Hypocreaterpenes A and B, cadinane-type sesquiterpenes from a marine-derived fungus, Hypocreales sp.

Phytochemistry Letters 6 (2013) 392–396

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Hypocreaterpenes A and B, cadinane-type sesquiterpenes from a marine-derived fungus, Hypocreales sp. Hong Zhu a,b, Xin-xing Hua b,c, Ting Gong a,b, Jie Pang c, Qi Hou a,b, Ping Zhu a,b,* a

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China c Fujian Agriculture and Forestry University, Fuzhou, Fujian 350000, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 February 2013 Received in revised form 12 April 2013 Accepted 14 April 2013 Available online 13 May 2013

Two new cadinane-type sesquiterpenes, hypocreaterpenes A (1) and B (2), along with five known compounds (3–7) were isolated from a marine-derived fungus Hypocreales sp. strain HLS-104 isolated from a sponge Gelliodes carnosa. Their structures were determined by a combination of spectroscopic methods. All compounds were tested for the inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-treated RAW264.7 cells. Among them, compounds 3 and 6 showed moderate anti-inflammatory activity with average maximum inhibition (Emax) values of 10.22% and 26.46% at 1 mM, respectively. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Marine-derived fungus Hypocreales sp. Secondary metabolites Hypocreaterpenes A and B Anti-inflammatory activity

1. Introduction Marine-derived fungi have become an important source of novel and bioactive secondary metabolites for drug discovery (Yang et al., 2012). So far, more than one thousand structurally unique and biologically active compounds have been isolated from marinederived fungi (Rateb and Ebel, 2011). Among the orders of marinederived fungi isolated for natural products research, Hypocreales is rarely encountered (Molnar et al., 2010). In our ongoing search for bioactive compounds from marine fungi, we isolated a strain HLS104 from the sponge Gelliodes carnosa (Fig. 1a) collected from the South China Sea (Liu et al., 2010). Based on the rDNA-ITS sequence (GenBank accession number FJ770062) analysis together with the morphologic traits, this strain was preliminarily grouped into the order of Hypocreales (Fig. 1b, c). Many fungal species in the order Hypocreales have proven to be pathogenic to higher organisms such as insects and plants (Tan and Zou, 2001; Isaka et al., 2005; Gunatilaka, 2006). Full genomic sequencing has revealed the exceptional chemical diversity of fungal metabolites from the order

* Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China. Tel.: +86 10 63165197; fax: +86 10 63037757. E-mail address: [email protected] (P. Zhu).

Hypocreales (Gao et al., 2011; Zheng et al., 2011). The extended investigation on the secondary metabolites of Hypocreales sp. HLS104 led to the isolation of seven compounds including two new cadinane-type sesquiterpenes.

2. Results and discussion 2.1. Comparison of the secondary metabolic profiles between the seawater and distilled water rice medium cultures of the HLS-104 Low water activity may influence the profile of the fungal secondary products (Sepcic et al., 2011). In the present report, the strain Hypocreales sp. HLS-104 was cultured on the rice media prepared with distilled water and seawater, respectively. The crude ethyl acetate extracts of the two kinds of solid cultures were dissolved by methanol and analyzed by HPLC (Fig. 2). To accumulate more compounds, the rice medium with seawater was used to cultivate HLS-104 strain and up to seven compounds were isolated. The seven compounds included two new sesquiterpenes, hypocreaterpenes A and B (1 and 2), and five known compounds (Fig. 3), 1R,6R,7R,10S-10-hydroxy-4(5)-cadinen-3-one (3) (Sanz et al., 2004), ergosta-5,7,22-trien-3b-ol (4) (Lv et al., 2008), 4-methoxy-3methyl-6-[(1E)-1-methyl-1-propenyl]-2H-pyran-2-one (5) (Ishikawa et al., 2003), (R)-5,6-dihydro-6-pentyl-2H-pyran-2-one (6)

