Antiviral isoindolone derivatives from an endophytic fungus Emericella sp. associated with Aegiceras corniculatum

Phytochemistry 72 (2011) 1436–1442

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Antiviral isoindolone derivatives from an endophytic fungus Emericella sp. associated with Aegiceras corniculatum Guojian Zhang, Shiwei Sun, Tianjiao Zhu, Zhenjian Lin, Jingyan Gu, Dehai Li ⇑, Qianqun Gu ⇑ Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, PR China

a r t i c l e

i n f o

Article history: Received 30 November 2010 Received in revised form 14 February 2011 Available online 23 May 2011 Keywords: Emericella Endophytic fungus Aegiceras corniculatum Myrsinaceae Isoindolone derivatives Antiviral activity

a b s t r a c t Chemical investigation of the endophytic fungus Emericella sp. (HK-ZJ) isolated from the mangrove plant Aegiceras corniculatum led to isolation of six isoindolones derivatives termed as emerimidine A and B and emeriphenolicins A and D, and six previously reported compounds named aspernidine A and B, austin, austinol, dehydroaustin, and acetoxydehydroaustin, respectively. Their structures were elucidated on the basis of NMR spectroscopic evidence while the anti-influenza A viral (H1N1) activities of eight compounds were also evaluated using the cytopathic effect (CPE) inhibition assay. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Endophytes are microorganisms that spend the whole or part of their life cycle colonizing tissues of their host plants without causing apparent symptoms of disease (Schulz and Boyle, 2006; Strobel et al., 2004). Their relationships with host plants range from symbiotic to slightly pathogenic (Schulz et al., 2002). Moreover, there is supposed to be an equilibrium between microorganism virulence and plant defense. In this balanced system, host plant provides nutrients to the endophyte, and in return, the endophyte produces bioactive substances to enhance the growth and competitiveness of the host in its natural habitat (Esser, 2009; Freeman and Rodriguez, 1993; Saikkonen et al., 1998). For this reason, endophytes have been identified as a prolific source of biologically active small molecules, thus representing potential leads for the development of new pharmaceutical agents (Zhang et al., 2006). Mangrove endosymbionts have been considered as a source of compounds possessing physiological activities due to their strong competitiveness in the special ecological niche residing in tidal mudflats. Aegiceras corniculatum is a shrub or small tree mangrove in the Myrsinaceae family with a wide distribution in coastal and estuarine areas ranging from India through South East Asia to southern China, New South Wales, New Guinea, and Australia (Clarke, 1993; Li and Lee, 1997; Paijmans and Rollet, 1977). Our previous work on the chemical investigation of endophytic fungi from this plant afforded the identification of cytotoxic polyketides ⇑ Corresponding authors. Tel.: +86 532 82032065; fax: +86 532 82033054. E-mail addresses: [email protected] (D. Li), [email protected] (Q. Gu). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.04.014

(Lin et al., 2008). The present work within the scope of an antiinfluenza screening program led to the isolation of a fungus authenticated as Emericella sp. (HK-ZJ) from the inner bark of A. corniculatum in the suburb of HaiKou, People’s Republic of China. The genus Emericella is one of the sexual states of Aspergillus (Geiser, 2009). Several species of this genus are saprobes, whereas others are either pathogenic or endophytic on living plants (Berbee, 2001; Thongkantha et al., 2008). With a increase in the number of chemical studies on Emericella sp. has come the discovery of various bioactive natural products including antitumor indole alkaloids and quinones (Kralj et al., 2006; Wang et al., 2007a), neuritogenic and antimicrobial polyketides (Nozawa et al., 1987), cytotoxic sesterterpenes (Wei et al., 2004), aflatoxins and sterigmatocystin (Frisvad and Samson, 2004), as well as xanthones with antimicrobial, immunostimulant and calmodulin inhibition activities (Figueroa et al., 2009; Fujimoto et al., 2006; Pornpakakul et al., 2006). In this paper, the first isolation, structural elucidation and biological evaluation of novel isoindolone derivatives from the fungal endophyte Emericella sp. (HK-ZJ) is reported. 2. Results and discussion Fermentation of Emericella sp. (HK-ZJ) was performed in flask liquid culture. Bioassay-guided (anti-H1N1 activity) fractionation of the EtOAc extract (from both broth and mycelium) led to isolation of eight isoindolone derivatives 1–8 and four austin-like meroterpenoids 9–12. Emerimidine A (1) was obtained as a pale amorphous powder. Its molecular formula was deduced to be C10H11NO4 by HRESIMS:

G. Zhang et al. / Phytochemistry 72 (2011) 1436–1442

m/z 210.0756 [M+H]+, indicating six degrees of unsaturation. The IR absorptions at 1680 cm1 (carbonyl) and 3300 cm1 (N–H stretch) denoted a secondary amide moiety. The UV spectral data at 223 (4.50), 259 (4.28), and 298 (3.79) nm indicated the existence of a benzoyl group. The 1H NMR spectrum of 1 (Table 1) showed signals ascribable to a methylene (dH 4.15), two aromatic methoxyls (dH 3.71 and dH 3.81), an aromatic proton (dH 6.70) and an NH amide proton (dH 8.30). The lack of any end groups together with the required degrees of unsaturation suggested a bicyclic aromatic lactam. Further signals of 13C NMR (Table 1) spectrum [dC 170.3 (C), 153.7 (C), 153.7 (C), 139.4 (C), 127.7 (C), 124.5 (C), 96.3 (CH), and 42.5 (CH2)] indicates the presence of a core isoindolone structure that was supported by a literature precedent (Suemitsu et al., 1995; Stierle et al., 1993). The locations of the substituent groups on the aromatic ring were established based mainly by HMBC and NOESY correlations (Fig. 2). The long-range coupling of H-7 to an amide carbon C-1 (dC 170.3) indicated connectivity between the benzene ring and the moieties. The two aromatic methoxy groups (dH 3.71 and 3.81) were accommodated at C-5 and C-6, respectively based on analysis of HMBC correlations from H-7 to C-5, 5-OCH3 to C-5 and 6-OCH3 to C-6, as well as the NOESY correlations from 6-OCH3 to H-7 and 5-OCH3. Finally, the structure was determined by the assignment of the remaining hydroxyl group to C-4, accounting for the proposed molecular formula. Therefore compound 1 was elucidated as 4-hydroxy-5,6-dimethoxy-2,3dihydro-1H-isoindol-1-one. Emerimidine B (2) was isolated as a colorless solid. Its HRESIMS suggested the same molecular formula as that of 1. The NMR signals of 2 were principally similar to those of 1 (Table 1), except for noticeable upfield chemical shifts of C-6 (4.4 ppm) and C-4 (11.4 ppm). This indicated that compound 2 possessed the same functional groups as 1 but with a different connectivity upon the aromatic ring. Moreover, the HMBC correlations from H2-3 to C4, 4-OCH3 to C-4 and from H-7 to C-6, 6-OCH3 to C-6 together with the NOESY (Fig. 2) correlations from 4-OCH3 to H2-3 and from 6OCH3 to H-7 signified the attachment of the two methoxy groups to C-4 and C-6, respectively. The structure of 2 was thus established as 5-hydroxy-4, 6-dimethoxy-2, 3-dihydro-1H-isoindol-1one. Emeriphenolicin A (3) was obtained as pale yellow oil. The ESIMS molecular ion cluster at m/z 466/468 [M+H]+ (rel. int. 3:1) indicated the presence of chlorine. Further positive HRESIMS analysis showed a pseudomolecular ion peak at m/z 466.2345 [M+H]+ and established a molecular formula of C25H36NO5Cl. Comparison of the 1D NMR spectra of 3 with that of 2 suggested that 3 showed

Table 1 1 H (600 MHz) and Position

13

C (150 MHz) NMR spectroscopic dataa for 1 and 2 DMSO-d6.

1 1

1 2 3 3a 4 5 6 7 7a 4-OCH3 5-OCH3 6-OCH3 4-OH 5-OH a b c

Hb

2 13 c

C

1

Hb

170.3 s 8.30 s 4.15 s

6.70 s

3.71 s 3.81 s 9.60 br s

42.5 t 124.5 s 153.7 s 139.4 s 153.7 s 96.3 d 127.7 s 60.0 q 55.8 q

13 c

C

170.2 s 8.29 s 4.31 s

3.83 s

42.4 t 123.0 s 142.3 s 141.8 s 149.3 s 100.7 d 128.1 s 59.3 q

3.83 s

56.1 q

6.94 s

9.18 br s

Chemical shifts (relative to TMS) are in (d) ppm. Assignments were aided by HMQC and HMBC. Assignments were made by DEPT HMQC and HMBC.

