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, 4, 5, 6, 7, 8, 9, 10, 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.

Figure 1
figure 1

(a) Structure of JBIR-52 (1) (b) correlations in DQF-COSY (bold line) and constant time heteronuclear multiple bond correlation (arrow) spectra of 1.

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Streptomyces sp. ML55 was cultured on a rotary shaker (220 r.p.m.) at 27 °C 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 ([α]D +40.0, c 0.07, 29 °C in MeOH, UV (MeOH) λmax (ɛ) 225 (22 500), 336 nm (4050)). The IR spectrum of 1 revealed the characteristic absorptions of esters (νmax 1750, 1280 cm−1), amide (νmax 1645 cm−1), hydroxyl and/or amide NH (νmax 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.

Table 1 13C and 1H NMR data for 1
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The sequence from an oxymethine proton 2-H (δH 5.46) to 11-H (δH 1.64), which in turn coupled to two methyl protons 12-H (δH 0.95) and 13-H (δH 0.94), through 10-H (δH 1.84, 1.72) in the DQF-COSY spectrum established a 3-methyl-1-oxybutyl moiety. A doublet methyl proton 27-H (δH 1.33) and a low-field shifted methine proton 9-H (δH 3.32), which were spin–spin coupled to each other, were each long-range coupled to an ester carbonyl carbon C-8 (δC 170.2) and a ketone carbonyl carbon C-1 (δC 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 (δH 8.58), 23-H (δH 6.97) and 24-H (δH 7.36) indicated the presence of a 1,2,3-trisubstituted benzene ring moiety. An amide proton 21-NH (δH 7.97) was coupled to an aldehyde proton 25-H (δH 8.52), which was considered to connect directly with this amide nitrogen atom from its 13C chemical shift (δC 159.4). The aldehyde proton and the aromatic proton 23-H were long-range coupled to an aromatic carbon C-21 (δC 127.2). These results suggested that a formamide group is substituted at the position of C-21. The aromatic proton 24-H was long-rangecoupled to a carbonyl carbon C-18 (δC 170.2) at the peri position, indicating that this carbonyl carbon is substituted at C-19 (δC 112.7). The aromatic protons 22-H and 24-H in addition to a phenolic hydroxyl proton 20-OH (δH 12.58) were long-range coupled to an aromatic carbon C-20 (δC 150.6). By taking into consideration these 13C chemical shifts of aromatic carbons, an oxygen atom should be substituted at the C-20 position. Other 1H–13C long-range couplings (Figure 1b) and the UV spectrum of 1 also suggested the existence of the 3-(formylamino)-2-hydroxybenzoyl moiety, which is the same chromophore as those of the antimycin-related compounds.12, 13, 14, 15

The sequence from an amide proton 6-NH (δH 7.10) to a methyl proton 26-H (δH 1.38) through an α-methine proton 6-H (δH 5.29) and an oxymethine proton 7-H (δH 5.60) was observed in the DQF-COSY spectrum of 1. In addition to these correlations, the long-range couplings from the methine proton 6-H to a carbonyl carbon C-5 (δC 168.8) and the amide carbonyl carbon C-18 were observed. These results established a threonine residue and its connectivity with the chromophore moiety.

The remaining substructure was also determined by the interpretation of 1H–1H spin couplings and 1H–13C long-range couplings as follows. The long-range coupling between an oxymethine proton 4-H (δH 5.30) and an ester carbonyl carbon C-3 (δC 167.7) along with the sequence from 4-H to 16-H (δH 0.94) through 14-H (δH 2.05), which was additionally coupled to a methyl proton 17-H (δH 0.99), and 15-H (δH 1.57, 1.31), established a 3-methyl-2-oxypentanoic acid moiety (Figure 1b). The connectivity of these partial structures was elucidated by the long-range couplings between 2-H and C-3, 4-H and C-5 and 7-H and C-8. In this manner, the planar structure of 1 was determined as shown in Figure 1. JBIR-52 is structurally related to JBIR-06 in which the dimethyl residue is replaced by a methyl residue at the position of C-9.

To evaluate the inhibitory activity of 1 against GRP78 expression induced by 2-deoxyglucose as an ER stress, we used reporter gene assay system utilizing luciferase gene described previously.5 The human fibrosarcoma HT1080 cells transformed with the luciferase reporter gene driven by the GRP78 promoter when treated with 10 mM of 2-deoxyglucose, produced four times more luciferase than did the untreated control. In this evaluation system, 1 reduced the expression of the reporter gene with an IC50 value of 137 nM, which is almost the same as that of JBIR-06 (IC50 value, 262 nM14). Contrary to the weak activity of 12-membered macrocyclic derivatives, 15-membered macrocyclic derivatives such as prunustatin A showed more potent activities (IC50 values, 1.9 nM12). It has been reported that GRP78 protects epithelial cells from ATP depletion.18 These results suggested that the coordinate action of ER and mitochondrial respiration exerts protective action against both ER and mitochondrial stress. Hence, the activities of GRP78 suppression by these compounds were considered to depend on respiratory inhibition due to salicylic acid moiety.14 These results provide us the interesting information that the size of macrocyclic structure determines the inhibitory activity of GRP78 expression by salicylic acid function. Studies in the detailed biological activities are now underway.