1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.04.008

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Fig. 1. (a) Sponge Gelliodes carnosa; (b) Colony appearance of Hypocreales sp. HLS-104; (c) Mycelium of the fungus under fluorescence microscope.

suggesting the existence of hydroxyl and carbonyl groups. The 13C (Table 1) and DEPT NMR spectra of 1 showed 15 carbon signals, including one carbonyl (dC 199.4), one double bond (dC 134.0, 152.8), two oxygenated quaternary carbons (dC 75.8; 71.3), three methines (dC 54.2; dC 46.2; dC 26.6), three methylenes (dC 36.9; dC 18.9; dC 35.7) and four methyl groups (dC 15.4; dC 18.8; dC 24.0; dC 30.8). According to the above data, compound 1 should be a bicyclic cadinane-type sesquiterpene (He et al., 1997). A downfield broad singlet at dH 6.72 (H-5) in the 1H NMR spectrum (Table 1) indicated the presence of a double bond conjugated with a carbonyl group (C-3). H-5 had a long-range coupling with a methyl group at dC 15.4 in the HMBC spectrum, indicating that this methyl (CH3-11) was attached to the other end of the double bond. The doublets of two methyl groups and the multiplet of one methine were assigned to an isopropyl unit. An additional methyl group was shifted relatively downfield to dH 1.54 and appeared as a singlet, indicating it was bonded to C-10 where a hydroxyl group was attached. Moreover, since H-2 have correlation with quaternary carbons dC 71.3 and dC 75.8 in HMBC spectrum, another oxygen should be linked to C-6. Therefore, the planar structure of 1 was determined as 6,10-dihydroxy-4(5)-cadinen-3one. The assignments of the 1H and 13C NMR signals, as well as the location of each substituent on the cadinane-type sesquiterpene were performed on the basis of HMBC correlation data and also by comparison with the data of compound 3. The relative stereochemistry of 1 was deduced from a combination of coupling constant analyses and the 1D, 2D-NOESY spectra (Fig. 4). When irradiating H-5 at dH 6.72, the integration values of H-7 (dH 1.57) and H-12 (dH 2.03) were enhanced, which showed that H-1 and OH-6 positioned trans to each other. According to the analog 3D molecular model and the coupling

Fig. 2. Comparison of the secondary metabolic HPLC profiles between the seawater and distilled water rice medium cultures of the HLS-104 (Grace Apollo C18 250 mm  4.6 mm, 5 mm; 0–60 min, 10–100% methanol in water, 60–70 min, 100% methanol; 1 mL/min; detection, 254 nm).

(Nair and Carey, 1979), (E)-3-(4-hydroxyphenyl)-2-propenoic acid (7) (Niwa et al., 2001) (Fig. 3). 2.2. Structure elucidation of compounds 1 and 2 Hypocreaterpene A (1) was obtained as yellow oil. The molecular formula was established as C15H24O3 on the basis of HR-ESI-MS data (m/z 275.1627 [M+Na]+, calcd for C15H24NaO3, 275.1618), which implies four degrees of unsaturation. The IR spectrum exhibited absorption bands at ymax 3337 and 1664 cm1

15

OH

H

O 3 4

2

1 6

5

11

10 7

R2

9 8

R1 13

12

14

1: R1 =OH R2 =H 2: R1 =H R2 =OH 3: R1 =H R2 =H

H HO

O

(E)

5

4

O O

O

H

O 6 Fig. 3. Structures of compounds 1–7.

