New antifungal ent-labdane diterpenes against Candida glabrata produced by microbial transformation of ent-polyalthic acid.

Candida glabrata, the most common non-albicans Candida species and one of the primary causes of candidemia, exhibits decreased susceptibility to azoles and more recently to echinocandins. Polyalthic acid 1, a furan diterpene, has been shown promising biological potential and in this study ent-polyalthic acid derivatives with antifungal activity against Candida glabrata were produced by microbial transformation. Incubation of 1 with Aspergillus brasiliensis afforded two known (compounds 5 and 10) and eight new derivatives (compounds 2-4, 6-9 and 11). The most common reaction was hydroxylation, but isomerization of the double bond and acetylation were also detected. None of the tested compounds showed cytotoxicity against HeLa, MCF-7 and MCF-10A cell lines showing IC50 values ranging from 62.6 µM to > 500 µM. Compounds 1, 5, 6, 8 and 11 showed fungistatic effects (ranging from 34.1 µM to 39.5 µM) on C. glabrata at lower concentrations than fluconazole (163.2 µM). Compounds 1, 6 and 8 were more potent fungicides (ranging from 79.0 to 143.6 µM) than fluconazole, which showed fungicidal effect at concentrations higher than 163.2 µM. These results suggest that ent-polyalthic acid and some of its derivatives could be used as lead compounds to develop new antifungal agents.

Design and biological evaluation of tetrahydro-ß-carboline derivatives as highly potent histone deacetylase 6 (HDAC6) inhibitors.

Various diseases are related to epigenetic modifications. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, and thus are involved in the regulation of gene expression. First generation histone deacetylase inhibitors (HDACi) are unselective, hinder all different kinds of zinc dependent HDACs and additionally cause several side effects. Subsequently, selective HDACi are gaining more and more interest. Especially, selective histone deacetylase 6 inhibitors (HDAC6i) are supposed to be less toxic. Here we present a successful optimization study of tubastatin A, the synthesis and biological evaluation of new inhibitors based on hydroxamic acids linked to various tetrahydro-ß-carboline derivatives. The potency of our selective HDAC6 inhibitors, exhibiting IC50 values in a range of 1-10 nM towards HDAC6, was evaluated with the help of a recombinant human HDAC6 enzyme assay. Selectivity was proofed in cellular assays by the hyperacetylation of surrogate parameter α-tubulin in the absence of acetylated histone H3 analyzed by Western Blot. We show that all synthesized compounds, with varies modifications of the rigid cap group, were selective and potent HDAC6 inhibitors.

Diarylheptanoid Glycosides of Morella salicifolia Bark.

A methanolic extract of Morella salicifolia bark was fractionated by various chromatographic techniques yielding six previously unknown cyclic diarylheptanoids, namely, 7-hydroxymyricanol 5-O-ß-d-glucopyranoside (1), juglanin B 3-O-ß-d-glucopyranoside (2), 16-hydroxyjuglanin B 17-O-ß-d-glucopyranoside (3), myricanone 5-O-ß-d-gluco-pranosyl-(1→6)-ß-d-glucopyranoside (4), neomyricanone 5-O-ß-d-glucopranosyl-(1→6)-ß-d-glucopyranoside (5), and myricanone 17-O-α-l-arabino-furanosyl-(1→6)-ß-d-glucopyranoside (6), respectively, together with 10 known cyclic diarylheptanoids. The structural diversity of the diarylheptanoid pattern in M. salicifolia resulted from varying glycosidation at C-3, C-5, and C-17 as well as from substitution at C-11 with hydroxy, carbonyl or sulfate groups, respectively. Structure elucidation of the isolated compounds was achieved on the basis of one- and two-dimensional nuclear magnetic resonance (NMR) as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) analyses. The absolute configuration of the glycosides was confirmed after hydrolysis and synthesis of O-(S)-methyl butyrated (SMB) sugar derivatives by comparison of their ¹H-NMR data with those of reference sugars. Additionally, absolute configuration of diarylheptanoid aglycones at C-11 was determined by electronic circular dichroism (ECD) spectra simulation and comparison with experimental CD spectra after hydrolysis.

