Three Bianthraquinone Derivatives from the Mangrove Endophytic Fungus Alternaria sp. ZJ9-6B from the South China Sea

1. Introduction

A wide variety of anthraquinone derivatives isolated from plants, animals and marine fungi have served as candidates for various therapeutic uses [1–3]. Anthraquinones inhibit the proliferation of human breast, colon and lung cancer cells [4]. They also displayed inhibitory ability towards protein kinase, NADH oxidase, quinone reductase and calmodulin [5–8]. Several laboratories have investigated anthraquinones as antibacterial agents [9].

The alterporriol family of bianthraquinone derivatives were first reported from Alternaria porri by Suemitsu et al. in 1984 [10]. Over the last 27 years, nine additional alterporriols have been reported from fungi. All the alterporriols except alterporriols G-J were described from Alternaria sp. [5,11–13]. In terms of the underlying monomers, alterporriols can occur as either homodimers or heterodimers. With regard to the coupling positions of the monomers, alterporriols A, B, D, E, I and J feature a C-5–C-5′ linkage, alterporriol C shows a C-1–C-7′ connection, and G and H possess a C-7–C-5′ linkage [5,11–13].

As part of our ongoing program to search for new bioactive natural products from the South China Sea [14–16], an endophytic fungus Alternaria sp. ZJ9-6B has been isolated from the fruit of the marine mangrove Aegiceras corniculatum in Zhanjiang, Guangdong, China. Chemical investigation of this fungus led to the isolation of nine metabolites, including three new anthraquinone derivatives 1–3 and six known compounds 4–9 (Figure 1). It is interesting that compounds 1–3 all possess dimeric structures with a C-2–C-2′ linkage. In this report, we describe the isolation, structural elucidation and biological activity of these new metabolites.

2. Results and Discussion

The methanol extract of the dried mycelium was subjected to a combination of column chromatography on silica gel, Sephadex LH-20 and C₁₈ reversed phase silica gel.

Compound 1 was isolated as a red amorphous powder. HR-EIMS at m/z = 586.1471 [M]⁺ indicated the molecular formula C₃₂H₂₆O₁₁ (calcd. for C₃₂H₂₆O₁₁, 586.1470). Compound 1 exhibited strong optical rotation ${[\mathit{\text{α}}]}_{\mathit{\text{D}}}^{20}$ (c = 1.0, MeOH) which indicated the possibility of an asymmetric centre and/or axial chirality (Figure 2). The IR spectrum (KBr) exhibited a weak shoulder at 1652 cm⁻¹ and an intense band at 1638 cm⁻¹ for carbonyl groups. The UV spectrum displayed bands at 224, 280 and 437 nm, suggesting a quinonoid chromophore. The ¹H NMR spectrum (Table 1) showed a pair of chelated hydroxyl resonances (δ_H = 13.61 and 13.15 ppm), four aromatic protons (δ_H = 7.67, 7.55, 6.92 and 6.88 ppm), two methoxyl protons (δ_H = 3.68 and 3.66 ppm), two singlet methyls (δ_H = 2.18 and 1.07 ppm), two methylene protons (δ_H = 2.53 and 2.72 ppm, δ_H = 2.20 and 2.34 ppm), and oxygenated methine (δ_H = 3.51 ppm). The ¹³C NMR spectrum displayed four carbonyl signals (δ_C = 183.6, 187.8, 181.1 and 186.7 ppm), twenty signs of aromatic carbons, one quaternary carbon (δ_C = 69.0 ppm), one methine (δ_C = 70.1 ppm) and two methylenes (δ_C = 29.1 and 36.1 ppm). These data implied that compound 1 possessed a bianthranquinone scaffold, including an anthraquinone unit and a tetrahydroanthraquinone unit (Figure 1) [5,13]. The unsubstituted carbons for two aromatic rings of the anthraquinone unit were located at C-8′ (δ_C = 130.3 ppm; δ_H = 7.67 ppm, d, J = 0.8 Hz), C-5′ (δ_C = 110.5 ppm; δ_H = 7.55 ppm, d, J = 0.8 Hz) and C-3′ (δ_C = 103.8 ppm; δ_H = 6.921 ppm, s) by the HMBC correlations (Figure 3). In the tetrahydroanthraquinone unit, one aromatic proton at H-3 (δ_H = 6.88 ppm, s) and the protons in the alicyclic ring, including one oxygenated methine H-5 (δ_H = 3.51 ppm, ddd, J = 5.4, 5.5, 12.5 Hz) and two methylene protons H-6 (δ_H = 2.53 and 2.72 ppm) and H-7 (δ_H = 2.20 and 2.34 ppm) were observed.

