Syntheses and biological evaluation of 2-amino-3-acyl-tetrahydrobenzothiophene derivatives; antibacterial agents with antivirulence activity.

Developing new compounds targeting virulence factors (e.g., inhibition of pilus assembly by pilicides) is a promising approach to combating bacterial infection. A high-throughput screening campaign of a library of 17 500 small molecules identified 2-amino-3-acyl-tetrahydrobenzothiophene derivatives (hits 2 and 3) as novel inhibitors of pili-dependent biofilm formation in a uropathogenic Escherichia coli strain UTI89. Based on compounds 2 and 3 as the starting point, we designed and synthesized a series of structurally related analogs and investigated their activity against biofilm formation of E. coli UTI89. Systematic structural modification of the initial hits provided valuable information on their SARs for further optimization. In addition, small structural changes to the parent molecules resulted in low micromolar inhibitors (20-23) of E. coli biofilm development without an effect on bacterial growth. The hit compound 3 and its analog 20 were confirmed to prevent pili formation in a hemagglutination (HA) titer assay and electron microscopy (EM) measurements. These findings suggest that 2-amino-3-acyl-tetrahydrobenzothiophenes may serve as a new class of compounds for further elaboration as antibacterial agents with antivirulence activity.

Methyl 5-chloro-4,5-didehydrojasmonate (J7) inhibits macrophage-derived chemokine production via down-regulation of the signal transducers and activators of transcription 1 pathway in HaCaT human keratinocytes.

Jasmonates are lipid-based stress hormones that are critical for the defense of plants against insects. Two naturally occurring jasmonates, jasmonic acid and methyl jasmonate, have recently been explored for their efficacy as anti-cancer agents. Furthermore, certain synthetic jasmonates (e.g., the cyclopentenone isoprostane J2) exert anti-inflammatory actions in lipopolysaccharide (LPS)-challenged murine macrophages via down-regulation of chemokines and other inflammatory mediators. Chemokines participate in the development and progression of many inflammatory disorders, such as atopic dermatitis (AD) and Crohn's disease, as exemplified by the role of macrophage-derived chemokine (MDC/CCL22) in the pathology of AD. The current study therefore investigated the impact of jasmonate derivatives (jasmonic acid and methyl jasmonate) and their synthetic analogues (J2 and J7) on the expression of MDC in interferon (IFN)-γ- and tumor necrosis factor (TNF)-α-stimulated HaCaT human keratinocytes, as well as the attendant mechanism of action. Jasmonic acid, methyl jasmonate, and J2 failed to inhibit the cytokine-stimulated production of MDC. By contrast, J7 suppressed the mRNA and protein expression levels of MDC in a dose-dependent manner. Moreover, J7 diminished the activation of signal transducers and activators of transcription 1 (STAT1), but had no inhibitory effect on the nuclear factor kappa B (NF-κB) or mitogen-activated protein kinase (MAPK) pathways. These results demonstrate that J7 impairs IFN-γ- and TNF-α-induced inflammatory chemokine production by targeting the STAT1 pathway.

In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates.

A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. However, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resistant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new jasmonate analogs may serve as potential anti-inflammatory leads.

Cytotoxic cytochalasins from the endozoic fungus Phoma sp. of the giant jellyfish Nemopilema nomurai.

Four new cytochalasin derivatives (1-4), together with cytochalasin B (5), were isolated from the fungus Phoma sp. obtained from the giant jellyfish Nemopilema nomurai. The planar structure and relative stereochemistry were established by analysis of 1D and 2D NMR data. The absolute configuration was defined by the modified Mosher's method. The compounds showed significant cytotoxicity against a small panel of human solid tumor cell lines (A549, SK-OV-3, SK-MEL-2, XF 498, and HCT15) with IC(50) values in the range of 0.5-30 µM. The cytochalasin B (5) showed obvious cytotoxicity with IC(50) of 7.9 µM against HeLa human cervical carcinoma cells.

Evaluation of endogenous fatty acid amides and their synthetic analogues as potential anti-inflammatory leads.