OH (E)

HO

O 7

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Fig. 4. The key HMBC and NOESY correlations for 1 and 2.

constants in the 1H NMR, dH 2.36 (J = 13.8, 17.5 Hz) was assigned as H-2b because the dihedral angle of H-2b and H-1 should be close to 1808, thus, dH 2.87 (J = 4.5, 17.5 Hz) assigned as H-2a. The NOE correlation between H-7 and H-2b was observed, it showed that H1 and H-7 necessarily positioned trans to each other, which also confirmed the isopropyl group at C-7 located at cis orientation. The equatorial configuration of the CH3-15 was confirmed by observed NOE correlation between H-15 and H-13(14). On the other hand, the NOE correlation between H-15 and H-1 also gave proof for the CH3-15 located at cis orientation. The molecular model for 1 was consistent with the NOESY result. 1R,6R,7S,10R- and 1S,6S,7R,10Scould be two possible candidate relative configurations of 1. An extension of the helicity rule (Djerassi et al., 1962) could be used to determine the absolute configuration of 1. The molecule was viewed from the line on the plane of the a, b-unsaturated ketone ring along the C5 5C–C5 5O grouping. And it was responsible for the negative Cotton effect (p–p* transition), which was good in accordance with the measured negative CD Cotton effect at lmax 251 nm of 1 (Fig. 5). Considering the CD spectrum, the absolute configuration of compound 1 was deduced to be 1S,6S,7R,10S-6,10dihydroxy-4(5)-cadinen-3-one. Hypocreaterpene B (2) was also isolated as yellow oil. The molecular formula was established as C15H24O3 on the basis of

Table 1 1 H (500 MHz) and

HR-ESI-MS data (m/z 275.1634 [M+Na]+, calcd for C15H24NaO3, 275.1618). The UV, IR, 1H and 13C NMR spectra of 2 (Table 1) resembled those of 1, and only differed from 1 by having two additional methines and losing one methylene and one quaternary carbon. These changes suggested a hydroxyl group was attached at C-8 or 9 in 2 instead of at C-6 in 1. The HMBC correlations of H-1 at dH 2.04 and H-15 at dH 1.26 with methine at dC 70.9 suggested that one hydroxyl group was located at C-9 (dC 70.9). Therefore, the structure of compound 2 was determined to be 9,10-dihydroxy4(5)-cadinen-3-one. The relative stereochemistry of 2 was also deduced from a combination of coupling constant analyses and the NOE spectrum (Fig. 4). Based on NOE, compound 2 was determined to be 1S,6S,7S,9R,10S- or 1R,6R,7R,9S,10R-9,10-dihydroxy-4(5)-cadinen3-one. On the basis of the helicity rule, a negative Cotton effect at 241 nm (Fig. 5) indicated the absolute configuration of compound 2 was 1R,6R,7R,9S,10R-9,10-dihydroxy-4(5)-cadinen-3-one. 2.3. Biological assays All compounds were assayed for their anti-inflammatory, cytotoxicity, antivirus and antibacterial activities. However, the results showed that all compounds were inactive (IC50 >50 mg/mL)

13

C (125 MHz) NMR spectroscopic data for compounds 1 and 2. 2

1 Position 1 2a 2b 3 4 5 6 7 8 9 10 11 12 13 14 15

dc, type

dH, mult (J/Hz)

dc, type

dH, mult (J/Hz)

54.2, CH 36.9, CH2 36.9, CH2 199.4, C 134.0, C 152.8, CH 75.8,C 46.2, CH 18.9,CH2 35.7, CH2 71.3, C 15.4, CH3 26.6, CH 18.8, CH3 24.0, CH3 30.8, CH3

2.07, m 2.87, dd (4.5, 17.5) 2.36, dd (13.8,17.5)

42.3, CH 37.3, CH2 37.3, CH2 200.5, C 132.1, C 151.6, CH 40.1, CH 44.6, CH 33.8, CH2 70.9, CH 72.3, C 15.5, CH3 26.7, CH 22.6, CH3 23.5, CH3 23.2, CH3

2.04, m 2.62, dd (3.5, 15.0) 2.50, dd (15.0,15.0)

