Aspernolides L and M, new butyrolactones from the endophytic fungus Aspergillus versicolor

1 Introduction

Bioactive secondary metabolites from endophytic fungi, isolated from higher plants, are a major focus of natural product research [1], [2], [3], [4]. They have been utilized as drugs and/or lead compounds in the pharmaceutical industry [1], [2], [3], [4], [5]. The specific habitats, metabolic pathways, and bioactivities of the endophytic fungi make them a good source of structurally novel and/or biologically active secondary metabolites [5], [6]. Fungi of the genus Aspergillus (Moniliaceae) have been reported as prolific producers of bioactive compounds as xanthones, butyrolactones, anthraquinones, polyketides, and alkaloids [1], [2], [5], [6], [7], [8], [9], [10], some of them possess various biological activities [11], [12], [13], [14]. In the course of our search for secondary metabolites from endophytic fungi, we investigated the fungal strain Aspergillus versicolor isolated from the roots of Pulicaria crispa (Asteraceae), which was collected from Gabal Al-aquiq, Al Madinah Al Munawwarah, Saudi Arabia. As a result, two new butyrolactones, namely aspernolides L (2) and M (4), and four known compounds (1, 3, 5, and 6) were isolated from this strain (Figure 1). The structures of these compounds were elucidated on the basis of a comprehensive analysis of 1D and 2D NMR spectra.

Figure 1:

Structures of isolated compounds 1–6.

2 Materials and methods

3 Results and discussion

The culture of A. versicolor was extracted with EtOAc. The extract was subjected repeatedly to SiO₂, sephadex, and RP₁₈ CC to afford two new and four known compounds. Compounds 1, 3, 5, and 6 were identified by combined spectroscopic analyses as 1-O-acetylglycerol [16], butyrolactone I [17], butyrolactone VI [1], [2], and (+) alantrypinone [18], [19], respectively. The spectroscopic data for these compounds were in good agreement with those reported in the literature.

Compound 2 was obtained as a yellow gum. Its molecular formula C₂₈H₃₄O₈was determined by HRESIMS pseudo-molecular ion peak atm/z499.2329 (calculated for 499.2332 [M+H]⁺, C₂₈H₃₅O₈), indicating 12 degrees of unsaturation. The ¹H and ¹³C NMR spectral data of 2 revealed that 8 of the 12 units of unsaturation were attributed to two phenyl moieties (Figures S3 and S4). In addition, two carbonyls and two olefinic carbons account for three degrees of unsaturation. The IR spectrum showed absorption bands of hydroxyl, carbonyl group, and C–H aromatic at 3436, 1729, and 1667 cm⁻¹, respectively. The ¹³C, DEPT, and HSQC spectra of 2 revealed the presence of 28 carbon resonances: three methyls, one methoxy (δ_C 53.5, 5-OCH₃), five methylenes, an oxymethylene δ_C 65.5 (C-1″′), seven methines, and 11 quaternary carbons, including two carbonyls at δ_C 169.8 (C-5) and 168.1 (C-1), two oxygen-bonded aromatic carbons at δ_C 157.9 (C-4′) and 152.5 (C-4″), and an oxygenated aliphatic carbon (δ_C 84.7, C-4), which suggested that 2 had a butyrolactone skeleton. The ¹H NMR and ¹H-¹H COSY spectra displayed four ortho-coupled aromatic protons of a 1,4-disubstituted benzene moiety at δ_H 7.50 (d, 2H,J=8.2 Hz, H-2′,6′) and 6.87 (d, 2H,J=8.2 Hz, H-3′,5′) (Figure 2). They correlated to the carbons at δ_C 128.8 and 115.7, respectively, in the HSQC spectrum (Figure S5, Table1).Three aromatic signals at δ_H 6.43 (d, J=2.2 Hz, H-2″)/δ_C 131.4, 6.49 (d, J=8.0 Hz, H-5″)/116.0, and 6.47 (dd, J=8.0, 2.2 Hz, H-6″)/128.7 characteristic for a 1,3,4-tri-substituted benzene ring were observed. The observed HMBC cross peaks of H-2′ and H-6′/C-1′ and C-4′, H-3′ and H-5′/C-1′, C-4′, and C-6′, H-2″/C-1″, C-3″, C-4″, and C-6″, H-5″/C-1″ and C-3″, and H-6″/C-1″ and C-2″ confirmed these moieties (Figure 3). Furthermore, a side chain consisting of an oxygenated quaternary carbon (δ_C 73.9 C-9″), two methylenes at δ_H 1.59 (t, J=6.8 Hz, H-8″)/δ_C 39.6 (C-8″) and 2.31 (t, J=6.8 Hz, H-7″)/δ_C 28.1 (C-7″), and two singlet methyls at δ_H 1.23 (H-10″)/26.6 (C-10″), and 1.24 (H-11″)/26.4 (C-11″) was observed characteristic for 3-hydroxy-3-methylbutyl moiety. This was confirmed by the observed ¹H-¹H COSY and HMBC correlations (Figure 3). The HMBC correlations of H-7″/C-2″ and C-4″, H-8″/C-3″, and H-2″/C-7″ indicated its attachment at C-3″ (Figure S6). A methylene group at δ_H 3.42 and 3.38 (2H, each d, J=14.9 Hz, H-6)/δ_C 38.1 (C-6) was observed. The HMBC cross peaks of H-6/C-4, C-1″, C-2″, and C-6″ indicated that this methylene was connected to C-4 and C-1″ of the tri-substituted phenyl residue. The singlet signal at δ_H 3.76 showed HSQC cross peak to the carbon at δ_C 53.5, attributable to a methoxy group. It had HMBC cross peak to C-5 (δ_C 169.8). Furthermore, the oxygen-bearing methylene group at δ_H 4.14 (dd, J=14.5, 6.8 Hz, H-1″′A) and 3.90 (dd, J=14.5, 5.3 Hz, H-1″′B)/δ_C 65.5 (C-1″′), two multiplet methylenes at δ_H 1.62 (H-2″′)/31.9 (C-2″′) and 1.25 (H-3″′)/21.7 (C-3″′), and a triplet methyl at δ_H 0.84 (J=6.6 Hz, H-4″′)/14.0 (C-4″′), indicated the presence of a butyloxy group in 2. The HMBC cross peaks of H-1″′ to C-2 confirmed the connectivity of this group at C-2. The configuration at C-4 of 2 was deduced to be 4R based on biosynthetic considerations and its specific rotation ([α]_d +48 (c 0.05, MeOH)). It was reported that the configuration at C-4 could be assigned on the basis of the optical rotation′s sign. Butyrolactones with R-configured C-4 had positive optical rotation values such as butyrolactones I-VIII [20], [21]; however; those with negative optical rotation values possessed S-configured C-4 as in isobutyrolactone II and V [22]. Thus, the structure of2was assigned as methyl (R)-4-butoxy-2-(4-hydroxy-3-(3-hydroxy-3-methylbutyl)benzyl)-3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylate and named aspernolide L.