A series of endogenous fatty acid amides and their analogues (1-78) were prepared, and their inhibitory effects on pro-inflammatory mediators (NO, IL-1ß, IL-6, and TNF-α) in LPS-activated RAW264.7 cells were evaluated. Their inhibitory activity on the pro-inflammatory chemokine MDC in IFN-γ-activated HaCaT cells was also examined. The results showed that the activity is strongly dependent on the nature of the fatty acid part of the molecules. As expected, the amides derived from enone fatty acids showed significant activity and were more active than those derived from other types of fatty acids. A variation of the amine headgroup also altered bioactivity profile remarkably, possibly by modulating cell permeability. Regarding the amine part of the molecules, N-acyl dopamines exhibited the most potent activity (IC(50) ∼2 µM). This is the first report of the inhibitory activity of endogenous fatty acid amides and their analogues on the production of nitric oxide, cytokines (IL-1ß, IL-6, and TNF-α) and the chemokine MDC. This study suggests that the enone fatty acid-derived amides (such as N-acyl ethanolamines and N-acyl amino acids) and N-acyl dopamines may be potential anti-inflammatory leads.

Anti-inflammatory effect of methyl dehydrojasmonate (J2) is mediated by the NF-κB pathway.

Inflammation as a major defense mechanism against pathogens is modulated by diverse microbial products. A variety of plant and microbial products interacting with Toll-like receptors initiate a wide spectrum of responses from phagocytosis to cytokine production, which modulates inflammation. Jasmonates are fatty acid-derived cyclopentanones produced by plants and lower eukaryotes that play an important role in the defense against insects. In this study, we are set up to define the molecular targets of J2 action. While the lipopolysaccharide (LPS) stimulation of macrophage cell line RAW264.7 induced TNF-α, IL-6, iNOS, and COX-2 that were associated with an increase in miR-155 and miR-146a, the J2 suppressed the induction of these inflammatory cytokines and enzymes as well as miR-155 in a dose-dependent manner. To assess the associations of miR-155 with inflammatory markers, we overexpressed miR-155 and found attenuation of COX-2 suppression with J2 treatment. Furthermore, J2 inhibited NF-κB, p65, and IκB but had no or only minimal effects on the mitogen-activated protein kinase pathway. In conclusion, the present study demonstrates that J2 suppresses LPS stimulation of RAW264.7 cells by targeting NF-κB pathways.

Bicyclic alpha,omega-dicarboxylic acid derivatives from a colonial tunicate of the family Polyclinidae.

In the course of our search for bioactive metabolites from a colonial tunicate of the family Polyclinidae, six new (1-6) cyclic fatty acid derivatives were isolated. Their planar structures were established on the basis of NMR and MS spectroscopic analyses. The relative configuration was determined by NOESY experiment. Compounds 1-6 represent a fused bicyclic skeleton possibly derived from alpha,omega-dicarboxylic acids such as eicosanedioic acid or docosanedioic acid via a Diels-Alder type of cyclization. Compounds 1-4 and 6 showed mild cytotoxicity against a panel of five human solid tumor cell lines.

Two enone fatty acids isolated from Gracilaria verrucosa suppress the production of inflammatory mediators by down-regulating NF-kappaB and STAT1 activity in lipopolysaccharide-stimulated RAW 264.7 cells.

Gracilaria verrucosa is a common marine red alga that has anti-oxidant and anti-cancer properties. Recently, we reported that anti-inflammatory constituents of G. verrucosa operate through an unknown mechanism. For this reason, we isolated two enone fatty acids from G. verrucosa and investigated their molecular mechanism in LPS-stimulated RAW264.7 cells. We found that the two compounds inhibited the production of inflammatory markers (nitric oxide, TNF-alpha, and IL-6) in a dose-dependent manner. We next studied the effects of G. verrucosa compounds on LPS-induced signaling pathways. The two compounds suppressed NF-kappaB reporter activity by interfering with nuclear translocation of NF-kappaB and suppressed JAK/STAT (p-STAT1) signaling. These results suggest that G. verrucosa inhibits the production of inflammatory mediators (NO, TNF-alpha, and IL-6) by suppressing the activation of NF-kappaB and the phosphorylation of STAT1.

Anti-inflammatory sesquiterpenoids from a sponge-derived Fungus Acremonium sp.

In the course of our search for bioactive metabolites from a sponge-derived fungus Acremonium sp., new sesquiterpenoids (1-4) were isolated along with known derivatives by bioactivity-guided fractionation. The unique cyclic skeleton of compounds 2 and 3 is unprecedented. The absolute configurations were determined by modified Mosher's method and CD spectroscopy, along with comparison of (1)H and (13)C NMR spectroscopic data and specific optical rotation values with those reported. The anti-inflammatory activity of the isolated compounds (1, 5, 7-13) was evaluated by measuring their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in RAW 264.7 murine macrophage cells. Among the compounds tested, compounds 7 and 9 significantly inhibited the production of NO and TNF-alpha at the concentration of 100 microM, while compounds 11 and 12 showed selective inhibition of NO production at the same concentration.

