Introduction

Exploration of phylogenetically diverse and unique organisms from rare or extreme ecosystems to obtain novel structural patterns with medicinally relevant bioactivities becomes attractive challenge in natural product chemistry. The deep sea has emerged as a new source for the discovery of lead compounds, which may supply as new drug templates against human diseases such as infectious diseases and cancer. Deep-sea organisms survive under extreme conditions in the absence of light, low levels of oxygen and intensely high pressures,1 whereas these extreme conditions are regarded to affect the pathways to derive primary and secondary metabolites to produce a greater genetic diversity and structurally unique metabolites. Recent publication showed that the deep sea is extraordinarily rich in biodiversity with respect to microorganisms. Microorganisms derived from deep-sea have been uncovered as a new source for the discovery of drug lead compounds with the development of methods for sampling, identification and successful culturing technologies. However, a limited number of metabolites have been isolated from truly deep-sea microorganisms until now.2 Genus Shewanella is one of the typical deep-sea bacterial genera.3 Although the ecological and genomic functions of Shewanella species are investigated comprehensively, few report related to the secondary metabolites. Shewanella piezotolerans WP3 is a psychrotolerant and psychrophilic strain obtained from west Pacific deep-sea sediment at a depth of 1914 m.4 It is characterized by growing optimally at 15–20 °C and with a broad pressure optimum extending from atmospheric pressure to about 20 MPa, as well as relatively rapid growth and broad range of physical adaptations.5 In the course of our study on the chemical diversity derived from the microorganisms derived from deep-sea, the bacterium Shewanella piezotolerans WP3 was selected for chemical examination. Herein, we report the isolation, structure elucidation and cytotoxicity of 15 indole alkaloids, including 3 new indole alkaloids to be isolated from the fermentation broth.

Results and Discussion

Structural elucidation of new compounds

Chemical examination of the EtOAc extract of the supernatant of S. piezotolerans WP3 was conducted by chromatographic separation, whereas the spectroscopic data analysis resulted in the identification of 3 new compounds (1a/1b and 2) together with 12 known alkaloids (Figure 1).

Figure 1
figure 1

Structures of alkaloids from S. piezotolerans WP3.

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Shewanellines A and B (1a/1b) were obtained as a pale brown solid. Both compounds showed duplicated NMR spectra with two peaks in chiral-phase HPLC chromatography, assuming a pair of enantiomers. The molecular formula of 1a/1b was determined as C17H14N2O2 from the HR-ESI-MS data (m/z=277.0983 [M–H]−). The 1H NMR spectrum displayed two aromatic ABCD spin systems for two ortho-substituted aromatic rings. In addition, two D2O exchangeable protons at δH=11.09 (br, NH-12) and δH=10.59 (br, NH-1), and an olefinic proton at δH=6.93 (br, H-11) correlating to NH-12 weakly in COSY spectrum were observed. The COSY, HSQC and HMBC correlations enabled to establish an indole moiety and a 2-oxo-indole moiety (Figure 2). Their linkage through C-3 (δC=81.2) and C-10 (δC=112.7) bond was determined by the HMBC correlation between H-11 and C-3. Additional HMBC correlation between the methoxy protons at δH=3.13 (3H, s) and C-3 clarified C-3 to be substituted by a methoxy group. Thus, the structures of 1a/1b were determined as 3-methoxylated analog of 3-hydroxy-3,3′-biindoline-2-one (3).6 The chiral-phase HPLC detection revealed 1a/1b to be an enantiomeric mixture, which was further separated on the semi-preparative chiral-phase HPLC to obtain 1a and 1b. The specific rotation ([α]D=+147.5) of 1a was closely similar to those of known analogs with 3S configuration,7 whereas the specific rotation ([α]D=−146.2) of 1b was compatible to those of 3R analogs with negative specific rotation.8 Thus, 1a was estimated to be 3S configuration, whereas 1b was in agreement with 3R configuration.