1437

a similar tri-substituted isoindolone skeleton to that of 2, and the difference was represented as one extra triprenyl side-chain attached to C-5 in 3 (Tables 2 and 3). The terpenoid moiety was elucidated from 2D NMR spectroscopic data, including HMQC, 1 H–1H COSY, and HMBC experiments (Fig. 2). From the 1H–1H COSY spectrum, it was possible to establish the proton sequence from H2-10 to H-20 , H2-40 to H-60 through H2-5, and H2-80 to H-100 through H2-90 . The two methyl groups attached at C-30 and C-70 were determined on the basis of the key HMBC correlations from H3-150 to C-20 , C-30 and C-40 ; and H3-140 to C-60 , C-70 and C-80 . The connections from C-20 to C-40 through C-30 and from C-60 to C-80 through C-70 were also confirmed by the HMBC experiment. Moreover, indicated by the chemical shifts of C-100 and C-110 (Tanaka et al., 2002; Wang et al., 2007b), as well as the crucial HMBC correlations from H3-120 /130 to C-110 , H2-80 to C-100 , and H2-90 to C-110 , the chlorine and hydroxyl group were positioned at C-110 and C-100 , respectively. Eventually, the connectivity of the above two fragments was determined by the key HMBC correlations from H2-10 to C-5. The appearance of the two vinyl methyl carbons upfield at 16.4 and 16.0 ppm in the 13C NMR spectrum 0 0 established the stereochemistry of the two double bonds D2 ,6 as E (in the case of Z geometry the vinyl methyl carbon should resonate around 23 ppm because of the absence of a c effect) (Nishino and William, 1976). The absolute configuration at C-100 was presumed tentatively to be R on the basis of comparison of the specific rotation (½a25 D +17.4) with that of 7-(7-chloro-6R-hydroxy-3, 7-dimethyl-2-octenyloxy) coumarin (½a25 D +27.3) (Ohashi et al., 1995). Emeriphenolicin B (4) was obtained as colorless oil. The molecular formula was deduced to be C24H34NO5Cl due to the ESIMS molecular ion cluster at m/z 474/476[M+Na]+ (rel. int. 3:1) and HRESIMS at m/z 452.2207 [M+H]+. The UV kmax absorptions at 221 (4.50), 245 (4.21), and 295 (3.66) nm together with the IR absorptions at 3285 and 1682 cm1 suggested that 4 was an analog of 3. Except for the absence of the aromatic methoxy group at dC 60.6, the 1D NMR spectroscopic data of 4 (Tables 2 and 3) displayed considerable similarity to 3, implying that the 4-OCH3 present in compound 3 was substituted by a hydroxyl group to generate the structure of 4. The specific rotation (½a25 D +18.7) indicated the same absolute configuration as 3. The similar UV and IR spectra suggested that emeriphenolicin C (5) was also an analog of 3. The NMR spectra of 5 (Table 2 and 3) were very similar to those of 3 except for the presence of a broad singlet (110 -OH) at dH 4.03 and a noticeable upfield shift of C-110 (-4.0 ppm), consistent with the presence of a hydroxyl group at C-110 instead of a chlorine. Moreover, the HRESIMS exhibited a pseudomolecular ion peak at m/z 448.2687 [M+H]+, confirming the molecular formula of C25H37NO6. The 100 R configuration of 5 was tentatively proposed on the basis of comparison of its specific rotation (½a25 D +8.7) with that of the tetraprenylbenzoquinone, capilloquinone (½a25 D 15.0) (Cheng et al., 2010) and the chromene derivative named 13-(6-hydroxy-2,8-dimethyl-2H-1-benzopyran2-yl)-2,6,10-trimethyl-(3R,6E,10E)-6,10-tridecadiene-2,3-diol (½a25 D +11.0) (Iwashima et al., 2005), for both of which the absolute configuration have been unambiguously determined. Compounds 6–8 were all obtained as pale yellow powder. Their molecular formulae were determined as C24H33NO4, C24H33NO4 and C23H31O4 from HRESIMS data at m/z 400.2486 [M+H]+, 400.2485 [M+H]+ and 386.2314 [M+H]+, respectively. Examination of their 1D NMR spectra (Tables 2 and 3) indicated that they are also isoindolone-sesquiterpene analogs sharing the same terpenoid side-chain with a variation only in the isoindolone moiety. With the exception of two olefinic carbons substitution for two oxygenated ones, the terpenoid moiety of compound 6 displayed very close spectroscopic data to those of 5. In further consideration of the nine degrees of unsaturation, compound 6 was postulated to have one