Figure 2:

¹H and ¹³C NMR spectra of compounds 2 and 4(DMSO-d₆, 500 and 125MHz).

Figure 3:

Some key ¹H-¹H COSY (―) and HMBC (H→C) correlations of 2and 4.

Compound 4 was also obtained as a yellow gum with a molecular formula C₂₉H₃₂O₉ determined by its HRESIMS pseudo-molecular ion peak at m/z 525.2121 [M+H]⁺ (calculated C₂₉H₃₃O₉, 525.2125), requiring 14 degrees of unsaturation. Compound 4 was 26 mass units and 2 degrees of unsaturation more than 2. The 1D and 2D NMR spectral data (Table 1) revealed that the structure of 4 was very similar to those of 2except the absence of the signals associated with the 3-hydroxy-3-methylbutyl, butyloxy, and 1,4-disubstituted benzene moieties. It had a 1,3,4-tri-substituted phenyl moiety at δ_H 6.87 (d, J=2.5 Hz, H-2′)/δ_C 128.8, 6.51 (d, J=7.5 Hz, H-5′)/115.8, and 7.49 (dd, J=7.5, 2.5 Hz, H-6′)/131.6 instead of the 1,4-disubstituted phenyl moiety in 2 (Figures S9 and S10). It was confirmed by the HMBC cross peaks of H-2′ to C-4′ and C-6′, H-5′ to C-1′, C-3′, and C-4′, and H-6′ to C-1′, C-4′, and C-5′. Its connectivity at C-3 was established by the HMBC cross peaks of H-2′ and H-6′ to C-3. Compared with 2, the two additional degrees of unsaturation, along with the observed ¹H and ¹³C NMR signals, indicating the presence of two 2,2-dimethyltetrahydro-2H-pyran-3-ol rings fused to the two tri-substituted phenyl rings in 4, which was further confirmed by the HMBC correlations of H-7′ to C-2′, C-4′, and C-9′, H-8′ to C-3′, C-10′, and C-11′, H-10′ and H-11′ to C-8′ and C-9′, H-7″ to C-2″, C-4″, and C-9″, H-8″ to C-3″, C-10″, and C-11″, and H-10″ and H-11″ to C-8″ and C-9″, establishing their attachment at C_3′-C_4′ and C_3″-C_4″, respectively (Figure S12). The configuration of the secondary alcohols at C-8′ and C-8″ was assigned as S-configured based on the comparison of the ¹H and ¹³C NMR chemical shifts as well as optical rotation value ([α]_d +85 (c 0.03, MeOH)) with those of previously reported butyrolactones [1], [2], [23], [24]. This unambiguously led to the elucidation of 4 as methyl (R)-4-hydroxy-3-((S)-3-hydroxy-2,2-dimethylchroman-6-yl)-2-(((R)-3-hydroxy-2,2-dimethylchroman-6-yl)methyl)-5-oxo-2,5-dihydrofuran-2-carboxylate and named aspernolide M.

4 Conclusions

Six compounds (1–6) were isolated and characterized from the EtOAc extract of the Aspergillus versicolor isolated from the roots of Pulicaria crispa (Asteraceae), two of them are new natural products (2and 4). Compounds 1, 5, and 6 are reported for the first time from this fungus.