A novel C₂₅ sterol peroxide from the endophytic fungus Phoma sp. EA-122

1 Introduction

Endophytic fungi are microorganisms that live in the internal tissues of their host without causing any apparent disease symptoms [1]. They have been proven to be excellent sources of bioactive natural products [2]. The genus Phoma comprises filamentous fungi that inhabit the soil and plant materials, while about 140 species have been defined and recognized. Previous studies within this genus led to the isolation of a variety of novel structures and compounds with interesting bioactivities. Notable examples include epoxyphomalin A and B, prenylated polyketides with potent cytotoxicity [3], while TMC-264 is a novel antiallergic heptaketide [4], and fusidienol A inhibits ras farnesyl-protein transferase [5]. Our investigation of the endophytic fungus Phoma sp. EA-122, which was isolated from the leaves of Eupatorium adenophorum, an invasive plant in Yunnan province, P. R. China, has resulted in the isolation of a novel C₂₅ sterol peroxide, phomasterol A (1), along with two known compounds 3β-hydroxy-(22E, 24R)-ergosta-5,8,22-trien-7-one (2) and (22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol (3) (Figure 1). Herein, we report the isolation and structural elucidation of the new compound, as well as its biological activity.

Figure 1:

Structures of compounds 1–3.

2 Results and discussion

Phomasterol A (1), exhibited a pseudomolecular ion peak (m/z 423.2503 [M+Na]⁺, calcd 423.2511), corresponding to the molecular formula C₂₅H₃₆O₄, with eight degrees of unsaturation. Its IR data indicated the presence of a hydroxy group (3441 cm^–1) and an α, β-unsaturated keto group (1670, 1626 cm^–1). The ¹H NMR spectrum of compound 1 displayed three singlets for methyls at δ_H 0.89, 0.91, and 2.24 and a doublet for a methyl at δ_H 1.12 (3H, d, J=6.6 Hz), four olefinic proton signals at δ_H 6.02 (1H, d, J=15.9 Hz), 6.28 (1H, d, J=8.5 Hz), 6.54 (1H, d, J=8.5 Hz) and 6.77 (1H, dd, J=15.9, 9.1 Hz). The ¹³C and DEPT spectra revealed 25 carbon signals, including four methyls, seven methylenes, nine methines (with four olefinic ones) and five quarternary carbons (with one carbonyl and two oxyquarternary carbons). In addition, an α,β-unsaturated ketone moiety was assumed by the diagnostic NMR signals at δ_C 130.0, 155.8, and 201.9, and the chemical shift values of two oxygenated quaternary carbons at δ_C 83.4 and 80.6 indicated the presence of an epidioxy group. These observations, in combination with the molecular formula, indicated the presence of an epidioxy group and five rings in 1.

Structural elucidation of 1 was based on COSY and HMBC data (Figure 2). The hydroxy group attached at C-3 was designated by the HMBC correlations from H-3 (δ_H 3.78 ppm, m) to C-1 and C-5. The angular tcorrelations from Me-18 (δ_H 0.89 ppm, s) to C-12, C-13, C-14, and C-17, and Me-19 (δ_H 0.91 ppm, s) to C-1, C-5, C-9, and C-10. The above data suggested an epoxyergostan scaffold similar to that of (22E,24R)-5α,8α-epidioxy-ergosta-6,22-dien-3β-ol (4) [6]. The key differences between the two compounds were that 1 had three carbon atoms <4, and the signals at δ_H 0.81, 0.82 (each 3H, d, J=6.8 Hz), 1.47 (1H, dq, J=13.5, 6.8 Hz), and at δ_C 19.7, 20.0, and 33.1 for the isopropyl group were absent in 1. Instead, the corresponding resonances of an acetyl moiety (δ_H 2.24, δ_C 26.7, and 201.9) were observed, suggesting an isopropyl moiety was cleaved from the side chain of 4 and the resulting compound was oxygenated at C₂₄ to yield 1, which was further supported by significant HMBC correlations from Me-25 (δ_H 2.24 ppm, s) to C-23 and C-24.

Figure 2:

Selected 2D NMR correlations of 1.

The relative configuration of 1 was established by a combination of coupling constant and ROESY experiment. In the ¹H NMR spectrum, The E-geometry of the Δ²²-double bond was deduced from the large coupling constant observed for H-22 and H-23 (J=15.9 Hz), whereas, the Z-geometry of the Δ⁶-double bond was deduced from the coupling constants observed for H-6 and H-7 (J=8.5 Hz). In the ROESY spectrum (Figure 2), H-3 showed a correlation with H-4α, while H-4β and H-12β showed correlations with 19-Me, indicating that H-3 was α-oriented, while Me-19 was in the opposite orientation (β). The ROE correlations of Me-18/H-12β, H-20 suggested that Me-18 and H-20 were β-oriented. The ROE correlations of Me-21/H-12α, H-17 and H-17/H-14, H-9 indicated that Me-21, H-17, H-14 and H-9 were α-oriented. Consequently, the structure of 1 was established and named phomasterol A.

The known compounds were identified as 3β-hydroxy-(22E, 24R)-ergosta-5,8,22-trien-7-one (2) [7] and (22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol (3) [8] by comparison with literature data.

There is a structural similarity of 1 to ursolic acid, a triterpene with significant inhibitory activity against protein-tyrosine phosphatases (PTPs). Members of the PTP family are important components of cell signal transduction chains that are involved in the regulation of cell function. Malfunctions of PTPs have been related to human diseases such as cancer, diabetes and immune disorders. As such, PTPs have become a key pharmaceutical target in the treatment of diseases [9]. Phomasterol A (1) was evaluated as an inhibitor of two PTPs: MEG2 and PTP1Bc and was found to exhibit moderate inhibitory activities with identical IC₅₀ values of 25 μM, while the IC₅₀ values of the positive control (ursolic acid) were 1.75 and 2.63 μM, respectively.

3 Experimental