Natural products are among the most important resources of the clinically used cancer chemotherapeutic agents. Many compounds with anticancer properties have been isolated from natural sources since actinomycin was discovered in 1940’s. More than 60% of the currently known compounds with anti-neoplasic activity are natural products or their derivatives.1, 2, 3, 4, 5, 6 As part of the search for new and biologically active secondary metabolites produced by actinomyces from unexplored and underexplored ecological niches, an endophytic actinomycete Streptomyces sp. neau50 was isolated from healthy root of soybean. Initial screening of the crude extract exhibited cytotoxicity against certain cancer cell lines and inhibitory activity against phytopathogenic fungi. Subsequent isolation resulted in borrelidin as the major active component. To further investigate minor amount of active constituents in this strain, the fermentation was scaled up, and detailed fractionation of the crude extract led to the isolation of a novel diphenyl ether compound, methyl 2-hydroxy-4-(2-hydroy-3-methoxy-5-methylphenoxy)-6-methylbenzoate (1), together with a known one, methyl 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)-6-methylbenzoate (2). In this paper, we describe the isolation, structure elucidation and bioactivities of 1 and 2.

The producing strain, Streptomyces sp. neau50, was isolated by moist incubation and desiccation method from healthy soybean root in Harbin, Heilongjiang province, China.7 The 16S rRNA was sequenced for taxonomic classification (GenBank Accession No: GQ 494994).

The strain was maintained in the medium containing 10 g glucose, 3 g maltose, 3 g yeast extract, 0.5 g K2HPO4 3H2O, 0.5 g MgSO4 7H2O, 0.5 g NaCl, 1 g KNO3 and 20 g agar in 1.0 l tap water, pH 7.0. Slant culture was incubated for 6–7 days at 28°C. The seed medium consisted of 4 g glucose, 10 g maltodextrin, 4 g yeast extract, 2 g CaCO3 in 1.0 l water, pH 7.2–7.4. All the media were sterilized at 121°C for 20 min.

Fermentation was carried out in a 50-l first seed fermentor (containing 30 l seed culture) and a 500-l second fermentor (containing 300 l producing medium), successively. The producing medium comprised 1% glucose, 4% soluble amylum, 0.5% yeast extract, 2.5% soybean powder, 0.5% peptone, 0.2% CaCO3, 0.8% MgSO4 7H2O, 0.6% FeSO4 7H2O, 0.2% ZnSO4 7H2O, 0.2% MnSO4 H2O, 0.05% CoCl2 6H2O, 0.2% Na2MoO4 2H2O, pH 7.0. The fermentation was performed at 28°C for 7 days stirred at 100 r min−1 with an aeration rate of 30 m3 of air per hour.

The final 300 l of fermentation broth was filtered to afford the mycelial cake. After it was washed with water, the mycelia was extracted twice with 100 l of EtOH for about 24 h. The EtOH extract was diluted to about 30% EtOH and subjected to a Diaion HP-20 resin column, which was eluted with 30, 40, 50, 60, 70 and 80% EtOH (each concentration eluted 2 bed volumes). The eluents at 70 and 80% EtOH were pooled and concentrated in vacuo at 50°C. Then part of the concentrated material (32 g) was chromatographed on a silica gel column, and successively eluted with a stepwise gradient of petroleum ether–acetone (100:0–50:50, v/v) to obtain five fractions (fractions I–V) based on the TLC profiles. Fraction III was chromatographed on another silica gel column and eluted with petroleum ether–acetone (90:10–70:30, v/v) to give three subfractions. Subfraction II was then subjected to a Sephadex LH-20 column (GE Healthcare, Glies, UK) and eluted with EtOH to obtain fractions A and B. Finally, fraction B was fractionated by semi-preparative HPLC with column Zorbax SB-C18 (5 μm, 250 × 9.4 mm) on Agilent 1100 system (Agilent, Palo Alto, CA, USA). It was eluted with CH3OH–H2O (85:15, v/v) at 1.5 ml min−1. Two main compounds 1 (tR 11.8 min, 8 mg) and 2 (tR 12.7 min, 11 mg) (Figure 1) were isolated.

