New Rev-export inhibitor from Alpinia galanga and structure–activity relationship

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, an HIV-1 viral regulatory protein, disclosed 1′-acetoxychavicol acetate (ACA, 1) as a new inhibitor for nuclear export of Rev from the roots of Alpinia galanga. Both analysis for mechanism of action with biotinylated probe (2) and several synthesized analogs established crucial portions in 1 for Rev-export inhibitory activity.     

Acetoxybenzhydrols as highly active and stable analogues of 1′S-1′-acetoxychavicol,a potent antiallergic principal from Alpinia galanga

Through SAR studies on 1′S-1′-acetoxychavicol acetate (1) against Type I antiallergic activity by indexing release of β-hexosaminidase, a marker of antigen-IgE-mediated degranulation in RBL-2H3 cells, more stable and potent analogue, 4-(methoxycarbonyloxyphenylmethyl)phenyl acetate (16), has been developed. The compound 16 also strongly inhibited the antigen-IgE-mediated TNF-α and IL-4 production.     

1′-Acetoxychavicol Acetate Isolated from Alpinia galanga Ameliorates Ovalbumin-Induced Asthma in Mice

The World Health Organization reports that 235 million people are currently affected by asthma. This disease is associated with an imbalance of Th1 and Th2 cells, which results in the upregulation of cytokines that promote chronic inflammation of the respiratory system. The inflammatory response causes airway obstruction and can ultimately result in death. In this study we evaluated the effect of 1′-acetoxychavicol acetate (ACA) isolated from Alpinia galanga rhizomes in a mouse model of ovalbumin (OVA)-induced asthma. To generate the mouse model, BALB/c mice were sensitized by intraperitoneal injection of OVA and then challenged with OVA inhalation for 5 days. Mice in the vehicle control group were sensitized with OVA but not challenged with OVA. Treatment groups received dexamethasone, 25 mg/kg/day ACA, or 50 mg/kg/day ACA for 5 days. Asthma-related inflammation was assessed by bronchoalveolar lavage fluid cell counts and histopathological and immunohistochemical analysis of lung tissues. Our results showed that ACA reduced the infiltration of white blood cells (especially eosinophils) and the level of IgE in the lungs of mice challenged with OVA and suppressed histopathological changes such as airway remodeling, goblet-cell hyperplasia, eosinophil infiltration, and glycoprotein secretion. In addition, ACA inhibited expression of the Th2 cytokines interleukin (IL)-4 and IL-13, and Th1 cytokines IL-12α and interferon-γ. Because asthmatic reactions are mediated by diverse immune and inflammatory pathways, ACA shows promise as an antiasthmatic drug candidate.     

Synthesis and antibacterial activity of a series of novel 9-O-acetyl- 4′-substituted 16-membered macrolides derived from josamycin

A series of novel 9-O-acetyl-4′-substituted 16-membered macrolides derived from josamycin has been designed and synthesized by cleavage of the mycarose of josamycin and subsequent modification of the 4′-hydroxyl group. These derivatives were evaluated for their in vitro antibacterial activities against a panel of Staphylococcus aureus and Staphylococcus epidermidis. 15 (4′-O-(3-Phenylpropanoyl)-9-O-acetyl-desmycarosyl josamycin) and 16 (4′-O-butanoyl-9-O-acetyl-desmycarosyl josamycin) exhibited comparable activities to josamycin against S. aureus (MSSA) and S. epidermidis (MSSE).     

The cytotoxicity of 8-O-4′ neolignans from the seeds of Crataegus pinnatifida

Nine new 8-O-4′ neolignans, named pinnatifidanin B I–IX (1–9), together with 9 known analogs (10–18) were isolated from the seeds of Crataegus pinnatifida. The structures of 1–18 were determined by spectroscopic methods, including 1D, 2D NMR, CD and HRESIMS analysis. Compounds 8–11, 17 and 18 displayed potent cytotoxic activities against human cancer cell lines, and most interestingly, none of the 6 compounds displayed inhibitory activity against human lung cell line (Mrc5). The 6 cytotoxic compounds are considered to be potential as antitumor agents, which could significantly inhibit the cancer cell growth in a dose-dependent manner and are probably safer than positive control drug.     

Characterization of a novel β-glucosidase-like activity from a soil metagenome

We report the cloning of a novel β-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known β-glucosidases. The putative β-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed d-glucosyl-β-(l–4)-d-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42°C using p-nitrophenyl-β-d-glucoside as the substrate. A CaCl₂ concentration of 1 mM was required for optimal activity. The putative β-glucosidase had an apparent K _m value of 0.19 mM, a V _max value of 4.75 U/mg and a k _cat value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.     

