Marine-derived fungi have recently become a research focus as one of the richest sources of new and bioactive secondary metabolites in the marine environment [1,2]. The mangrove plant Scyphiphora hydrophyllacea Gaertn. F. (Rubiaceae) growing in tropical and subtropical intertidal habitats is a rich source of new iridoids, which have been found to possess cytotoxic activity [3,4,5,6]. The metabolites produced by endophytic fungi from S. hydrophyllacea collected in Hainan attracted our attention [7], a new fatty acid glycoside and two new meroterpenes have been found from the fungus A1 of S. hydrophyllacea [8,9]. Further phytochemical investigation of the secondary metabolites from the culture broth of the fungus A1 led to the isolation of four new meroterpenes, guignardones F–I (1–4) (Figure 1), and two known compounds, guignardones A (5) and B (6) [10]. This paper reports the isolation and structural elucidation, as well as biological activities and plausible biogenetic pathway of these compounds.

Guignardone F (1) was isolated as colorless needle crystals, and possessed a molecular formula of C₁₇H₂₄O₅ as established by HREIMS (m/z 308.1625 [M]⁺), indicating six degrees of unsaturation. The IR spectrum displayed hydroxyl (3463 cm⁻¹), conjugated carbonyl (1657 cm⁻¹) and double bond (1606 cm⁻¹) absorptions. In the ¹³C NMR and DEPT spectra (Table 1), a total of 17 carbon resonances were found and classified into three methyls, five methylenes (one oxygenated), three methines (one oxygenated), and six quaternary carbons (one α,β-unsaturated carbonyl unit, three oxygenated). In the HMBC spectrum (Figure 2), the correlations from H₂-8 (δ_H 3.03, 1.92) to C-1 (δ_C 200.2), C-2 (δ_C 114.7), and C-3 (δ_C 173.9), from H-4 (δ_H 4.46) to C-2 and C-6 (δ_C 81.8), from H₂-5 (δ_H 2.31, 1.91) to C-1 and C-3, and from H₂-7 (δ_H 3.80, 3.45) to C-4 (δ_C 81.3), C-5 (δ_C 42.5), and C-6 were found and led to the establishment of partial structure 1a, which was identical to that of guignardone B (6) [10]. In the ¹H-¹H COSY spectrum, a proton spin system (H-8/H-9/H-14/H-13/H-12) was found as drawn with bond lines in Figure 2. In addition, the HMBC cross-peaks from H₃-11 (δ_H 1.35) to C-9 (δ_C 46.1), C-10 (δ_C 80.5), and C-12 (δ_C 39.3), and from both H₃-16 (δ_H 1.17) and H₃-17 (δ_H 1.45) to C-14 (δ_C 54.1) and C-15 (δ_C 86.9) determined the establishment of substructure 1b. Careful comparison of the ¹³C NMR data of 1 with those of 6, the upfield shift of C-10 (Δ −10.2 ppm) and the downfield shift of C-15 (Δ +14.0 ppm) were observed. So it was proposed that C-3 connected with C-15 via an ether bond to form a seven-membered ring in 1, instead of the C-3 connected with C-10 via an ether bond to form a six-membered ring in 6. This also gave a good explanation for the obvious difference (Δ 10.8 ppm) of chemical shifts between the 16-CH₃ and the 17-CH₃, since their stereochemistry circumstance was quite different in the structure of 1. Thus, the gross structure of 1 was elucidated and further confirmed by X-ray crystallography which also established its relative configuration (Chart 1). Therefore, the structure of 1 was established as shown in Figure 1, named Guignardone F (1).

Guignardone G (2) was obtained as a colorless oil. Its molecular formula was determined as C₁₆H₂₂O₄ from [M]⁺ ion peak at m/z 278.1513 in the HREIMS, with six degrees of unsaturation. IR absorptions implied the presence of hydroxyl (3440 cm⁻¹), conjugated carbonyl (1653 cm⁻¹) and double bond (1622 cm⁻¹) groups. The ¹³C NMR and DEPT spectra (Table 1) revealed 16 carbon signals, including one α,β-unsaturated carbonyl group, one terminal double bond, one oxygenated quaternary carbon, four methines (two oxygenated), four methylenes, and two methyls. Apart from one carbonyl and two double bonds, the remaining degrees of unsaturation indicated that 2 possessed a tricyclic system. Detailed analysis of its ¹H NMR and ¹³C NMR spectra showed that compound 2 was analogous to guignardone A (5) [10], except for the absence of a methylene (C-7), a quaternary carbon (C-6) in 5 and the presence of a methine (C-6) in 2. In addition, the ¹³C NMR signals of C-4 upshifted from δ_C 78.5 to δ_C 66.7 and C-5 from δ_C 44.0 to δ_C 36.9. Base on the above evidence, it was supposed that the C-7 of 5 was degraded and the corresponding ring was broken to form the structure of 2, which was confirmed by 2D NMR experiments. The relative configuration of 2 was assigned by a ROESY experiment and comparison with 5. H-9 (δ_H 1.95) showed correlations to both H₃-11 (δ_H 1.32) and H₂-16 (δ_H 4.64), which indicated that when H-9 and CH₃-11 possess α-orientation, H-14 (δ_H 2.27) will possess β-orientation, as in accordance with the relative configuration of 5 (Figure 3). The α-orientation of H-4 (δ_H 4.35) was also tentatively established as same as that of 5 for the biogenetic pathway consideration. H-6 (δ_H 4.48, dd, J = 11.9, 5.4 Hz) exhibited ROESY correlation with H-4, and the large vicinal coupling constant (J = 11.9 Hz) established the pseudoaxial position for H-6. So H-6 was established as α-oriented. Thus, the structure of 2 was established and named guignardone G.

