JBIR-52, a new antimycin-like compound, from Streptomyces sp. ML55

Abstract

GRP78/Bip is a molecular chaperone in the endoplasmic reticulum (ER) induced by ER stress that promotes protein folding and has an important role as a survival factor in solid tumors by providing resistance to both chemotherapy and hypoglycemic stress.1 Thus, specific downregulators of GRP78 expression can reasonably be expected to become promising drugs in cancer chemotherapy.2 In the course of our screening program for downregulators of GRP78 expression, we have isolated versipelostatin A-F,3–11 prunustatin A,12,13 JBIR-04, -0514 and JBIR-06.15 Further screening resulted in the isolation of a new inhibitor designated as JBIR-52 (1, Figure 1) from culture of a JBIR-06 producer, Streptomyces sp. ML55.15,16 In this paper, we report the isolation, structure elucidation and brief biological activity of a new member of antimycin, 1. Streptomyces sp. ML55 was cultured on a rotary shaker (220 r.p.m.) at 27 1C for 5 days in 500-ml Erlenmeyer flasks containing 100 ml of a production medium consisting of 2% glycerol (Nacalai Tesque, Kyoto, Japan), 1% molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan), 0.5% casein (Kanto Chemical, Tokyo, Japan), 0.1% polypepton (Nihon Pharmaceutical, Tokyo, Japan), 0.4% CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan) (pH 7.2 before sterilization). The mycelium from the culture broth (2 l) was extracted with Me2CO (400 ml). After concentration in vacuo, the residue was extracted twice with EtOAc. The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo. The dried residue (1.99 g) was applied to normal-phase MPLC (Purif-Pack SI-60, size:60, Moritex, Tokyo, Japan) and developed with a n-hexane–EtOAc linear gradient system (0–100% EtOAc), and peak detection was carried out by UV absorption at 254 nm. The 60–75% EtOAc eluate (470 mg) was further chromatographed on normal-phase MPLC (Purif-Pack SI-60, size:20, Moritex) with n-hexane–EtOAc (80:20). A portion (44.5 mg) of the fraction (388 mg) including both 1 and JBIR-06 was finally purified by preparative reversed-phase HPLC using an L-column2 ODS (20 i.d. 150 mm, Chemical Evaluation Research Institute, Tokyo, Japan) with 60% CH3CN–H2O containing 0.1% formic acid (flow rate, 9.5 ml min 1) to yield 1 (1.7 mg, retention time (Rt) 28.0 min) and JBIR-06 (2.3 mg, Rt 37.0 min). Compound 1 was obtained as a white powder ([a]D +40.0, c 0.07, 29 1C in MeOH, UV (MeOH) lmax (e) 225 (22 500), 336 nm (4050)). The IR spectrum of 1 revealed the characteristic absorptions of esters (nmax 1750, 1280 cm 1), amide (nmax 1645 cm 1), hydroxyl and/or amide NH (nmax 3400 cm 1) groups. The HR electron spray ionization MS spectrum of 1 gave the (M+H)+ ion at m/z 549.2429 (calcd. for C27H37N2O10, 549.2448) consistent with a molecular formula of C27H36N2O10. Direct connectivity between protons and carbons was established by the heteronuclear single quantum coherence spectrum and the 13C and 1H NMR spectral data for 1 are shown in Table 1. The observed double-quantum-filtered (DQF)-COSY and constant time heteronuclear multiple bond correlation (HMBC)17 spectra established four partial structures. The sequence from an oxymethine proton 2-H (dH 5.46) to 11-H (dH 1.64), which in turn coupled to two methyl protons 12-H (dH 0.95) and 13-H (dH 0.94), through 10-H (dH 1.84, 1.72) in the DQF-COSY spectrum established a 3-methyl-1-oxybutyl moiety. A doublet methyl proton 27-H (dH 1.33) and a low-field shifted methine proton 9-H (dH 3.32), which were spin–spin coupled to each other, were each long-range coupled to an ester carbonyl carbon C-8 (dC 170.2) and a ketone carbonyl carbon C-1 (dC 202.1), which in turn long-range coupled to 2-H and 10-H. These HMBC correlations indicated the successive connectivity of C-2, C-1, C-9 and C-8 as shown in Figure 1b. Thus, a 2,6-dimethyl-3-oxo-4-oxyheptanoic acid moiety was elucidated as a partial structure of 1 (Figure 1b). The proton sequence between the aromatic protons 22-H (dH 8.58), 23-H (dH 6.97) and 24-H (dH 7.36) indicated the presence of a 1,2, 3-trisubstituted benzene ring moiety. An amide proton 21-NH (dH 7.97) was coupled to an aldehyde proton 25-H (dH 8.52), which was