Introduction

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, as it can kill tumor cells selectively.1 TRAIL-induced apoptosis initiated by the death receptor pathway involves the engagement of death receptors, formation of a death-inducing signaling complex, proteolytic activation of caspase-8 and, consequently, activation of caspase-3. However, considerable numbers of cancer cells are resistant to TRAIL. Overcoming TRAIL resistance and understanding the mechanisms underlying such resistance are, therefore, very important in anticancer drug discovery.2

In the course of our screening program for bioactive natural products from actinomycetes,3, 4, 5 we collected soil and seawater samples from different areas of Japan. The crude extract of terrestrial Streptomyces sp. IFM 11307 drew our attention due to striking yellow bands on TLC, which gave a blue color reaction with 2 N NaOH and strong orange fluorescence under UV light (366 nm). Here, we report the isolation and structural elucidation of four new pyranonaphthaquinones (1–4) and a phenazine derivative (5) from the culture extract of Streptomyces sp. IFM 11307. Compounds 1–5 were evaluated for their activity in overcoming TRAIL resistance in human gastric adenocarcinoma (AGS) cell lines, as we are interested in screening studies targeting signaling molecules related to cancer diseases.6

Materials and methods

General experimental procedures

Optical rotations were measured with a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured by ATR (attenuated total reflection) on a JASCO FT-IR 230 spectrophotometer (JASCO). UV spectra were measured on a Shimadzu UV mini-1240 spectrometer (Shimadzu, Kyoto, Japan). The NMR spectra were recorded on JEOL JNM-A500 and JEOL JNM-ecp600 spectrometers (JEOL, Tokyo, Japan) with a deuterated solvent, the chemical shift of which was used as an internal standard (see supporting information). Mass spectra were recorded on an AccuTOF-T100LP (JEOL) mass spectrometer.

Identification of the Streptomyces sp. IFM 11307

The strain IFM 11307 was isolated on humic acid-vitamin agar,7 from a soil sample collected from Yoro-keikoku, Ichihara-shi, Chiba prefecture, Japan in 2007. It was identified as Streptomyces sp. and deposited at the Medical Mycology Research Center, Chiba University, Japan with the code number IFM 11307. Identification of the strain was carried out by sequence analysis of 16S rRNA gene using the DDBJ-BLAST search.

Fermentation of the Streptomyces sp. IFM 11307

The Streptomyces sp. IFM 11307 was cultivated from the glycerol stock on the Waksman agar medium at 28 °C for 3 days. Pieces of agar (1 cm2) cultures were used to inoculate 4 × 500 cm3 Sakaguchi flasks each containing 100 ml of the Waksman medium under shaking (200 r.p.m.) at 28°C for 5 days. The subculture was used to inoculate 16 × 3 l round flask each containing 750 ml of the Waksman medium under the same conditions.

Extraction and isolation

The culture broth (12 l) was centrifuged at 3500 r.p.m. for 20 min then extracted three times with ethyl acetate. The organic layer was concentrated in vacuo to dryness to give 2.81 g of crude extract. The mycelial cake was extracted three times with acetone. After removal of acetone, the aqueous solution was extracted three times with EtOAc to yield 0.60 g residue. As the TLC of both extracts from the culture filtrate and mycelia showed the same composition, they were combined and concentrated under reduced pressure. The crude extract of Streptomyces sp. IFM 11307 (3.41 g) was subjected to silica gel 60 N flash column chromatography (ϕ 25 × 600 mm) using gradient of CHCl3/MeOH to afford six fractions. Fraction II (224 mg) was subjected to Sephadex LH-20 (GE Healthcare BioScience, Uppsala, Sweden; ϕ 15 × 600 mm, CHCl3/MeOH, 3:2) to give three subfractions Iia–IIc. Subfraction IIb (69 mg) was purified by preparative HPLC (Nomura Chemical Co., Ltd, Seto, Japan; Develosil ODS HG-5, 10 × 250 mm) to give compounds 1 (1.5 mg, Rt=26.5 min) and 3 (2.7 mg, Rt=28.3 min). The mobile phase was gradients of CH3CN/H2O at flow rate of 2.0 ml min−1. Subfraction IIa (35 mg) was applied to preparative TLC (six plates, 20 × 20 cm, CHCl3/5%MeOH) to yield compound 2 (2.4 mg). Compounds 4 (6.5 mg) and 5 (2.9 mg) were isolated from fraction III (130 mg) by Sephadex LH-20 chromatography (ϕ 15 × 600 mm, MeOH) followed by preparative TLC (five plates, 20 × 20 cm, CH2Cl2/15%MeOH). Compound 6 (8.3 mg) was precipitated when acetone was added to fraction IV and the mixture was kept overnight. It was finally purified by Sephadex LH-20 chromatography (ϕ 15 × 600 mm, MeOH).

