Lin Yongcheng, Wan Jun, Zhou Shining, Gareth Jones E B^#
(Department of Appied Chemistry, Zhongshan University, Guangzhou 510275; ^#Department of Biology and Chemistry, City University of Hong Kong, China)

Received Feb.16, 2001. Supported by the National Natural Science Foundation of China (29672053, 20072058), the Natural Science Foundation of Guangdong Province, China (950026 and 980317) and a strategic grant at City University of Hong Kong (7000650).

Abstract Two new isocoumarins avicennin A(1) and B (4) with two analogues(2, 3) and vermopyrone(5) were isolated from the mangrove endophytic fungus (strain #2533) from the South China Sea. Their structures were elucidated using spectroscopic methods, primarily 2D NMR techniques.
Keyword mangrove endophytic fungus, isocoumarins, metabolites.

Endophytes are all organisms inhabiting plant materials that some time in their life, and can colonise internal plant tissues without causing apparent harm to their host ^[1]. Endophytic fungi are normally restricted to healthy plant parts where symptoms of fungal infection cann't be detected. It was hypothesized that the endophyte-host interaction was a balanced antagonism probably involving secondary metabolites produced by the partners ^[2] Endophytic fungi are now recognized as potential producers of novel secondary metabolites, which can be used as possible biocontrol agents and drugs .^[3] Although a variety of new compounds have been discovered from fungi isolated from the marine environment recently,^{[4 - 8]} there is no records of metabolites or novel compounds from endophytic fungi of mangrove plants to date. We have first isolated and characterized such fungal metabolites from leaves of the mangrove plants from the coasts of South China Sea ^[9]. In this paper, we report two new compounds, avicennin-A (1) and B (4) and two analogues (2) and (3), and (5) obtained from an endophytic fungus isolated from young leaves of the mangrove plant Avicennia marina in the Pearl River Estuary of Southern China.

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1. RESULTS AND DISCUSSTION          
A 220L fermentation broth was concentrated and extracted with ethyl acetate. The extract was repeatedly chromatographed on silica gel columns. avicennin-A (1) was obtained as colorless needles, mp. 197-199^oC. 1 has the molecular formula C₁₁H₉ClO₅ as determined by FABMS and elemental analysis. The isotopic peak of molecular ion showed that 1 contained chlorine atom and this was confirmed by X-ray photoelectron spectroscopy. The numbers of hydrogen and carbon atoms in the ¹H and ¹³C NMR spectra were in agreement with the molecular formula.
    The IR spectrum of 1 showed the presence of carbonyl (s) and hydroxyl group (s) with bands at 1690 cm^-1and at 3436 cm^-1. The ¹³C NMRspectra of 1 exhibited eleven carbon signals, except two methyl and a carbonyl group, the other eight signals were all unsaturated carbons (equivalent to four double bonds). The seven unsaturation equivalents required by the molecular formula indicated the compound has two rings. In the ¹HNMR spectrum, the hydrogen-bond hydroxyl proton at d11.37 showed that it was adjacent to the carbonyl group. These implied that 1 was a substituted isocoumarin. The HMBC spectrum confirmed the deduction (see Fig.1). Although no any correlation signal orienting of the hydroxyl goup (at d6.60 ) and chlorine was observed, they could be assigned by the relative up-field shift of the carbon (C-7) bearing methoxyl group. According to the empirical formula, ^[10] an adjacent OH has great effect to the chemical shift of a carbon atom to d12.7, but an adjacent Cl has nearly no effect (+ d0.2). When a chlorine atom attaches to C-6, the calculated shift is 147.4ppm. While in the case of a hydroxy at C-6, as 1, the calculated shift of C-7 is 159.9-(12.7×2) = d134.5. This is near the observed value (d133.2). Hence, the structure of avicennin A was elucidated to be 5-chloro- 6, 8-dihydroxy-7-methoxy-3-methylisocoumarin (1).

Fig.1 The HMBC correlations of 1,3 and 4

    2 and 3 were analogues of 1 and had identical ¹H, ¹³CNMR, MS and melting point data to that published for two isocoumarines from Streptomyces mobaraensis^[11].
    4 was colorless plates, mp 159-160ºC, [a]= -53.8º. It had the molecular C₁₂H₁₄O₄ as revealed by FBMS and elementary analysis. Most of spectral data for 4 were similar to those of 1, e.g. a hydroxyl proton signal at d_H11.20, ester carbonyl group signal at d_C 170.6, (IR at 1663cm^-1) and methoxyl group (d_H3.89,d_C 56.3). Considered with the unsaturation of 4 to be one less than that of 1, 4 was suggested to be a dihydroisocoumarin. The HMBC established the structure of 4 (Fig. 1). The correlations between C-3 and H-4, H-11, and between H-4 and C-9, C-10 respectively constructed the lactone ring. The correlations from C-10 to H-12 and H-6, and from H-12 to C-5 and C-6 assigned the substitution pattern in the aromatic ring.
    The steric conformation of the lactone ring in this formulation was deduced from the diaxial relationship of protons at positions 3 and 4 (J_BX=12Hz) found in the ¹HNMR of 4. Hence, the structure of avicennin B was elucidated to be 3,4-hydro-8-hydroxy-7-methoxy-3a,5-dimethyl -isocoumarin (4).
    The structure of 5 was elucidated by its spectra. It is the known compound, vermopyrone, a metabolite of Gliocladium vermoesenii. The biosynthesis study of vermopyrone showed that it was consistent with a polykitide origin with extra methyl groups introduced from methionine. ^[12-13] Although it has been well-known that 1-4 were also derived biosynthetically from an acetate unit, it seems that there was biosynthetically no direct relationship between 5 and 1-4.
    It is probably a feature that isocoumarins, the chlorine-containing isocoumarin and the dihydroisocoumarin coexisted in this endophytic fungus. Anderson and coworkers's systematic survey of the Xylariaceous fungi led to isolation of seven dihydroisocoumarins and other metabolites, but nor any isocoumarins was found from those fungi.^[11] On the other hand, Eaton et al found four isocoumarins without dihydroisocoumarins from Streptomyces mobaraensis.^[12] As our knowledge, it has not been reported that isocoumarins, chlorine-containing isocoumarin and dihydroisocoumarins were simultaneously isolated from a fungus.