Phytochemical study of Juglans regia L. leaves.

A methanolic extract of Juglans regia L. leaves was fractioned by various chromatographic techniques yielding a total of 40 metabolites belonging to megastigmane, tetralone, phenylpropanoid, neolignane and juglone glycosides. Ten unknown megastigmane glucoside derivatives (juglanionosides A-K, 1-10) and six unknown tetralone glucoside derivatives (juglanosides J-O, 11-16) together with 24 known compounds - among them 16 described for the first time in Juglans - were isolated. As characteristic structural feature, the previously undescribed compounds showed acylation of the sugar units with sinapic, ferulic, coumaric, benzoic or salicylic acid. Their chemical structures were elucidated on the basis of 1D and 2D NMR techniques, HRESIMS as well as CD spectroscopy. Absolute stereochemistry was revealed by mild alkaline hydrolysis and comparison of CD and polarimetric data to literature values.

Synthesis, Crystal Structure and Biological Activity Screening of Novel N-(α-Bromoacyl)-α-amino Esters Containing Valyl Moiety.

Three novel N-(α-bromoacyl)-α-amino esters: methyl 2-(2-bromo-3-methylbutanamido)pentanoate (1), methyl 2-(2-bromo-3-methylbutanamido)-2-phenylacetate (2) and methyl 2-(2-bromo-3-methylbutanamido)-3-phenylpropanoate (3) were synthesized. Single crystal X-ray diffraction data are reported for compounds 1 and 2. The cytotoxicity, antiinflammatory and antibacterial activity of compounds 1-3 were investigated. Additionally, the physico-chemical properties of studied compounds were calculated and an in silico toxicological study of compounds 1-3 was performed. The low level of cytotoxicity and absence of antibacterial and anti-inflammatory activity of 1-3 in tested concentrations might be a beneficial prerequisite for their incorporation in prodrugs.

Revisited anti-inflammatory activity of matricine in vitro: Comparison with chamazulene.

The proazulene matricine (1) is present in chamomile flower heads and has been proven to exhibit strong in vivo anti-inflammatory activity. In contrast to other secondary metabolites in chamomile preparations like its degradation product chamazulene (2), no plausible targets have been found to explain this activity. Therefore we revisited 1 regarding its in vitro anti-inflammatory activity in cellular and molecular studies. Using ICAM-1 as a marker for NF-κB activation, it was shown that ICAM-1 protein expression induced by TNF-α and LPS, but not by IFN-γ, was remarkably inhibited by 1 in endothelial cells (HMEC-1). Inhibition was concentration-dependent in a micromolar range (10-75 µM) and did not involve cytotoxic effects. At 75 µM expression of the adhesion molecule ICAM-1 was down to 52.7 ± 3.3% and 20.4 ± 1.8% of control in TNF-α and LPS-stimulated HMEC-1, respectively. In contrast, 2 showed no activity. Quantitative RT-PCR experiments revealed that TNF-α-induced expression of the ICAM-1 gene was also reduced by 1 in a concentration-dependent manner, reaching 32.3 ± 6.2% of control at 100 µM matricine. Additional functional assays (NF-κB promotor activity and cytoplasm to nucleus translocation) confirmed the inhibitory effect of 1 on NF-κB signaling. Despite the fact that 1 lacks an α,ß-unsaturated carbonyl and is thus not able to act via a Michael reaction with electron rich SH groups of functional biological molecules, data gave strong evidence that 1 inhibits NF-κB transcriptional activity in endothelial cells by an hitherto unknown mechanism and this may contribute to its well-known anti-inflammatory activity in vivo.

Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

In vitro structure-toxicity relationship of chalcones in human hepatic stellate cells.