Fluorometric microculture cytotoxicity assay (FMCA)

AGS cells were seeded in a 96-well culture plate (6 × 103 cells per well) in 200 μl of RPMI medium containing 10% fetal bovine serum. Cells were incubated at 37 °C in a 5% CO2 incubator for 24 h. Then the test samples with or without TRAIL (Wako, Osaka, Japan, 100 ng ml−1) at different doses were added to each well. After 24 h incubation, the cells were washed with phosphate-buffered saline (PBS), and 200 μl of PBS containing fluorescein diacetate (10 μg ml−1) was added to each well. The plates were then incubated at 37 °C for 1 h, and fluorescence was measured in a 96-well scanning spectrofluorometer at 538 nm, following excitation at 485 nm.

(2 S ,9S,10 S ,3′ S ,4′ S ,6′ S )-griseusin E (1): yellow solid; [α]22D+54.9 (c 0.07, MeOH); UV (MeOH) λmax (log ɛ) 424 (3.1), 252 (3.6) and 211 (4.1) nm; CD (MeOH) λext (Δɛ) 452 (+0.2), 381 (+0.1), 334 (1.4), 273 (+4.4) and 214 (+6.9) nm; IR νmax (ATR) ca 3394, 2921, 2852, 1732, 1646, 1456, 1035 and 800 cm−1; 1H and 13C NMR data in Table 1; (+)-HRESIMS m/z 499.1227 [M+Na]+ (calcd. for C23H24O11Na, 499.1216); (−)-HRESIMS m/z 475.1191 [M–H]− (calcd. for C23H23O11, 475.1240).

Table 1 1H and 13C NMR data for compounds 1 and 2
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(2 S ,10 R ,3′ S ,4′ S ,6′ S )-4′-deacetyl-griseusin B methyl ester (2): yellow solid; [α]22D+173.8 (c 0.06, MeOH); UV (MeOH) λmax (log ɛ) 421 (3.5), 249 (4.0) and 211 (4.5) nm; CD (MeOH) λext (Δɛ) 436 (+2.6), 355 (+2.4), 289 (7.2), 252 (+28.1) and 214 (+9.0) nm; IR νmax (ATR) ca 3587, 2917, 2849,1637, 1456, 1275, 1086 and 799 cm−1; 1H and 13C NMR data in Table 1; (+)-HRESIMS m/z 441.1151 [M+Na]+ (calcd. for C21H22O9Na, 441.1162).

(2S,9S,10S,3′S,4′S,6′S)-4′-deacetyl-griseusin A (3): [α]22D+148.2 (c 0.05, MeOH); UV (MeOH) λmax (log ɛ) 421 (3.5), 249 (4.0) and 211 (4.5) nm; CD (MeOH) λext (Δɛ) 459 (+6.1) 365 (+1.4), 320 (13.2), 260 (+48.6) and 213 (+51.3) nm; 1H NMR (600 MHz, CDCl3) δ 11.9 (1H, s, 4-OH), 7.69 (1H, m, H-6), 7.66 (1H, m, H-7 ), 7.30 (1H, d, J=7.4 Hz, H-5), 5.27 (1H, d, J=2.5 Hz, H-9), 4.79 (2H, m, H-3′, H-10), 4.19 (1H, m, H-6′), 4.16 (1H, m, H-4′), 3.03 (1H, dd, J=4.6, 14.0 Hz, Ha-11), 2.75 (1H, d, J=14.0 Hz, Hb-11), 2.07 (1H, m, Ha-5′), 1.93 (1H, ddd, J=2.7, 3.5, 14.1 Hz, Hb-5′), 1.26 (3H, d, J=6.5 Hz, H3-7′); (+)-HRESIMS m/z 425.0811 [M+Na]+ (calcd. for C20H18O9Na, 425.0849).

