HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 1, 2014, pp. 189-196
Received, 29th October, 2013, Accepted, 26th November, 2013, Published online, 29th November, 2013.
DOI: 10.3987/COM-13-12874

■ Novel Cytotoxic Metabolites from the Marine-derived Fungus Trichoderma citrinoviride

Xiao Hu, Mei-Wei Gong, Wei-Wei Zhang, Qiu-Hong Zheng, Qin-Ying Liu, Li Chen,* and Qi-Qing Zhang*

College of Chemistry and Chemical Engineering, Institute of Biomedical and Pharmaceutical Technology , Fuzhou University, No. 523, Gongye Road, Fuzhou City, 350002, China

Abstract

Three new compounds, trichoderiol C (1), citrinoviric acid (4), and penicillenol D (5), together with seven known compounds, trichoderiol A (2), lignoren (3), penicillenol B1 (6), penicillenol B2 (7), cyclo-(Leu-Pro) (8), cyclo-(Ile-Pro) (9), and cyclo-(Phe-Pro) (10), were isolated from the marine-derived fungus Trichoderma citrinoviride. The structures of these compounds were elucidated using spectroscopic methods, including 1D and 2D nuclear magnetic resonance and high-resolution mass spectrometric analyses. Among these compounds, 4 and 5 showed moderate cytotoxic effects on the A-375 cell line, with IC50 values of 85.7 and 32.6 μM, respectively.

Numerous natural products with novel structures and distinct biological activities have been discovered as secondary metabolites of marine-derived microbes.1 Some of these natural products have been used as drugs. For example, echinocandins, ergot alkaloids, cyclosporine, and lovastatin have been used as antifungal, analgesic, immunosuppressive, and cholesterol-lowering drugs,2 respectively. To search for new anticancer compounds, more than 300 microbial strains isolated from sediment samples that were collected from the Min River estuary in China were screened for cytotoxicity against the A-375 cell line. Among these strains, the fungus Trichoderma citrinoviride showed significant cytotoxic activity. The crude extract of T. citrinoviride was separated using chromatography on silica gel and Sephadex LH-20 columns. The crude extract was then purified via reversed-phase high-performance liquid chromatography (HPLC) to yield three new compounds, trichoderiol C (1), citrinoviric acid (4), and penicillenol D (5), together with seven known compounds, trichoderiol A (2),3 lignoren (3),4 penicillenol B1 (6),5 penicillenol B2 (7),5 cyclo-(Leu-Pro) (8),6 cyclo-(Ile-Pro) (9),7 and cyclo-(Phe-Pro) (10).8 In the present study, we report the isolation, structural elucidation, and bioactivities of compounds 1–10.

Compound 1, which is trivially named as trichoderiol C, was obtained as yellow oil and was analyzed to have the molecular formula C15H26O2 through positive high-resolution electrospray ionization mass spectroscopy (HRESIMS) (m/z: 239.2003 [M + H]+, Calcd. for C15H27O2, 239.2011). Its nuclear magnetic resonance (NMR) data (Tables 1 and 2), combined with distortionless enhancement by polarization transfer (DEPT) and heteronuclear multiple quantum coherence (HMQC) spectrum analyses, revealed 15 carbon signals, including four methyls, five methylenes, three methines, and three quaternary carbons. The plane structure of 1 was revealed via COSY and HMBC spectrum analyses (Figure 2). The COSY correlations of H-4 with H-3 and H-5, H-7 with H-3 and H-8, and H-12 with H-11 and H-13 demonstrated the connections from H-5 to H-8 and from H-11 to H-13. The HMBC correlations of H-8 with C-3, C-6, and C-7, H-9 with C-5, C-6, and C-7, and H-10 with C-2 and C-3 confirmed the presence of the bridged-ring part. The HMBC correlations of H-15 with C-13, C-14, and C-16 and H-16 with C-13, C-14, and C-15 confirmed the presence of the long-chain part. Finally, the HMBC correlations of H-10 with C-11 and H-11 with C-2 and C-3 linked the two independent parts. The relative stereochemistry of 1, which contains four asymmetric carbons, was settled based on observable NOE signals between H-8 and H-10 (Figure 3). The Z configuration was assigned to the C-13 and C-14 double bond through the NOE correlation of H-13 with H-16. Therefore, the structure of 1 was deduced as shown in Figure 1.