6.72,s 1.57, m 1.60, m 1.65, m 1.79, 2.03, 1.00, 0.93, 1.54,

d, (1.2) m d (7.0) d (7.0) s

6.64, s 2.93, d (8.5) 1.71, m (3.0) 1.74–1.76, m 3.55, dd (4.5, 11.8) 1.76, 1.68, 0.97, 0.91, 1.26,

br s (1.2, 2.7) m d (6.0) d (6.0) s

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3.2. Fungal material, fermentation and extraction

10

1

Mol. CD

5

2

0

251 nm, -2.9497

241 nm, -1.29182

-5

200

250

300 Wavelength [nm]

350

Strain HLS-104 isolated from a sponge Gelliodes carnosa produced white to cream mycelia surrounding the host. The ITS rDNA of this strain was subjected to a BLAST search in GenBank. The strain was tentatively designated as Hypocreales sp. by its high similarity value. The fungus HLS-104 was first cultivated on YPD agar plates (YPD; Yeast extract 10 g; Peptone 10 g; Glucose 20 g; Natural seawater 1 L; pH 7.0) at 28 8C for 3 days, then mycelium was inoculated into 500 mL Erlenmeyer flasks, each containing 100 mL of liquid YPD medium. The flasks were incubated at 28 8C on a rotary shaker (180 r/min) for 5 days. Seed culture (10 mL) was transferred into fifty 500 mL Erlenmeyer flask (each Erlenmeyer flask contains 100 g rice and 100 mL natural seawater, the contents were soaked for 3–5 h before autoclaving at 121 8C for 30 min (Li et al., 2009) at 28 8C for 35 days. The fermented material was extracted with ethyl acetate by ultrasound repeatedly, and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (45 g).

Fig. 5. The circular dichroism (CD) spectra of 1 and 2.

3.3. Isolation against human cancer cell lines including lung adenocarcinoma (A549), stomach cancer (BGC-823), ovarian carcinoma (A2780), hepatoma (Bel7402), and human colon cancer (HCT-8) cell lines. Meantime, all compounds showed no activity against HIV-1 replication, as well as against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa at a concentration of 0.1 mg/mL. Fortunately, some of them showed the inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-treated RAW264.7 cells. As shown in Fig. 6, compound 3 and 6 were effective against the nitric oxide (NO) production and showed moderate inhibition with Emax values of 10.22% and 26.46% at 1 mM, respectively.

3. Experimental 3.1. General experimental procedures Optical rotations were measured on a JASCO P-2000 digital polarimeter. UV, CD, and IR spectra were recorded using a Cary 300 spectrometer, a JASCO J-815 CD spectrometer, and a Nicolet 5700 FT-IR spectrometer (FT-IR microscope transmission), respectively. 1 H and 13C NMR spectra were obtained at 500 and 125 MHz, using a Bruker AVANCE500-III spectrometer. HR-ESI-MS data were measured using an Agilent 1100 LC/MSD Trap SL LC/MS/MS spectrometer.

The crude extract was first subjected to silica gel column chromatography using petroleum ether/EtOAc gradient elution and separated into six fractions with different polarity. The third fraction was separated again by silica gel column chromatography using petroleum ether/CH2Cl2/CH3OH gradient elution and Sephadex LH-20 column chromatography using 1:1 CH2Cl2/CH3OH as eluent. Purification of the resulting subfractions by semipreparative HPLC afforded hypocreaterpene A (6.0 mg, 65% CH3OH in H2O, tR 37.8 min) and hypocreaterpene B (11.3 mg, 60% CH3OH in H2O, tR 35.6 min). 3.3.1. Hypocreaterpene A (1) Pale yellow oil; ½a20 D +1.3 (c 0.1, MeOH); UV (MeOH) lmax 239 nm. IR (KBr) ymax 3337, 2973, 2927, 2899, 1664, 1450, 1379, 1330, 1273, 1089, 1050, 881, 804, 669 cm1. 1H and 13C NMR spectroscopic data, see Table 1; HR-ESI-MS m/z 275.1627 [M+Na]+ (calcd for C15H24NaO3, 275.1618). 3.3.2. Hypocreaterpene B (2) Pale yellow oil; ½a20 D 14.5 (c 0.1, MeOH); UV (MeOH) lmax 246 nm. IR (KBr) ymax 2956, 2929, 2871, 1719, 1664, 1508, 1459, 1375, 1234, 1171, 1141, 1087, 1066, 1034, 989, 939, 917, 885, 800, 755, 669 cm1. 1H and 13C NMR spectroscopic data, see Table 1; HR-ESI-MS m/z 275.1634 [M+Na]+ (calcd for C15H24NaO3, 275.1618).

