In the course of our research on protein tyrosine phosphatase (PTP) 1B inhibitors, we identified a new 2,4′-linked tetrahydroxanthone dimer (1), named secalonic acid F1 (Figure 1a) in the culture broth of the Okinawan freshwater fungus Aspergillus sp. TPU1343. This fungus produced the unusual bis-tetrahydroxanthone, asperdichrome.1 PTP1B plays an important role as a negative regulator in the insulin and leptin signaling pathways. Therefore, a PTP1B inhibitor is anticipated to become a new type of clinical application for Type 2 diabetes mellitus and obesity.2, 3, 4, 5 We herein describe the fermentation, isolation, structural elucidation, and biological activity of compound 1.

Figure 1
figure 1

(a) Structures of compounds 1 and 2 produced by the freshwater fungus Aspergillus sp. TPU1342 and of related compounds. (b) 1H–1H COSY and key HMBC correlations for compound 1.

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Aspergillus sp. TPU1343, maintained on a PDA plate, was inoculated into a 100 ml Erlenmeyer flask containing 50 ml of seed medium (2.0% glucose (Wako, Osaka, Japan), 0.50% polypeptone (Wako), 0.050% MgSO4·7H2O (Wako), 0.20% yeast extract (BD, Franklin Lakes, NJ, USA), 0.10% KH2PO4 (Wako), and 0.10% agar (Wako) in freshwater and adjusted to pH 6.0 before sterilization).1 The flask was shaken reciprocally at 25 °C for 3 days to obtain the seed culture, which was then transferred to the production medium (3.0% sucrose (Wako), 3.0% soluble starch (Wako), 1.0% malt extract (BD), 0.30% Ebios (Asahi Food & Healthcare, Tokyo, Japan), 0.50% KH2PO4, and 0.050% MgSO4·7H2O in freshwater and adjusted to pH 6.0 before sterilization) and cultured at 25 °C for 7 days under agitation.

Acetone (20 l) was added to the culture broth (20 l) and filtered. The filtrate was concentrated in vacuo to remove acetone and extracted three times with EtOAc. The EtOAc extract was concentrated in vacuo to dryness, and the extract (35 g) was suspended in 30% CH3OH in H2O and applied on an ODS column (100 g). The ODS column was eluted stepwise with 30, 50, 70, 85 and 100% CH3OH in H2O (200 ml each × 2) to separate 10 fractions (Fr. 1–Fr. 10). Fr. 8 (the second 200 ml of the 85% CH3OH eluate) was concentrated to give a dark black oil (241 mg), which was purified by preparative HPLC (column; inertsil ODS-3 (GL Science, Tokyo, Japan), 10 × 250 mm; mobile phase, 70% CH3CN containing 0.05% TFA; detection, UV at 254 nm; flow rate, 2.0 ml min–1) to afford 2.4 mg of compound 1 and 13 mg of secalonic acid F (2)1, 6 (Figure 1a).

Compounds in the secalonic acid family typically possess a 2,2′-linked bis-tetrahydroxanthone skeleton as secalonic acids A, E and F (2) (Figure 1a).7 Secalonic acid F (2) is a heterodimer consisting of two epimeric monomers at C-5/C-5′, and secalonic acids A and E have homodimeric structures with single isomers (Figure 1a).

Secalonic acid F1 (1) was obtained as a yellow oil ([α]D22=+75.2, c 0.10, CHCl3), and showed UV absorptions at 201, 249 and 337 nm, and IR bands at 3424, 1740, 1616, 1437, 1234 and 1044 cm–1, similar to 2.1, 6 The molecular formula of 1 was deduced as C32H30O14 by HREIMS data (m/z 638.1630 [M]+, Δ−0.6 mmu), which was the same as that of 2. The 1H and 13C NMR spectra of 1 (in CDCl3) indicated two sets of signals (Table 1). Two proton signals corresponding to 5-H (δH 4.15, s) and 5′-H (δH 3.85, d (J=11.1 Hz)) suggested that compound 1 possessed a heterodimeric tetrahydroxanthone skeleton with the same configuration as that of 2. Marked differences between 1 and 2 were observed in their HMBC spectra. HMBC correlations from H-3 (δ 7.74) to C-4′ (δ115.5) and from H-3′ (δ 7.49) to C-2 (δ 118.7) were observed in the spectrum of 1, which indicated the linkage between two monomeric units at the C-2 and C-4′ positions.

