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
Full size table

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