Two New Derivatives of Griseofulvinfrom the Mangrove EndophyticFungus Nigrospora sp. (StrainNo.1403) from Kandelia candel (L.)Druce

Xuekui Xia1,2, Qing Li1, Jia Li1, Changlun Shao1, Jianye Zhang3,Yonggang Zhang2, Xin Liu2, Yongcheng Lin1, Changheng Liu2,Zhigang She1

1 School of Chemistry and Chemical Engineering, Sun YatsenUniversity, Guangzhou, P.R. China

2 Key Laboratory for Applied Microbiology of Shandong Province,Biotechnology Center of the Shandong Academy of Sciences,Jinan, P.R. China

3 Research Department, Cancer Center, Sun Yatsen University,Guangzhou, Guangdong, P.R. China

Abstract!

Two new compounds, methyl 3-chloro-6-hydroxy-2-(4-hy-droxy-2-methoxy-6-methylphenoxy)-4-methoxybenzoate (1)and (2S,5′R,E)-7-hydroxy-4,6-dimethoxy-2-(1-methoxy-3-oxo-5-methylhex-1-enyl)-benzofuran-3(2H)-one (2), together withfour known compounds, griseofulvin (3), dechlorogriseofulvin(4), bostrycin (5), and deoxybostrycin (6), were isolated fromthe marine endophytic fungus Nigrospora sp. (No. 1403) col-lected from the South China Sea. The structures were elucidatedby spectroscopic methods, 1D, 2D NMR, and HREIMS. Com-pounds 5 and 6 showed moderate antitumor and moderate anti-microbial activity.

Key wordsTrichosphaeriaceae · Nigrospora sp. · Kandelia candel (L.) Druce· metabolites · cytotoxicity · antimicrobial activity

Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica

Marine-derived fungi have been proven to be a rich source ofchemically diverse natural products with a broad range of biolog-ical activities [1,2]. In our continuing search for secondary me-tabolites from marine fungi with cytotoxicity or novel chemicalstructures [3,4], we have previously isolated five anthraquinonesfrom a mangrove endophytic fungus of Halorosellinia sp.(No. 1403) [5]. Further investigation for metabolites of this fungalstrain has now led to the isolation of six compounds. We reportherein the isolation and structures of compounds 1–6 (l" Fig. 1)and their cytotoxic and antimicrobial activities.Compound 1 was obtained as a colorless powder. The molecularformulawas assigned as C17H17O7Cl based on HR‑EI‑MS. In the IRspectrum, two strong absorption bands at 3505 and 3463 cm−1

showed two hydroxyl groups, and bands at 1654 cm−1 indicatedone carbonyl group. Seventeen signals in the 13C NMR were clas-sified by the DEPT spectra, attributable to four methyl, threemethine, and ten quaternary carbons, and disclosed a carbonylfunctionwhich appeared at δC 170.8. This carbonyl groupwas hy-drogen-bonded with a hydroxyl group at C-7, as evidenced by aproton signal at δH 11.54. The 1H NMR revealed a methyl (s,2.24), three oxymethyl groups (s, 3.86, 3.74, 3.44), three benzeneprotons (s, 6.31; d, 6.25, 6.21), one phenolic hydroxyl group (s,4.73), and another phenolic hydroxyl group (s, 11.54). The posi-tion of the methoxy and hydroxyl groups of ring A was estab-lished through the observed HMBC (l" Table 1) correlations: 6-OH and C-1, C-5, C-6, H-5 and C-1, C-3, C-4, C-6, CH3-8 and C-4,CH3-9 and C-7. In ring B, the position of the methyl and methoxygroups was established by CH3-8′and C-2′, CH3-7′ and C-1′, C-5′,C‑6′. Therefore, compound 1 was identified as methyl 3-chloro-6-hydroxy-2-(4-hydroxy-2-methoxy-6-methylphenoxy)-4-me-thoxy-benzoate.Compound 2 was obtained as a white solid; the molecular for-mula was assigned as C17H18O7 based on the HR‑EI‑MS. Seven-teen signals in the 13C NMR were classified by the DEPT spectra,including one methyl, three oxymethyls, one methylene, threemethines, and nine quaternary carbons. The 1H NMR revealed amethyl doublet (d, 0.98), one methylene (dd, 2.38, 3.02), threemethyls (s, 3.61, 3.84, 3.94), and three methines (m 2.84; s, 5.50)including one benzene proton (s, 6.12), and a hydroxy (br s, 5.29).In the 1H-1H COSY, the correlation from H-4′ to H-6′ could be de-tected. To analyze the HMBC spectrum of 2 (l" Table 2), we as-sembled the overall structure of 2. So the structure of compound2 was confirmed as (2S,5′R,E)-7-hydroxy-4,6-dimethoxy-2-(1-methoxy-3-oxo-5-methylhex-1-enyl)-benzofuran-3(2H)-one.