Table 1 13C (100 MHz) and 1H (400 MHz) NMR data for 1 in CDCl3
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To date, penicillixanthones A and B have been reported as the 2,4′-linked homodimers of secalonic acids A and E, respectively, from the culture broth of Penicillium thomii8 and Setophoma terrestris9 (Figure 1a). Penicillixanthone B was also identified as an antibacterial metabolite from the marine-derived Penicillium sp.10 Comparisons of 1H and 13C data for 1 with the reported values for penicillixanthones revealed that data for partial structure I (Figure 1b) were similar to those for penicillixanthone B, while data for partial structure II (Figure 1b) were identical to those for penicillixanthone A (Table 1). Thus, the structure of 1 including its relative configuration was assigned as shown in Figure 1a.

The absolute configuration of 1 was proposed to be the same as 2 because heterodimers 1 and 2 were obtained from the same culture broth of strain TPU1343. This was supported by the positive n→π* CD bands of 1 and 2 at 326 nm (Δɛ=+8.6) and 330 nm (Δɛ=+8.6), respectively, due to the R configurations at the C-10 and C-10′ positions.1, 11, 12 Thus, the absolute configuration of 1 was elucidated as (5 S, 6 S, 10 R, 5′ R, 6′S, 10′R) (Figure 1a).

Penicillixanthone A (2,4′-linkage, Figure 1a) was previously reported to be transformed from secalonic acid A (2,2′-linkage) in polar solvents such as CH3CN and pyridine.13 Qin et al.14 demonstrated that secalonic acid A in DMSO was isomerized to 2,4′- and 4,4′-linked derivatives at room temperature for 10–15 h. Therefore, the transformation of 2 to 1 was examined in CH3CN, DMSO and CH3OH. The solution of compound 2 in CH3CN, DMSO or CH3OH (1 mg ml–1) was kept at room temperature, and each solution was monitored on 0, 24 and 48 h by HPLC. Compound 2 in CH3CN was stable for 48 h (Supplementary Figure S1A), and a small peak corresponding to 1 appeared after 24–48 h in DMSO (Supplementary Figure S1B). The isomerization of 2 in DMSO was markedly slower than the reported transformation of secalonic acid A in DMSO.14 Although CH3OH was used for ODS column chromatography of the EtOAc extract and to dissolve Fr. 8 for HPLC separation, the conversion of 2 to 1 in CH3OH was negligible, even after 48 h (Supplementary Figure S1C). During the isolation of compounds 1 and 2, these compounds were dissolved in CH3OH only for a few hours. Therefore, it is unlikely that compound 1 was transformed from 2 during the separation procedures. Secalonic acid F1 (1) must exist in the fermentation broth of strain TPU1343.

Compound 1 was evaluated for its PTP1B inhibitory activity using the enzyme assay method.15 PTP1B activity was inhibited by 1 with an IC50 value of 5.9 μm (Supplementary Table S1). A positive control, oleanolic acid16 (Tokyo Chemical Industry, Tokyo, Japan), showed an IC50 value of 1.1 μm in the same experiment.

Various cellular functions are controlled by PTPs composed of >100 members including PTP1B,17 and, thus, selectivity against PTP1B over other PTPs is an important property. The inhibitory activities of 1 against T-cell PTP (TCPTP), one of the non-transmembrane PTPs, CD45 tyrosine phosphatase (CD45), one of the receptor-like PTPs, and Vaccinia H-1-related phosphatase (VHR), one of the dual-specificity phosphatases, were examined.17, 18 Compound 1 had IC50 values of 6.9 and 6.2 μm against TCPTP and VHR, respectively, similar to PTP1B, while the inhibitory activity of 1 against CD45 was weaker (IC50=14 μm) than those against other PTPs (Supplementary Table S1).