Violapyrones H and I, new cytotoxic compounds isolated from Streptomyces sp. associated with the marine starfish Acanthaster planci.

Two new α-pyrone derivatives, violapyrones H (1) and I (2), along with known violapyrones B (3) and C (4) were isolated from the fermentation broth of a marine actinomycete Streptomyces sp. The strain was derived from a crown-of-thorns starfish, Acanthaster planci, collected from Chuuk, Federated States of Micronesia. The structures of violapyrones were elucidated by the analysis of 1D and 2D NMR and HR-ESIMS data. Violapyrones (1-4) exhibited cytotoxicity against 10 human cancer cell lines with GI50 values of 1.10-26.12 μg/mL when tested using sulforhodamine B (SRB) assay. This is the first report on the cytotoxicity of violapyrones against cancer cell lines and the absolute configuration of violapyrone C.

1. Introduction

Marine actinomycetes, isolated from the surface of marine algae and invertebrates, have received increased attention as a potential source because they produce a variety of new bioactive secondary metabolites compared to terrestrial microorganisms [1,2]. As a part of our ongoing research for the discovery of bioactive metabolites from marine bacteria, we isolated a marine actinomycete Streptomyces sp. 112CH148 from a crown-of-thorns starfish, Acanthaster planci. A. planci has a long history in the scientific literature but only few studies have been done on its microbial symbionts [3,4,5]. We tried to isolate bioactive strains from the starfish and found that among the isolates, the strain 112CH148 produces unusual 3,4,6-trisubstituted α-pyrone derivatives.

α-Pyrones are an important class of lactones having a broad spectrum of biological activities, such as potent anticancer [6], antimicrobial [7,8], antifungal [9,10], antioxidant [11,12,13], androgen like [14], HIV-1 protease inhibitory [15,16] and pheromonal effects [17]. Here, we report the isolation, structure determination of the new 3,4,6-trisubstituted α-pyrone derivatives, violapyrones H (1) and I (2), and the cytotoxicity of violapyrones (1–4) (Figure 1).

Figure 1

Structures of violapyrones H (1), I (2), B (3) and C (4).

2. Results and Discussion

2.1. Isolation of Compounds

The bacterial strain 112CH148 was isolated from the crown-of-thorns starfish, Acanthaster planci, collected from Chuuk, Federated States of Micronesia and identified as Streptomyces sp. by 16S rRNA sequencing. The strain was cultured in Bennett’s medium (salinity 32 g/L, pH 7.02 before sterilization) at 28 °C for 7 days. Then, the fermentation broth was extracted with EtOAc. Thereafter, two new violapyrones (1,2) and two known violapyrones (3,4) were isolated from the EtOAc extract by stepwise gradient open column chromatography followed by reversed-phase HPLC separations.

2.2. Structure Determination

Violapyrone H (1) was isolated as a yellowish, amorphous solid. The molecular formula C14H22O3 was deduced from the [M + Na]+ peak at m/z 261.1466 (calcd for 261.1467) in the HR-ESIMS, which required four degrees of unsaturation. The IR absorptions at 3341 and 1674 cm−1 indicated the presence of hydroxyl (OH) and carbonyl (CO) groups, respectively. The UV maximum at 290 nm and 13C NMR data indicated typical α-pyrone moiety [12,18]. The 13C NMR and HSQC spectra displayed three oxygenated quaternary carbons (δC 164.8–169.9), an olefinic methine carbon (δC 102.0), an sp2 quaternary carbon (δC 98.7), five methylene carbons (δC 28.1–40.1), an sp3 methine carbon (δC 29.7), a methyl carbon (δC 8.4) and an isomethyl carbon (δC 23.1) (Table 1). Analysis of the 1H–1H COSY spectrum suggested two spin systems: one from H2-7 at δH 2.46 to H2-8 at δH 1.64 and another from H2-9 at δH 1.34 to H3-13 at δH 0.88. Their connectivity with C-9 (δC 30.4) was established by a long-range HMBC correlation of H2-8 with C-9 (Figure 2), constructing an aliphatic chain. The position of the methyl group (δH 1.84, s) at C-3 was readily determined by its HMBC correlations with two oxygenated quaternary carbons C-2 (δC 169.9) and C-4 (δC 169.5), and as well as with the quaternary carbon C-3 (δC 98.7). Similarly, the olefinic methine proton (δH 5.96, s, H-5) showed HMBC cross-peaks with C-3, C-4, C-6 and C-7. From these HMBC correlations, together with the fact that 1 needed to form a ring to satisfy the unsaturation number, an α-pyrone ring was constructed (Figure 2). In addition, the HMBC correlation between H-5 and C-7 confirmed the connectivity of the α-pyrone ring to the aliphatic chain (Figure 2). From these data analysis, the structure of 1 was determined as a previously unreported 3,4,6-trisubstituted α-pyrone, and 1 was named violapyrone H.