Fig. 1 Structures of compounds 1–6.

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The absolute configurations of 2 were deduced by comparison ofits CD data with those of 3 and 4 (l" Fig. 2). The absolute configu-rations at the stereogenic center at C (2, 5′) are (S, R) in all cases.CD spectrum of 2 (in CHCl3; λ in nm [θ inmdeg]) showed bands at245 (sh, 1.93), 255.4 (1.71), 282.4 (1.14), 291.8 (sh, 1.23). The CDspectrum of 3 showed bands at 243 (1.36), 291 (1.36), and 4showed bands at 244.4 (0.76), 285.2 (1.00).Compounds 3 and 4 were identified as griseofulvin [6] and de-chlorogriseofulvin [7], respectively, by the comparison of theirspectroscopic data with those in the literature. Compounds 5

and 6 compared with those in the literature readily allowed theiridentification as bostrycin [8–10] and deoxybostrycin [10–12],respectively.Compounds 5 and 6 were tested against six human tumor celllines using the MTT method. As shown in l" Table 3, compound5 significantly suppressed the growth of A549, Hep-2, Hep G2,KB, MCF-7, and MCF-7/Adr with IC50 values of 2.64, 5.39, 5.90,4.19, 6.13, and 6.68 µg ·mL−1, respectively. Compound 6 alsoinhibited the in vitro growth of all the tumor cell lines whenIC50 = 2.44, 3.15, 4.41, 3.15, 4.76, and 5.46 µg ·mL−1 respectively.The antimicrobial action of the compounds 2, 3, 4, 5, and 6 ex-pressed as MICs (µg ·mL−1) is summarized in l" Table 4, using amodified version of the 2-fold serial dilutions method as Fromt-ling et al. [13]. Compounds 5 and 6 showed strong antimicrobialactivities against Staphylococcus aureus, Escherichia coli, Pseudo-monas aeruginosa, Sarcina ventriculi, Bacillus subtiliswith an IC50of 3.13 µg ·mL−1, and inhibited Candida albicans with an IC50 of12.5 µg ·mL−1, while compound 2 showed no inhibition againstthe growth of Staphylococcus aureus, Escherichia coli, and Bacillussubtilis, and compound 3 and 4 showed no inhibition against allof the six bacteria strains. Ampicillin and nystatin were used aspositive controls.

Table 1 1H and 13C NMR (500 and 125MHz, δ value, J in Hz) spectral data andHMBC correlations of 1 (in CDCl3).

Position 13C NMR 1H NMR HMBC (C to H)

1 101.2 (C) H-5, 6-OH

2 156.4 (C)

3 106.6 (C) H-5

4 160.1 (C) H-5, 8

5 95.6 (CH) 6.31 (s) 6-OH

6 162.2 (C) H-5, 6-OH

7 170.8 (C) H-9

8 56.3 (CH3) 3.86 (s)

9 52.2 (CH3) 3.74 (s)

1′ 150.9 (C) H-3′, 5′, 7′

2′ 149.8 (C) H-3′, 8′,

3′ 99.3 (CH) 6.21(d, 2.4) H-5′

4′ 138.7 (C) H-3′

5′ 109.4 (CH) 6.25 (d, 2.4) H-3′, 7′

6′ 129.9 (C) H-5′, 7′

7′ 17.1 (CH3) 2.24 (s) H-5′

8′ 56.3 (CH3) 3.44 (s)

4′-OH 4.73 (s)

6-OH 11.54 (s)

Fig. 2 CD Spectra of compounds 2–4 (recorded in CHCl3 at ambienttemperature).

Table 2 1H and 13C NMR (500 and 125MHz, δ value, J in Hz) spectral data andHMBC correlations of 2 (in CDCl3).