(2 S ,10 R ,3′ S ,4′ S ,6′ S )-4′-deacetyl-griseusin B (4): yellow solid; [α]22D+162.4 (c 0.03, MeOH); UV (MeOH) λmax (log ɛ) 427 (3.0), 248 (3.9) and 212 (4.2) nm; CD (MeOH) λext (Δɛ) 453 (+0.36), 345 (+0.69), 289 (−1.7), 252 (+7.1) and 213 (+1.6) nm; 1H NMR (600 MHz, CDCl3) δ 12.11 (1H, s, 4-OH), 7.60 (1H, m, H-7), 7.58 (1H, m, H-6), 7.25 (1H, d, J=7.9 Hz, H-5), 4.68 (1H, d, J=3.9 Hz, H-3′), 4.46 (1H, m, H-10), 4.31(1H, m, H-6′), 4.15 (1H, m, H-4′), 2.88 (1H, dd, J=2.8, 17.2 Hz, Ha-9), 2.51 (1H, dd, J=8.1, 17.2 Hz, Hb-9), 2.82 (1H, dd, J=3.1, 15.0 Hz, Ha-11), 2.69 (1H, d, J=15.0 Hz, Hb-11), 2.10 (1H, m, Ha-5′), 1.90 (1H, m, Hb-5′), 1.25 (3H, d, J=6.0 Hz, H3-7′); (+)-HRESIMS m/z 427.0992 [M+Na]+ (calcd. for C20H20O9Na, 427.1005).

Yorophenzine (5): yellow solid; [α]22D+47.3 (c 0.09, MeOH); UV (MeOH) λmax (log ɛ) 449 (3.3), 370 (3.7) and 251 (4.2) nm; CD (MeOH) λext (Δɛ) 287 (−0.77), 258 (−0.14), 244 (+0.15), 225 (−0.33) and 207 (+0.91) nm; IR νmax (ATR) ca 3722, 2921, 2852, 1698, 1456, 1036 and 669 cm−1; 1H NMR (600 MHz, CD3OD) δ 8.80 (1H, d, J=8.5 Hz, H-7), 8.49 (1H, d, J=8.5 Hz, H-9), 8.32 (1H, d, J=7.4 Hz, H-2), 8.08 (1H, t, J=8.5 Hz, H-8), 7.99 (1H, d, J=7.4 Hz, H-3), 4.73 (1H, dd, J=4.3, 8.2 Hz, H-2′), 4.05 (3H, s, 1′-OMe), 3.84 (1H, dd, J=8.2, 14.0 Hz, H-3′β), 3.49 (1H, dd, J=4.3, 14.0 Hz, H-3′α), 1.96 (3H, s, H3-5′) p.p.m.; 13C NMR (125 MHz, CD3OD) δ 173.2 (C-4′), 168.4 (6-COOH), 167.9 (C-1′), 167.7 (1-COOH), 144.8 (C-4), 144.6 (C-9a), 142.7 (C-10a), 139.5 (C-5a), 137.9 (C-7), 138.5 (C-4a), 135.9 (C-9), 135.3 (C-2), 132.4 (C-8), 127.1 (C-3), 127.2 (C-1), 125.4 (C-6), 54.5 (C-2′), 53.1 (1′-OCH3), 35.2 (C-3′), 22.7 (C-5′) p.p.m.; (+)-HRESIMS m/z 466.0692 [M+Na]+ (calcd. for C20H17N3O7SNa, 466.0685); (−)-HRESIMS m/z 442.0695 [M–H]− (calcd. for C20H16N3O7S, 442.0708).

