Microbial metabolites used as potential pesticides have attracted great interest from the agricultural and food community due to their potential activity and low toxicity.1, 2 Several microbial metabolites, such as the avermectin and milbemycin families, have been proven to be potent preventatives and treatment against a variety of pests such as insects and parasites. During the course of our screening program for new natural pesticides and antiparasitic veterinary drugs, two novel macrocyclic lactones, three milbemycins and six new doramectin congeners have been isolated from Streptomyces avermitilis NEAU1069.3, 4, 5, 6 In the effort to improve the doramectin yield, a mutant S. avermitilis NEAU1069-3 was obtained through the treatment of the spores of S. avermitilis NEAU1069 with UV and N-methyl-N′-nitro-N-nitrosoguanidine. Compared with the wild-type strain, S. avermitilis NEAU1069-3 showed significantly different phenotypes such as the morphology of aerial mycelia and the metabolite HPLC profiles. Therefore, the secondary metabolites of mutant S. avermitilis NEAU1069-3 were investigated, leading to two new doramectin analogs 1 and 2 (Figures 1 and 2). The discovery of new doramectin analogs 1 and 2 in the mutant S. avermitilis NEAU1069-3 may shed new insight into the biosynthesis of doramectins. Here we described the fermentation, isolation and structural elucidation of these two new doramectin analogs.

Figure 1
figure 1

Structures of 1, 3 and key 1H–1H COSY and HMBC correlations of 1.

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Figure 2
figure 2

Structures of 2, 4 and key HMBC correlations of 2.

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The culture and fermentation of mutant S. avermitilis NEAU1069-3 were conducted according to the procedure as described in the literature.5 The fermentation broth (30 liters) was filtered. The resulting cake was washed with water, and both filtrate and wash were discarded. Methanol (10 liters) was used to extract the washed cake. The MeOH extract was evaporated under reduced pressure to 2 liters at 45 °C and the resulting concentrate was extracted three times using an equal volume of EtOAc. The combined EtOAc phase was concentrated under reduced pressure to yield 26 g of oily substances. The residual oily substance was chromatographed on silica gel (Qingdao Haiyang Chemical Group, Qingdao, China; 100–200 mesh) and eluted with a petroleum ether–acetone mixture (100:0–50:50, v/v). The fractions eluted with the petroleum ether–acetone mixture (90:10, v/v) were combined and evaporated to obtain fraction I, and the fractions eluted with the petroleum ether–acetone mixture (85:15, v/v) were pooled and concentrated to give fraction II. The fraction I was subjected to Sephadex LH-20 (GE Healthcare, Glies, UK) gel column eluting with MeOH to give subfraction I. The semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5 μm, 250 × 9.4 mm i.d.; Agilent, Palo Alto, CA, USA) was applied to obtain pure compounds. The eluates were monitored using a photodiode array detector at 254 nm, and the flow rates were 1.5 ml min−1 at room temperature. The subfraction I was further separated by semi-preparative HPLC using a solvent containing a CH3OH–H2O mixture (90:10, v/v) to obtain compound 1 (tR 14.5 min, 17 mg). The fraction II was subjected to Sephadex LH-20 gel column eluting with MeOH to give subfraction II, which was subsequently purified by semi-preparative HPLC using a solvent containing a CH3OH–CH3CN–H2O mixture (48:45:7, v/v/v) to obtain compound 2 (tR 24.5 min, 13 mg).