Xanthohumol (XN), the major prenylated chalcone from hops (Humulus lupulus L.), has received much attention within the last years, due to its multiple pharmacological activities including anti-proliferative, anti-inflammatory, antioxidant, pro-apoptotic, anti-bacterial and anti-adhesive effects. However, there exists a huge number of metabolites and structurally-related chalcones, which can be expected, or are already known, to exhibit various effects on cells. We have therefore analyzed the effects of XN and 18 other chalcones in a panel, consisting of multiple cell-based assays. Readouts of these assays addressed distinct aspects of cell-toxicity, like proliferation, mitochondrial health, cell cycle and other cellular features. Besides known active structural elements of chalcones, like the Michael system, we have identified several moieties that seem to have an impact on specific effects and toxicity in human liver cells in vitro. Based on these observations, we present a structure-toxicity model, which will be crucial to understand the molecular mechanisms of wanted effects and unwanted side-effects of chalcones.

Effect of choline carboxylate ionic liquids on biological membranes.

Choline carboxylates, ChCm, with m=2-10 and choline oleate are known as biocompatible substances, yet their influence on biological membranes is not well-known, and the effect on human skin has not previously been investigated. The short chain choline carboxylates ChCm with m=2, 4, 6 act as hydrotropes, solubilizing hydrophobic compounds in aqueous solution, while the longer chain choline carboxylates ChCm with m=8, 10 and oleate are able to form micelles. In the present study, the cytotoxicity of choline carboxylates was tested using HeLa and SK-MEL-28 cells. The influence of these substances on liposomes prepared from dipalmitoylphosphatidylcholine (DPPC) was also evaluated to provide insights on membrane interactions. It was observed that the choline carboxylates with a chain length of m>8 distinctly influence the bilayer, while the shorter ones had minimal interaction with the liposomes.

Aqueous extracts of the marine brown alga Lobophora variegata inhibit HIV-1 infection at the level of virus entry into cells.

In recent years, marine algae have emerged as a rich and promising source of molecules with potent activities against various human pathogens. The widely distributed brown alga Lobophora variegata that is often associated with tropical coral reefs exerts strong antibacterial and antiprotozoal effects, but so far has not been associated with specific anti-viral activities. This study investigated potential HIV-1 inhibitory activity of L. variegata collected from different geographical regions, using a cell-based full replication HIV-1 reporter assay. Aqueous L. variegata extracts showed strong inhibitory effects on several HIV-1 strains, including drug-resistant and primary HIV-1 isolates, and protected even primary cells (PBMC) from HIV-1-infection. Anti-viral potency was related to ecological factors and showed clear differences depending on light exposition or epiphyte growth. Assays addressing early events of the HIV-1 replication cycle indicated that L. variegata extracts inhibited entry of HIV-1 into cells at a pre-fusion step possibly by impeding mobility of virus particles. Further characterization of the aqueous extract demonstrated that even high doses had only moderate effects on viability of cultured and primary cells (PBMCs). Imaging-based techniques revealed extract effects on the plasma membrane and actin filaments as well as induction of apoptosis at concentrations exceeding EC50 of anti-HIV-1 activity by more than 400 fold. In summary, we show for the first time that L. variegata extracts inhibit HIV-1 entry, thereby suggesting this alga as promising source for the development of novel HIV-1 inhibitors.

In-vitro stability and metabolism of a tacrine-silibinin codrug.

OBJECTIVES: A tacrine-silibinin codrug showed promising results in pharmacological and toxicity testing, superior to an equimolar mixture of tacrine and silibinin. The aim of this study was to get more information about its stability, possible degradation products, metabolites, and especially its active principle in vitro and in vivo. METHODS: The stability of the codrug was analysed under in-vitro assay conditions. Additionally, its metabolism was investigated using pooled human liver microsomes. Metabolites were identified via liquid chromatography-high resolution electrospray ionization mass spectrometry. Furthermore, the influence of one of the main cleavage products, tacrine hemi succinamide, on viability and mitochondria of hepatic stellate cells was analysed. KEY FINDINGS: The codrug remained stable in culture medium (Dulbecco's modified Eagle's medium) over an incubation period of 24 h, whereas exposition to microsomal enzymes led to rapid cleavage of the ester bond to form silibinin and a tacrine hemi succinamide. In addition, glucuronidated metabolites of both silibinin and the codrug were detected. For the tacrine hemi succinamide, no effects were observed with regard to cell viability and mitochondrial impairment. CONCLUSIONS: This study helps understand and interpret previous results concerning the effects and the absence of toxicity of the tacrine-silibinin codrug and supplies important information for further identification of the active principles of the codrug in vivo.