Figure 2
figure 2

Key HMBC and COSY correlations of 1a/1b and 2. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Shewanelline C (2) presented as a pair of inseparable rotational isomers. The NMR spectroscopic data enabled to distinguish their structural features, whereas the proton integrates in 1H NMR revealed the ratio of 1:0.96. Compound 2 had a molecular formula of C16H11N3O3 as determined by HR-ESI-MS (m/z=294.0873 [M+H]+). The NMR data of 2a exhibited two ABCD spin systems, which were closely related to those of 1, whereas 2D NMR data analyses declared the structure of 2 consisting with two moieties. One substructure was identified as 3-substituted indoline-2-one on the basis of the HMBC correlations of the aromatic proton at δH=7.11 (d, J=7.2 Hz, H-5) to C-3 (δC=52.6), C-7 (δC=128.5) and C-9 (δC=142.8), the aromatic proton at 6.86 (d, J=7.2 Hz, H-8) to C-4 (δC=125.8) and C-6 (δC=121.4), the NH proton at δH=10.61 (s) to C-2 (δC=173.9), C-3 and C-4, and H-3 (δH=6.30, s) to C-2, C-4 and C-9. The remaining NMR data (Table 1) presented an aromatic ABCD spin system attributed to an ortho-phenyl group. The HMBC correlations from H-14 (δH=8.07, d, J=7.7 Hz) to C-12 (δC=162.6) and C-18 (δC=139.4) and from H-17 (δH=7.27, d, J=8.3 Hz) to C-13 (δC=113.6) in addition to the NOE interaction between H-17 and the second NH (δH=11.86, s) indicated the presence of a quinazoline-2,4-dione moiety. The linkage of the two partial structures through C-3 and N-11 bond was confirmed by the HMBC correlation from H-3 to C-12 and C-10 (δC=148.2) and between H-14 and C-12.

Table 1 1H NMR data (500 MHz, DMSO-d6) for compounds 1a/1b, and 2a/2b, δ in p.p.m., J in Hz
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The NMR data of 2b were closely similar to those of 2a, except for the downfield shifted C-10 (δC=150.6) and C-3 (δC=53.5) and the upfield shifted C-12 (δC=160.3) in comparison with the corresponding carbons of 2a. Analyses of 1D and 2D NMR data including the NOE interaction between H-17 (δH=7.27, d, J=8.3 Hz) and the second NH (δH=11.58, s) revealed the gross structure of 2b to be the same as that of 2a. The chiral-phase HPLC analysis revealed 2a and 2b presenting two shoulder peaks, which were inter-exchangeable during separation. Thus, the NMR distinction between 2a and 2b was assumed to be derived from the axial chirality of C3-N11 bond. This was partly supported by the NOE interaction between HN-19 and H-5 in 2b, which was absent in 2a (Figure 3). Based on the optical rotation computation,9 the specific rotation of 2 at [α]D25=–125.4 (c=0.12, MeOH) was compatible to the value of −182 for 3R congener as calculated at the B3LYP/aug-cc-pVDZ / /B3LYP/6-31G(d) levels. Thus, the absolute configuration of 2 was estimated to be 3R.

Figure 3
figure 3

The NOE differentiation between 2a and 2b. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Based on the spectroscopic comparison (Supporting Information), the known compounds were identical to isatin (5),10 (S)-3-hydroxy-3,3′-biindoline-2-one (3),6 trisindoline (4),11 quinazoline-2,4-dione (6),12 spiro[indoline-3,2′-quinazoline]-2,4′(3′H)-dione (7),13 isatan (8), isoindigo (9),14 3-(2-oxopropyl)-3-hydroxyindolin-2-one (10),15 (Z) and (E) 3-(2-methylpropylidene)-indol-2-one (11/12).16

