HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 8, 2011, pp. 1853-1858
Received, 12th April, 2011, Accepted, 13th May, 2011, Published online, 20th May, 2011.
DOI: 10.3987/COM-11-12234

■ Penicitrinols F—I, New Citrinin Derivatives from the Marine-Derived Fungus Penicillium citrinum

Li Chen,* Wei Liu, Kai Huang, Xiao Hu, Zhe-Xiang Fang, Jiu-lin Wu, and Qi-Qing Zhang*

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

Abstract

Four new citrinin derivatives, namely, penicitrinols F, G, H, and I (1–4), along with four known compounds, namely, 4,6-dihydroxy-2,3-dimethyl-benzaldehyde (5), pennicitrinone A (6), dicitrinone B (7), and dicitrinone C (8), were isolated from the marine-derived fungus Penicillium citrinum. Their structures were established using spectroscopic methods.

Penicillium citrinum is a rich source of various citrinin derivatives.1,2 Our previous chemical investigation of P. citrinum resulted in the isolation of three new citrinin derivatives, namely, penicitrinols C–E,3 Our continuing search for bioactive compounds from this organism has further resulted in the isolation of another four new derivatives, namely, penicitrinols F–I (1–4), along with four known analogues (5–8).2,4 In this paper, we report the isolation and structural elucidation of these metabolites.
Bioactive ethyl acetate extract of P. citrinum was seperated by chromatography on Si gels and Sephadex LH-20 columns and purified by reversed-phase HPLC to yield the eight compounds (1–8).
Compound 1 was established to have the molecular formula C15H20O4 by its HRESIMS. The 1H NMR spectrum of 1 indicated the presence of an exchangeable proton (δ 9.13), an aromatic proton (δ 6.18), three protons attached to oxygenated carbons (δ 5.31, 4.51, and 3.98), and four methyl groups (δ 3.41, 1.95, 1.19, and 1.10). The 13C NMR and DEPT data indicated the presence of the following: six aromatic carbons, two of which were oxygenated (δ 155.1 and 148.7) and one was protonated (δ 100.8); three oxymethine carbons (δ 100.2, 73.5, and 60.3); one methylene carbon (δ 36.3); one methine carbon (δ 33.7); and four methyl carbons (δ 55.1, 21.5, 17.7, and 9.9). The connectivities of these groups and carbons were deduced from the COSY and HMBC spectra (Figure 2). The NOESY correlations between H-3/H-12, H-4/H-11, H-1/H-11, and H-1/H-15 observed in 1 indicated that H-1, H-4, H-15, and CH3-11 were on the same side, whereas CH3-12 and OCH3 were on the reverse side. Therefore, the structure of 1 was elucidated as shown (Figure 1).

Compound 2 has the same molecular formula (C15H20O4) as 1, based on HRESIMS data (m/z: 263.1266 [M – H]ˉ). The NMR (1D NMR, COSY, HMQC, and HMBC) data revealed that 2 and 1 possessed the same planar structure. However, the NOESY correlations of H-1 with H-11 and OCH3, as well as H-4 with H-11 suggested that 2 and 1 were stereoisomers with different C-15 configurations.
Compound 3 was obtained as a yellow gum. Its molecular formula, C16H22O4, was established using HRESIMS (m/z 277.1451 [M – H]ˉ). The analysis and comparison of the 1D NMR of 3 with those of 1 and 2 (Tables 1 and 2) suggested that the planar structure of 3 had one methyl group (δH 1.52, 3H; δC 23.3 q) more than 1 or 2, which was supported by the MS data. The COSY and HMBC spectra suggested that this methyl group was connected to C-15. The NOESY correlations between H-3/H-12, H-4/H-11, H-1/H-3, and H-1/H-16 observed in 3 indicated that H-4, CH3-11, and OCH3 were on the same side. On the other hand, H-1, H-3, CH3-12 and CH3-16 were located on the reverse side.
Compound 4 was obtained as a yellow gum. Its molecular formula, C15H20O4, was established using negative HRESIMS (m/z 263.1275, [M – H]ˉ, calcd for C15H19O4, 263.1283). Comparing the 1D and 2D NMR data of 4 with those of 3 (Tables 1 and 2), an exchangeable proton (δH 6.44) in 4 replaced the O-methyl group (δH 3.28, 3H; δC 49.0 q) in 3. The similar NOESY correlations indicated that the configuration of 4 was the same as that of 3.

