NPC 2011 Vol. 6 No. 12 1905 - 1906 Natural Product Communications Antimicrobial Chemical Constituents from the Endophytic Fungus Phomopsis sp. from Notobasis syriaca Hidayat Hussaina,c*, Michel Kenne Tchimenea, Ishtiaq Ahmeda, Kathrin Meierb, Michael Steinertb, Siegfried Draegerb, Barbara Schulzb and Karsten Krohna,* a Department of Chemistry, Universität Paderborn, Warburger Straβe 100, 33098 Paderborn, Germany Institute of Microbiology, Technical University of Braunschweig, Spielmannstraße 7, 31806 Braunschweig, Germany c Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, Birkat Al-Mouz, Nizwa 616, Sultanate of Oman b Hidayat110@gmail.com; k.krohn@upb.de Received: May 14th, 2011; Accepted: October 31st, 2011 Phomosine K (1), a new phomosine derivative, has been isolated from Phomopsis sp., in addition to six known compounds: phomosine A (2), phenylalanine amide (3), 2-hydroxymethyl-4β,5α,6β-trihydroxycyclohex-2-en (4), (-)-phyllostine (5), (+)-epiepoxydon (6), and (+)-epoxydon monoacetate (7). Preliminary studies showed that compound 1 had strong antibacterial activity, while compounds 4-7 showed good antifungal, antibacterial, and algicidal properties, except compounds 4 and 6, which lacked antifungal activity. Key words: Fungal metabolites, Phomopsis sp., phomosines, epoxydones, antimicrobial activity. Endophytic fungi are a rich source of novel organic compounds with interesting biological activities [1]. As part of a program on the isolation of biologically active compounds, we have now investigated Phomopsis sp. (internal strain no. 8966), isolated from the plant Notobasis syriaca and isolated a number of metabolites (Figure 1). OH R HO 3 O OH 3' 1' NH2 NH2 5' 1 O 3 1 R = Me 2 R = CHO O O O RO HO OH O HO O HO OH OH Phomosine K (1), a white solid, showed an [M]+ at m/z = 332.1267 (HREIMS), corresponding to the molecular formula C18H20O6. The IR spectrum of 1 showed the presence of an ester carbonyl group (1647 cm-1) and a hydroxyl group (3410 cm-1). The 1H NMR spectrum showed signals for four methyl groups at δ 2.14 (s, 3H, CH3-2'), 2.15 (s, 3H, CH3-5), 2.16 (s, 3H, CH3-5'), and 2.30 (s, 3H, CH3-2), three phenolic OH groups at δ 2.14 (s, 3H, CH3-2'), 2.15 (s, 3H, CH3-5), and 2.16 (s, 3H, CH3-5'), 2.30 (s, 3H, CH3-2), three phenolic OH groups at δ 11.87, 7.29, and 6.54 (s, 1H, OH), one methoxy signal at δ 3.94 (s, 3H, CO2CH3) and two aromatic protons at δ 5.84 (s, 1H, 6'-H), and 6.46 (s, 1H, 4'-H). The 13C NMR and DEPT spectrum indicated the presence of five CH3, two CH, and eleven quaternary carbons. The structure of 1 was determined by comparison of its NMR spectroscopic data with those of phomosine A (2) [2]. A fourth methyl group appeared at δ 2.14 (s, 3H), instead of an aldehydic proton at δ 10.43, as found in compound 2. The fourth methyl group at C-2 was further confirmed from HMBC correlations of CH3-2 to C-1, C-2, and C-3. Thus, the structure of 1, was established as methyl 2,4-dihydroxy5-(3-hydroxy-2,5-dimethylphenoxy)-3,6-dimethylbenzoate. The known compounds phomosine A (2) [2], phenyl- O O 5 4 O 5 6 R= H 7 R = Ac Figure 1: Chemical structure of compounds 1-7. alanine amide [3], 2-hydroxymethyl-4β,5α,6β-trihydroxycyclohex-2-en (4) [4], (-)-phyllostine (5) [5a], (+)epiepoxydon (6) [5], and (+)-epoxydon monoacetate (7) [5b] were identified by comparison with published data. The isolated compounds 4-7 were tested in an agar diffusion assay for their antifungal, antibacterial, and algicidal properties towards Microbotryum violaceum, Escherichia coli, Bacillus megaterium, and Chlorella fusca (Table 1). Metabolite 1 was tested for antibacterial activity in a MIC (minimum inhibitory concentration)-assay in liquid medium against Legionella pneumophila Corby, Escherichia coli K12 and Bacillus megaterium (Table 2). Experimental Plant material: College. Experimental procedures were quite similar to those reported earlier [1b]. Microbiological methods and culture conditions were as previously described [6a,b]. Culture, extraction, and isolation: The endophytic fungus Phomopsis sp. was isolated from Notobasis syriaca. It was 1906 Natural Product Communications Vol. 6 (12) 2011 Table 1: Biological activities of pure metabolites 4-7 against microbial test organisms in agar diffusion assay. Compound antibacterial Eca 7 10 7 9 14 18 0 0 0 4 5 6 7 Penicillin Tetracycline Nystatin Actidione Acetone antibacterial Bm PI 8 PI 7 0 PI 7 18 18 0 0 0 antialgal Chl 6 10 5 9 0 PI 10 0 35 0 antifungal Mv 0 7 0 5 0 0 20 50 0 a Chlorella fusca (Chl), Microbotryum violaceum (Mv), Escherichia coli (Ec), and Bacillus megaterium (Bm). Application of pure substances at a concentration of 0.05 mg (50 µL of 1 mg/mL). The radius of zone of inhibition was measured in mm. PI = partial inhibition, i.e. there was some growth within the zone of inhibition. Table 2: Antibacterial activity of 1 in a MIC assay in liquid medium against bacterial test organisms. Compound 1 µg / mL Lpa 100 50 25 12.5 + + + - Test organisms Ec + - Bm - cultivated at room temperature for 28 days [7,8] on biomalt solid agar medium. The culture medium was then extracted with ethyl acetate to afford 5.0 g of a residue after removal of the solvent under reduced pressure. The extract was separated into 2 fractions by CC on silica gel, using gradients of dichloromethane / ethyl acetate (85:15, 50:50, 0:100). The less polar fraction 1 (1.5 g) contained mainly fatty acids and lipids. The remaining fraction was further purified by silica gel CC and preparative TLC with n-hexane / ethyl acetate (10:1 to 5:1) to give pure compounds 1 (11 mg), 2 (40 mg), 3 (4 mg), 4 (6 mg), 5 (5 mg), 6 (5 mg), and 7 (5 mg). Phomosine K (1) MP: 199°C. UV (CHCl3): λ max (log ε) = 277 (3.70), 310 (3.50) nm. IR (KBr): ν = 3430 cm-1, 2953, 1650, 1620, 1580, 1440, 1372, 1090, 1020, 795. Hussain et al. H NMR (500 MHz, CDCl3): δ = 2.14 (s, 3H, CH3-2'), 2.15 (s, 3H, CH3-5), 2.16 (s, 3H, CH3-5'), 2.30 (s, 3H, CH3-2), 3.94 (s, 3H, CO2CH3), 5.84 (s, 1H, 6'-H), 6.46 (s, 1H, 4'-H), 6.54 (s, 1H, OH), 7.29 (s, 1H, OH), 11.87 (s, 1 H, 6-OH). 13 C NMR (125 MHz, CDCl3): δ = 8.2 (CH3-5), 15.4 (CH3-2), 20.9 (CH3-2'), 21.7 (CH3-5'), 51.9 (CO2CH3), 104.5 (C-1), 105.8 (C-6'), 108.4 (C-3), 110.8 (C-4'), 112.8 (C-2'), 131.1 (C-6), 133.1 (C-5), 142.2 (C-5'), 152.5 (C-4), 156.6 (C-1'), 156.6 (C-3'), 160.5 (C-6), 172.4 (C-1a). EIMS (70 eV, 200°C): m/z (%) = 332.1 (55) [M+], 298 (100), 255(25), 180 (69), 108 (96). HREIMS: m/z 332.1215 (calcd. 332.1260 for C18H20O6). 1 Assays for biological activity: For the agar diffusion assays, the compounds were dissolved in acetone at a concentration of 1 mg/mL. Fifty μL of the solutions (50 µg) was pipetted onto a sterile filter disk (Schleicher & Schuell, 9 mm), which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organism [6a]. The fungal test organisms were Escherichia coli, Bacillus megaterium, and Microbotryum violaceum. Commencing at the middle of the filter disk, the radius of the zone of inhibition was measured in mm. The MICassays were conducted in microtiter plates in liquid media. Legionella pneumophila Corby was tested in YEB medium [6c]; Escherichia coli and Bacillus megaterium in NB medium. The metabolites were dissolved in methanol. In the wells, the bacterial test organisms were initially at a concentration of 2 x 107 cells / mL; final concentrations of the metabolites were 12.5 – 100 µg / mL: incubation of L. pneumophila was for 3 days with 5% CO2 at 37°C, and that of B. megaterium and E. coli for one day at 24°C. 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