Guignardone H (3) was isolated as a colorless oil. The HRTOFMS of 3 showed a pseudo-molecular ion peak at m/z 293.1745 [M + H]⁺ corresponding to the molecular formula C₁₇H₂₄O₄, implying six degrees of unsaturation. The ¹H NMR and ¹³C NMR spectra of 3 (Table 1 and Table 2) were strikingly similar to those of 2, with the only obvious difference being the presence of a methoxy (δ_H 3.47, 3H, s; δ_C 58.3) group in 3. The HMBC correlations of the methoxy protons to C-6 (δ_C 79.1) and from H-6 (δ_H 3.70) to the methoxy carbon indicated that the methoxy group located at C-6. β-orientations of OH-4 and H-14 and α-orientations of H-9 and CH₃-11 were established by ROESY correlations and comparison with 2 and proved to be the same as 2. There was no ROESY correlation between H-6 (δ_H 3.70, dd, J = 6.8, 3.6 Hz) and H-4 (δ_H 4.24), and the proton coupling constants between H-6 and H₂-5 (δ_H 2.35, 2.23) were 6.8 and 3.6 Hz, differed from those of H-6 in 2. Hence, H-6 was deduced to be β-oriented. Thus, the structure of 3 was established as shown (Figure 1) and named guignardone H.

Guignardone I (4) was obtained as a colorless oil. The molecular formula was deduced as C₁₇H₂₆O₅ on the basis of its HREIMS ion peak at m/z 310.1783 [M]⁺ with five degrees of unsaturation. The ¹H NMR and ¹³C NMR data (Table 1 and Table 2) of 4 closely resembled those of 3, except for the disappearance of an exocyclic double bond and the presence of an oxygenated quarternary carbon (C-15, δ_C 72.9) and a methyl group (C-16, δ_C 28.6) in 4. Therefore, it was supposed that compound 4 may derived from 3 by hydroxylation at the exocyclic double bond, which was confirmed by the HMBC correlations of H₃-16 (δ_H 1.20) with C-14 (δ_C 51.1), C-15 (δ_C 72.9), and C-17 (δ_C 27.5). The ROESY correlations and chemical shifts at chiral centers of 4 were similar to those of 3, which showed the same relative configurations at C-4, C-6, C-9, C-10, and C-14.

A possible biogenetic pathway of compounds 1–6 was proposed as shown in Scheme 1. Guignardone A (5), a major product isolated in this experiment, was taken as the biogenetic precursor. Two ether bonds were suitable for the enzymatic opening that resulted in a 3,10-dihydroxy intermediate (i) and a 4,7-dihydroxy intermediate (ii). The five-membered ring and the connected isopropanol group of intermediate i could turn upside-down around the bond C-8–C-9 to generate 1. The intermediate ii underwent an oxidation and decarboxylation to give an intermediate (iii). Afterward, the intermediate (iii) converted to its C-1 ketone isomer. This assumption reasonably accounted for the simultaneous occurrences of β-orientation of OH-6 in 2 and α-orientation of OCH₃ in 3.

All compounds were tested for antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus. Guignardone I (4) exhibited inhibition zones of 9.0 and 11.0 mm in diameter (the diameter of sterile filter paper discs was 6 mm) toward MRSA and S. aureus at 65 µM, respectively. Guignardone B (6) gave an inhibition zone of 8.0 mm against MRSA at 65 µM. The other compounds have not show any antibacterial action under the same conditions. 10 µL 0.08 mg mL⁻¹ kanamycin sulfate was used as positive control, for which the diameter of the inhibition zone was 30 mm.

Four new meroterpenes, guignardones F–I (1–4), together with two known compounds guignardones A (5) and B (6), were isolated from the endophytic fungus A1 Guignardia sp. of mangrove plant Scyphiphora hydrophyllacea. The plane structure of guignardone G was same as coibanol A, one of the three tricyclic meroterpenes isolated from an endophytic fungus Pycnoporus sanguineus. The H-9 and CH₃-11 possess α-orientations in compounds 1–6, different from the β-orientation in coibanols A–C [12]. The plane structure of guignardone H was same as tricycloalternarene F, another tricyclic meroterpene isolated from an endophytic fungus Guignarda mangiferae, the stereochemistry of which has not been clarified [13]. Guignardones A (5) and B (6) with an additional tetrahydrofuran ring in their structure have been isolated from a fungus Guignardia mangiferae associated with normal Ilex cornuta leaves [10]. Especially, compound 1 possessed a novel structure, beside an additional tetrahydrofuran ring, the six-membered ring possessing an oxygen atom was changed to a seven-membered ring. Antibacterial tests demonstrated that guignardone I (4) showed modest inhibitory effects on Staphylococcus aureus and MRSA, diameters of inhibition zones of which were 9.0 and 11.0 mm, respectively. Guignardone B (6) exhibited weak antibacterial activity against MRSA with a diameter of inhibition zones of 8.0 mm.