α-Glucosidase inhibitory and nitric oxide production inhibitory activities of alkaloids isolated from a twig extract of Polyalthia cinnamomea

The first phytochemical investigation of Polyalthia cinnamomea led to the isolation and identification of two new oxoprotoberberine alkaloids, (−)-(13aS)-polyalthiacinnamines A and B, together with eleven known compounds. The structures of the new compounds were elucidated by extensive spectroscopic methods. The absolute configuration of miliusacunine E and consanguine B was established by X-ray diffraction analysis using Cu Kα radiation and ECD spectra, whereas the absolute configurations of polyalthiacinnamines A and B were established by comparison of their ECD spectra and specific rotations with those of miliusacunine E and consanguine B. Compounds 1–4, 6, and 8 exhibited α-glucosidase inhibitory activities (IC₅₀ values ranging from 11.3 to 57.9 µM) better than a positive control (acarbose, IC₅₀ 83.5 μM). Compound 2 also exhibited NO production inhibitory activity with an IC₅₀ value of 24.4 μM (indomethacin, a positive control, IC₅₀ = 32.2 μM).     

Synthesis of C,N′-linked bis-heterocycles using a deprotometalation–iodination–N-arylation sequence and evaluation of their antiproliferative activity in melanoma cells

Benzothiophene, benzofuran, benzothiazole and benzoxazole were deprotometalated using the lithium–zinc combination prepared from ZnCl₂·TMEDA (TMEDA = N,N,N′,N′-tetramethylethylenediamine, 1 equiv) and lithium 2,2,6,6-tetramethylpiperidide (LiTMP, 3 equiv). Subsequent interception of the 2-metalated derivatives using iodine as electrophile led to the iodides in 81%, 82%, 67% and 42% yields, respectively. These yields are higher (10% more) than those obtained using ZnCl₂·TMEDA (0.5 equiv) and LiTMP (1.5 equiv), except in the case of benzoxazole (10% less). The crude iodides were involved in the N-arylation of pyrrole, indole, carbazole, pyrazole, indazole, imidazole and benzimidazole in the presence of Cu (0.2 equiv) and Cs₂CO₃ (2 equiv), and using acetonitrile as solvent (no other ligand) to provide after 24 h reflux the expected N-arylated azoles in yields ranging from 33% to 81%. Using benzotriazole also led to N-arylation products, but in lower 34%, 39%, 36% and 6% yields, respectively. A further study with this azole evidenced the impact of 2,2,6,6-tetramethylpiperidine on the N-arylation yields. Most of the C,N′-linked bis-heterocycles thus synthesized (in particular those containing benzimidazole) induced a high growth inhibition of A2058 melanoma cells after a 72 h treatment at 10⁻⁵ M.     

Characterization of α-amylase inhibitor from rice bean with inhibitory activity against midgut α-amylases from Spodoptera litura

The α-amylase inhibitor (α-AI) activity varied from 7.529 to 10.766 (IU/g) in 13 rice bean with different genotypes. BRS-2 exhibited the highest α-AI activity (55.3%). Rice bean α-AI was purified to homogeneity by 80% ammonium sulfate precipitation, dialysis, ion exchange chromatography on DEAE-Sepharose and gel filtration through Superdex-75. Its homogeneity was confirmed by SDS-PAGE under reducing conditions showing a single band protein of molecular weight 25 kDa. The inhibitor was purified to 75.9 fold with final yield of 28.0% with specific activity of 660.2 IU. Inhibition studies carried out at pH from 2.2 to 9.0 revealed pH optimum at pH 6.9 (69.3%). The maximum α-AI activity was found at 37°C (68.8 %) and the lowest was revealed at 100°C (37.0%). Optimum inhibitory activity was expressed during pre-incubation of enzyme with inhibitor at pH 6.9 and 37°C. Isoelectric focusing of purified inhibitor showed a single band near pH 4.7. The first 6 amino acids in the N-terminus were recorded as Ala-Ser-Ser-Arg-Phe-Cys (ASSRFC). The purified inhibitor inhibited the α-amylase from the larval midgut of Spodoptera litura up to 86.6%. The α-amylase inhibitors are important seed storage proteins because of their potentiality for exploitation in pest control and crop defense against insect infestation. Their expression at high levels can confer resistance in transgenic legumes, which could be exploited for crop improvement.     

Synthesis and PKCθ inhibitory activity of a series of 5-vinyl phenyl sulfonamide-3-pyridinecarbonitriles

A series of 5-vinyl phenyl sulfonamide-3-pyridinecarbonitriles were prepared and evaluated as PKCθ inhibitors. Optimization resulted in the identification of compound 15 with an IC₅₀ value 0.44 nM for the inhibition of PKCθ with 150-fold selectivity over PKCδ.     