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G. Zhang et al. / Phytochemistry 72 (2011) 1436–1442

O MeO 6 R2

R1

O 7a 1 NH 4 R1

R4

NH

12' 11'

3

R 3 9'

13'

3'

7'

O 1'

5'

R2

15'

14'

3: R 1 = OMe, R2 = OMe, R3 = Cl, R4 = OH 4: R 1 = OMe, R2 = OH, R 3 = Cl, R 4 = OH 5: R 1 = OMe, R2 = OMe R 3 = OH, R4 = OH

1: R 1 = OH, R2 = OMe 2: R 1 = OMe, R 2 = OH

O

O

O

R1

O

R1

OH

R1

O O

NH O

O R2 6: R 1 = OH, R 2 = OMe 7: R 1 = OMe, R 2 = OH 8: R 1 = OH, R 2 = OH

O

O O

O

O

O 9: R1 = OAc 10: R 1 = OH

O

R2

11: R1 = OAc, R 2 = H 12: R1 = OAc, R 2 = OAc

Fig. 1. Structures of compounds 1–12.

O

O

O

O

NH

NH

HO

O

O

OH 1

2 O O NH

OH O Cl

O 3

O O NH O O

6 Fig. 2. Selected COSY (), HMBC (?) and NOESY (M) correlations of 1–3 and 6.

more double bond than 5. Moreover, the extra double bond was positioned at C-100 by analysis of the key HMBC correlations [dH 1.67 (H-120 )/dH 1.60 (H-130 ) to124.3 (C-100 ) and 131.4 (C-110 )] (Fig. 1). The structure of the isoindolone fragment and the arrangement of the sesquiterpene skeleton as well as the connection of the two moieties via an ether linkage were confirmed by further 2D NMR experiments (Fig. 1). The E-geometry of the two double 0 0 bonds D2 ,6 was identified from the NOESY correlations between 0 0 H-2 /H-4 , H-10 /H3-150 , H-60 /H-80 and H3-140 / H-50 . This was in agreement with the presumption that the sesquiterpene moiety was probably derived from farnesyl pyrophosphate. Consequently, compound 6 was elucidated as 6-hydroxy-4-methoxyl-5-[(2E,6E)3,7,11-trimethyl-2,6,10-dodecatrien-1-yl)oxy]-2,3-dihydro-1H-iso indol-1-one. As the HRESIMS data suggested, aspernidine A (7) possessed the same molecular formula as 6. Further careful comparison of their 13 C NMR spectroscopic data (Table 2 and 3) indicates that the aromatic methoxy carbon of 7 displayed a noticeable upfield shift by 4.3 ppm, suggesting the presence of the 4-hydroxy-6-methox-

yl- isoindolone fragment. In comparison with 6 and 7, the 1H and 13 C NMR spectra (Table 2 and 3) of aspernidine B (8) gave no methoxyl signals, indicating the presence of a diphenol structure. The molecular formula was further confirmed by HRESIMS data. Thus compound 8 was deduced as 4,6-dihydroxy-5-[(2E,6E)-(3,7,11-trimethyl-2,6,10-dodecatrien-1-yl)oxy]-2,3-dihydro-1H-isoindol-1one. In regards to the absolute configuration at C-100 for compounds 3–5, it seems that the comparisons of optical rotation with related compounds can not provide sufficient evidence. Consequently Mosher method was applied to 3 to generate further information. However, the reaction failed to produce the intended Mosher ester; instead, a series of unexpected side products was detected by TLC. This result can be explained by an easy epoxidation at C-100 and C110 under the treatment with pyridine-DMAP (Ohashi et al., 1995) followed by the subsequent epoxy ring opening. This also reduces any concern that compounds 3–5 are artifacts of the isolation protocol according to which MeOH and CHCl3 were used in the silica gel chromatographic separation of the initial crude extract. In order to solve this question, a small scale fermentation (200 mL) was re-performed. The presence of compounds 3–5 in the initial EtOAc extract was tested by HPLC (supplementary data), confirming a natural occurrence for 3–5. Additionally, according to a literature report (Andreas et al., 2001), biohydrolysis of trialkyl oxiranes in microorganisms frequently proceeded in an enantio-convergent fashion and thus led to the corresponding (R)-configurated vicinal diols. This consists with the 100 R configuration of 5. There are presumed to be close biogenetic relationships among compounds, a mixed biosynthetic route involving polyketide and mevalonate pathways is proposed in Fig. 3. Isoindolone derivatives distribute broadly in natural products of microbial origin. This family of secondary metabolites display large diversity in structure and biological activity. Some of them are phytotoxins such as zinnimidine and porritoxin (Horiuchi et al., 2003; Suemitsu et al., 1995; Stierle et al., 1993), whereas others, for instance, stachybotrins and staplabin (Minagawa et al., 2002; Shinohara et al., 1996) are lead compounds with antiviral and plasminogen activation activities. Compounds 1–8 were tested for in vitro activity against H1N1 replication in MDCK cells. However, only compounds 1 and 2 showed moderate inhibitory effects with IC50 values of 42.07 lg/mL and 62.05 lg/mL (ribavirin as a positive control, IC50 24.60 lg/mL).