Position 13C

NMR

1H NMR HMBC

(C to H)

1

2 90.5 (C) H-2′, 4′, 5′

3 193.3 (C) H-5′

4* 156.1 (C) H-5,10

5 89.1 (CH) 6.12 (s)

6* 152.4 (C) H-5,11

7 160.1 (C) H-5

8 105.1 (C)

9 124.4 (C) H-5

10 56.4 (CH3) 3.94 (s)

11 57.0 (CH3) 3.84 (s)

1′ 171.7 (C) H-2′, 7′

2′ 101.6 (CH) 5.50 (s)

3′ 197.5 (C) H-2′, 4′

4′ 40.3 (CH2) 3.02 (dd, 3.0,16.0); 2.38 (dd, 4.0, 16.0) H-2′, 5′, 6′

5′ 36.8 (CH) 2.84 (m) H-4′, 6′

6′ 14.5 (CH3) 0.98 (d, 7.0) H-4′, 5′

7′ 56.8 (CH3) 3.61 (s)

7-OH 5.29 (br s)

* Could exchange

Table 3 Six human cancer cell line inhibition values (IC50 µg ·mL−1) for com-pounds 5 and 6a.

Cancer cell lineb Compounds (IC50 µg·mL−1)

5a 6 Cisplatinc

A549 2.64 2.44 2.88

Hep- 2 5.39 3.15 1.02

Hep G2 5.90 4.41 5.79

KB 4.19 3.15 0.26

MCF-7 6.13 4.76 1.94

MCF-7/Adr 6.68 5.46 1.63

a Compounds 5 and 6 in DMSO; b cancer type: A549 (lung carcinoma); Hep-2 (laryn-

geal carcinoma); Hep G2 (hepatoma cell line); KB (oral cavity epidermis squamo-

cellular carcinoma cell line); MCF-7 (breast adenocarcinoma); MCF-7/Adr (adriamycin-

resistant human breast adenocarcinoma); c Cisplatin: positive control

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Materials and Methods!

Melting points were detected on a Fisher-Johns hot-stage appara-tus andwere uncorrected. The NMR data were recorded on a Var-ian INOVA-500 MHz spectrometer, using chloroform or dimeth-ylsulfoxide as the solvent and TMS as the internal standard. Massspectra were acquired on a VG‑ZAB mass spectrometer. CD spec-tra were acquired by a Jasco J-810 circular-dichroism spectrome-ter. IR spectra were obtained on a Nicolet 5DX-FTIR spectropho-tometer. Column chromatography was carried out on silica gel(200–300 mesh; Qingdao Haiyang Chemicals) and Sephadex LH-20 (Amersham Biosciences). Precoated silica gel plates (Yan Tai ZiFu Chemical Group Co.; G60, F-254) were used for thin-layerchromatography (TLC).3‑(4,5‑Dimethyl‑2‑thiazolyl)-2,5‑diphenyl‑2H‑tetrazolium bro-mide (MTT) was purchased from Sigma Chemical Co. Ampicillinand nystatin were purchased from AppliChem Co. Cis-diaminedi-choroplatinum (Cisplatin) was purchased from Merck & Co., Inc.Other routine laboratory reagents were obtained from commer-cial sources of analytical grade. Compounds 1–6 were isolated asthe second metabolites from cultures of the endophytic fungusstrain 1403; all of these compounds and positive controls had apurity > 98%.The fungus strain (No. 1403), identified by our team as Trichos-phaeriaceae, Nigrospora sp. (Genebank accession number:HQ891110), was isolated from decayed wood of Kandelia candel(L.) Druce, collected from Mai Po, Hong Kong, and a salt lake inthe Bahamas. A voucher specimen (registration number:No. 1403) has been stored in the Department of Biology andChemistry, City University of Hong Kong, Hong Kong, the Schoolof Chemical and Chemistry Engineering, Zhongshan University,Guangzhou, PR China.The cultivation of the fungus was performed as previously re-ported [5].The cultures (150 L) were filtered through cheesecloth. The fil-trate was concentrated to 5 L below 50°C and extracted threetimes by shaking with 5 L of ethyl acetate. The combined organicextracts were subjected to a reduced pressure silica gel column(60 × 7 cm), eluting with a gradient of petroleum ether to ethylacetate to yield eight fractions (Fr.1–Fr.8), containing 3.7, 2.3,

