Unsaturated fatty acid amides, which formed from an unsaturated fatty acid and an amine, have diverse biological potency including the modulatory effect on isolated gastrointestinal tract, the effect on transactivational activity of peroxisome proliferator-activated receptors (PPARs) and the cholesterol acyltransferase inhibitory activity.1, 2, 3 At present, more than 20 unsaturated fatty acid amides have been isolated from Zanthoxylum piperitum, such as α-sanshool, β-sanshool, hydroxyl-γ-sanshool and ZP-amide A–F, which possessed unsaturated aliphatic acids conjugated with isobutylamine or its derivatives.2, 3, 4, 5, 6 To our knowledge, natural compounds possessing a 12-carbon polyketide conjugated with l-isoleucine (or derivatives), for example, curvularides A–E and coronatine, have been obtained from microbial resources.7, 8 In the course of hunting for new biologically active compounds from microbial sources, two new unsaturated fatty acid amides, maoxianamides A (1) and B (2), were isolated from Streptomyces maoxianensis sp. nov. Herein, we report details of the isolation, structure elucidation and biological activity of the two new compounds.

The producing strain S. maoxianensis sp. nov. was isolated from a soil sample collected from a pine forest in Songpinggou, Maoxian, southwest China, which has been reported in the previous paper.9 The GenBank/EMBL/DDBJ database accession number of the 16S ribosomal RNA sequence of strain is KF887908 and it was deposited in the China General Microbiological Culture Collection Center (CGMCC) with accession CGMCC No. 4.7139.

This strain was grown and maintained on the medium containing 4 g yeast extract, 4 g glucose, 10 g malt extract and 20 g agar in 1.0 l tap water, pH 7.0–7.2 and incubated for 6–7 days at 28 °C. The strain of stock culture was transferred into 1 l Erlenmeyer flasks containing 25% volume of the seed medium and incubated at 28 °C for 24 h, shaken at 150 r.p.m. Then, 1 l of the culture was transferred into a 50 l fermentor containing 30 l of producing medium consisting of 10 g glucose, 40 g soluble amylum, 5 g yeast extract, 25 g soybean powder, 5 g peptone, 2 g CaCO3, 8 g MgSO4·7H2O, 6 g FeSO4·7H2O, 2 g ZnSO4·7H2O, 2 g MnSO4·H2O, 0.5 g CoCl2·6H2O, 2 g Na2MoO4·2H2O, pH 7.0–7.2. The fermentation was carried out at 28 °C for 6 days and stirred at 100 r.p.m. with an aeration rate of 900 l of air per hour.

The fermentation broth (30 l) was centrifuged to separate mycelial cake and supernatant. The mycelial cake was extracted with MeOH (5 l) and the supernatant was subjected to a Diaion HP-20 resin (Mitsubushi Chemical, Tokyo, Japan) column eluting with 95% EtOH (5 l). The MeOH extract and the EtOH eluents were evaporated under reduced pressure to yield a mixture (33 g) at 50 °C. The mixture was chromatographed on a silica gel column (Qingdao Haiyang Chemical Group, Qingdao, China; 100–200 mesh), and successively eluted with a stepwise gradient of CHCl3/MeOH (100:0–50:50, v/v) to obtain three fractions (Fr.1–Fr.3) based on the TLC profiles. The Fr.2 was subjected to another silica gel column eluted with CHCl3/MeOH (95:5–60:40, v/v) to give one fraction (Fr.2–1). Fr.2–1 was further isolated by preparative HPLC (Shimadzu LC-8 A, Shimadzu-C18, 5 μm, 250 × 20 mm inner diameter; 20 ml min−1; 220 /254 nm; Shimadzu, Kyoto, Japan) eluting with a stepwise gradient MeOH/H2O (10–70%, v/v 30 min) to obtain five subfractions (Fr.2–1–1 to Fr.2–1–5) based on the retention time. Then, Fr.2–1–2 (tR 12.1 min) was purified by semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5 μm, 250 × 9.4 mm inner diameter; 1.5 ml min−1; 220 nm; Agilent, Palo Alto, CA, USA) eluting with CH3CN/H2O (12:88, v/v) to obtain maoxianamide A (1) (tR 29.0 min, 31 mg). Fr.2–1–3 (tR 10.3 min) was also isolated by semi-preparative HPLC eluting with CH3CN/H2O (17:83, v/v) to obtain maoxianamide B (2) (tR 18.6 min, 6.3 mg). 1H and 13C NMR spectra were measured with a Bruker DRX-400 (400 MHz for 1H and 100 MHz for 13C) spectrometer (Rheinstetten, Germany). The ESIMS and HRESIMS spectra were taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Milford, MA, USA).