Compound 1 (Figure 1) was obtained as colorless oil. Its molecular formula was determined to be C43H60O9 on the basis of HRESIMS (found: m/z 743.4093 [M+Na]+, calculated for C43H60O9Na, 743.4130), indicating 14 degrees of unsaturation. The IR spectrum of 1 showed absorption bands assignable to the hydroxyl group (3414 cm−1) and carbonyl group (1703 cm−1). The 1H NMR (400 MHz, CDCl3) data (Table 1) showed two downfield proton signals at δH 7.39 (1H, s), 6.62 (1H, s), one trans-double bond at δH 6.09 (1H, dd, J=15.0, 10.0 Hz), 5.46 (1H, dd, J=15.0, 10.0 Hz), one methoxy group at δH 3.53 (3H, s), an aromatic methyl at δH 2.24 (3H, s), two olefinic methyls at δH 2.07 (3H, br s), 1.57 (3H, br s), and three aliphatic doublet methyls at δH 1.28 (3H, d, J=6.2 Hz), 1.17 (3H, d, J=6.9 Hz) and 0.92 (3H, d, J=7.2 Hz). Its 13C NMR and DEPT data (Table 1) displayed an ester carbonyl at δC 169.7 (s), a ketal at δC 95.9 (s), an acetal at δC 95.0 (d), one methoxy carbon at δC 56.8 (q), seven oxygenated methines at δC 83.0 (d), 78.3 (d), 77.3 (d), 76.2 (d), 68.5 (d), 67.9 (d) and 67.9 (d), three aliphatic methines at δC 38.7 (d), 30.1 (d) and 41.0 (d), six methyls at δC 15.3 (q), 18.2 (q), 19.6 (q), 17.7 (q), 15.6 (q), and 16.6 (q) in addition to nine aliphatic methylenes and 14 sp2 carbons. Comparison of the 1H and 13C NMR data of 1 with those of the doramectin analog (3, Figure 1) reported in the literature5 suggested that 1 was similar to 3. The difference between 1 and 3 is that a double bond was present in C-22 and C-23 in 1, which is supported by the 18 mass unit difference from 3. The 1H–1H COSY correlation (Figure 1) of δH 5.54 and δH 5.73, and the observed HMBC correlation from C-24 methyl group (δH 0.92) to δC 136.1 (C-23; Figure 1) further confirmed the structural assignment of 1. As a result, the gross structure of 1 was established as shown in Figure 1. The relative stereochemistry was assigned by analogy with 3.

Table 1 13C (100 MHz) and 1H (400 MHz) NMR assignments for 1 and 2 in CDCl3
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Compound 2 (Figure 2) was also obtained as colorless oil with [α]+72.7° (c 0.08, EtOH). Its molecular formula was determined to be C44H66O12 by HRESIMS (found: m/z 809.4445 [M+Na]+, calculated for C44H66O12Na, 809.4446). The IR spectrum showed absorption bands due to the hydroxyl group (3506 cm−1) and carbonyl group (1734 cm−1). The 1H and 13C NMR spectra in connection with HMQC experiment of 2 showed 64 proton and 44 carbon signals, and the multiplicity of carbon signals was classified into one carbonyl (δ 173.8), three sp2 quaternary carbons, five sp2 methines, one ketal (δ 99.7), one acetal (δ 95.0), an oxygen-bearing quaternary carbon (δ 80.6), an oxygen-bearing methylene (δ 68.2), ten oxygenated methines, two methoxy carbons (δ 57.8, 56.6), four sp3 methines and five methyl carbons in addition to ten aliphatic methylenes by analysis of HMQC data. All the carbons and the corresponding proton signals were assigned by extensive analysis of the HMQC spectrum. The similarity of the NMR data between 2 and the known compound (4, Figure 2)5 and selamectin7 indicated that 2 was also an analog of selamectin. The difference between 2 and 4 is that compound 2 has the furan ring moiety as that of selamectin, which is supported by the 14 mass unit difference from 4. This conclusion was further supported by the HMBC correlation from 8a-H2 (δH 4.63/4.70) to C-6 (δC 77.4) and C-8 (δC 139.8) (Figure 2). The relative configuration of 2 was also assigned by the analogy with 4 and selamectin.

Avermectins have attracted extensive attention due to the impressive anthelmintic and insecticidal activity. Until now, six avermectins including abamectin, doramectin, aprinomectin, ivermectin and selamectin have been successfully commercialized, and they are considered to be the most widely used drugs in animal health and agriculture.4, 5, 6 Similar to selamectin that exhibits high insecticidal activities,8 compounds 1 and 2 possess a monosaccharide subunit attached at C13. Therefore, the bioactivities of 1 and 2 were evaluated. Unfortunately, compounds 1 and 2 exhibited weak acaricidal and insecticidal activities even at a concentration of 100 μg ml−1.

In conclusion, two new doramectin analogs were obtained from the culture broth of the mutant S. avermitilis NEAU1069-3. Along with previously obtained analogs of doramectin, avermectin and milbemycin from S. avermitilis NEAU1069, the discovery of compounds 1 and 2 may have important roles in understanding and perfecting the proposed biosynthetic pathways of doramectins.