Ental Section. In addition to compounds 1?, six recognized secondary metabolites such as N-methylphenyldehydroalanyl-L-prolin-anhydrid (5) [18,19], cyclo-trans-4-OH-(D)-Pro-(D)-Phe (six) [20], cyclo(D)-Pro-(D)-Val (7) [20], rubralide C (8) [21], 5-epialtenuene (9) [22] and altenuene (10) [22]Mar. Drugs 2013,(Figure 1), have been also isolated, and their structures had been elucidated by comparing the NMR data with these of literature reports [18?2]. 2.2. Biological Activities of your Isolated Compounds The isolated compounds 1?0 had been evaluated for the brine shrimp (Artemia salina) lethal activity and antibacterial activity. Amongst them, compound two was located to possess potent lethality with LD50 11.two M, that is much more active than that on the positive handle colchicine (with LD50 92.1 M), but the other tested compounds only displayed weak activity. In the antibacterial assay, compounds 1, 3, 5?, and constructive manage chloromycetin displayed inhibitory activity against Escherichia coli, causing the 10.0, 9.0, 8.0, eight.0, 7.0, ten.0, and 15.0 mm zones of inhibition at 20 g/disk, respectively. three. Experimental Section 3.1. Common NMR Spectra was recorded on a Bruker Avance-500 MHz spectrometer (500 MHz for 1H and 125 MHz for 13C) and chemical shifts were recorded as values.5-Bromo-1H-imidazole-2-carboxylic acid custom synthesis Low and higher ESI-Mass spectra had been performed on a VG Auto spec 3000 spectrometer. Optical rotations have been obtained on Optical Activity A-55 polarimeter. UV Spectra were measured in methanol on a Lengguang Gold S54 spectrophotometer. Silica gel (SiO2; one hundred?00 mesh, 200?00 mesh and GF254) for column chromatography and preparative thin-layer chromatography were made by Qingdao Haiyang Chemical Group Corporation. RP-18 reverse-phase silica gel (40?3 ) and Sephadex LH-20 had been purchased from the Merck Corporation.Mal-PEG4-OH site All solvents were distilled to use.PMID:23341580 three.2. Fungal Material The fungal strain Penicillium pinophilum SD-272 was isolated from the sediment sample collected in the estuary from the Pearl River in South China Sea, in October 2010. The fungal identification was accomplished by analysis of the ITS region of its rDNA as described previously [23]. The sequence data obtained from the fungus has been submitted to GeneBank with accession quantity KC 427134. A voucher specimen was stored at the Crucial Laboratory of Experimental Marine Biology of your Institute of Oceanology, Chinese Academy of Sciences. 3.3. Fermentation One particular hundred 1000-mL Erlenmeyer flasks, every single includes 300 mL liquid medium (sucrose two , peptone 0.5 , yeast extract 0.three , monosodium glutamate 1 , mannitol two , potato flour 0.four , seawater, pH 6.5), had been sterilized at 116 ?for 20 min and cooled to room temperature subsequently. A C 2 piece of mycelium (size three cm ) developing on malt agar plate was inoculated into 1000-mL Erlenmeyer flask. Static fermentation was then performed at 28 ?for 35 days. CMar. Drugs 2013, 11 3.4. Extraction and IsolationThe culture broth was filtrated making use of filter paper and separated into mycelia and culture broth. The air-dried mycelia had been immersed in acetone-H2O (4:1) with ultrasonic processor for 20 min after which extracted three occasions with ethyl acetate, even though the culture broth was stirred for three times with ethyl acetate and then concentrated to acquire an organic extract. Because the two extract show comparable HPLC and TLC profiles, they were combined to afford a crude extract (50.two g) for additional purification. The crude extract was subjected to vacuum liquid chromatography (VLC) on silica gel eluting with step solve.