Fungi has been demonstrated to be a rich source of secondary metabolites with various structural features and diverse biological activities [1, 2]. Azaphilones are a structurally variable class of fungal metabolites with highly oxygenated pyranoquinone bicyclic core from numerous fungal genera (such as Penicillium, Aspergillus, Chaetomium, Phomopsis, Emericella, Monascus) [3], which displayed a wide range of impressive biological activities such as cytotoxic, antibacterial, and antiviral activities [1, 2, 4]. The extract of fungus Penicillium sp. CPCC 400786 showed anti-HIV activities with inhibition rates of 99.9% at a concentration of 100 μg ml−1. As part of our ongoing search for bioactive natural products, the EtOAc extract of this fungi was investigated, which led to the isolation of a new azaphilone derivative, peniazaphilin A (1), along with a known isocoumarin, (R)-3-methyl-6-hydroxy-8-methoxy-3,4-dihydroisocoumarin (2) (Fig. 1). Their structures and absolute configurations were elucidated by extensive spectroscopic analyses. Compounds 1 and 2 were tested for anti-HIV activities, and displayed weak anti-HIV activities. Herein, we report the isolation, structural elucidation, and biological activities for these two compounds.

The fungus Penicillium sp. CPCC 400786 was isolated from the soil, collected in Goddess Peak, Chongqing, China, in September 2006, and classified basing on morphological and molecular (ITS1-5.8S-ITS2 rRNA gene sequence) analyses. The strain was deposited at the China Pharmaceutical Culture Collection (No.: CPCC 400786).

The fungal strain was spread onto slants of modified potato dextrose agar (PDA) medium (potato 200 g, glucose 20 g, distilled water 1 l, KH2PO4 3 g, MgSO4 0.73 g, vitamin B1 10 mg, agar 8 g, pH 6–6.3), and incubated at 28 °C for 8–10 days. The agar plugs were inoculated into 250 ml Erlenmeyer flasks containing 50 ml of PDA liquid medium at 28 °C on a rotary shaker (180 rpm) for 48 h to prepare the seed culture. Fermentation was carried out in Fernbach flasks (500 ml), each containing 80 g of rice. Distilled H2O (120 ml) was added to each flask, and the contents were soaked overnight before autoclaving at 121 °C for 30 min. After cooling to room temperature, each flask was inoculated with 8 ml of the seed culture, and incubated at 28 °C for 30 days.

The fermented material (13 flasks) was extracted repeatedly with EtOAc (4 × 2 l), and the organic solvent was evaporated to dryness under vacuum to yield the crude extract (4.1 g), which was initially subjected to silica gel (Qingdao Marine Chemical Inc. Qingdao, China) column chromatography (CC) eluting with CH2Cl2–acetone gradient (100:0–50:50) to produce seven fractions (Fr.1–Fr.7). Fraction Fr.3 (300 mg) was applied to Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) CC eluting with CHCl3–CH3OH (50:50) to give six fractions (Fr.3.1–Fr.3.6), fraction Fr.3.4 (140 mg) was further purified by reversed-phase semi-preparative HPLC eluting with CH3OH–H2O (40:60) at 4 ml min−1 to obtain 2 (28 mg, tR = 14.8 min). Fraction Fr.4 (280 mg) was subjected to Sephadex LH-20 CC eluting with CHCl3–CH3OH (50:50) to afford five fractions (Fr.4.1–Fr.4.5), fraction Fr.4.4 (50 mg) was isolated by reversed-phase semi-preparative HPLC eluting with CH3OH–H2O (55:45) at 2 ml min−1 to yield 1 (1.4 mg, tR = 7.8 min).

Peniazaphilin A (1): White powder; [α]25D –47.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε): 228 (3561), 298 (499) nm; IR (νmax): 3351, 2935, 1679, 1403, 1203, and 1138 cm−1; CD (MeOH) Δε (nm): +1.31 (218), −3.04 (251), +0.24 (323); ESIMS m/z 283.3 [M−H]; HRESIMS m/z 307.1146 [M+H]+ (calcd for C14H20O6Na, 307.1152); 1H and 13C NMR data, see Table 1.