The cytotoxic effects of compounds 1–4 were evaluated using the MTT method on A-549, HL-60, hela, and K562 cancer cell lines. Unfortunately, the results showed that all of their IC50 values were larger than 100 μM.

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. ESI-MS were obtained by an AGILENT 1200/Q-TOF 6510 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 P. citrinum 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 was cultured under static conditions at 28 ˚C for 30 days in 1000-mL conical flasks containing the 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 supernatant from mycelia. The former was extracted two times with EtOAc to yield an EtOAc solution that was concentrated under reduced pressure to give a crude extract (32.0 g).
Purification. The crude extract (32.0 g) was separated into 1L fractions on a Si gel column using a step gradient elution of petroleum ether, CH2Cl2, and MeOH. Fraction 5 (2.1 g) eluted with petroleum ether/CH2Cl2 (1:3) was further purified on a Si gel column using a step gradient elution. Subfraction 5-4 (437 mg) eluted with petroleum ether/EtOAc (2:1) was purified on a Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give compound 8 (52.3 mg). Subfraction 5-6 (326 mg) eluted with petroleum ether/EtOAc (1:1) was purified on a Sephadex LH-20 (CH2Cl2/MeOH, 1:1) and a reversed-phase column (MeOH/H2O, 2:1) to give compound 7 (48.6 mg). Fraction 6 (1.5 g) eluted with CH2Cl2 was further purified on a Sephadex LH-20 (CH2Cl2/MeOH, 1:1) and a reversed-phase column (MeOH/H2O, 3:2) to give compound 6 (15.8 mg). Fraction 9 (5.6 g) eluted with CH2Cl2/MeOH (50:1) was further separated on a Sephadex LH-20 (CH2Cl2/MeOH, 1:1). Subfraction 9-8 (650 mg) was purified by a reversed-phase column (MeOH/H2O, 3:2) and semipreparative HPLC (45% MeCN), yielding compounds 1 (2.8 mg), 2 (4.2 mg), 3 (36.5 mg), 4 (40.2 mg), and 5 (5.8 mg).
Penicitrinol F (1): white solid; [α]25D −9.7° (c 0.4, MeCN); UV λmax (MeCN) nm (log ε): 282 (2.71); 1H and 13C NMR (see Tables 1 and 2); HRESIMS m/z 263.1297 [M – H]ˉ (Calcd for C15H19O4: 263.1283).
Penicitrinol G (2): white solid; [α]25D +16.9° (c 0.6, MeCN); UV λmax (MeCN) nm (log ε): 285 (2.90); 1H and 13C NMR (see Tables 1 and 2); HRESIMS m/z 263.1266 [M – H]ˉ (Calcd for C15H19O4: 263.1283).
Penicitrinol H (3): yellow gum; [α]25D –69.4° (c 0.6, MeCN); UV λmax (MeCN) nm (log ε): 284 (3.03); 1H and 13C NMR (see Tables 1 and 2); HRESIMS m/z 277.1451 [M – H]ˉ (Calcd for C16H21O4: 277.1440).
Penicitrinol I (4): yellow gum; [α]25D –79.5° (c 1.0, MeCN); UV λmax (MeCN) nm (log ε): 287 (2.87); 1H and 13C NMR (see Tables 1 and 2); HRESIMS m/z 263.1275 [M – H]ˉ (Calcd for C15H19O4: 263.1283).
Biological assay. Cytotoxic activity was evaluated by the MTT method using A-549, HL-60, hela, and K562 cell lines.3 The cell lines were grown in RPMI-1640 supplemented with 10% FBS under a humidified atmosphere of 5% CO2 and 95% air at 37 ˚C. Those cell suspensions (200 μL) at a density of 5×104 cell mL−1 were plated in 96-well microtiter plates and incubated for 24 h at the above condition. The test compound solution (2 μL in DMSO) at different concentrations was added to each well and further incubated for 72 h in the same condition. Then 20 μL of the MTT solution (5 mg/ml in RPMI-1640 medium) was added to each well and incubated for 4 h. The old medium containing MTT (150 μL) was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 540 nm.

ACKNOWLEDGEMENTS
This research was supported by the Natural Science Foundation of Fujian Province (2009J05075), the Scientific Research Foundation in Fuzhou University (2009-XY-16, 022229), Scientific Key Research Project of Fujian Province (2010N0015), Key Laboratory of Biomedical Material of Tianjin and Scientific Major Research Project of Fujian Province (2010NZ0001-1).

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