New diketopiperazine derivatives with cytotoxicity from Nocardiopsis sp. YIM M13066

The strain YIM M13066 was first cultured in Petri dishes with ca. 20 ml MP agar medium for 7 days to obtain seed cultures. The seeds were inoculated on MP agar media (20 l) by streaking plate method and cultured for 10 days at 28 °C. The fermented culture was extracted with EtOAc–MeOH (85:15, volume per volume, 20 l) at room temperature to obtain a crude extract. The crude extract was partitioned with equal volume EtOAc (0.5 l) and H₂O. The EtOAc portion was concentrated under vacuum to afford the EtOAc extract (7.8 g). The extract was subjected to column chromatography over Sephadex LH-20 (25–100 μm; GE Healthcare, Sweden; column dimensions: 200 × 20 mm) eluted with MeOH and pooled on the basis of TLC detection to obtain Fr.1–10. Fr.7 (1.1 g) was separated by MPLC (310 × 2.6 mm) over RP-18 silica gel (80 g) to afford Fr.7a–7h. Compounds 5 (30.0 mg) and 6 (40 mg) were obtained by crystallization from Fr.7d and Fr.7e, respectively. Fr.7f was purified by HPLC (SunFire Prep C₁₈ OBD, 19 × 150 mm, 15 ml min⁻¹, UV 365 nm) eluted with 70% acetonitrile to afford 4 (t_R 11.7 min, 6.8 mg) and 3 (t_R 13.5 min, 7.1 mg). Fr.10 (108.0 mg) was separated by MPLC over RP-18 silica gel (80 g) to afford Fr.10a–10j. Fr.10e was purified by HPLC (SunFire Prep C₁₈ OBD, 19 × 150 mm, 15 ml min⁻¹, UV 365 nm) eluted with 40% acetonitrile to yield 1 (t_R 7.1 min, 29.2 mg) and 2 (t_R 9.2 min, 20.1 mg), respectively.

Nocazine G (2) was obtained as a yellow amorphous powder. Its molecular formula was determined as C₂₁H₂₀N₂O₄ according to its HRESI-MS at m/z=365.1503 [M+H]⁺ (calcd 365.1496). The NMR spectra of 2 were very similar to those of nocazine A.⁸ The difference is that the 18-OCH₃ in nocazine A was replaced by 18-OH in 2. The Z configurations of both Δ⁶ and Δ³⁽¹⁴⁾ double bonds were determined by the NOE correlations between 5-OCH₃ (δ 4.04) and H-20 (δ 8.05), 1-N-CH₃ (δ 3.00), and H-13 (δ 7.19). Furthermore, the Z configurations of the two double bonds in 2 also can be deduced from the similarity of the chemical shifts of H-7 (δ 6.80) and H-14 (δ 7.22) to those of nocazine A and B⁸ (in CDCl₃). Therefore, compound 2 was determined as 3-((Z)-4-hydroxybenzylidene)-5-methoxy-6-((Z)-4-methoxybenzylidene)-1-methyl-3,6-dihydropyrazin-2(1H)-1.

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