Table 1 NMR data of peniazaphilin A (1) in DMSO-d6

Peniazaphilin B (2): Pale yellow powder; [α]25D –122.8 (c 0.5, MeOH); UV (MeOH) λmax (log ε): 214.5 (24,050), 263.5 (14,499), and 298 (7874) nm; IR (νmax): 3181, 1685, 1612, 1587, 1351, 1248, 1082, and 841 cm−1; CD (MeOH) Δε (nm): −5.97 (231), +0.47 (250.5), −4.81 (269.5); ESIMS m/z 209.1 [M+H]+; HRESIMS m/z 209.0806 [M+H]+ (calcd for C11H13O4, 209.0808); 1H NMR(600 MHz, CDCl3) δ: 6.40 (1 H, d, J = 2.4 Hz, H-7), 6.28 (1 H, d, J = 2.4 Hz, H-5), 4.50 (1 H, m, H-3), 3.84 (3 H, s, H-10), 2.81 (1 H, dd, J = 16.2, 11.4 Hz, H-4a), 2.74 (1 H, dd, J = 16.2, 3.6 Hz, H-4b), 1.43 (3 H, d, J = 0.6 Hz, H-9); 13C NMR (150 MHz, CDCl3) δ: 163.6 (C-1), 73.7 (C-3), 36.2 (C-4), 143.9 (C-4a), 106.5 (C-5), 162.5 (C-6), 98.6 (C-7), 163.5 (C-8), 105.5 (C-8a), 20.6 (C-9). 55.9 (C-10).

Compound 1 was isolated as white powder and gave a HRESIMS ion peak at m/z 307.1146 [M+Na]+, corresponding to a molecular formula of C14H20O6 with five degrees of unsaturation. The IR absorption bands at 3351 and 1679 cm–1 indicated the presence of hydroxyl and carbonyl groups. The 13C NMR and DEPT spectra displayed 14 carbon resonances (Table 1), which consisted of five quaternary carbons (δC 199.7, 146.6, 129.9, 106.8, and 76.1, including one carbonyl and two olefinic carbons), two methine carbons (δC 76.3, 72.4, two oxygenated), five methylene carbons (δC 68.3, 58.8, 34.9, 33.2, and 23.7, including two oxygenated), and two methyl carbons (δC 57.0, 18.8, including one oxygenated). The HMBC correlations (Fig. 2) of H-1/C-2, C-3, C-7, and C-9; H-13/C-3, C-4, and C-5; H-6/C-2, C-4, C-5, C-7, and C-8; H-8/C-2, C-6, C-7, C-9, and C-14 indicated the presence of an isochromenone group. Furthermore, the HMBC correlations for H-10/C-8, C-9, C-11, and C-12; H-12/C-9, C-10, and C-11 along with the 1H–1H COSY correlations of H-10/H-11/H-12, suggested the tetrahydrofuran ring. Thus, the planar structure of 1 was determined to be a new azaphilone analog.

Fig. 1
figure 1

Structures of 1 and 2

Fig. 2
figure 2

key COSY (▬) and HMBC (→) correlations of 1

The relative configuration of 1 was established by NOESY spectrum. The NOESY correlations (Fig. 3) from H-6α to H-13, from H-6β to H-5 and H-8 indicated that H-5 and H-8 were syn-orientated, and H-13 was on the opposite side. The NOESY correlations from H-8 to H-10 was observed, but the distance of H-8 and H-10 were both close no matter that the configuration of C-9 was either R or S (Figure S1). In the CD spectrum (Figure S12), the positive Cotton effect at 218 nm, and negative Cotton effect at 251 nm were nearly identical to those of pestafolide A [5], suggesting the 4R, 5S, 8R configurations of 1. The C-9 was far away from the chromophore group, and has no effect on the CD spectrum of 1, so that the absolute configuration of C-9 remains unsolved. The structure of 1 was established as peniazaphilin A.

Fig. 3
figure 3

key NOESY (↔) correlations of 1

Compound 2 was obtained as pale yellow powder. Its molecular formula of C11H12O4 with six degrees of unsaturation was established by a HRESIMS ion peak at m/z 209.0806 [M+H]+. Interpretation of 1H NMR, 13C NMR, DEPT, 1H−1H COSY, HSQC, and HMBC spectroscopic data (Fig. 3) assigned the planar structure of compound 2, which was the same to known compound, 3-methyl-6-hydroxy-8-methoxy-3,4-dihydroisocoumarin (2a) [6], the optical rotation of 2 was similar to that of 2a, indicating these two compounds were the same one. However, the CD spectrum or X-ray diffraction data of 2a was lacking, its NMR data and optical rotation were not enough for determining the absolute configuration. So, the reported stereochemistry of 2a was inaccurate. For the purpose of confirming the absolute configuration, the CD spectrum (Figure S22) was performed, and showed the negative Cotton effect at 231 and 269.5 nm, and positive Cotton effect at 250.5 nm, which was nearly identical to those of angelicoin A [7]. Therefore, the absolute configuration of 2 was assigned as the 3R configuration.

Compounds 1 and 2 were evaluated for anti-HIV activities (efavirenz as the positive control) [8]. Compounds 1 and 2 showed weak anti-HIV activities with the IC50 values of 60.4 and 69.3 μM, respectively, while the CC50 values of 1 and 2 were both more than 100 μM.

In summary, we isolated and fully characterized via extensive spectroscopic analyses a new azaphilone analog and a known isocoumarin from the fungus Penicillium species. These two compounds showed low anti-HIV activities.