Fig. 6. (a) Cell viability assessed by MTT assays; (b) The effects of the compounds on the production of NO by LPS-induced RAW 264.7 cells.

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3.4. Biological assays Anti-inflammation, cytotoxicity, antibacterial and antivirus bioassays were performed with compounds of purity >90% by HPLC. 3.4.1. Anti-inflammation bioassays The anti-inflammation activity of the pure compounds was evaluated based on the reported procedures (Zhang et al., 2012). 3.4.2. Cytotoxicity bioassays MTT assay was adopted for the cytotoxicity assay in vitro to the human cancer cell lines A549, BGC-823, A2780, Bel7402 and HCT-8 based on the reported procedures (Gong et al., 2009). The dose– response curves were fitted with Sigma plot and IC50s were determined with 5-FU (Sigma, 98% pure) as a positive control. 3.4.3. Antibacterial bioassays The antibacterial activity of the pure compounds was evaluated against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using the agar plate method (Afolayan and Meyer, 1997). The positive control was ampicillin. 3.4.4. Antivirus bioassays A cell-based VSVG/HIV-1 pseudotyping system was used for evaluating the anti-HIV replication activity of the compounds as described previously (Fan et al., 2009). Acknowledgments This work was supported by grants from the Mega-project for Inovative Drugs (Nos. 2009ZX09301-003-4-1 and 2012ZX09301002001-005) and the Fundamental Research Funds for the Central Universities (2012N06). We are grateful to Dr. Li Li in our Institute for the help of determination of the compounds absolute configuration by CD. We thank Ms. Wan-Qi Zhou in our Institute for the in vitro anti-cancer assay. Special thanks to Dr. Wei Yuan and Miachael S. Elder in Stephen F. Austin State University, USA, for their proof-reading of the manuscript both academically and grammatically. References Afolayan, A.J., Meyer, J.J., 1997. The antimicrobial activity of 3,5,7-trihydroxyflavone isolated from the shoots of Helichrysum aureonitens. J. Ethnopharmacol. 57, 177–181.

