measurement of nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264. iNOS-negative counterparts

measurement of nitric oxide in lipopolysaccharide (LPS)-stimulated RAW 264. iNOS-negative counterparts [12]. In this respect the discovery of iNOS inhibitors is important for the treatment of inflammatory diseases as well as the prevention of cancers. During the course of our Gossypol search for bioactive natural products from marine-derived actinomycete strains the crude extract of our strain CNY-325 exhibited significant activity in screens associated with tumor induction. This strain isolated from a Chilean marine sediment was identified as a sp. based on 16S rDNA gene sequence analysis. Bioassay-guided separation of the crude extract using diverse chromatographic methods yielded dechlorothienodolin (1) and thienodolin (2) (Physique 1). Physique 1 Chemical structures of dechloro-thienodolin (1) and thienodolin (2). The molecular formula of dechloro-thienodolin (1) was assigned as C11H8N2OS by interpretation of combined HRESIMS and 13C NMR spectral data. The IR spectrum of 1 showed an absorption band at 1650 cm?1 which suggested the presence of an amide group. The distinct chemical shifts and coupling constants of four aromatic proton signals (H-4~H-7; δ 7.74 dd = 8.2 1.3 Hz 7.14 ddd = 8.2 8.2 1.3 Hz 7.23 ddd = 8.2 8.2 1.3 Hz 7.48 dd = 8.2 1.3 Hz respectively) in the 1H NMR spectrum illustrated the presence of a 1 2 disubstituted benzene Gossypol ring. The 1H NMR spectrum of 1 displayed an olefinic proton H-4 which showed an HMBC correlation to a quaternary olefinic carbon (C-3a δ 123.7). A long Gossypol range HMBC correlation from H-3 to three quaternary olefinic carbons (C-3a δ 123.7; C-8a δ 144.3; C-2 δ 131.5) and to a primary amide carbonyl carbon (C-9 δ 164.3) were also observed. These data in conjunction with the molecular formula revealed the structure of 1 1 as dechloro-thienodolin. This assignment was confirmed by comparison of previously reported spectroscopic data. Thienodolin (2) was reported as a plant growth-regulating substance from [13]. In 2004 Engqvist [13 34 35 Tryptophan is FJX1 an essential amino acid. It was reported that 3-hydroxyanthranilic acid one of the tryptophan metabolites in human beings significantly enhanced both indoleamine 2 3 (IDO) expression and IDO activity and inhibited NO production and iNOS expression via its enhancement of heme oxygenase-1 expression. [36]. In this study we described for the first time the inhibitory effects of plausible halogenated tryptophan analogues on iNOS expression and NO production via down-regulation of upstream signaling molecules including NF-κB and STAT1. In summary thienodolin can be considered as a potential drug candidate for anti-inflammation and cancer chemoprevention with a unique mechanism of action. Experimental General experimental procedures IR Perkin-Elmer 1600 FT-IR spectrometer; UV Varian Cary UV-visible spectrophotometer; NMR Varian Inova spectrometers; Low-resolution LC-MS (Hewlett-Packard series 1100 LC/MS system) and high resolution mass TOF spectral data were acquired at the Scripps Research Institute La Jolla CA. Collection and phylogenetic analysis of strain CNY-325 The marine-derived strain CNY-325 was isolated from a Chilean marine sediment (Valparaiso V region Chile). NCBI blast analysis of the partial (1480 base pairs) 16S rDNA gene sequence of CNY-325 indicated that this strain shared only 93% sequence identity to sp. GZ33 and thus is likely to be a new species. Cultivation and extraction sp. Gossypol (strain CNY325) was cultured in 2.8 L Fernbach flasks each containing 1 L of a seawater-based medium (10 g starch 4 g yeast extract 2 g peptone 1 g CaCO3 40 mg Fe2(SO4)3-4H2O 100..