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G. Zhang et al. / Phytochemistry 72 (2011) 1436–1442 Table 2 1 H (600 MHz) NMR spectroscopic dataa for 3–8. H

3b

4b

2 3 7 10 20 40 a 40 b 50 a 50 b 60 80 a 80 b 90 a 90 b 100 120 130 140 150 4-OCH3 6-OCH3 4-OH 6-OH 100 -OH 110 -OH

6.62 4.38 7.14 4.60 5.55 2.09 2.05 2.11 2.06 5.13 2.25 2.02 1.73 1.44 3.48 1.54 1.58 1.60 1.67 3.99 3.90

s s s d (6.6) t (6.6) m m m m t (7.7) m m m m d (12.1) s s s s s s

5c

6.37 4.36 7.01 4.68 5.49 2.12 2.07 2.13 2.10 5.13 2.26 2.04 1.74 1.45 3.48 1.55 1.59 1.60 1.66

s s s m t (7.3) m m m m t (7.7) m m m m dd (9.2, 5.5) s s s s

8.49 4.35 6.98 4.49 5.46 2.02 1.99 2.04 2.01 5.09 2.15 1.86 1.61 1.13 3.03 0.97 1.03 1.55 1.62 3.91 3.83

3.92 s 6.36 s

s s s d (6.9) t (6.9) m m m m t (6.9) m m m m ddd (9.2, 5.5, 1.4) s s s s s s

6b

7b

6.76 s 4.40 s 7.17 s 4.67 d (11.1) 5.51 t (11.1) 2.10 m 2.05 m 2.11 m 2.07 m 5.09 t (8.1) 2.09 m 2.03 m 1.99 m 1.96 m 5.08 t (8.1) 1.674 s 1.60 s 1.60 s 1.668 s 3.98 s

6.91 4.35 7.00 4.67 5.50 2.03 1.97 2.10 2.03 5.06 2.07 2.07 2.03 1.95 5.07 1.67 1.58 1.59 1.64

s s s d (7.7) t (7.7) m m m m t (8.1) m m m m t (8.1) s s s s

3.92 s 6.24 s 6.01 s

2.36 d (5.5)

8c 8.28 4.10 6.62 4.52 5.52 2.01 1.95 2.08 2.03 5.03 2.06 2.06 2.02 1.90 5.03 1.62 1.53 1.54 1.57

s s s d (10.4) t (10.4) m m m m t (8.5) m m m m t (8.1) s s s s

9.46 s 9.26 s

4.26 d (5.9) 4.03 s

Assignments were aided by 1H–1H COSY and HMQC. a The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm. b NMR data in CDCl3. c NMR data in DMSO-d6.

Table 3 13 C (150 MHz) NMR spectroscopic data

a

for 3–8.