Compound 4, which is trivially named as citrinoviric acid, was obtained as white powder and was analyzed to have the molecular formula C14H20O6 using negative HRESIMS (m/z: 283.1183 [M – H]–, Calcd. for C14H19O6, 283.1182). The IR absorptions at 3432, 1736, and 1634 cm−1, indicated the presence of a carboxylic acid moiety and an alkenyl moiety.9 Its NMR data (Tables 1 and 2), combined with DEPT and HMQC spectrum analyses, revealed 14 carbon signals, including three methyls, four methylenes, two methines, and five quaternary carbons. The plane structure of 4 was revealed using COSY and HMBC spectrum analyses (Figure 2). The COSY correlations of H-3 with H-4, H-7 with H-6 and H-8, and H-9 with H-8 and H-10 demonstrated the connections from H-3 to H-4 and from H-6 to H-10. The HMBC correlations of H-11 with C-1, C-2, and C-3, and H-3, H-4, and H-6 with C-5 confirmed the presence of a long chain with eleven carbons. The residual HMBC correlations of H-14 with C-12 and C-13 suggested the short carbon chain from C-14 to C-12 via C-13. Since the degree of unsaturation of 4 was five, a cycle was necessary considering the three carbonyl groups and an alkenyl group determined. Furthermore, taking into account the molecular formula C14H20O6 and the chemical shifts of C-1 (177.4 s), C-2 (83.1 s), and C-13 (96.4 s), the two carbon chains should be connected via two oxygen atoms, which formed a cyclic acetal structure. The relative stereochemistry of 4 was determined based on the NOE correlation of H-11 with H-14 (Figure 3). The double bond E configuration was confirmed through the NOE correlation of H-8 with H-10. Therefore, the structure of 4 was deduced as shown in Figure 1.

Compound 5, which is trivially named as penicillenol D, was obtained as yellow oil and was analyzed to have the molecular formula C17H29NO4 using negative HRESIMS (310.2015 [M – H]–, Calcd. for C17H28NO4, 310.2018). FT-IR microscopy data suggested the presence of the hydroxyl group (3433), the carbonyl group (1712), the amide group (1640), and the alkenyl group (1618). Its NMR data (Tables 1 and 2), combined with DEPT and HMQC spectrum analyses, revealed 17 carbon signals, including four methyls, seven methylenes, one methine, and five quaternary carbons. The COSY correlations from H-16 to H-17 confirmed the presence of a long-chain part, including nine carbons. The COSY correlations of H-6 with H-7 verified the presence of an ethyl group. Further careful comparison of the molecular formular and the similar NMR spectral data (Tables 1 and 2) of 5 with those of penicillenols A1,5 showed 5 differed from the known compound at the chemical shifts of C-5 (90.3 s, 21.6 ppm downfield shifting than A1, 68.7 d), C-6 (27.2 t, 39.4 ppm highfield shifting than A1, 66.6 d), C-7 (7.6 q, 10.2 ppm highfield shifting than A1, 17.8 q), and an additional methylene group (27.0 t) suggesting that 5 owned the similar plane structure as penicillenols A1 with exception of the position of a hydroxyl group and the longth of a carbon chain. This result was confirmed by the very similar FT-IR data of these two compounds and the HMBC correlations from H-18 to C-2 and C-5, H-6 to C-4 and C-5, H-7 to C-5, and H-17 to C-8, C-9, and C-10. Therefore, the structure of 5 was deduced as shown in Figure 1.
The new compounds (1, 4, and 5) were tested for their cytotoxic effects on the A-375 cell line using the MTT method.10 Compounds 4 and 5 showed moderate cytotoxicity against the A-375 cell line, with IC50 values of 85.7 and 32.6 µM, respectively.
Trichoderma species are important sources of many bioactive compounds. However, chemical investigations of T. citrinoviride focused only on the cellulase11 and antifeedant12 activities until now. To the best of our knowledge, this study is the first to reveal the antitumor activity of metabolites from T. citrinoviride. Moreover, the metabolite of the carboxylic acid and alkaloid is almost never found in T. citrinoviride.