Figure 1
figure 1

Structures of compounds 1 and 2.

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Compound 1 was obtained as colorless oil with UV (EtOH) λmax nm (log ɛ): 211 (4.29), 264 (3.92), 299 (3.52). The absorption peaks in the IR spectrum of 1 suggested the presence of hydroxyl (3447 cm−1) and conjugated carbonyl (1656 cm−1) groups. Its molecular formula was established as C17H18O6 on the basis of HRESIMS, which gave a quasi-molecular ion at m/z 341.1009 [M+Na]+. 1H NMR spectrum of 1 exhibited two methyl singlets at δH 2.28, δH 2.50, four aromatic protons at δH 6.27, δH 6.42, δH 6.50 and δH 6.58, two methoxy groups at δH 3.91 and δH 3.93, in addition to two hydroxyl signals at δH 5.32 and δH 11.65. Its 13C NMR spectrum displayed 17 carbon resonances, including an ester carbonyl carbon, four sp2 methines, eight sp2 quaternary carbons, two methyls and two methoxy carbons. The detailed analysis of the 1H NMR and 13C NMR data (Table 1) of 1 indicated that it had two benzene rings. The full structure of 1 was established by the correlated signals in the HMBC spectrum. A methocarbonyl group was confirmed by the long-range coupling from the methoxy signal at δH 3.93 to δC 172.1 (Figure 2). The downfield proton signal of a hydroxyl at δH 11.65 (7-OH) showed the OH group was in ortho position to the methocarbonyl. In addition to an oxygen atom attachment to C-5 revealed by the 13C chemical shift of C-5 (δC 162.5), the substituents of tetra-substituted ring A were assigned by the HMBC correlations from H3-8 to C-2, C-3, C-4, from δH 11.65 to C-2, C-6, C-7 and from H-6 to C-5. The observed crossing peaks in HMBC spectrum of 1 between δH 3.91 and C-11, δH 5.32 (10-OH) and C-9, C-10, C-11, H3-15 and C-12, C-13, C-14, H-12 and C-11, H-14 and C-9 demonstrated the presence of another tetra-substituted benzene ring and established the substituents of ring B as shown in Figure 2. The ether linkage of the two benzene rings through C-4 and C-9 was confirmed by the molecular formula of C17H18O6. Furthermore, the 1H and 13C NMR data of 1 were very similar to those of gerfelin,8 except the two methoxy groups in 1. This finalized the study and assigned the structure of 1 as methyl 2-hydroxy-4-(2-hydroxy-3-methoxy-5-methylphenoxy)-6-methylbenzoate.

Table 1 1H and 13C NMR data of compounds 1 and 2
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Figure 2
figure 2

Key HMBC correlations of 1.

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Compound 2 was also isolated as colorless oil with UV (EtOH) λmax nm (log ɛ): 217 (4.50), 264 (4.12), 302 (3.72). The IR spectrum of 2 showed the presence of hydroxyl (3447 cm−1) and conjugated carbonyl (1655 cm−1) groups. The 1H NMR spectrum of 2 indicated five aromatic protons, two aromatic methyls, a downfield OH signal and a methoxy group. In the 13C NMR spectrum of 2, there were 12 sp2 carbons in addition to three methyl carbons (one oxygenated one) and one carbonyl. These suggested the presence of two benzene rings in 2. Comparison of the NMR data (Table 1) of 2 with those of cordyol B,9 showed that compound 2 was identical to the aglycone of cordyol B and elucidated the structure of 2 as methyl 2-hydroxy-4- (3-hydroxy-5-methylphenoxy)-6-methylbenzoate.

We examined the inhibitory activity of compounds 1 and 2 against the growth of human lung adenocarcinoma cell line A549 using the CCK-8 colorimetric method as described in our preceding paper.10 The result showed that compounds 1 and 2 inhibited the growth of A549 cells dose-dependently with an IC50 value of 5.3 and 15.2 μg ml−1, respectively.