Acyloxybutadiene tricarbonyl iron complexes as enzyme-triggered CO-releasing molecules (ET-CORMs): a structure-activity relationship study.

A series of η(4)-acyloxycyclohexadiene-Fe(CO)(3) complexes was prepared and fully characterized by spectroscopic methods including single crystal X-ray diffraction. For this purpose a new synthetic access to differently acylated 1,3- and 1,5-dienol-Fe(CO)(3) complexes was developed. The enzymatically triggered CO release from these compounds was monitored (detection of CO through GC and/or by means of a myoglobin assay) and the anti-inflammatory effect of the compounds was assessed by a cellular assay based on the inhibition of NO-production by inducible NO synthase (iNOS). It was demonstrated that the properties (rate of esterase-triggered CO release, iNOS inhibition, cytotoxicity) of the complexes strongly depend on the substitution pattern of the π-ligand and the nature of the acyloxy substituent.

Tacrine-silibinin codrug shows neuro- and hepatoprotective effects in vitro and pro-cognitive and hepatoprotective effects in vivo.

A codrug of the anti-Alzheimer drug tacrine and the natural product silibinin was synthesized. The codrug's biological and pharmacological properties were compared to an equimolar mixture of the components. The compound showed potent acetyl- and butyrylcholinesterase inhibition. In a cellular hepatotoxicity model, analyzing the influence on viability and mitochondria of hepatic stellate cells (HSC), the toxicity of the codrug was markedly reduced in comparison to that of tacrine. Using a neuronal cell line (HT-22), a neuroprotective effect against glutamate-induced toxicity could be observed that was absent for the 1:1 mixture of components. In subsequent in vivo experiments in rats, in contrast to the effects seen after tacrine treatment, after administration of the codrug no hepatotoxicity and no induction of the cytochrome P450 system were noticed. In a scopolamine-induced cognitive impairment model using Wistar rats, the codrug was as potent as tacrine in reversing memory dysfunction. The tacrine-silibinin codrug shows high AChE and BChE inhibition, neuroprotective effects, lacks tacrine's hepatotoxicity in vitro and in vivo, and shows the same pro-cognitive effects in vivo as tacrine, being superior to the physical mixture of tacrine and silibinin in all these regards.

Xanthohumol uptake and intracellular kinetics in hepatocytes, hepatic stellate cells, and intestinal cells.

Xanthohumol (XN) is the major prenylated chalcone of hops and hence an ingredient of beer. Despite many advances in understanding of the pharmacology of XN, one largely unresolved issue is its low bioavailability in the human organism. Also, not much is known about its actual concentrations and pharmacokinetics in liver and intestinal cells. Therefore, the uptake, intracellular distribution, and kinetics of XN were studied in various cell types, namely, hepatocellular carcinoma cells (HuH-7), hepatic stellate cells (HSC), primary cultured hepatocytes, and colorectal adenocarcinoma cells (Caco-2). Fluorescent microscopy allowed for the first time visualization and tracing of the uptake and intracellular distribution of XN. A rapid accumulation of XN concentrations that were up to >60-fold higher than the concentration present in the ambient culture medium was observed. Fluorescence recovery after photobleaching experiments revealed that most XN molecules are bound to cellular proteins, which may alter properties of cellular factors.

Hyperforin is a modulator of inducible nitric oxide synthase and phagocytosis in microglia and macrophages.