A contiguous sequence of coupled signals from H-5 to H-7 in the ¹H-¹H COSY spectrum combined with the HMBC correlations from H-5 to C-6, C-7 and C-8a, from H-6 to C-5, C-8, and C-10a, and from H-7 to C-5, C-8, C-8a, C-9 and C-11, established the substructure of the cyclohexene ring (Figure 3).

HMBC correlations from each of 4-OH (δ_H = 13.15 ppm) and 4′-OH (δ_H = 13.63 ppm) to C-4, C-3, C-4a and to C-4′, C-3′, C-4′a, respectively, indicated their location at C-4 and C-4′ of the bianthraquinone scaffold.

The presence of a bond connecting C-2 and C-2′ was suggested by the absence of two sets of ortho-coupled doublets and the presence of two singlets H-3 (δ_H = 6.88 ppm, s) and H-3′ (δ_H = 6.92 ppm, s). Moreover, HMBC correlations of H-3 with C-1, C-2, C-2′, C-4, C-4a and C-10, and of H-3′ with C-1′, C-2, C-2′, C-4′, C-4′a and C-10′, respectively (Figure 3) provided evidence for C-2–C-2′ linkage of 1. The HMBC spectrum showed correlations of the methoxyl proton (δ_H = 3.66 ppm, s, 3H, H-12) with C-1 and C-3. Likewise, correlations were observed from methoxyl proton (δ_H = 3.69 ppm, s, 3H, H-12′) to C-1′. These allowed us to assign two methoxyl groups to C-1 and C-1′.

The relative configuration of the chiral centers of C-5 and C-8 were deduced by 2D ¹H-¹H NOESY experiments (Figure 3) and the analysis of ¹H-¹H coupling constants (Table 1). A NOESY correlation between CH₃-11 (δ_H = 1.07 ppm, s) and H-5 suggested that they were on the same side of the cyclohexene ring. The axial position of H-5 was confirmed by coupling constant J_H-5,H-6a = 12.5 Hz, indicating CH₃-11 to also have an axial orientation. Therefore, compound 1 was determined as (5S*,8R*)-4,4′,5,7′,8-pentahydroxy-1,1′-dimethoxy-6′,8-dimethyl-5,6,7,8-tetrahydro-[2,2′-bianthracene]-9,9′,10,10′-tetraone. We propose the trivial name alterporriol K.

Compounds 2 and 3 were obtained as a mixture after separating with Sephadex LH-20 chromatography. Upon HPLC analysis, the mixture exhibited two partially overlapped peaks (area ratio ca. 4:1). Then compounds 2 and 3 were isolated with re-separation by preparative HPLC, respectively. Compound 2 was a red amorphous powder, ${[\alpha ]}_{\text{D}}^{25}\hspace{0.17em}=\hspace{0.17em}+30$ (c = 1.0, MeOH). The HR-ESI-TOF-MS exhibited a peak at m/z = 601.1340 [M – H]⁻ indicating a molecular formula of C₃₂H₂₆O₁₂ (calcd. for C₃₂H₂₅O₁₂, 601.1346). Comparison of the ¹H and ¹³C NMR spectral data of 2 (Table 2) with that of 1 showed a close structural relationship between both compounds, except for the presence of an additional oxymethine group proton (δ_H = 4.03, d, J = 6.7 Hz, H-8) and an additional hydroxyl group signal (δ_H = 2.17 ppm, s, 6-OH) and the absence of the methylene signals corresponding to H-7 in the ¹H NMR spectrum of 2. The substructure of the cyclohexene ring was established by HMBC correlations from H-8 to C-6, C-7, C-8a, C-9, C-10a and C-11 and ¹H-¹H COSY correlations between H-5 and H-6 (Figure 3). NOE difference and analysis of ¹H-¹H coupling constants (Table 2) enabled the relative of configuration of 2 to be deduced. In the NOE experiment, when the methyl signal CH₃-11 (δ_H = 1.16, s) was irradiated, no enhancement of signals H-6 and H-8 was observed. Meanwhile, irradiation of H-8 caused an enhancement of H-6. These data suggested that CH₃-11 with H-6 and H-8 was trans-configuration in the cyclohexene ring (Figure 3). A large coupling constant for H-5a/H-6 (J_H-5a,H-6 = 10.0 Hz) indicated an axial location for H-6, likewise suggesting an axial location for H-8. Thus, the relative configuration at C-6, C-7, and C-8 was 6S*, 7R* and 8R*. Compound 2 was finally defined as (6S*,7R*,8R*)-4,4′,6,7,7′,8-hexahydroxy-1,1′-dimethoxy-6′,7-dimethyl-5,6,7,8-tetrahydro-[2,2′-bianthracene]-9,9′,10,10′-tetraone, named as alterporriol L.