Compounds 13 and 14 were a pair of inseparable mixture with the ratio of 10:1. However, the NMR spectra are able to distinct both compounds. The 1H MMR spectrum of 13 exhibited an aromatic ABCD spin system at δH=7.59 (1H, d, J=7.4 Hz, H-5), 6.97 (1H, dd, J=7.5, 7.4 Hz, H-6), 7.21 (1H, dd, J=7.7, 7.5 Hz, H-7) and 6.85 (1H, d, J=7.7 Hz, H-8), an olefinic proton at δH=6.58 (1H, d, J=10.4 Hz, H-10), a methine at δH=3.04 (1H, m, H-11), a methylene at δH=1.54 (1H, m, H-12a) and 1.48 (1H, m, H-12b), and two methyl at δH=1.12 (3H, d, J=6.6 Hz, H-14) and 0.88 (3H, t, J=7.5 Hz, H-13), in addition to a NH proton at δH=10.49 (1H, s, NH-1). The COSY correlations from H-11 to H3-14 and H2-12 and between H2-12 and H3-13 established a butyl group, which was linked to an olefinic carbon C-10 (δC=146.1) on the basis of the COSY correlation between H-11 and H-10. The presence of a 3-substituted indoline-2-one was evident from the ABCD spin system for an ortho-phenyl group, in association with the HMBC interactions from NH-1 to C-2 (δC=168.2), C-3 (δC=127.2), C-4 (δC=121.8), C-8 (δC=109.8) and C-9 (δC=142.2), and from H-5 to C-3 and C-8. The linkage of C-10 to C-3 through an olefinic bond was based on additional HMBC interactions from H-10 to C-2 and C-3. The NOE interaction between H-11 and H-5 was indicative of 3E geometry.

Analyses of 1D and 2D NMR data and comparison of the NMR data revealed 14 possessing the same gross structure as 13. The distinction was attributed to the NOE interaction between H-5 (δH=7.50, d, J=7.4 Hz) and H-10 (δH=6.79) instead of the interaction between H-5 and H-11, confirming 14 to be a 3Z isomer. Literature survey informed that 13 was documented by a patent cooperation treaty (PCT) without detailed structure and lacking chemical and physical properties,17 whereas the NMR data of 14 differed from those reported for (3′-methylbutylidene)-2-indolinone.18 Thus, compounds 13 and 14 were regarded as new natural products.

Compounds 3, 7, 8, 10 and 11 were isolated for nature for the first time. Application of 2D NMR data, the previously unreported 1H and 13C NMR data of 11–14 were assigned.

Cytotoxic results

Compounds 1–4, 7, 9 and 11–14 were selected to test for the cytotoxicities toward the human promyelocytic leukemia cell line (HL-60) and human hepatocellular carcinoma cell line (Bel-7402). Primary assay of the compounds with the concentration of 25 μg ml−1 revealed compounds 2, 4 and 9 showing potent inhibition against HL-60, whereas their IC50 values were presented in Table 2. It is noted that compounds 2 and 4 also exhibited significant inhibition against Bel-7402, whereas 9 showed weak inhibition against Bel-7402.

Table 2 IC50 values of 2, 4 and 9 toward HL-60 and Bel-7402 (24 h)a
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Methods

General experiments

Optical rotations were recorded on a Rudolph Research Analytical AUTOPOL IV polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). 1D and 2D NMR spectra (chemical shifts in p.p.m., coupling constants in Hz) were recorded on Bruker Avance DRX 500 NMR spectrometer (Bruker, Billerica, MA, USA) using DMSO-d6 as solvent. NMR spectra were referenced to the solvent signal. ESI mass spectra were obtained on a ThermoFinnigan LTQ Orbitrap-XL mass spectrometer (Thermo Finnigan, Waltham, MA, USA). HR-ESI-MS spectra were determined on a ThermoFinnigan LTQ-Orbitrap FT-ESI-MS. For HPLC analysis, 20 μl samples were injected into a Shimadzu LC-20AD HPLC system equipped with a SPD-M20A detector (Shimadzu, Kyoto, Japan), employing a linear gradient from 0.1% phosphoric acid to MeOH (HPLC grade) at 40 min. Routine detection was at 254 nm. A Shim-pack VP-ODS column (150 × 4.6 mm, Shimadzu) was used as the separation column, and the temperature of the column oven was set at 25 °C. Semi-preparative HPLC was performed on Alliance Series III pump, Model 201 UV detector, and Grace Allsphere ODS-2 5 μm column (250 × 10 mm) (Alltech, Lexington, KY, USA). TLC was performed on TLC plates precoated with Si 60 F254 (Merck, Darmstadt, Germany). The compounds were detected from their UV absorbance and by spraying the TLC plates with anisaldehyde reagent.

