Cytochalasin P₁, a new cytochalasin from the marine-derived fungus Xylaria sp. SOF11

1 Introduction

Marine-derived fungus has been recognized as a productive and important source of novel natural compounds that could potentially be used or modified as anticancer, antibacterial, and antiviral agents. Up to date, more than 1000 structurally interesting compounds have been isolated and characterized from marine fungi [1]. Among these compounds, a promising example is halimide, which demonstrated potent cytotoxic activities against human cancer cells [2]. A derivative of halimide, plinabulin (NPI-2358), is undergoing clinical trials as potential anticancer drug [3].

We focused our efforts on the investigation of bioactive secondary metabolites from the South China Sea-derived fungi with the intention of developing drug candidates. In the past few years, we have reported new cytochalasins from Xylaria sp. SCSIO 156, cytotoxic cycloheptapeptides from Acremonium persicinum SCSIO 115, immunosuppressive mycophenolic acid derivatives from Penicillium sp. SOF07 and halogenated anthraquinones from Aspergillus sp. SCSIO F063 [4], [5], [6], [7]. Recently, a marine-derived fungus strain Xylaria sp. SOF11 attracted our attention because that the crude extract of Xylaria sp. SOF11 was lethal to brine shrimp (Artemia salina) and displayed cytotoxicity against three human tumor cell lines (A549, HCT15, and HEP3B). Chemical investigation of the fermentation products of Xylaria sp. SOF11 has resulted in the isolation of a new cytochalasin, named cytochalasin P₁ (1), as well as four known analogs, cytochalasin P (2) [8], 19, 20-epoxycytochalasin N (3), 19, 20-epoxycytochalasin D (4) [9], and zygosporin G (5) [10] (Figure 1). Herein, we report the isolation, structure elucidation, and cytotoxicity of the new compound.

Figure 1:

Structures of compounds 1–5.

2 Results and discussion

Compound 1 was obtained as a white powder. Its molecular formula was determined to be C₃₀H₃₉NO₈ based on the quasimolecular ion peak at m/z 540.2612 [M–H]⁻ (calculated 540.2603) in HR-ESI-MS spectrum, indicating 12 degrees of unsaturation. The ¹H NMR spectrum of 1 displayed five methyl group proton signals at δ_H 0.91 (d, J=6.5 Hz, H-11), 1.13 (d,J=7.0 Hz, H-22), 1.17 (s, H-12), 1.49 (s, H-23), 2.09 (s, acetyl), 2 olefinic proton signals at δ_H 5.63 (dd, J=15.8, 10.5 Hz, H-13), 5.60 (m, H-14), 2 pair of ortho-coupled aromatic proton signals at δ_H 7.13 (2H, d, J=7.0 Hz, H-2′, 6′), 7.29 (2H, d, J=7.0 Hz, H-3′, 5′), and several aliphatic proton signals between δ_H 1.89–3.55 (Table 1). Analysis of the ¹³C NMR and HSQC data for 1 revealed 3 carbonyls (C-1, C-17, and acetyl), 4 quaternary carbons, including 2 oxygen-bearing quaternary carbons (C-6 and C-18), 16 methines, including 3 oxymethines (C-7, C-19, and C-20) and 7 aromatic methines (C-2′, C-3′, C-4′, C-5′, C-6′, C-13, and C-14), 2 methylenes (C-10 and C-15) and five methyls (C-11, C-12, C-22, C-23, and acetyl).

The ¹H and ¹³C NMR data of 1 were similar to those of the known cytochalasin P (2), except that the signals of the double bond at δ_H 5.10 (1H, dd, J=16.2, 2.0 Hz, H-19), 6.08 (1H, dd, J=15.5, 3.0 Hz, H-20) and at δ_C 128.8 (C-19), 135.0 (C-20) in 2 were absent in 1. Instead, two oxymethine signals were observed at δ_H 3.48 (1H, br s, H-19) and δ_C 54.3 (C-19), 3.09 (1H, d, J=1.5 Hz, H-20), and δ_C 59.1 (C-20), suggesting the double bond at C-19 and C-20 in 2 was replaced by an epoxide bond in 1. In the HMBC spectrum (Figure 2), the correlations of H-21/C-19, C-20, and H-23/C-19 substantiated the above conclusion.

Figure 2:

Key HMBC and NOESY correlations for compound 1.

The relative configuration of 1 was established by a combination of coupling constant and NOESY experiment. In the ¹H NMR spectrum, the large coupling constant (J=12.0 Hz) for H-7 and H-8 suggested the trans orientation of the proton pair. The E-geometry of the Δ¹³-double bond was deduced from the large coupling constant observed for H-13 and H-14 (J=15.8 Hz). In the NOESY spectrum (Figure 2), the NOE correlation of H-3/H₃-11 and H₃-12, H₃-12/H-7, H-4/ H-5 and H-8 indicated that H-3, H₃-11, H-7, and H₃-12 were in the same orientation (α); while H-4, H-5 and H-8 were in the opposite direction (β). The NOE correlation of H-7/H-13, H-14/H-8 and H-15b, H-15a/H-22, H-16/H-23 indicated that H-13 and H-22 were α-oriented; H-16 and H-23 were β-oriented. The NOE correlation of H-19/H-13 and H-21, H-13/H-20 indicated that H-19, H-20, and H-21 were α-oriented. Thus, compound 1 was established to be a new cytochalasin, and named cytochalasin P₁.

Compounds 1–5 were biologically evaluated for in vitro cytotoxicity against four human tumor cell lines (SF-268, MCF-7, NCI-H460, and HepG-2) by using the SRB method with cisplatin as the positive control. Compounds 1–5 showed cytotoxic activities against SF-268 and MCF-7 cell lines, with the IC₅₀ values varying between 0.33 and 4.17 μM, as listed in Table 2. However, Compounds 1–5 exhibited no cytotoxic activities against NCI-H460 and HepG-2 cell lines, those tentative results revealed that the tested compounds had excellent selective cytotoxic activities.

3 Experimental

4 Cytotoxicity assay

The cell growth inhibitory activities of compounds 1–5 against the human tumor cell lines (SF-268, MCF-7, NCI-H460, and HepG-2) were determined using the SRB method [11]. Briefly, cells (180 μL) with a density of 3×10⁴ cells/mL of media were seeded onto 96-well plates and incubated for 24 h at 37 °C, 5% CO₂. Various concentrations of compounds (20 μL) were added to the plate wells, and plates were further incubated for 72 h. After incubation, cell monolayers were fixed with 50% (w/v) trichloroacetic acid (50 μL) and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by washing repeatedly with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution (200 μL) for OD determination at 570 nm using a microplate reader. Cisplatin was used as a positive control. All data were obtained in triplicate and are presented as means±S.D. IC₅₀ values were calculated with the Sigma Plot 10.0 software using a nonlinear curve-fitting method.