Maoxianamide A (1) was obtained as colorless oil with UV (EtOH) λmax nm (log ɛ): 202 nm (4.32) and [α]25D+44 (c 0.25, EtOH). It exhibited a molecular formula of C17H31NO6, as deduced from the HRESIMS at m/z 368.2039 [M+Na]+ (calcd as 368.2044 for C17H31NO6Na) in combination with 13C NMR data (Table 1). The IR spectrum of 1 showed absorption bands for hydroxyl (at 3389 cm−1) and carbonyl (at 1653 cm−1) groups. Analysis of 1H NMR spectrum of 1 revealed the presence of three olefinic protons at δH 5.68 (1H, m), 5.68 (1H, d, J=9.2 Hz), 5.25 (1H, br d, J=8.8 Hz), three aliphatic oxygenated methine protons at δH 4.75 (1H, d, J=8.9 Hz), 3.66 (1H, m), 3.22 (1H, dd, J=10.4, 1.9 Hz), six downfield methylene protons at δH 3.49 (2H, d, J=5.2 Hz), 3.39 (2H, m), 2.92 (1H, dd, J=5.5, 6.1 Hz), 2.95 (1H, dd, J=6.0, 5.4 Hz), an olefinic methyl at δH 1.76 (3H, d, J=1.2 Hz), one aliphatic singlet methyl at δH 1.25 (3H, s), one aliphatic triplet methyl at δH 1.00 (3H, t, J=7.4 Hz), in addition to proton signals at δH 1.76 (1H, m), 1.64 (1H, m), 1.56 (1H, m) and 1.25 (1H, m). The 13C NMR and DEPT135 spectra (Table 1) of 1 showed 17 resonances attributable to an amide carbonyl carbon at δC 176.3, three sp2 methines at δC 136.9, 127.6 and 125.3, one sp2 quaternary carbon at δC 141.5, three oxygenated methines at δC 81.1, 71.3 and 70.1, one oxygenated quaternary carbon at δC 76.0, one oxygenated methylene at δC 67.2, four methylenes at δC 37.2, 36.5, 34.0, 25.2 and three methyl carbons at δC 23.7, 23.7, 11.7. The 1H–1H COSY correlations (Figure 1) of H2-1′/H2-2′/H-3′/H2-4′ in 1 established connectivity from H-1′ atom along the chain through to C-4′ atom. The correlations between H2/H3, H5/H6/H7, H9/H10H11 protons in the 1H–1H COSY spectrum indicated the three structural units of C-2–C-3, C-5–C-7 and C-9–C-11. The observed HMBC correlations (Figure 1) from H3-12 to C-3, C-4, C-5, from H3-13 to C-7, C-8, C-9 established the linkage of C-2–C-11. The amide carbonyl group was connected with C-2 by the HMBC corrections from H-2 and H-3 to C-1 (δC 176.3). The connection of C-1 and C-1′ through a NH group was evident from the correlation of H2-1′ to C-1 in the HMBC spectrum, and the 1H and 13C chemical shifts of C-1′. Taken the molecular formula of C17H31NO6 into account, five hydroxyl groups were situated at C-2, C-8, C-9, C-3′ and C-4′, respectively. On the basis of the above spectroscopic data, a gross structure of 1 was established, and the 1H and 13C resonances in 1 were assigned (Table 1). The NOESY correlation between H3-12 and H-2 demonstrated the geometry of the C-3–C-4 double bond was E. The geometry of Δ6,7 was also assigned as E by the large coupling constant (J=15.8 Hz) between H-6 and H-7 in the 1H NMR spectrum obtained in pyridine-d5. The other stereochemistry of 1 remained unassigned.