EXPERIMENTAL
General Experimental Procedures. Optical rotations were obtained from a Shenguang SGW-1 digital polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. 1H-NMR, 13C-NMR, DEPT spectra and 2D-NMR were recorded on a BRUKER BIOSPIN AVANCE III spectrometer using TMS as the internal standard. HRESIMS were obtained by an Agilent Q-TOF 6520 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column (ODS-A, 10 × 250 mm, 5 µm) at 5 mL/min.
Fungal Material. The fungus T. citrinoviride was isolated from marine sediments collected from Langqi Island, Fujian, China. It was identified according to its morphological characteristics and ITS by Beijing Sunbiotech Co. Ltd, and preserved in our laboratory at −80 °C. The producing strain was prepared on Martin medium and stored at 4 °C.
Fermentation and Extraction. The fungus T. citrinoviride was cultured under static conditions at 28 °C for 30 d in 1000 mL conical flasks containing a liquid medium (400 mL/flask) composed of glucose (10 g/L), maltose (20 g/L), mannitol (20 g/L), monosodium glutamate (10 g/L), KH2PO4 (0.5 g/L), MgSO4∙7H2O (0.3 g/L), yeast extract (3 g/L), and seawater. The fermented whole broth (60 L) was filtered through cheese cloth to separate the supernatant from the mycelia. The former was extracted two times with EtOAc to obtain an EtOAc solution, whereas the latter was extracted three times with acetone. The acetone solution was concentrated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted two times with EtOAc to give another EtOAc solution. Both EtOAc solutions were combined and concentrated under reduced pressure to obtain a crude extract (46.5 g).
Purification. The crude extract (46.5 g) of the fungus T. citrinoviride was separated into six fractions on a silica gel column using a step-gradient elution of petroleum ether, CH2Cl2, and MeOH. Fraction A (5.6 g) was further purified on a silica gel column using a step-gradient elution of CH2Cl2 and MeOH to obtain four subfractions. Subfraction A-1 (1.8 g) was subjected to Sephadex LH-20 (CHCl3:MeOH, 1:2), followed by semipreparative HPLC (50% MeCN, 0.1% TFA), to yield compounds 1 (12 mg), 2 (6 mg), 3 (23 mg), and 8 (10 mg). Subfraction A-2 (0.9 g) was subjected to Sephadex LH-20 (CHCl3:MeOH, 1:2), followed by semipreparative HPLC (40% MeCN, 0.1% TFA), to yield compounds 4 (9 mg) and 10 (9 mg). Fraction B (5.2 g) was further purified on a silica gel column using a step-gradient elution of CH2Cl2 and MeOH to obtain five subfractions. Subfraction B-1 (1.7 g) was subjected to Sephadex LH-20 (CHCl3:MeOH, 1:2), followed by semipreparative HPLC (85% MeCN, 0.1% TFA), to yield compounds 5 (21 mg), 6 (19 mg), and 7 (13 mg). Subfraction B-2 (1.2 g) was subjected to Sephadex LH-20 (CHCl3:MeOH, 1:2), followed by semipreparative HPLC (35% MeCN), to yield compound 9 (15 mg).
Trichoderiol C (1): yellow oil (CHCl3); [α]23D -28.8 (c 0.10, MeOH); 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 239.2003 [M + H]+, calcd for C15H27O2, 239.2011); IR (KBr) νmax 3395, 2962, 2925, 2848, 1659, 1454, 1377, 1095, 1021, 955, 915, 882 cm-1.
Citrinoviric acid (4): white powder (MeOH); [α]20D +96.7 (c 0.03, CHCl3); 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 283.1183 [M – H]–, calcd for C14H19O6, 283.1182); IR (KBr) νmax 3432, 2958, 2921, 2848, 1736, 1634, 1462, 1381, 1266, 1098, 1025, 800 cm-1.
Penicillenol D (5): yellow oil (CHCl3); [α]20D -60.0 (c 0.13, CHCl3); 1H and 13C NMR data (see Tables 1 and 2); HRESIMS (m/z: 310.2015 [M – H]–, calcd for C17H28NO4, 310.2018); IR (KBr) νmax 3433, 2956, 2925, 2856, 1712, 1640, 1618, 1462, 1377, 1344, 1258, 1095 cm-1.
Biological Assays. The cytotoxic activity for the A-375 cancer cell line was evaluated by the MTT method. Doxorubicin was used as the reference drug.

ACKNOWLEDGEMENTS
This research was supported by the Chinese National Natural Science Fund (21102015 and 31201034), Natural Science Foundation of Fujian Province (2012J05138), the Scientific Research Foundation in Fuzhou University (2009-XY-16 and 022229), and Scientific Key Research Project of Fujian Province (2010N0015).

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