Upon activation, microglia, the immunocompetent cells in the brain, get highly phagocytic and release pro-inflammatory mediators like nitric oxide (NO). Excessive NO production is pivotal in neurodegenerative disorders, and there is evidence that abnormalities in NO production and inflammatory responses may at least support a range of neuropsychiatric disorders, including depression. Although extracts of St. John's wort (Hypericum perforatum L.) have been used for centuries in traditional medicine, notably for the treatment of depression, there is still considerable lack in scientific knowledge about the impact on microglia. We used N11 and BV2 mouse microglia, as well as RAW 264.7 macrophages to investigate the effects of St. John's wort extract and constituents thereof on NO production Moreover, flow cytometry and fluorescence microscopy were employed to analyze the influence on phagocytosis, transcription factor activation states, and cell motility. We found that extracts of St. John's wort efficiently suppress lipopolysaccharide-induced NO release and identified hyperforin as the responsible compound, being effective at concentrations between 0.25 and 0.75 microM. The reduced NO production was mediated by diminished inducible nitric oxide synthase expression on the mRNA and protein level. In addition, at similar concentrations, hyperforin reduced zymosan phygocytosis to 20-40% and putatively acted by downregulating the CD206 macrophage mannose receptor and modulation of cell motility. We found that the observed effects correlate with a suppression of the activated state of Nf-kappaB and phospho-CREB, while c-JUN, STAT1, and HIF-1alpha activity and cyclooxygenase-2 expression remained unaffected by hyperforin. These results reveal that hyperforin influences pro-inflammatory and immunological responses of microglia that are involved in the progression of neuropathologic disorders.

Xanthohumol, a chalcon derived from hops, inhibits hepatic inflammation and fibrosis.

Xanthohumol (XN) is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. In this study, we first investigated the effects of XN on hepatocytes and hepatic stellate cells (HSC), the central mediators of liver fibrogenesis. XN inhibited the activation of primary human HSC and induced apoptosis in activated HSC in vitro in a dose dependent manner (0-20 microM). In contrast, XN doses as high as 50 microM did not impair viability of primary human hepatocytes. However, in both cell types XN inhibited activation of the transcription factor NFkappaB and expression of NFkappaB dependent proinflammatory genes. In vivo, feeding of XN reduced hepatic inflammation and expression of profibrogenic genes in a murine model of non-alcoholic steatohepatitis. These data indicate that XN has the potential as functional nutrient for the prevention or treatment of non-alcoholic steatohepatitis or other chronic liver disease.

Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates.

Five tacrine-ferulic acid hybrids (6a-e) were designed and synthesized as multi-potent anti-Alzheimer drug candidates. All target compounds have better acetylcholinesterase inhibitory activity and comparable butyrylcholinesterase inhibitory activity in relation to tacrine. Interestingly, 6d showed a reversible and non-competitive inhibitory action for acetylcholinesterase indicating interaction with the peripheral anionic site, whereas a reversible but competitive inhibitory action for butyrylcholinesterase. The antioxidant study revealed that four target compounds have, compared to Trolox, high ability to absorb reactive oxygen species.

Design, synthesis and pharmacological evaluation of hybrid molecules out of quinazolinimines and lipoic acid lead to highly potent and selective butyrylcholinesterase inhibitors with antioxidant properties.

A set of hybrid molecules were synthesized out of lipoic acid, alpha,omega-diamines of different lengths serving as spacers, and cholinesterase (ChE) inhibiting [2,1-b]quinazolinimines. Depending on the length of the alkylene spacer the amide hybrids are inhibitors of acetylcholinesterase (AChE) with inhibitory activities of 0.5-4.6microM and inhibitors of butyrylcholinesterase (BChE) with activities down to 5.7nM, therefore greatly exceeding the inhibitory activities of the parent quinazolinimines by factors of up to 1000. Due to increasing activity at BChE with increasing length of the alkylene spacer approximately 100-fold selectivity toward BChE is reached with a hepta- and an octamethylene spacer. Kinetic measurements reveal competitive and reversible inhibition of both ChEs by the hybrids. Furthermore, cell viability and antioxidant activity (using the ORAC-fluorescein assay) of several hybrids were evaluated, showing cytotoxicity at concentrations from 3.7 to 10.2microM and antioxidant properties are in the range of 0.4-0.8 Trolox equivalents (lipoic acid=0.6).