Results and discussion

The producing Streptomyces sp. IFM 11307 was isolated from soil sample collected from Chiba prefecture, Japan. The strain was determined to belong to the genus Streptomyces on the basis of 16S rRNA analyses. It showed 100% identity to Streptomyces fimicarius and 99.8% to Streptomyces griseus. Well-grown agar cultures of Streptomyces sp. IFM 11307 served to inoculate 4 × 500 cm3 Sakaguchi flasks, each containing 100 ml of the Waksman medium.8 The flasks were incubated at 28 °C while shaking at 200 r.p.m. for 5 days. The seed culture (10 ml) was used to inoculate 16 × 3-l flasks, each containing 750 ml of the same medium, which were incubated using similar conditions. After centrifugation and extraction of the culture broth (12 l), working up of the crude extract resulted in the isolation of six compounds (1–6; Figure 1).

Figure 1
figure 1

Structures of isolated compounds 1–6.

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Structure elucidation

Compound 1 was isolated as yellow solid that gave an orange fluorescence under UV light at 366 nm and a blue color reaction with 2 N NaOH. The ESI mass spectrum of compound 1 displayed in the positive ion mode a signal at m/z 499 [M+Na]+. In the negative ion mode m/z 475 [M–H]− was visible, suggesting a molecular weight of 476 Da. The molecular formula was estimated as C23H24O11 from the (+)-HRESI-MS m/z 499.1227 [M+Na]+ (calcd. 499.1216, Δ +1.1 mmu). The UV spectrum of compound 1 showed absorption at λmax 424, 252 and 211 nm, indicating the presence of a peri-hydroxy naphthoquinone moiety. The 1H NMR spectrum of compound 1 was recorded in CD3OD (Table 1) and showed three aromatic protons forming an ABC spin system (H-5, H-6 and H-7; δH 7.21 (d, 8.2), 7.60 (t, 8.2) and 7.54 (d, 8.2)), several oxymethine protons (between δH 5.18 and 4.20), one methoxy group [δH 3.64 (s)], four methylene protons as well as two methyl groups. The 13C NMR spectrum of compound 1 depicted resonance for two quinone carbonyl groups (δC 190.2, 183.9), two ester carbonyl groups (δC 173.1 and 172.9), one oxygenated sp2 carbon (δC 162.9), three sp2 aromatic methine carbons (δC 137.7, 125.7 and 119.7) and four sp2 quaternary carbons (δC 146.9, 140.9, 133.2 and 116.7). In the aliphatic region one quaternary carbon (δC 99.1), five methine carbons connected to oxygen atoms (δC 71.2, 68.9, 68.6, 63.6 and 60.7), one methoxy group (δC 52.3), two methylene signals (δC 37.5 and 36.5) and two methyl groups (δC 21.2 and 20.7) were observed. Analysis of the 2D NMR spectra (1H-1H COSY, HMQC and HMBC) of compound 1 gave two units A and B (Figure 2). The juglone moiety (unit A) in compound 1 was confirmed by the following HMBC correlations: H-7 (δH 7.54) to C-8 (δC 183.9), C-8a (δC 140.9), C-5 (δC 125.7) and C-3a (δC 116.7); H-6 (δH 7.60) to C-4 (δC 162.9) and C-7a (δC 133.2); and H-5 (δH 7.21) to C-3a (δC 116.7) and C-7 (δC 119.7). The partial structure of unit B was indicated by the HMBC couplings from H3-7′ (δH 1.08) to C-6′ (δC 63.6) and C-5′ (δC 37.5); H2-5′ (δH 1.80 and 1.75) to C-3′ (δC 68.6); H-4′ (δH 5.18) to C-2 (δC 99.1); 4′-OCOCH3 (δH 2.00) to the acetate carbonyl group (δC 173.1); H-3′ (δH 4.70) to C-4′ (δC 71.2) and C-2 (δC 99.1); H3-13 (δH 3.64) to C-12 (δC 172.9); and H-10 (δH 4.45) to C-12 (δC 172.9). The 1H-1H COSY NMR correlations between H-9–H-10–H2-11 and H-3′–H-4′–H2-5′–H-6′–H3-7′ supported structure of unit B. The connectivity between units A and B was confirmed by the HMBC correlations of the oxymethine proton H-9 (δH 4.50) to the quinone carbonyl C-8 (δC 183.9) and the two sp2 quaternary carbons C-2a (δC 146.9) and C-8a (δC 140.9). From these observations, the structure of compound 1 was elucidated as shown in Figure 1 and named as griseusin E. Its relative stereochemistry was determined by the NOE and coupling constants (Figure 3). The NOE observed between the vicinal protons H-3′ and H-4′ supported the conformation of C-3′ and C-4′ as depicated in Figure 3. This conformation was supported by the coupling constant of J=4.1 Hz between H-3′ and H-4′. Additionally, NOE correlations between H-4′ and H-5′a, and H-6′ and H-5′b suggested the configuration of the upper pyrane ring in griseusin E (1) shown in Figure 3. The cis relation of H-9 and H-10 was confirmed by the NOE between H-9 and H-10 (J9,10=2.2 Hz). The absolute configuration of griseusin E (1) was determined using CD spectroscopy. It was reported that CD spectral analyses has been used as a tool to assign the absolute configuration of griseusins A and B by comparison with the related (+)-9-deoxygriseusin B and actionorhodin.9, 10 The CD spectrum of compound 1 (see supporting information) showed positive cotton effects at λext 452, 381, 273 and 214 nm and one negative sign at λext 334. In contrast, the structurally related (−)-griseusins A had negative cotton effects at λext 460, 390 and 250 nm and positive sign at λext 289 nm. Furthermore, the optical rotation of compound 1 ([α]22D+54.9) has opposite sign to the reported value of griseusin A ([α]23D–147.8).11 These interesting findings suggested that compound 1 is the (+)-enantiomer of the structurally related (−)-griseusin A with configuration of 2S,9S,10S,3′S,4′S,6′S. The absolute configuration of compound 1 was also confirmed by comparing the CD data with different steroisomers of griseusins12 and the synthetically known (+)-griseusin A.13