2.0, 1.4, 1.6, 2.0, 1.7, and 2.3 g of material, respectively. Fr.3 wasfurther purified by Sephadex LH-20 (120 × 1.5 cm column) elutedwith mixtures of petroleum-ether-CHCl3-MeOH (2:1:1), yield-ing compounds 1 (5.0mg), 2 (7.0mg), 3 (15.0mg), 4 (32.0mg),and Fr.6 was purified by Sephadex LH-20 (120 × 1.5 cm column)elutedwith themixtures of CHCl3-MeOH (1:1) to give compound5 (40mg) and 6 (50mg).Compound 1: m.p. 158–159°C; [a]D20 = 0 (acetone c 1.8); IR (KBr):vmax 3505, 3463, 3092, 2957, 2848, 1654, 1607, 1488, 1432, 1386,1324, 1250, 1194, 1152, 1097, 1069, 1015, 972 cm−1; HR‑EI‑MS:m/z 391.0577 [M + Na]+; 1H‑NMR (500MHz, CDCl3), 13C‑NMR(125MHz, CDCl3), and HMBC data, see l" Table 1.Compound 2: m.p. 153–154°C; [a]D20 = 176.19 (CDCl3, c 0.028); CD(c 8.4 · 10−5, CHCl3) λmax (θ in mdeg) (sh, 1.93), 255.4 (1.71), 282.4(1.14), 291.8 (sh, 1.23); HR‑EI‑MS: 334.1049, (calc. for C17H18O7,334.1047). 1H‑NMR (500MHz, CDCl3), 13C‑NMR (125MHz,CDCl3), and HMBC data, see l" Table 2.

Supporting informationDescriptions of the in vitro cytotoxicity assay and the antimicro-bial assay are available as Supporting Information.

Acknowledgements!

We wish to acknowledge financial support from the NationalNatural Science Foundation of China (20972197), the NaturalScience Foundation of Guangdong province of China(9151027501000055), and the Science & Technology Plan Projectof Guangdong Province of China (2010B030600004). We also ac-knowledge the support of the University-industry cooperationprojects of Guangdong Province and Ministry of Education(2008B090500171), Guangzhou Project of Science & TechnologyPlanning (2010J1-E331), the Fundamental Research Funds for theCentral Universities (11lgjc01), Prize Fund for Excellent MiddleAged and Young Scientists of Shandong Province (BS2009HZ004).

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Table 4 Tests of MIC (µg ·mL−1) for compounds 2, 3, 4, 5, and 6 against sixbacteria.

Strains Compounds (MIC µg·mL−1)

2 3 4 5 6 Ampa Nysa

Staphylococcusaureus(ATCC 27154)

–c – – 3.13 3.13 50 NTb

Escherichia coli(ATCC 25922)

– – – 3.13 3.13 25 NT

Pseudomonasaeruginosa(ATCC 25668)

NT – – 3.13 3.13 12.5 NT

Sarcina ventriculi(ATCC 29068)

NT – – 3.13 3.13 3.1 NT

Candida albicans(ATCC 10231)

NT – – 12.5 12.5 NT 1.56

Bacillus subtilis(ATCC 6633)

– – – 3.13 3.13 50 NT

a Ampicillin (Amp), Nystatin (Nys): positive control; b NT, not determined; –c no inhi-

bition zone

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received February 12, 2011revised March 29, 2011accepted March 31, 2011

BibliographyDOI http://dx.doi.org/10.1055/s-0030-1271040Published online April 21, 2011Planta Med 2011; 77: 1735–1738© Georg Thieme Verlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Changheng LiuKey Laboratory for Applied Microbiology of Shandong ProvinceBiotechnology Center of the Shandong Academy of SciencesNo. 19, Keyuan RoadJinan 250014P.R. ChinaPhone: + 86/531/82605355Fax: + 86/531/82965634liuchh@keylab.net

Prof. Zhigang SheSchool of Chemistry and Chemical EngineeringSun Yatsen UniversityNo. 135, West Xingang RoadGuangzhou 510275P.R. ChinaPhone: + 862084034096Fax: + 862084034096cesshzhg@mail.sysu.edu.cn

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