Djerassi, C., Records, R., Bunnenberg, E., Mislow, K., Moscowitz, A., 1962. Inherently dissymetric chromophores. Optical rotatory dispersion of a,b-unsaturated ketones and conformational analysis of cyclohexenones. J. Am. Chem. Soc. 84, 870–872. Fan, X., Zi, J., Zhu, C., Xu, W., Cheng, W., Yang, S., Guo, Y., Shi, J., 2009. Chemical constituents of Heteroplexis micocephala. J. Nat. Prod. 72, 1184–1190. Gao, Q., Jin, K., Ying, S.H., Zhang, Y., Xiao, G., Shang, Y., Duan, Z., Hu, X., Xie, X.Q., Zhou, G., Peng, G., Luo, Z., Huang, W., Wang, B., Fang, W., Wang, S., Zhong, Y., Ma, L.J., St Leger, R.J., Zhao, G.P., Pei, Y., Feng, M.G., Xia, Y., Wang, C., 2011. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet. 7, e1001264. Gong, T., Wang, D.X., Chen, R.Y., Liu, P., Yu, D.Q., 2009. Novel benzil and isoflavone derivatives from Millettia dielsiana. Planta Med. 75, 236–242. Gunatilaka, A.A., 2006. Natural products from plant-associated microorganisms: distribution, structural diversity, bioactivity, and implications of their occurrence. J. Nat. Prod. 69, 509–526. He, K., Zeng, L., Shi, G., Zhao, G.X., Kozlowski, J.F., McLaughlin, J.L., 1997. Bioactive compounds from Taiwania cryptomerioides. J. Nat. Prod. 60, 38–40. Isaka, M., Kittakoop, P., Kirtikara, K., Hywel-Jones, N.L., Thebtaranonth, Y., 2005. Bioactive substances from insect pathogenic fungi. Acc. Chem. Res. 38, 813–823. Ishikawa, M., Amaike, M., Itoh, M., Warita, Y., Kitahara, T., 2003. Synthesis of the racemate and both enantiomers of massoilactone. Biosci. Biotechnol. Biochem. 67, 2210–2214. Li, Y., Ye, D., Chen, X., Lu, X., Shao, Z., Zhang, H., Che, Y., 2009. Breviane spiroditerpenoids from an extreme-tolerant Penicillium sp. isolated from a deep sea sediment sample. J. Nat. Prod. 72, 912–916. Liu, W.C., Li, C.Q., Zhu, P., Yang, J.L., Cheng, K.D., 2010. Phylogenetic diversity of culturable fungi associated with two marine sponges: Haliclona simulans and Gelliodes carnosa, collected from the Hainan Island coastal waters of the South China Sea. Fungal Divers. 42, 1–15. Lv, Z.M., Jiang, Y.T., Wu, L.J., Liu, K., 2008. Chemical constituents from dried sorophore of cultured Cordyceps militaris. Zhongguo Zhong Yao Za Zhi 33, 2914–2917. Molnar, I., Gibson, D.M., Krasnoff, S.B., 2010. Secondary metabolites from entomopathogenic Hypocrealean fungi. Nat. Prod. Rep. 27, 1241–1275. Nair, M.S., Carey, S.T., 1979. Metabolites of pyrenomycetes: XII. Polyketides from the Hypocreales. Mycologia 71, 1089–1096. Niwa, T., Doi, U., Kato, Y., Osawa, T., 2001. Antioxidative properties of phenolic antioxidants isolated from corn steep liquor. J. Agric. Food Chem. 49, 177–182. Rateb, M.E., Ebel, R., 2011. Secondary metabolites of fungi from marine habitats. Nat. Prod. Rep. 28, 290–344. Sanz, J., Soria, A.C., Garcia-Vallejob, M.C., 2004. Analysis of volatile components of Lavandula luisieri L. by direct thermal desorption-gas chromatography–mass spectrometry. J. Chromatogr. A 1024, 139–146. Sepcic, K., Zalar, P., Gunde-Cimerman, N., 2011. Low water activity induces the production of bioactive metabolites in halophilic and halotolerant fungi. Mar. Drugs 9, 43–58. Tan, R.X., Zou, W.X., 2001. Endophytes: a rich source of functional metabolites. Nat. Prod. Rep. 18, 448–459. Yang, K.L., Wei, M.Y., Shao, C.L., Fu, X.M., Guo, Z.Y., Xu, R.F., Zheng, C.J., She, Z.G., Lin, Y.C., Wang, C.Y., 2012. Antibacterial anthraquinone derivatives from a sea anemone-derived fungus Nigrospora sp. J. Nat. Prod. 75, 935–941. Zhang, T., Sun, L., Liu, R., Zhang, D., Lan, X., Huang, C., Xin, W., Wang, C., Du, G., 2012. A novel naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in RAW264.7 macrophages. Mol. Pharmacol. 9, 671–677. Zheng, P., Xia, Y., Xiao, G., Xiong, C., Hu, X., Zhang, S., Zheng, H., Huang, Y., Zhou, Y., Wang, S., Zhao, G.P., Liu, X., St Leger, R.J., Wang, C., 2011. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine. Genome Biol. 12, R116.