Two New Anisic Acid Derivatives from Endophytic Fungus Rhizopycnis vagum Nitaf22 and Their Antibacterial Activity

Rhizopycnis acids A (1) and B (2), two new anisic acid derivatives, were obtained from the ethyl acetate extract of the fermentation cultures of Rhizopycnis vagum, an endophytic fungus isolated from the healthy tissues of Nicotiana tabacum. The structures of the two compounds were determined through a series of 1D and 2D NMR and HRMS spectral analyses. Both compounds were the first anisic acid derivatives containing methylbutanoic/methylbutenoic acid group found in fungi. 1 and 2 displayed antibacterial activity against six tested bacteria with IC50 values in the range 16.1~81.3 μg/mL.

Chemical and biological investigation for the endophytic fungus Rhizopycnis vagum Nitaf22 from N. tabacum, have led to the isolation of bioactive dibenzo-α-pyrone derivatives [7]. In our ongoing search for new bioactive metabolites from this fungus, two new anisic acid derivatives containing methylbutanoic or methylbutenoic acid group (Figure 1) were isolated. They were screened for antibacterial activities. Herein, we reported the isolation, structural elucidation, and antibacterial activity of these two compounds.
Chemical and biological investigation for the endophytic fungus Rhizopycnis vagum Nitaf22 from N. tabacum, have led to the isolation of bioactive dibenzo-α-pyrone derivatives [7]. In our ongoing search for new bioactive metabolites from this fungus, two new anisic acid derivatives containing methylbutanoic or methylbutenoic acid group (Figure 1) were isolated. They were screened for antibacterial activities. Herein, we reported the isolation, structural elucidation, and antibacterial activity of these two compounds.

Structural Elucidation of the Compounds
Compounds 1 and 2 ( Figure 1) were purified from EtOAc extract of R. vagum fermentation cultures through vacuum liquid chromatography, MPLC over silica gel, Sephadex LH-20, and reverse-phase semi-preparative HPLC.
Compound 1 was obtained as a light yellow amorphous powder. It had the molecular formula C 13 H 14 O 5 (seven degrees of unsaturation), based on analysis of its HRESIMS at m/z 249.0771 [M − H] − (calcd for C 13 H 13 O 5 , 249.0768) ( Figure S1). The IR spectrum showed absorption for hydroxyl at 3500 cm −1 , intense and sharp absorption for carbonyl at 1681 cm −1 , and absorptions for phenyl at 1427, 1503 and 1604 cm −1 ( Figure S2). The 1 H NMR spectrum (Table 1) displayed the signals of 1,3,4-trisubstituted aromatic system at δ H 7.93 (d, J = 8.6 Hz), 7.80 (s) and 7.03 (d, J = 8.6 Hz), an olefinic signal at δ H 6.85 (t, J = 7.5 Hz), a methoxy signal at δ H 3.92, a methene signal at δ H 3.53 (d, J = 7.5 Hz), and a singlet methyl single at δ H 1.93 ( Figure S3). Corresponding to 1 H NMR spectrum, the 13 C NMR spectrum (Table 1) Figure S4). The placement of the above groups was confirmed by analysis of HMBC spectrum ( Figure S5). The correlations from H-2 to C-1, C-4, and C-6; from H-5 to C-3 and C-4; and from H-6 to C-1 and C-4, verified the presence of 1,3,4-trisubstituted aromatic system. Furthermore, a carboxyl group was attached to C-1 by the correlations from H-2/H-6 to C-7. Based on the chemical shift of C-4 (δ C 162.7) and 4-OCH 3 (δ H , C 3.92, 56.3), and NOESY correlation between 4-OCH 3 and H-5, the methoxy was linked to C-4 ( Figure S6). These correlations established a 3-substituted anisic acid skeleton of 1. The HMBC correlations from H 2 -1 to C-2 and C-3 and from H 3 -5 to C-2 , C-3 and C-4 , suggested a methylbutenoic acid substituent. The HMBC correlations from H 2 -1 to C-2, C-3 and C-4 confirmed this substituent being located at C-3 of the aromatic ring ( Figure 2). The planar structure of 1 was then determined as shown in Figure 1. The E configuration of the double bond in the side chain was concluded from the NOESY correlation between H 3 -5 and H 2 -1 . Therefore, the structure of 1 was elucidated as (E)-3-(3-carboxybut-2-en-1-yl)-4-methoxybenzoic acid, which was named as rhizopycnis acid A.  Compound 2 was isolated as a yellow amorphous powder, and has the molecular formula C13H16O5 (six degrees of unsaturation), as deduced from the HRESIMS at m/z 251.0930 [M − H] − (calcd for C13H15O5, 251.0925) ( Figure S7). The IR spectrum showed similar absorptions to those attributed to the hydroxyl (3500 cm −1 ), carbonyl (1681 cm −1 ), and phenyl (1443, 1505, 1606 cm −1 ) groups found in 1 ( Figure S8). The NMR spectral data of 2 are also similar to those of 1, but differed in the side chain ( Figures S9 and S10). The signals for the double bond in 1 were replaced by one methylene (δC, 34.9) and methine (δC, 40.5) in 2. In addition, the methyl group was shifted upfield and appeared as a doublet at δH 1.18 (d, J = 7.0 Hz). These suggested 2 was a C2′/C3′ saturated derivative of 1. This was corroborated by analysis of the HMBC spectrum, in which correlations were observed from H2-1′ to C-2′ and C-3′ and from H3-5′ to C-2′, C-3′ and C-4′ (Figures 2 and S11). Thus, the planar structure of 2 was determined as shown in Figure 1 [11]. Therefore, the structure of 2 was elucidated as (S)-3-(3-carboxybutyl)-4-methoxybenzoic acid, which was named as rhizopycnis acid B.
The structures of 1 and 2 incorporated methylbutanoic/methylbutenoic acid and anisic acid moieties. A similar compound, 3-(3-methylbut-2-en-1-yl)-4-methoxybenzoic acid, was previously isolated from the stems of a perennial plant Wigandia urens (Hydrophyllaceae) [12]. The differences were ascribed to a different side chain locating at C-3 (Figure 1). To the best of our knowledge, these three compounds were the only anisic acid derivatives containing an isopentyl group in natural products. The anisic acids with their structures containing methylbutanoic or methylbutenoic acid group (1 and 2) represent a new class of natural products found in fungi.