2. EXPERIMENTAL 
2. 1 General
NMR data were recorded on a Varian Inova 500NB NMR spectrometer, mass spectra on a VG-ZAB-HS mass spectrometer, IR spectra on Bruker EQUINOX 55 spectrophotometer, UV spectra on a Shimadzu UV-2501PC spectrophotometer, optical rotations on a Horiba High Sensitivity Polarimeter SEPA-300, elemental analyses on a Elementar Vario EL CHNS-O elemental analyzer and on a S-520 scan electronic microscope/Link ISIS-300 X-ray photoelectron spectroscope (SEM/EDS).
2. 2 Fungal strain
A strain of the fungus (#2533), an endophytic fungus, was isolated from mangrove young avicennia leaf from the South China Sea Coast, and was stored in Department of Applied Chemistry, Zhongshan University Guangzhou, P. R. China.
2. 3 Culture conditions
Starter cultures (from Professor E. B. G. Jones and Dr. L. L. P. Vrijmoed) were maintained on cornmeal seawater agar. Plugs of agar supporting mycelial growth were cut and transferred aseptically to a 250mL Erlenmeyer flask containing 100mL liquid medium (glucose 5 g/L, peptone 1g/L, yeast extract 0.5 g/L, beef extract 0.5g/L, natural sea water 50ml/L). The flask was incubated at 30^oC on a rotary shaker for 5-7 days. The mycelium was aseptically transferred to 500ml Erlenmeyer flasks containing culture liquid (200mL). The flasks were incubated at 30^oC for 45 days.
2. 4 Extraction and separation of metabolites
The cultures (220 L) were filtered through cheesecloth. The filtrate was concentrated to 5 L below 50^oC, and extracted three times by shaking with an equal volume of ethyl acetate. The combined extracts were chromatographed on silica gel using a gradient elution from petroleum to ethyl acetate to obtain3 (55mg), 2 (20mg), 5(60mg), 4 (50mg) and 1(120 mg) in turn from the 10-20% ethyl acetate/petroleum ether fraction.
1 colorless needles: mp. 197-199^oC. IR (KBr) 3436, 3010, 2955, 2850, 1690, 1646, 1560, 1472, 1345, 1229, 1174, 1095, 795 cm^-1; UV:l_max(CHCl₃) 247 nm (e51571), 359nm (e2540). ¹H NMR (CDCl₃,TMS), ¹³C NMR (CDCl₃) and 2D NMR see Table 1. FABMS m/z 259 (M+2)⁺, 257(M)⁺. Anal. Found: C 51.18, H 4.01,Calcd. (for C₁₁H₉ Cl O₅): C 51.26, H 3.88.
2 colorless needles, mp198-200^oC. FBMS 237(M+1). ¹H NMR (CDCl₃,TMS),d2.26(CH₃ ), 3.94(CH₃ ), 3.90 (CH₃ ), 6.33(s, 1H), 6.18(s, 1H), 11.06(s, 1H). ¹³C NMR (CDCl₃) d166.4(C),160.0(C),154.7(C),154.3(C), 153.3 (C), 135.1 (C), 134.5(C), 104.8(C), 98.0(CH), 60.8(CH₃), 56.1(CH₃), 19.36(CH₃) (lit.,^[10] mp199^oC).
3 colorless needles, mp194-196^oC, FBMS 223(M+1). ¹H NMR (CDCl₃,TMS),d2.25(CH₃-11 ), 4.03(CH₃-12 ), 6.15(s, H-4), 6.39 (s, H-5), 6.46(s, OH-6), 11.31(s, OH-8). ¹³C NMR (CDCl₃)d166.6(C-1), 156.3(C-6), 153.7 (C-8), 153.2(C-3), 134.5(C-5), 132.6 (C-7), 104.2(CH-4), 101.2 (C-10), 100.3(C-9), 60.9 (CH₃-12), 19.3(CH₃-11). the HMBC correlations see Fig.1 (lit.^[10] mp 194 ^oC).
4 colorless plates, mp 159-160^oC, [a]_D²² = -53.8º. EIMS M/z 222(M)+, 207, 204, 193, 189, 176.  IR (KBr) 3328, 3266, 3050, 3014, 2981, 2894, 1663, 1596, 1476, 1385, 1262, 1110, 740 cm^-1. UV: l_max (CHCl₃) 256 nm (e5471), 340nm (e4502). ¹H NMR (CDCl₃, TMS), ¹³C NMR (CDCl₃) and 2D NMR see Table 1. Anal. Found: C65.23, H 6.62, Calcd.(for C₁₂H₁₄ O₄): C 64.87, H 6.31.
5 colorless needles, mp 138-140^oC, FBMS 183(M+1). ¹H NMR (CDCl₃,TMS),d2.01(CH₃ ), 2.55(CH₃ ), 3.96 (CH₃ ), 7.07(s, 1H). ¹³C NMR (CDCl₃) d191.6(C), 164.0 (C), 163.2(C),153.1(C), 109.7 (C), 98.0(CH), 56.7(CH₃), 25.8(CH₃), 9.4(CH₃) (lit.,^[11] mp 139-141).

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