Figure 2
figure 2

HMBC ( ) and 1H-1H-COSY () NMR correlations of compounds 1, 2 and 5. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Figure 3
figure 3

Selected NOE correlations and coupling constants of compounds 1 and 2. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Compound 2 was obtained as yellow solid. The molecular weight of compound 2 was determined to be 418 Da from the positive ion mode of (+)-ESI mass spectroscopy. The molecular formula was determined to be C21H22O9 by the (+)-HRESI-MS m/z 441.1151 [M+Na]+ (calcd. 441.1162, Δ –1.1 mmu). The 1H NMR of compound 2 (Table 1) recorded in CDCl3 showed signals assigned to a hydrogen-bonded phenolic hydroxyl group [δH 12.13 (s)] and protons of a 1,2,3-trisubstituted aromatic spin system (H-5, H-6 and H-7; δH 7.24 (d, 7.9), 7.59 (t, 7.9) and 7.56 (d, 7.9)) characteristic of the juglone moiety. In the aliphatic region, four oxymethine protons (H-3′, H-4′, H-6′ and H-10; δH 4.66 (d, 4.2), 4.07 (m), 4.24 (m) and 4.42 (m)), one methoxy group [δH 3.64 (s)], six methylene proton signals (between δH 2.86 and 1.88) as well as one methyl doublet [δH 1.35 (d, 6.3)] were observed. The 13C NMR spectrum of compound 2 revealed the presence of three carbonyls and eight sp2 aromatic carbons. In addition, compound 2 contained 10 sp3 carbons including four oxygenated methines, one ketal, one methoxy group, three methylenes and one methyl signal. It was clear that the NMR data of compound 2 matched those of griseusin E (1). However, the doublet in the proton spectrum of compound 1 at H-9 [δH 4.50 (d, 2.2)] had disappeared and a new resonance arising from 2H was observed [δH 2.86 (dd, 3.2, 16.2) and 2.39 (dd, 11.8, 16.2)]. The corresponding change in the 13C NMR spectrum was a shift from δC 60.7 to 28.4. Furthermore, the acetate group in griseusin E (1) was not observed in compound 2. The connectivity of all protons and carbons was established by 1H-1H COSY, HMQC and HMBC data. The HMBC correlation of the –OCH3 group (δH 3.74) to the carbonyl signal at C-12 (δC 171.1) confirmed the presence of the methyl ester group in compound 2. After comparing these data with the literature,11 the constitution of compound 2 was elucidated as the methyl ester of 4′-deacetyl-griseusin B (4). The relative configuration of compound 2 was established by analyses of the NOE spectra. The relative stereochemistry at C-3′, C-4′, C-6′ and C-10 was assigned on the basis of the NOE correlations between H-3′ and H-4′ (J3′,4′=4.1 Hz), H-4′ and H-5′b, H-6′ and H-5′a, and H-10 and H-9 (Figure 3). The specific rotation value ([α]22D+173.8) and the CD spectrum of compound 2 (see supporting information) were of the opposite sign to that of 4′-deacetyl-griseusin B ([α]23D–162).11 These data conclusively reveal that compound 2 is the mirror image of the natural 4′-deacetyl-griseusin B having the 2S,10R,3′S,4′S,6′S configuration.