C

3b

4b

5c

6b

7b

8c

1 3 3a 4 5 6 7 7a 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 4-OCH3 6-OCH3

171.6 s 43.3 t 127.3 s 148.9 s 143.5 s 155.4 s 101.4 d 128.2 s 69.9 t 119.9 d 142.2 s 39.6 t 26.2 t 124.6 d 134.9 s 36.6 t 29.8 t 78.5 d 76.1 s 29.2 q 27.4 q 16.0 q 16.4 q 60.6 q 56.1 q

171.4 s 42.7 t 122.6 s 137.2 s 145.0 s 153.5 s 98.6 d 127.5 s 69.5 t 119.5 d 143.7 s 39.5 t 25.9 t 124.5 d 134.9 s 36.4 t 29.4 t 78.3 d 75.9 s 29.1 q 27.3 q 15.8 q 16.2 q

170.2 s 43.3 t 127.7 s 149.1 s 142.6 s 155.0 s 101.3 d 128.8 s 69.4 t 120.6 d 141.4 s 39.6 t 26.5 t 123.6 d 136.0 s 37.2 t 30.1 t 77.6 d 72.1 s 26.9 q 25.1 q 16.5 q 16.6 q 60.3 q 56.7 q

171.2 s 43.2 t 126.6 s 147.5 s 141.0 s 151.1 s 104.8 d 128.0 s 70.0 t 118.8 d 144.2 s 39.7 t 26.7 t 123.4 d 135.6 s 39.6 t 26.2 t 124.3 d 131.4 s 25.7 q 16.0 q 17.7 q 16.4 q 60.0 q

171.9 s 42.9 t 122.6 s 137.3 s 145.1 s 153.6 s 98.6 d 127.7 s 69.7 t 119.2 d 144.3 s 39.7 t 26.8 t 123.6 d 135.7 s 39.7 t 26.4 t 124.4 d 131.5 s 25.8 q 16.1 q 17.8 q 16.5 q

171.7 s 43.2 t 124.5 s 137.4 s 141.6 s 152.1 s 101.6 d 127.7 s 68.4 t 120.6 d 146.5 s 39.5 t 26.5 t 122.5 d 135.2 s 39.5 t 26.3 t 124.1 d 131.4 s 25.9 q 16.1 q 17.9 q 16.4 q

56.2 q

for compounds 9, 11 and 12 (Kataoka et al., 2011). In view of this, Emericella sp. (HK-ZJ) was supposed to have the capacity to protect its host plant against insect invaders by producing bioactive substances. This host-endophyte relationship needs to be further investigated. 2.1. Concluding remarks In conclusion, a fungal endophyte identified as Emericella sp. was isplated from the inner bark of A. corniculatum. Through a bioactivity-guided fractionation, isolation, and characterization of two isoindolones (1–2), four isoindolone-sesquiterpenoids (3–6), and six known meroterpenoids (7–12) was successfully achieved. To the best of our knowledge, meroterpenoids such as 3–8, derived from a sesquiterpenoid moiety and an isoindolinone fragment connected through an ether linkage, are very rare in nature. With this skeleton, aspernidine A and B (7–8) were the only two examples reported previously (Scherlach et al., 2010). Bioactivity evaluation demonstrated the moderate antiviral activity for compounds 1–2. 3. Experimental 3.1. General experimental procedure

56.3 q

All the assignments were made by DEPT, HMQC and HMBC experiments. a Chemical shifts are given in ppm. b NMR data in CDCl3. c NMR data in DMSO-d6.

Austin-like compounds 9–12 represent a class of meroterpenoids mainly isolated from Aspergillus and Penicillium genera (Chexal et al., 1976; Schurmann et al., 2010; Geris dos Santos and Rodrigues-Filho, 2002, 2003). It was previously reported that these derivatives exert notable toxicities to insects (Geris et al., 2008; Hayashi et al., 1994) and the very recently a blocking action on cockroach nicotinic acetylcholine receptors was demonstrated

Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on Beckman DUÒ 640 spectrophotometer, whereas IR spectra were acquired on a NICOLET NEXUS 470 spectrophotometer in KBr discs. 1H, 13C NMR and DEPT spectra and 2D-NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer using TMS as internal standard, with chemical shifts reported as d values. ESI-MS were measured on a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. TLC was performed on plates precoated with silica gel GF254 (10–40 lm). Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., People’s Republic of China), RP-18 gel (40–75 lm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used

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G. Zhang et al. / Phytochemistry 72 (2011) 1436–1442