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

Structures and key 1H–1H COSY, HMBC and NOESY correlations of maoxianamides A (1) and B (2).

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Maoxianamide B (2) was isolated as colorless oil with UV (EtOH) λmax nm (log ɛ): 201 nm (4.20) and [α]25D+57 (c 0.30, EtOH). Its molecular formula was determined to be C17H31NO6 on the basis of the HRESIMS at m/z 346.2222 [M+H]+ (calcd for C17H32NO6 346.2224). The IR spectrum of 2 showed absorption bands for hydroxyl (at 3364 cm−1) and carbonyl (at 1650 cm−1) groups. The 1H NMR spectrum of 2 display two olefinic protons (δH 5.45 (1H, d, J=8.4 Hz), 5.27 (1H, d, J=9.2 Hz)), four aliphatic oxygenated methine protons (δH 4.79 (1H, d, J=9.2 Hz), 4.79 (1H, m), 4.59 (1H, m), 3.85 (1H, t, J=6.8 Hz)), six downfield methylene protons (δH 3.46 (2H, d, J=5.9 Hz), 3.34 (2H, m), 2.12 (1H, dd, J=13.6, 3.8 Hz), 2.59 (1H, dd, J=13.6, 9.1 Hz)), two olefinic methyls (δH 1.83 (3H, d, J=0.9 Hz), 1.68 (3H, d, J=1.1 Hz)), one aliphatic triplet methyl (δH 0.87 (3H, t, J=7.6 Hz)), as well as three proton signals (δH 1.76 (1H, m), 1.59 (1H, m), 1.52 (1H, m)). The 13C NMR and DEPT135 spectra (Table 1) of 2 exhibited 17 carbon signals composing an amide carbonyl carbon (δC 176.1), two sp2 methines (δC 130.3, 127.3), two sp2 quaternary carbons (δC 139.9, 139.1), four oxygenated methines (δC 79.4, 71.2, 69.7, 67.1), one oxygenated methylene (δC 67.2), four methylenes (δC 41.6, 37.3, 33.9, 28.7) and three methyl carbons (δC 24.2, 12.0, 10.6). Comparison of the 1H and 13C NMR data of 2 with those of 1 suggested that 2 has the same skeleton as 1. The differences between 2 and 1 were that the C-7 hydroxyl group and Δ6,7 olefin in 1 were replaced by the C-6 hydroxyl group and Δ7,8 olefin in 2. The correlation of H-6/H-7 in the 1H–1H COSY spectrum (Figure 1) and the observed HMBC correlated signals from H3-13 to C-7, C-8, C-9 further confirmed the assignment. The geometry of Δ7,8 was assigned as E by the NOESY correlations between H-9 and H-7. The other relative stereochemistry of 2 was assigned by analogy with 1.

The antimicrobial activities of maoxianamides A (1) and B (2) using a disk diffusion assay was carried out against five microorganisms.10 Neither maoxianamides A (1) nor B (2) were found to be active against Micrococcus luteus, Bacillus subtilis, Candida albicans and Rhizoctonia solani, even at 100 μg per 7 mm paper disks. Maoxianamides A (1) and B (2) exhibited weakly inhibitory activity against Sclerotinia sclerotiorum with inhibition zones of 7 mm and 9 mm at 100 μg per 7 mm paper disks.

The cytotoxicity of maoxianamides A (1) and B (2) were assayed in vitro against the human lung carcinoma A549 cell line, human hepatoma carcinoma HepG2 cell line and human leukemia K562 cell line by the CCK8 method as described in our previous papers.11, 12 Maoxianamide A (1) exhibited cytotoxic activity with IC50 (half maximal inhibitory concentration) values of 55.3, 32.4 and 33.2 μg ml−1, respectively. The values of maoxianamide B (2) were 60.4, 37.7 and 40.6 μg ml−1.