Synthesis and PKCθ inhibitory activity of a series of 4-indolylamino-5-phenyl-3-pyridinecarbonitriles

A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCθ were synthesized as potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and variations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the discovery of compound 12d, which had an IC₅₀ value of 18 nM for the inhibition of PKCθ.     

Investigation of apigenin-7-O-β-glucoside hydrolysis by β-glucosidase from almonds

Summary The enzymatic transformation of apigenin-7-O--glucoside into apigenin is effected using -glucosidase from almonds. Kinetic studies gave the following values K_m=7.63×10^–4mol/dm³, v_max=10.37 mol mg^–1 min^–1 and E₁=65.21 kJ/mol when the substrate was pure apigenin-7-O--glucoside, while in case of apigenin-7-O--glucoside from dry extract of camomile ligulate flowers E₂ = 65.57 kJ/mol.Nomenclature S_o initial substrate concentration (g/100 cm³) - P product concentration (g/100 cm³) - k reaction rate constant (min^–1) - t reaction time (min)     

Synthesis of the (1→6)-linked thiodisaccharide of galactofuranose: Inhibitory activity against a β-galactofuranosidase

A new (1→6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized. Methyl (methyl α,β-d-galactofuranosid)uronate was employed as the starting compound, which was per-O-silylated with TBSCl and reduced with LiAlH₄ to afford methyl 2,3,5-tri-O-tert-butyldimethylsilyl-β-d-galactofuranoside (2β) as a key precursor for the preparation of methyl per-O-tert-butyldimethylsilyl-6-thio-β-d-galactofuranoside (12). The free thiol group of 12 was glycosylated and the product O-deprotected to afford the target β-d-Galf-S-(1→6)-β-d-Galf-OMe (14). The conformations of this thiodisaccharide were preliminarily studied using combined theoretical calculations and NMR data. Furthermore, the glycomimetic 14 showed to be a competitive inhibitor of the β-galactofuranosidase from Penicillum fellutanum (K_i = 3.62 mM).     

Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase

β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.     

Isolation of (-)-olivil-9′-O-β-d-glucopyranoside from Sambucus williamsii and its antifungal effects with membrane-disruptive action

In this study, we isolated (-)-olivil-9′-O-β-d-glucopyranoside (OLI9G), a phytochemical from the stem bark of Sambucus williamsii, and investigated the antifungal mechanism of OLI9G against Candida albicans. First of all, the antifungal susceptibility testing and hemolysis assay showed that OLI9G exerted a potent activity without hemolysis compared to the activity of amphotericin B. To investigate the mechanism of action of OLI9G, we first examined membrane depolarization using cyanine dye, 3,3′-dipropylthiacarbocyanine iodide (diSC₃5). The results showed that OLI9G significantly changed the fungal membrane potential. To further understand this activity on the membrane, we did the propidium iodide (PI) influx assay. From the results, OLI9G caused membrane permeabilization in the fungal membrane, and the three dimensional (3D) flow cytometric contour plot from the PI influx assay further showed that the cells had shrunk due to the membrane damage. Finally, the membrane-active mechanism of OLI9G was confirmed by synthesizing a model membrane, calcein-encapsulating large unilamellar vesicles (LUVs). The calcein leakage showed the membrane-disruptive effects caused by direct action of OLI9G. In conclusion, the current study suggests that OLI9G exerts its antifungal activity through a membrane-disruptive action.     

The Preparation of a 3′-(2-Cyanoethyl)phosphoramidite of 5′-O-(3-Thiopropyl)methylphosphorylthymidine

Abstract

A high-yielding three-step reaction sequence to a useful novel phosphoramidite, using 3′-O-acetylthymidine as starting material, is reported.     

Design and synthesis of a vialinin A analog with a potent inhibitory activity of TNF-α production and its transformation into a couple of bioprobes

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5',6'-dimethyl-1,1':4'1″-terphenyl-2',3',4,4″-tetraol (2) with a comparable inhibitory activity (IC(50)=0.02 nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process.     

GSK-3β inhibitory activities of novel dichroloresorcinol derivatives from Cosmospora vilior isolated from a mangrove plant

Cosmochlorins A (1), B (2), and C (3) were isolated from the endophytic fungus Cosmospora vilior IM2-155. The structures of 1, 2, and 3 were elucidated by a combination of extensive spectroscopic analyses, including extensive 2D NMR, HRESITOFMS, and chemical reactions. Compounds 1, 2, and 3 were evaluated for their biological activity. Compounds 1 and 2 partially restored the growth inhibition caused by hyperactivated Ca²⁺-signaling in mutant yeast and showed glycogen synthase kinase (GSK)-3β inhibition activity at IC₅₀ values of 62.5 and 60.6 μM, respectively. Further, compound 2 significantly increased osteoclast formation by more than 1.5-fold in RAW264.7 cells compared to receptor activator of nuclear factor-κB ligand (RANKL) alone.