisia absinthium L., Thuja occidentalis L. and Tanacetum vulgare L.

Neurotoxic thujones (α- and ß-diastereoisomers) are common constituents of plant essential oils. In this study, we employed a statistical approach to determine the contribution of thujones to the overall observed behaviour-modulating and toxic effects of essential oils (Salvia officinalis L., Artemisia absinthium L., Thuja occidentalis L. and Tanacetum vulgare L.) containing these monoterpene ketones. The data from three in vivo neuropharmacological tests on rats (open field, light-dark, and diazepam-induced sleep), and toxicity assays (brine shrimp, and antimicrobial activity against a panel of microorganisms), together with the data from detailed chemical analyses, were subjected to a multivariate statistical treatment to reveal the possible correlation(s) between the content of essential-oil constituents and the observed effects. The results strongly imply that the toxic and behaviour-modulating activity of the oils (hundreds of constituents) should not be associated exclusively with thujones. The statistical analyses pinpointed to a number of essential-oil constituents other than thujones that demonstrated a clear correlation with either the toxicity, antimicrobial effect or the activity on CNS. Thus, in addition to the thujone content, the amount and toxicity of other constituents should be taken into consideration when making risk assessment and determining the regulatory status of plants in food and medicines.

Essential-Oil Constituents and Alkanes of Cephalaria ambrosioides Roem. & Schult. (Family Caprifoliaceae, Subfamily Dipsacaceae) and (Chemo)taxonomic Discernment of the Subfamilies Dipsacaceae and Morinaceae.

Herein, the results of the first study of the volatile and alkane profiles of Cephalaria ambrosioides Roem. & Schult. (Caprifoliaceae, subfamily Dipsacaceae) were reported. The GC-FID and GC/MS analyses of the essential oils hydrodistilled from leaves and stems (CA1) and flowers (CA2) of C. ambrosioides allowed the identification of 284 different components. The main compounds of the studied oil samples were palmitic acid (24.3 and 32.5% for CA1 and CA2, resp.), hexahydrofarnesyl acetone (1.4 and 10.8% for CA1 and CA2, resp.), (Z)-hex-3-en-1-ol (7.0 and <0.1% for CA1 and CA2, resp.), and linoleic acid (1.9 and 6.5% for CA1 and CA2, resp.). Essential-oil compositional data of selected plant species belonging to the Dipsacaceae (15) and Morinaceae (2) subfamilies were used to resolve taxonomical ambiguities regarding the genus Cephalaria and its infrageneric relations, especially concerning the subfamily Morinaceae (formerly a genus within Dipsacaceae). The results of multivariate statistical analyses (25 different essential-oil samples) supported the exclusion of Morina species from the Dipsacaceae subfamily. The relative abundances of alkanes from n-, iso-, and anteiso-series followed a (distorted) Gaussian-like distribution and suggested that the biosyntheses of n- and branched alkanes in C. ambrosioides are possibly not controlled by the same elongase. Also, the obtained results suggested that there was a difference in the biosynthesis/accumulation of alkanes in the vegetative and reproductive parts of C. ambrosioides.

Structural diversity and possible functional roles of free fatty acids of the novel soil isolate Streptomyces sp. NP10.

Herein, a novel soil bacterium Streptomyces sp. NP10 able to grow outside usual streptomycetes optimum conditions (e.g., at 4 °C, pH 9 and high NaCl concentration), exhibiting atypical hemolytic, DNAse, and cellulolytic activities, is described. This strain produces and excretes into the growth medium large amounts of free long-chain fatty acids (FAs). A concurrent lipidomics study revealed a large structural diversity of FAs with over 50 different n- and branched-chain, (un)saturated, and cyclopropane FAs (C7-C30) produced by this strain. Two of these, i-17:0cy9-10 and a-18:0cy9-10, represent new natural products and the first ever identified branched cyclopropane FAs. Both free and bound lipid profiles of Streptomyces sp. NP10 were dominated by saturated branched chain FAs (i-14:0, a-15:0, and i-16:0). Although these free FAs showed only a moderate antimicrobial activity, our results suggest that they could have an ecophysiological role in interspecies signaling with another soil microorganism Pseudomonas aeruginosa. This work represents the first comprehensive report on the structural diversity and complexity of the free FA pool in Streptomyces. A naturally occurring streptomycete, such as Streptomyces sp. NP10, which secretes significant amounts of free long-chain FAs (non-cytotoxic) into the medium, could be useful in microbial biodiesel production.