Taxonomic studies

Shewanella piezotolerans WP3 was isolated from west Pacific deep-sea sediment (142° 30′ 08′′ E, 8° 00′ 11′′ N) at a depth of 1914 m. The species was identified by Dr Xiao3 through 16S rDNA detection.

Fermentation

The modified marine 2216E broth (5 g l−1 tryptone, 1 g l−1 yeast extract, 0.1 g l−1 FePO4, 34 g l−1 NaCl) was used for cultivation and fermentation. Shewanella piezotolerans WP3 was inoculated into a 1000-ml Erlenmeyer flask containing 400 ml 2216E medium and cultured at 20 °C for 24 h at 200 r.p.m. Then, 3000 ml of the resultant seed culture was pumped into a 100-l fermentor (Model D-100, B.Braun Biotech, Melsungen, Germany) with a working volume of 70 l for 48 h with a 0.6 v/v/m air flow rate at the same conditions.

Extraction and isolation

A 70 l of culture broth of S. piezotolerans WP3 was separated by centrifugation into the mycelia and supernatant. The supernatant was exhaustively extracted with EtOAc, and the total extract was concentrated in vacuum to yield a brown gum (10.84 g). The EtOAc extract was separated by MPLC with gradient elution using octadecylsilane (ODS) as stationary phase and MeOH/H2O as eluent to afford five fractions F1-F5. Fraction F2 (348.6 mg) was separated upon semi-preparative HPLC with a mobile phase of MeOH:H2O (7:3) to yield a mixture of 11/12 (3.5 mg), a mixture of 13/14 (2.5 mg), 4 (2.8 mg) and 9 (1.9 mg). Fraction F1 (431 mg) was followed by the same separation protocol as that for F2 with a mobile phase of MeOH:H2O (1:1) to obtain 7 (4.8 mg), 2 (4.6 mg), 3 (9.6 mg) and a mixture of 1a/1b (2.3 mg). Fraction F3 (200 mg) was separated upon ODS column eluted with MeOH:H2O (4:6) to afford 5 (6.8 mg), 6 (6.3 mg), 10 (1.4 mg) and 8 (3.1 mg). Compounds 1a/1b were further separated by chiral phase semi-preparative HPLC (chiralcel OD-RH, 0.46 × 15 cm) with MeOH-H2O (1:3) containing 0.04% trifluoroacetic acid (TFA) as a mobile phase to obtain 1a (Rt 32 min, 0.7 mg) and 1b (Rt 35 min, 0.5 mg).

Spectroscopic data

Shewanelline A (1a): amorphous solid; [α]D25=+147.5 (c=0.10, MeOH); UV (MeOH/H2O) λmax=215, 261 nm; 1H and 13C NMR data, see Table 1; ESI-MS m/z=301.1 [M+Na]+; HR-ESI-MS m/z=277.0983 [M–H]– (277.0977 calcd for C17H13O2N2).

Shewanelline B (1b): amorphous solid; [α]D25=−146.2 (c=0.09, MeOH); UV (MeOH/H2O) λmax=215, 261 nm; 1H and 13C NMR data, see Table 1; ESI-MS m/z=301.1 [M+Na]+; HR-ESI-MS m/z=277.0983 [M–H]– (277.0977 calcd for C17H13O2N2).

Shewanelline C (2): amorphous solid; the mixture rate (1:0.96); [α]D25=–125.4 (c=0.12, MeOH); UV (MeOH/H2O) λmax=199, 220, 311 nm; 1H and 13C NMR data, see Table 1; ESI-MS m/z=294.1 [M+H]+; HR-ESI-MS m/z=294.0873 [M+H]+ (294.0879 calcd for C16H12O3N3).

