Epothilones are a new class of 16-membered macrolides with taxol-like effects produced by myxobacterium Sorangium cellulosum.1, 2 In contrast to taxol, they are active against taxol-resistant tumors and are water soluble. Following the isolation and characterization of the major metabolites, epothilones A and B from the strain S. cellulosum So ce90, some natural and synthetic epothilone variants have been reported,3, 4, 5, 6, 7 and a few of them have undergone preclinical or clinical evaluations.8 As a result, ixabepilone, a lactam analog of epothilone B, has been approved for clinical use for treatment of certain forms of breast cancer.9 In our previous investigation, five new epothilone glycosides and two new epothilone congeners have been isolated from the epothilones A and B producing strain S. cellulosum strain So0157-2.10, 11 As part of our ongoing research on the metabolites produced by the S. cellulosum strain So0157-2, a new 16-membered macrolide, designed as epothilone O (1), was obtained from the 120 l fermentation broth of S. cellulosum strain So0157-2. Herein, we describe the isolation, structure elucidation and biological activity of the new compound.

The producing organism S. cellulosum strain So0157-2 was provided by Professor Yuezhong Li at the Shandong University, China, and was deposited in the China Center of Typical Culture Collection (CCTCC) with accession No.CCTCC M 208078. The culture and fermentation of S. cellulosum strain So0157-2 were conducted according to the procedure as described in the literature.10 The adsorber resin XAD-1600 (Rohm and Haas, Philadelphia, PA, USA; 2.4 l) was separated from the 120 l fermentation broth with a process filter. After washing the resin with water, the resin was eluted with four bed volumes of 95% ethanol. The ethanol eluent was diluted to about 30% ethanol and charged to a XAD-1600 resin column eluting with 30, 40, 50, 60 and 70% ethanol (two bed volumes each). The fractions eluting with 60 and 70% ethanol were pooled and evaporated at 50 °C to give a mixture, which was dissolved in CHCl3 and chromatographed on silica gel (100–200 mesh) eluting with petroleum ether/acetone solvent gradient from 70:30 to 40:60. The polar fraction eluting with petroleum ether/acetone (60:40) was further chromatographed on a Sephadex LH-20 (GE Healthcare, Glies, UK) column and eluted with ethanol and detected by TLC developed with petroleum ether/acetone (3:2) and visualized under UV light at 254 nm (Rf=0.46) to give a subfraction. The subfraction was further isolated by semipreparative HPLC (Agilent 1100 instrument, Agilent, Palo Alto, CA, USA; C18 column: Zorbax SB-C18, Agilent, 9.4 × 250 mm, 5 μm) using CH3CN/H 2O (40:60) with a flow rate of 1.5 ml min−1 at a room temperature to give compound 1 (tR 37.80 min, 3.5 mg).

Compound 1 was isolated as colorless oil. Its molecular formula was determined to be C27H41NO6S by the HRESIMS m/z 506.2572 [M-H]−, (calcd for C27H40NO6S, 506.2582), indicating 8 degrees of unsaturation. Compound 1 is thus an isomer of epothilone B. The UV spectrum of 1 showed absorption maxima at 248 nm. The IR spectrum indicated the presence of hydroxyl (3436 cm−1) and carbonyl (1636 cm−1) groups. In the 1H NMR spectrum (Table 1), signals for two aliphatic doublet methyls (δH 0.97 (d, J=6.7 Hz), 1.14 (d, J=7.1 Hz)), two aliphatic singlet methyls (δH 1.13 (s), 1.26 (s)), three olefinic methyls (δH 1.65 (s), 2.10 (d, J=1.0 Hz), 2.70 (s)), four oxygenated methine protons (δH 3.97 (m), 4.08 (m), 4.10 (m), 5.16 (dd, J=8.4, 4.0 Hz)) and three olefinic protons (δH 5.55 (t, J=6.5 Hz), 6.53 (s), 6.97 (s)) were easily identified. The 13C NMR and DEPT spectra (Table 1) displayed 27 carbon resonances, which corresponded to seven methyls (δC 12.3, 13.0, 15.1, 16.0, 19.1, 21.0, 21.3), four methylenes (δC 23.3, 29.1, 39.0, 39.2), nine methines (four oxygenated and three olefinic) and seven quaternary carbons (one ketone, one ester carbonyl, four olefinic). The above 1H and 13C NMR data indicated that compound 1 possessed a structural skeleton similar to epothilone D.3 Detailed comparisons of the NMR data of 1 with those of epothilone D revealed that the C-11-C-13 moieties of these two compounds were different. In the 1H–1H COSY spectrum, the correlations of H-14 (δH 2.03, 2.06) with H-15 (δH 5.16) and H-13 (δH 4.10), and the correlations of H-10 (δH 2.10, 2.24) with H-11 (δH 5.55) and H-9 (δH 1.47, 1.78) established the subunits of C-13-C-15 and C-9-C-11, respectively. The observed HMBC correlations from δH 1.65 (H-26) to C-11 (δC 128.0), C-12 (δC 136.5) and C-13 (δC 76.0) established the connectivity of the two subunits. Consequently, the planar structure of 1 was assigned as shown in Figure 1. The NOESY correlation between H-26 and H-10 indicated the geometry of Δ11 olefin was E. Except the configuration of C-13, the other chiral centers in 1 were assigned in analogy with epothilones A and B. The orientation of the 13-OH was assigned as α based on the correlation of H-13 and H-15 in the NOESY experiment.

Table 1 1H and 13C NMR data for compound 1 (in CDCl3)
Figure 1
figure 1

The structure and key 1H–1H COSY, HMBC and NOESY correlations of compound 1.

Epothilone O (1) was evaluated for cytotoxic activity against human lung carcinoma A549 using the CCK-8 colorimetric method as described previously.10 Bioassay result showed that compound 1 was cytotoxic with an IC50 of 7.9 μg ml−1.

Epothilone O (1): Colorless oil; [α]25D-6.25 (c 0.016, EtOH); UV (EtOH) λmax 248 (log ɛ 3.74) nm; IR (KBr): vmax 3436, 2925, 1636, 1460, 1374, 1267, 1050 cm−1; 1H and 13C NMR spectral data see Table 1; ESIMS: m/z 506 [M-H]−; HRESIMS: m/z 506.2572 [M-H]− (calcd for C27H40NO6S, 506.2582).