O 4 SCoA polyketide biosynthesis OH HO

3

COOH HO

O

HO

OH mevalonate biosynthesis

OH

OPP

O HO NH OH

m

et

hy la t

io n

HO

prenylation 1 and 2

O HO NH

methylation

O

6 and 7

OH 8 epoxydation O HO

O

NH O OH nucleophilic ring opening 3, 4 and 5 Fig. 3. Hypothetic biosynthesis pathway of 1–8.

for column chromatography (cc). Semiprepartive HPLC was performed using an ODS column [YMC-pak ODS-A, 10  250 mm, 5 lm, 4 mL/min]. 3.2. Collection and identification The endophytic fungus Emericella sp. (HK-ZJ) was isolated from mangrove inner bark collected in HaiKou, People’s Republic of China. It was identified according to its morphological characteristics and 18S rRNA sequence (NCBI Nucleotide ID: GU358698). This strain was preserved in our lab at -80 °C and working stocks were prepared on Potato Dextrose agar slants stored at 4 °C. Colonies on PDA agar grew well at 28 °C, attaining a diameter of 6 to 7 cm. in 14 days. At first the color of obverse side was white, but gradually became dark green and slightly brownish in the center with the development of hypha; reverse side was wheat yellowish; substratum was red-brownish. Conidial structures were produced abundantly and evenly distributed throughout the colony. Conidial heads were abundant and columnar. Conidiophores were short, a little sinuous, smooth, brownish and nonseptate.

3.3. Fermentation, extraction and isolation Spores were directly inoculated into 1 L Erlenmeyer flasks containing 300 mL fermentation media (mannitol 20 g, maltose 20 g, glucose 10 g, monosodium glutamate 10 g, KH2PO4 0.5 g, MgSO47H2O 0.3 g, yeast extract 3 g and corn steep liquor 1 g, dissolved in 1 L seawater, pH 6.5). The flasks were incubated under static conditions at 24 °C for 30 days. The whole broth (15 L) was filtered through cheesecloth to separate into supernatant and mycelia. The former was extracted with EtOAc (3  15 L) while the latter was extracted with acetone-H2O (4:1, v/v) (3  5 L). The acetone extract was evaporated under reduced pressure to afford an aqueous solution, and then extracted with EtOAc (3  3 L). The two EtOAc extracts were concentrated together in vacuo to give a crude gum (15.0 g). The total extract was subjected to VLC on a silica gel column using step gradient elution with MeOH– CHCl3 (0:100 ? 50:50). The collected materials were combined into 6 fractions based on TLC properties. The active fraction 5 (with an inhibition ratio of 68% against influenza A Virus under the concentration of 100 lg/mL) from the 20:1 CHCl3–MeOH eluent was further separated on a Sephadex LH-20 column with CHCl3–MeOH (1:1). The active fraction 5-3 (with an inhibition ratio of 80%

G. Zhang et al. / Phytochemistry 72 (2011) 1436–1442

against influenza A Virus under the concentration of 100 lg/mL) was separated by semipreparative HPLC employing isocratic elution with MeOH–H2O (25:75, 4.0 mL/min) to yield compounds 1 (3.2 mg) and 2 (6.3 mg). The fraction 5-2 was separated by ODS cc (MeOH–H2O gradient mixtures) into four subfractions. Subfractions 5-2-1 and 5-2-2 were further purified, respectively by extensive HPLC MeOH–H2O (70:30 and 65: 35; 4.0 mL/min), to give compound 4 (2.0 mg), 5 (3.3 mg) and 8 (7.0 mg); 3 (5.1 mg), 6 (3.0 mg) and 7 (4.4 mg). Fractions 5-2-3 and 5-2-4 were subjected to HPLC MeOH–H2O (75: 25, 4.0 mL/min) separately to afford 9 (6.0 mg), 10 (4.2 mg), 11 (10.3 mg) and 12 (9.2 mg). 3.3.1. Emerimidine A (1) White powder; UV (MeOH) kmax (log e) 224 (4.50), 259 (4.26), 298 (3.79); IR (KBr) mmax cm1 3423, 3378, 2921, 2851, 1677, 1623, 1480, 1458, 1340, 1255, 1113, 1049, 878, 762 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 210. 0756 [M+H]+ (calcd. for C10H12NO4, 210. 0766). 3.3.2. Emerimidine B (2) White powder; UV (MeOH) kmax (log e) 223 (4.50), 259 (4.28), 298 (3.79); IR (KBr) mmax cm1 3310, 2951, 2850, 1681, 1458, 1310, 1111, 1028, 761 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 210. 0758 [M+H]+ (calcd. for C10H12NO4, 210. 0766). 3.3.3. Emeriphenolicin A (3) Pale yellow oil; ½a25 D +17.4 (c 0.1, EtOH); UV (MeOH) kmax (log e) 220 (4.50), 245 (4.21), 295 (3.67); IR (KBr) mmax cm1 3325, 2957, 2923, 2853, 1729, 1695, 1472, 1458, 1369, 1338, 1122 cm1; for 1 H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 466.2345 [M+H]+ (calcd. for C25H37NO5Cl, 466.2340). 3.3.4. Emeriphenolicin B (4) Colorless oil; ½a25 D +18.7 (c 0.1, EtOH); UV (MeOH) kmax (log e) 221 (4.50), 245 (4.21), 295 (3.66); IR (KBr) mmax cm1 3285, 2951, 2925, 2856, 1682, 1616, 1457, 1361, 1213, 1119, 764 cm1; for 1 H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 462.2207 [M+H]+ (calcd. for C24H35NO5Cl, 452.2204).