Antibacterial Activity
Both compounds were tested for their antibacterial activity against six pathogenic bacteria. Compounds 1 and 2 were active against all the tested bacteria (Table 2), and 1 showed stronger inhibition. This suggested that the C-2′/3′ olefinic bond positively correlated to the antibacterial activity. To our knowledge, this is the first report of antibacterial activity for this type of natural product. Though these two compounds are not as active as the positive control streptomycin sulfate, they show potential as the antibacterial agents. The similar compound 3-(3-methylbut-2-en-1-yl)-4-methoxybenzoic acid was reported to exhibit anti-HIV activity against CCR5 receptor with IC50 value of 26 μM [12]. Therefore, other biological activities, including the anti-HIV activity of compounds 1 and 2, are necessary to study.  Figure S7). The IR spectrum showed similar absorptions to those attributed to the hydroxyl (3500 cm −1 ), carbonyl (1681 cm −1 ), and phenyl (1443, 1505, 1606 cm −1 ) groups found in 1 ( Figure S8). The NMR spectral data of 2 are also similar to those of 1, but differed in the side chain ( Figures S9 and S10). The signals for the double bond in 1 were replaced by one methylene (δ C , 34.9) and methine (δ C , 40.5) in 2. In addition, the methyl group was shifted upfield and appeared as a doublet at δ H 1.18 (d, J = 7.0 Hz). These suggested 2 was a C2 /C3 saturated derivative of 1. This was corroborated by analysis of the HMBC spectrum, in which correlations were observed from H 2 -1 to C-2 and C-3 and from H 3 -5 to C-2 , C-3 and C-4 ( Figure 2 and Figure S11). Thus, the planar structure of 2 was determined as shown in Figure 1 [11]. Therefore, the structure of 2 was elucidated as (S)-3-(3-carboxybutyl)-4-methoxybenzoic acid, which was named as rhizopycnis acid B.
The structures of 1 and 2 incorporated methylbutanoic/methylbutenoic acid and anisic acid moieties. A similar compound, 3-(3-methylbut-2-en-1-yl)-4-methoxybenzoic acid, was previously isolated from the stems of a perennial plant Wigandia urens (Hydrophyllaceae) [12]. The differences were ascribed to a different side chain locating at C-3 ( Figure 1). To the best of our knowledge, these three compounds were the only anisic acid derivatives containing an isopentyl group in natural products. The anisic acids with their structures containing methylbutanoic or methylbutenoic acid group (1 and 2) represent a new class of natural products found in fungi.

Antibacterial Activity
Both compounds were tested for their antibacterial activity against six pathogenic bacteria. Compounds 1 and 2 were active against all the tested bacteria (Table 2), and 1 showed stronger inhibition. This suggested that the C-2 /3 olefinic bond positively correlated to the antibacterial activity. To our knowledge, this is the first report of antibacterial activity for this type of natural product. Though these two compounds are not as active as the positive control streptomycin sulfate, they show potential as the antibacterial agents. The similar compound 3-(3-methylbut-2-en-1-yl)-4-methoxybenzoic acid was reported to exhibit anti-HIV activity against CCR5 receptor with IC 50 value of 26 µM [12]. Therefore, other biological activities, including the anti-HIV activity of compounds 1 and 2, are necessary to study.