Compound 3, obtained as a red amorphous power, ${[\alpha ]}_{\text{D}}^{25}\hspace{0.17em}=\hspace{0.17em}-90$ (c = 1.0, MeOH), had the molecular formula C₃₂H₂₆O₁₂, as determined by HR-ESI-TOF-MS at m/z = 601.1340 [M – H]⁻ (calcd. for C₃₂H₂₅O₁₂, 601.1346). The MS analysis revealed 3 to be an isomer of 2. The UV and IR absorptions, ¹H and ¹³C NMR data, HMBC and ¹H-¹H COSY correlations (Figure 3 and Table 2) of 3 were almost identical with those of 2, suggesting the two compounds to have identical carbon skeletons. However, in contrast with 2 in the NOE experiment of 3, irradiation of the methyl signal CH₃-11 (δ_H = 1.16 ppm, s) resulted in obvious enhancements of the signals for both H-6 and H-8. The coupling constant for H-5a/H-6 was measured to be J_H-5a,H-6 = 9.9 Hz suggesting axial locations for H-6 and, by extension, H-8. These data suggested that 2 and 3 are epimers at C-7. Therefore compound 3 was elucidated as (6S*,7S*,8R*)-4,4′,6,7,7′,8-hexahydroxy-1,1′-dimethoxy-6′,7-dimethyl-5,6,7,8-tetrahydro-[2,2′-bianthracene]-9,9′,10,10′-tetraone, named alterporriol M.

An interesting feature of the isolated bianthquinones is their optical activity. Ab initio calculations of CD spectra for several phenylanthraquinones with chiral axes have recently been reported [17,18]. Specific optical rotation of compounds 2 and 3 showed ${[\mathit{\text{α}}]}_{\mathit{\text{D}}}^{20}\hspace{0.17em}+60$ and −30, respectively. CD spectra of 2 and 3 recorded in methanol showed a near quasi-mirror image pattern (Figure 4). In the case of 2, the CD spectrum consists of a small positive band at 480 nm, followed by two moderate negative bands in the 300–400 nm regions, and a stronger negative band centered at 220 nm, then a stronger positive band at 264 nm. For 3, the sequence of bands is similar but their signs are inverted. The CD spectra of 2 and 3 reflect contributions from a chiral axis and three chiral centers. The chiral axis occurring within the chromophore is expected to dominate the observed CD spectrum [18,19] (Figure 4).The chiral center C-7 is expected to have a much smaller contribution to the observed CD spectrum. With these results, compounds 2 and 3 were deduced as two diastereomers and atropisomers [20].

The known compounds were identified as physcion (4), marcrospin (5), dactylariol (6), tetrahydroaltersolanol B (7), alternariol (AOH) (8) and alternariol methyl ether (AME) (9) by spectral analyses and comparison with reported literature data, respectively [21–24]. The structure of 7 was confirmed by X-ray diffraction analysis (Figure 5) and its crystallographic data was reported for the first time.

Compounds 1 and 2 were evaluated for their cytotoxicity against human breast cancer cell lines MDA-MB-435 and MCF-7 by MTT assay. Compound 1 had an IC₅₀ value of 26.97 μM against MDA-MB-435 and 29.11 μM against MCF-7 cells, respectively. Compound 2 showed activities against MDA-MB-435 (IC₅₀ = 13.11 μM) and MCF-7 (IC₅₀ = 20.04 μM). No biological studies were performed for compound 3 due to the limited yield.

3. Experimental Section

4. Conclusions

Alternaria sp. ZJ9-6B is a prolific producer of bioactive metabolites. Nine compounds have been isolated from this fungal strain, including three new alterporriols. These three alterporriols all possess dimeric structures with a C-2–C-2′ linkage. In the primary bioassay, compounds 1 and 2 showed moderate cytotoxic activity against human breast cancer cell lines.

Supplementary Data