Compounds 3 and 4 were also isolated as yellow solids. The molecular formula of compound 3 was established as C20H18O9 by the (+)-HRESIMS, which displayed a pseudomolecular ion at m/z 425.0811 [M+Na]+ (Δ −3.7 mmu). Compound 4 had a molecular formula of C20H20O9, revealed from the (+)-HRESIMS m/z 427.0992 [M+Na]+ (calcd. 427.1005, Δ −1.3 mmu). The spectral data (including NMR and MS) of compounds 3 and 4 were identical with those of 4′-deacetyl-griseusin A and B, respectively, which indicated the same relative stereochemistry. A further interesting observation was that compounds 3 and 4 also possess opposite signs of optical rotation values and CD spectra to those of (−)-4′-deacetyl-griseusin A and B. Therefore, compounds 3 and 4 are the (+)-enantiomers of the natural 4′-deacetyl-griseusin A and B and have configurations of 2S,9S,10S,3′S,4′S,6′S and 2S,10R,3′S,4′S,6′S, respectively. According to the literature, compounds 1–4 belong to the family of pyranonaphthoquinone antibiotics that containing a 1,7-dioxaspiro [5.5] undecane ring system fused to a juglone moiety.14 Several griseusins such as A–D and their derivatives have been isolated from alkaphilic Nocardiopsis sp. and S. griseus.12, 15 To the best of our knowledge, the isolated compounds 1–4 are new members, with (+) enantiomer, of this class of natural products.