Isatan (8): amorphous solid; UV (MeOH/H2O) λmax=200, 247, 256, 289 nm; 1H NMR (DMSO-d6, 500 MHz) δ=10.36 (1H, s, NH-1), 10.21 (1H, s, NH-12), 7.50 (1H, d, J=7.5 Hz, H-17), 7.25 (1H, dd, J=7.6, 7.5 Hz, H-15), 7.12 (1H, dd, J=7.6, 7.6 Hz, H-7), 7.00 (1H, dd, J=7.5, 7.5 Hz, H-16), 6.76 (1H, d, J=7.6 Hz, H-14), 6.74 (1H, d, J=7.6 Hz, H-8), 6.64 (1H, dd, J=7.6, 7.2 Hz, H-6), 6.15 (1H, d, J=7.2 Hz, H-5), 3.99 (1H, s, C3-OH), 1.15 (6H, d, J=6.6 Hz, H-12 and H-13); 13C NMR (DMSO-d6, 125 MHz) δ=177.0 (qC, C-2), 174.2 (qC, C-11), 143.3 (qC, C-13), 142.7 (qC, C-9), 129.6 (CH, C-7), 128.4 (CH, C-15), 128.3 (CH, C-4), 126.2 (CH, C-17), 125.8 (qC, C-18), 123.6 (CH, C-5), 121.1 (CH, C-6), 121.0 (CH, C-16), 109.6 (CH, C-8), 109.0 (CH, C-14), 75.5 (qC, C-3), 51.3 (CH, C-10); ESI-MS m/z=210.1 [M+Na]+; HR-ESI-MS m/z=188.1070 [M+H]+ (188.1075 calcd for C12H14ON).

Compounds 11/12: amorphous solid; the mixture rate (10:1); UV (MeOH/H2O) λmax=200, 247, 256, 289 nm; for compound 11, 1H NMR (DMSO-d6, 500 MHz) δ=10.48 (1H, s, NH-1), 7.56 (1H, d, J=7.6 Hz, H-5), 7.21 (1H, dd, J=7.8, 7.7 Hz, H-7), 6.97 (1H, dd, J=7.7, 7.6 Hz, H-6), 6.85 (1H, d, J=7.8 Hz, H-8), 6.61 (1H, d, J=10.0 Hz, H-10), 3.24 (1H, m, H-11), 1.15 (6H, d, J=6.6 Hz, H-12 and H-13); 13C NMR (DMSO-d6, 125 MHz) δ=168.3 (qC, C-2), 146.9 (CH, C-10), 142.1 (qC, C-9), 129.0 (CH, C-7), 126.2 (qC, C-3), 123.6 (CH, C-5), 121.6 (qC, C-4), 121.4 (CH, C-6), 109.8 (CH, C-8), 27.6 (CH, C-11), 21.6 (CH3, C-12 and C-13); ESI-MS m/z=210.1 [M+Na]+; HR-ESI-MS m/z=188.1070 [M+H]+ (188.1075 calcd for C12H14ON). For compound 12, 1H NMR (DMSO-d6, 500 MHz) δ=10.48 (1H, s, NH-1), 7.49 (1H, d, J=7.5 Hz, H-5), 7.16 (1H, dd, J=7.7, 7.6 Hz, H-7), 6.92 (1H, dd, J=7.6, 7.5 Hz, H-6), 6.82 (1H, d, J=10.5 Hz, H-10), 6.78 (1H, d, J=7.7 Hz, H-8), 4.00 (1H, m, H-11), 1.07 (6H, d, J=6.7 Hz, H-12 and H-13); 13C NMR (DMSO-d6, 125 MHz) δ=168.0 (qC, C-2), 148.3 (CH, C-10), 140.4 (qC, C-9), 128.6 (CH, C-7), 126.4 (qC, C-3), 123.5 (qC, C-4), 121.0 (CH, C-6), 119.5 (CH, C-5), 109.3 (CH, C-8), 25.4 (CH, C-11), 22.1 (CH3, C-12 and C-13); ESI-MS m/z=210.1 [M+Na]+; HR-ESI-MS m/z=188.1070 [M+H]+ (188.1075 calcd for C12H14ON).