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922, 668 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 386.2314 [M+H]+ (calcd. for C23H32NO4, 386.2331). 3.4. Anti-influenza A virus (H1N1) bioassay The antiviral activity against influenza A virus (H1N1) was evaluated by the CPE inhibition assay. Confluent MDCK cell monolayers were firstly incubated with influenza A virus for 1 h at 37 °C. After removing the virus dilution, cells were maintained in infecting media (RPMI 1640, 4 lg/mL trypsin) containing different concentrations of test compounds at 37 °C. Ribavirin was used as a positive control. After incubation for 2 days, the plates were washed with PBS and stained with 1% crystal violet (Sigma–Aldrich, USA) in 20% ethanol and 3.7% formaldehyde (Boster, China). Cell damage was quantified with respect to intensity of the stain retained by living cells in a microplate reader at 630 nm. Cell damage in the presence of inhibitors was calculated by setting mock infected cells to 0% damage and cells infected without inhibitor to 100% damage. The antiviral 50% inhibition concentration (IC50) was defined as the concentration achieving 50% cytoprotection against viral infection. Acknowledgements This work was financially supported by the Chinese National Science Fund (No. 30973627 and 30772640), the public projects of State Oceanic Administration (No. 2010418022-3), the program for Changjiang Scholars and Innovative Research Team in University (No. IRT0944) and the Shandong Provincial Natural Science Fund (No. ZR2009CZ016). The anti-influenza A virus (H1N1) assay was performed by Dr. Wei Wang at the laboratory of molecular pharmacology of Ocean University of China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2011.04.014. References

3.3.5. Emeriphenolicin C (5) Colorless oil; ½a25 D +8.7 (c 1.0, CHCl3); UV (MeOH) kmax (log e) 244 (4.51), 259 (4.19), 295 (3.67); IR (KBr) mmax cm1 3290, 2945, 2924, 2856, 1687, 1616, 1476, 1348, 1115 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 448.2687 [M+H]+ (calcd. for C25H38NO6, 448.2699). 3.3.6. Emeriphenolicin D (6) Pale yellow powder; UV (MeOH) kmax (log e) 225 (4.50), 259 (4.26), 298 (3.78); IR (KBr) mmax cm1 3204, 3061, 2922, 2844, 1677, 1614, 1475, 1351, 1297, 1221, 1102, 981, 774 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 400.2486 [M+H]+ (calcd. for C24H34NO4, 400.2488). 3.3.7. Emeriphenolicin E (7) Pale yellow powder; UV (MeOH) kmax (log e) 224 (4.50), 259 (4.26), 298 (3.78); IR (KBr) mmax cm1 3193, 3062, 2922, 2850, 1668, 1603, 1481, 1455, 1359, 1247, 1117, 925, 670 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 400.2485 [M+H]+ (calcd. for C24H34NO4, 400.2488). 3.3.8. Emeriphenolicin F (8) Pale yellow powder; UV (MeOH) kmax (log e) 224 (4.51), 259 (4.26), 298 (3.77); IR (KBr) mmax cm1 3356, 3339, 2966, 2921, 2853, 1650, 1607, 1486, 1453, 1351, 1249, 1206, 1072, 1019,

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