Compound 5 was obtained as well from fraction III as an optically active ([α]22D+47.3) yellow solid. It gave a positive color reaction with the Dragendorff's reagent and fluorescence under UV light at 254 nm. The molecular formula was determined as C20H17N3O7S by the (–)-HRESIMS m/z 442.0695 [M–H]− (calcd. 442.0708, Δ–1.4 mmu). Compound 5 was rapidly identified as phenazine class antibiotic by considering the UV data (maxima were visible at 449, 370 and 251 nm) and the characteristic low-field chemical shifts of the aromatic protons.3 The 1H NMR spectrum of compound 5 in CD3OD exhibited two ortho-coupled aromatic protons (δH 8.32 (1H, d, J=7.4 Hz) and 7.99 (1H, d, J=7.4 Hz)) and one 1,2,3-trisubstituted aromatic spin system (δH 8.80 (1H, d, J=8.5 Hz), 8.49 (1H, d, J=8.5 Hz) and 8.08 (1H, t, J=8.5 Hz)). The 1H-1H COSY NMR correlations between H-2–H-3 and H-7–H-8–H-9 supported this conclusion (Figure 2). The aliphatic region showed one methine signal (δH 4.73 (1H, dd, J=4.3, 8.2 Hz)), one methoxy group (δH 4.02 (3H, s)), one methylene (δH 3.84 (1H, dd, J=8.2, 14.0 Hz), 3.49 (1H, dd, J=4.3, 14.0 Hz)) and one methyl singlet (δH 1.97 (3H, s)). The 13C NMR spectrum of compound 5 revealed four carbonyl groups (δC 173.2, 168.4, 167.9 and 167.7), five sp2 aromatic methine carbons (δC 137.9, 135.9, 135.3, 132.4 and 127.1) and seven sp2 quaternary carbons (δC 144.8, 144.6, 142.7, 139.5, 138.5, 127.2 and 125.4). In the aliphatic pattern, one methine carbon (δC 54.5), one methoxy group (δC 53.1), one methylene signal (δC 35.2) and methyl group (δC 22.7) were observed. Interpretation of the HMBC correlations of compound 5 suggested the presence of para substituted phenazine-1,6-dicarboxylic acid (H-2/C-10a, C-4, 1-COOH; H-3/ C-4a, C-1; H-7/C-9, 6-COOH, C-5a; H-8/C-9a, C-6 and H-9/C-7, C-5a, C-10a). Several biosynthetic studies have demonstrated that phenazine-1,6-dicarboxylic acid is a universal precursor for may phenazine secondary metabolites.16, 17 The HMBC spectrum also showed correlations of the methyl signal (δH 1.96) to C-4′ (δC 173.2) and both of methylene (δH 3.84, 3.49) and methoxy groups (δH 4.05) to C-1′ (δC 167.9). The 1H and 13C chemical shifts in compound 5 suggested the presence of an amino acid. The NMR signals were consistent with an N-acetyl-cysteine methyl ester.18, 19 Because the aromatic proton H-2 (δH 8.32) and H2-3′ (δH 3.84, 3.49) coupled to C-4 (δC 144.8), the N-acetyl-cysteine methyl ester could be located at C-4 position. The absoulte streochemistry of compound 5 was proposed by comparing the CD spectra and the optical rtotation value with that of N-acetyl-L-cysteine and L-cysteine.20 Two positive cotton effects at 207 and 244, and the three negative peaks at 225, 258 and 287 nm confirmed the L-configuration of the N-acetyl-cysteine methyl ester moiety in compound 5. Thus, the structure of compound 5 was determined as shown in Figure 1, which we have named yorophenazine. From the literature, SB 212305 is the only phenazine antibiotic linked to N-acetyl-cysteine.18 The known compound 6 was easily identified as phenazine-1,6-dicarboxylic acid based on the NMR data and by comparison with the reference values.21

Biological activity

We evaluated the bioactivity of compounds 1–5 for their ability to overcome TRAIL resistance in AGS cells. Recently, this cell line has been widely used as a model system for evaluating cancer cell apoptosis and is reported to be refractory to apoptosis induction by TRAIL.22 To assess effects of the isolated secondary metabolites on cell viability in the presence and absence of TRAIL, AGS cells were treated with the indicated agents and subjected to the FMCA method.23 Luteolin was used as a positive control, producing about 44% more inhibition along with TRAIL than the agent alone at 17.5 μM.24 The assay results (Figure 4) showed that compound 1 at 0.5 and 1.5 μM exhibited 20% and 23% decreases, respectively, in cell viability in the presence of TRAIL (100 ng ml−1) compared with in the absence of TRAIL. Compound 2 at 0.1 and 0.5 μM caused 28% and 27% decreases, respectively, in cell viability in the presence of TRAIL (100 ng ml−1). Compound 3 at 0.1 μM proved to be the most active in this series, with 33% decrease in cell viability in the presence of TRAIL (100 ng ml−1). Combined treatment of TRAIL and compound 4 at 0.5 μM resulted in 19% more inhibition than the agent alone. Compound 5, however, did not produce any significant reduction in cell viability with TRAIL. These results suggest that pyranonaphthoquinones 1–4 had a synergistic effect in combination with TRAIL in AGS cell lines.

Figure 4
figure 4

Effect of the isolated compounds 1–4 on the cell viability of AGS cells in the presence and absence of TRAIL. The s.e. bar represents the means (n=3±s.d.). Abbreviations: AGS, human gastric adenocarcinoma; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. A full color version of this figure is available at The Journal of Antibiotics journal online.

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