Compounds 13/14: amorphous solid; the mixture rate (10:1); [α]D25 +6.0 (c 0.20, MeOH) for a mixture with the ratio of 10:1; UV (MeOH/H2O) λmax=199, 248, 256, 289 nm. For compound 13, 1H NMR (DMSO-d6, 500 MHz) δ=10.49 (1H, s, NH-1), 7.59 (1H, d, J=7.4 Hz, H-5), 7.21 (1H, dd, J=7.7, 7.5 Hz, H-7), 6.97 (1H, dd, J=7.5, 7.4 Hz, H-6), 6.85 (1H, d, J=7.7 Hz, H-8), 6.58 (1H, d, J=10.4 Hz, H-10), 3.04 (1H, m, H-11), 1.54 (1H, m, H-12a), 1.48 (1H, m, H-12b), 1.12 (3H, d, J=6.6 Hz, H-14), 0.88 (3H, t, J=7.5 Hz, H-13); 13C NMR (DMSO-d6, 125 MHz) δ=168.2 (qC, C-2), 146.1 (CH, C-10), 142.2 (qC, C-9), 129.0 (CH, C-7), 127.2 (qC, C-3), 121.8 (qC, C-4), 121.4 (CH, C-6), 123.5 (CH, C-5), 109.8 (CH, C-8), 34.4 (CH, C-11), 29.2 (CH2, C-12), 19.3 (CH3, C-14), 11.8 (CH3, C-13); (+)ESI-MS m/z=224.0 [M+Na]+; HR-ESI-MS m/z=202.1226 [M+H]+ (202.1232 calcd for C13H16ON). For compound 14, 1H NMR (DMSO-d6, 500 MHz) δ=10.46 (1H, s, NH-1), 7.50 (1H, d, J=7.4 Hz, H-5), 7.16 (1H, dd, J=8.1, 7.5 Hz, H-7), 6.92 (1H, dd, J=8.1, 7.4 Hz, H-6), 6.78 (1H, d, J=7.5 Hz, H-8), 6.79 (1H, d, J=10.2 Hz, H-10), 3.83 (1H, m, H-11), 1.37 (2H, m, H-12), 1.04 (3H, d, J=6.7 Hz, H-14), 0.84 (3H, t, J=7.4 Hz, H-13); 13C NMR (DMSO-d6, 125 MHz) δ=168.1 (qC, C-2), 147.4 (CH, C-10), 128.6 (CH, C-7), 121.0 (CH, C-6), 119.6 (CH, C-5), 109.3 (CH, C-8), 32.0 (CH, C-11), 29.3 (CH2, C-12), 19.8 (CH3, C-14), 12.1 (CH3, C-13); ESI-MS m/z=224.0 [M+Na]+; HR-ESI-MS m/z=202.1226 [M+H]+ (202.1232 calcd for C13H16ON).

Tumor cells culture

Human promyelocytic leukemia cell line HL60 and human hepatocellular cell line BEL-7402 were purchased from China Center for Type Culture Collection. HL60 cells were cultured in 25 cm2 tissue culture flasks at 37 °C under a humidified 5% CO2 atmosphere in RPMI medium 1640 (Thermo, Waltham, MA, USA) supplemented with 10% fetal bovine serum, penicillin (100 U ml−1) and streptomycin (0.1 mg ml−1). BEL-7402 cells were cultured in 10 cm plates in minimal essential medium at the same condition.

Cytotoxicity assay

The cytotoxicity assay was performed with the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) according to the kit instructions. Briefly, the 96-well tumor cell plates were incubated in the incubator for 48 h after adding different concentration of compound into the culture medium. Then, CCK-8 solution (10 μl) was added into each well of the 96-well plate, while the plates were incubated for 24 h in the incubator and the absorbance at 450 nm was measured using a micro-plate reader. Each experiment was tested in quadruplication, and the data were shown as mean±s.e.m. The inhibition ratio was calculated by the following equation: Inhibition ratio=[(A control–A treated)/A control] × 100. The IC50 was taken as the concentration at which it caused 50% inhibition of cell proliferation (50% reduction in the absorbance value in the treated cells, in respect to the control).