New jasmonate analogues as potential anti-inflammatory agents.

In an effort to develop new anti-inflammatory agents, methyl jasmonate analogues (2-20) were synthesized and evaluated for their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophage cells. The introduction of an enone functionality to the structure of a plant hormone (1) rendered the product (2) a significant anti-inflammatory activity. Analogues further derived from 2 (7, 9, 13, and 15) exhibited even more enhanced activity, and these compounds were much more potent than natural anti-inflammatory prostaglandins (PGA(1), PGA(2), and 15-deoxy-Delta(12,14)-PGJ(2)). Among them, compounds 9 and 15 showed the highest potency, while compounds 7 and 13 would be more desirable with respect to safety. This is the first study demonstrating the anti-inflammatory potential of jasmonate derivatives, and the present results suggest that alpha-haloenone jasmonates (7, 9, 13, and 15) may serve as potential anti-inflammatory leads.

Apoptotic activity of fatty acid derivatives may correlate with their inhibition of DNA replication.

The apoptogenic and DNA damaging effects of (E)-10-oxooctadec-8-enoic acid (S5C) and (E)-9-oxooctadec-10-enoic acid (S6C), two structurally related fatty acids isolated from Red Alga Gracilaria verrucosa, were compared and their apoptosis-inducing properties characterized against human lung carcinoma (A549) cells. Significantly, the two acids decreased the rates of proliferation and viability (IC50 of approximately 170 and approximately 140 microM) as well as evidence of the induction of apoptosis. Cell morphological changes observed under light microscopy confirmed apoptosis occurrence. The results from Annexin V/PI dual staining and the cell cycle arrest assay indicated that S5C and S6C induced an earlier apoptosis of A549 cells in a concentration-dependent manner. We found that they induced DNA damage and inhibited DNA replication followed by S-phase arrest. In addition, the very sensitive alkaline micro-gel electrophoresis technique (comet assay) was used to estimate the compound-induced DNA single- and double-strand breaks. These findings suggest that S5C and S6C induced A549 cell apoptosis and their effects are associated with DNA damage. Therefore, S5C and S6C have the potential to be developed into anticancer agents due to their relatively easy synthesis and structural manipulation.

Cytotoxic bromotyrosine derivatives from a two-sponge association of Jaspis sp. and Poecillastra sp.

Bioassay-guided chemical investigation of the lipophilic extract of a two-sponge association (Jaspis sp. and Poecillastra sp.) led to the isolation of two new bromotyrosine derivatives (1 and 2), along with known derivatives (3-12). Cyclobispsammaplin A (1) is a cyclic derivative of the previously reported bispsammaplin A (13), while psammaplin M (2) is composed of beta-alanine (or aspartic acid) unit. Compounds 3, 4, 6, 10, and 12 are isolated for the first time from a sponge belonging to the subclass Tetractinomorpha. Structure elucidation was performed by a combination of high resolution mass and 2D NMR (principally COSY, HMBC, HSQC, and NOESY) spectroscopy. Compounds 1-4, 6, 10, and 12 were evaluated for cytotoxicity against a small panel of five human solid tumor cell lines and their activity was compared in relevance to their structure.

Furan metabolites from the sponge-derived yeast Pichia membranifaciens.

Five new compounds, pichiafurans A-C (1-3) and pichiacins A and B (4 and 5), along with five known compounds (6-10), have been isolated from the yeast Pichia membranifaciens derived from a marine sponge Petrosia sp. Their structures were elucidated by 1D and 2D NMR and mass spectrometry techniques. Pichiafurans are rare examples of monofurano metabolites isolated from yeast.

Anti-inflammatory constituents of the red alga Gracilaria verrucosa and their synthetic analogues.

A chemical study on the anti-inflammatory components of the red alga Gracilaria verrucosa led to the isolation of new 11-deoxyprostaglandins ( 1- 4), a ceramide ( 5), and a C 16 keto fatty acid ( 6), along with known oxygenated fatty acids ( 7- 14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds 1- 5 were determined by Mosher's method. The anti-inflammatory activity of the isolated compounds ( 1- 14) was evaluated by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most potent activity. In the evaluation of these two compounds and derivatized analogues ( 15- 40), the anti-inflammatory activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential anti-inflammatory leads for the first time.