The genus Penicillium comprises >300 species,1 which produce a variety of novel bioactive compounds. Well-known drugs from this genus are penicillin antibiotics produced by P. chrysogenum2 and the antifungal metabolite griseofulvin produced by P. griseofulvum3 and P. patulum.4 As a part of an ongoing program aimed at exploring antibacterial natural products produced by the fungi obtained from South China,5, 6, 7 we have investigated the bioactive compounds of the fungus P. purpurogenum SC0070 isolated from a soil sample collected under a palm grove. We recently reported that cultures of P. purpurogenum SC0070 produced a rearranged sterol with anti-inflammatory and cytotoxic properties, penicillitone, and a new related sterol, penicillisterol.8 In this paper, continuing chemical investigation of the fungus has resulted in the isolation of two new anthraquinones, penipurdin A (1) and B (2), together with a known analog, questin (3). Details of the isolation, structure elucidation and biological activities of the compounds are presented here.

Solid-substrate fermentation cultures of Aspergillus versicolor SC0070 grown on wheat grains were extracted with 95% EtOH, and the resultant extract was sequentially partitioned with petroleum ether, CHCl3, EtOAc and n-BuOH. The CHCl3 and EtOAc extract were fractionated by silica gel column chromatography, followed by Sephadex LH-20 and/or reversed-phase HPLC, to afford compounds 1–3 (Figure 1).

Figure 1
figure 1

Structures of compounds 1–3 isolated from P. purpurogenum SC0070.

Penipurdin A (1) was obtained as an amorphous organge-red solid ([α]20D+33.3, c 1.14, MeOH). Its molecular formula was determined as C18H16O6 from HRESIMS (found m/z 329.1022 [M+H]+, calcd 329.1020) and NMR data. The 1H and 13C NMR spectra of 1 (Table 1) revealed the presence of two methyl groups including one O-methyl, one methylene, one O-methine, 12 aromatic carbons (four of which were protonated) and two conjugated ketone carbonyl groups (δC 182.6 and 186.6). These spectroscopic features suggested that 1 has the same anthraquinone skeleton as found in questin (3)9 that was isolated from the crude extract of the same fungus. The substituents and their location on the anthraquinone ring were established by the analysis of the 1H–1H COSY, HMBC and NOESY spectra of 1 (Figure 2a). The chelated hydroxyl group at δH 13.22 exhibited HMBC correlations to C-1, C-2 and C-9a, establishing the location of this hydroxyl group at C-1. 1H–1H COSY connectivities of H2-1' to H-2' (δH 3.88) and H-2' to H3-3' suggested the presence of a 2-hydroxypropyl group. HMBC correlations of H2-1' to C-2, C-3 and C-4 indicated that the 2-hydroxypropyl group was attached at C-1. For the left portion of 1, a pair of protons [δH 7.21 and 6.84 (each 1H, d, J=2.0 Hz)] were located at C-5 and C-7, respectively, on the basis of HMBC correlations from H-5 (δH 7.21) to the carbonyl carbon C-10 (δC 182.6). The methoxyl group at δH 3.90 was placed on C-8 based on its correlation with H-7 (δH 6.84) in the NOESY spectrum. Consequently, the remaining phenolic hydroxyl group was assigned to C-6, which was further supported by the chemical shift for C-6 at δC 164.6. The absolute configuration at C-2' of 1 was determined by the modified Mosher method.10 Treatment of 1 with (S)-MPA (methoxyphenylacetic acid) or (R)-MPA in the presence of DMAP (4-dimethylaminopyridine) afforded the (S)-MPA ester or (R)-MPA ester, respectively. The values of ΔδR,S (δR−δS) in the 1H NMR (Figure 2b) spectra suggested that the absolute configuration at C-2' of 1 was S. Therefore, the structure of 1 was proposed as shown in Figure 1, and named penipurdin A.

Table 1 1H- and 13C-NMR data of Penipurdin A (1) and B(2)
Figure 2
figure 2

(a) Key HMBC (solid arrows), 1H–1H COSY (bond lines) and NOESEY (dashed arrow) correlations of 1. (b) Observed chemical shift differences (Δδ=δR−δS, p.p.m., 400 MHz) for the (R)- and (S)-MPA esters of 1.

Penipurdin B (2) was isolated as a yellow powder. High-resolution ESIMS showed a peak at m/z 299.0916 [M+H]+ (m/z calcd 299.0914) corresponding to the molecular formula C17H15O5. The 1H and 13C NMR data of 2 were greatly similar to those of 1 except for the absence of the methoxyl (8-OMe, 1) and phenolic hydroxyl (6-OH, 1) groups, which were replaced by a chelated hydroxyl group and an aromatic proton, respectively. The gross structure of 2 was further confirmed by COSY and HMBC experiments and the S configuration at C-2' was assigned by comparison of the sign of the optical rotation value of 2 (+14.2) with that of penipurdin A (1; +33.3).

The known compound 3 was identified as questin by comparison of its NMR and MS data with those reported.9

The cytotoxicities of compounds 1, 2 and 3 were evaluated in vitro against the A549, HepG2 and Hela cell lines by MTT method.11 Compound 2 exhibited cytotoxic activity with IC50 values of 74.7, 3.9 and 15.7 μm, respectively. The IC50 values of compound 3 were 39.5, 6.2 and 14.9 μM, respectively. Compound 1 showed no cytotoxic activity to the three tested cell lines.