Table 1

1H and 13C NMR data of 1 and 2 in CD3OD.

Position	1		2
	δC, Type	δH, Mult. (J in Hz)	δC, Type	δH, Mult. (J in Hz)
2	169.9, C		169.4, C
3	98.7, C		98.9, C
4	169.5, C		168.9, C
5	102.0, CH	5.96, s	101.6, CH	5.97, s
6	164.8, C		164.9, C
7	34.4, CH2	2.46, t (7.5)	34.4, CH2	2.46, t (7.5)
8	28.1, CH2	1.64, m	28.1, CH2	1.64, m
9	30.4, CH2	1.34	30.3, CH2	1.33
10	28.3, CH2	1.34	30.2, CH2	1.35
11	40.1, CH2	1.19, m	33.0, CH2	1.30, m
12	29.7, CH	1.53, m	23.8, CH2	1.31, m
13	23.1, CH3 (× 2)	0.88, d (6.5)	14.5, CH3	0.90, t (6.5)
3-Me	8.4, CH3	1.84, s	8.4, CH3	1.85, s

Figure 2

Key HMBC and COSY correlations of 1 in CD3OD.

Violapyrone I (2) was also obtained as a yellowish amorphous solid and the molecular formula was determined to be C13H20O3 from the [M + Na]+ peak at m/z 247.1313 (calcd for 247.1310) in the HR-ESIMS. Preliminary, the NMR analysis showed a close similarity between the spectra of 1 and 2 (Table 1). However, the differences between these compounds were figured out from the molecular weight (CH2 less than 1) and the observation of different splitting pattern of the methyl signal H-13 at δH 0.90 (t, 6.5 Hz). In addition, a lack of one methyl carbon was observed in the 13C NMR data of 2 compare to 1. A detailed analysis of 1D and 2D spectra of 2 revealed the existence of a rigid α-pyrone ring same to 1. Furthermore, an aliphatic chain was assigned by COSY and HMBC correlations, consisting of 6 methylenes with a terminal methyl proton H-13 resonated at δH 0.90 (t, 6.5 Hz). Finally, the aliphatic chain was connected to the α-pyrone ring and complete assignments of the atoms in the structure of violapyrone I (2) were achieved by 1H–1H COSY and HMBC experiments. Thus, the structure of 2 was determined as a new 3,4,6-trisubstituted α-pyrone, and 2 was named violapyrone I.

The structures of 3 and 4 were determined straightforward as they were very similar to those of 1 and 2, and identified as the previously reported violapyrones B and C, respectively, by the comparison of their NMR results, MS data and optical rotation values with the literature (Supplementary Information) [18]. However, the absolute stereochemistry at C-11 of violapyrone C (4) was not determined in the previous report [18]. To determine the stereochemistry of C-11 in 4, we synthesized both (S)- and (R)-violapyrones C [19]. Optical rotation values of (S)- and (R)-violapyrones C were [α]D27 +49° (c 0.1, MeOH) and [α]D27 −53° (c 0.1, MeOH), respectively. The absolute stereochemistry of C-11 in violapyrone C (4) was determined to be S, because the optical rotation value [α]D27 +50° (c 0.1, MeOH) and 1H and 13C NMR data were consistent with those of synthetic (S)-violapyrone C (Supplementary Information).

2.3. Cytotoxic Properties

The cytotoxicity of violapyrones H (1), I (2), B (3) and C (4) was assessed by sulforhodamine B (SRB) assay [20] using human cancer cell lines. Violapyrones (1–4) showed growth inhibitory activity against cancer cell lines at the concentrations less than 26.12 μg/mL (Table 2). Recently, violapyrones (A–G) were reported to have antibacterial activities, but did not show any cytotoxicity against five cancer cell lines (BGC-823, gastric carcinoma; Hep-G2, liver carcinoma; NCI-H460, lung carcinoma; HeLa, cervical carcinoma; HCT-116, colon carcinoma) when tested using MTT method [18]. However, we found the cytotoxicity of violapyrones H (1) and I (2) as well as B (3) and C (4) against human cancer cell lines (HeLa; ACHN, renal carcinoma; HCT-15 and HCT-116, colon carcinomas; MDA-MB-231, breast carcinoma; NCI-H23 and NCI-H460, lung carcinomas; NUGC-3, stomach carcinoma; Hep-G2; PC-3, prostate carcinoma). Especially, compound 1 showed the highest activity against HCT-15 cell line with a GI50 value of 1.10 μg/mL. Moreover, it may be noteworthy that each compound has structural similarity, but showed different activities. Our results suggested that the length of the aliphatic side chain and the position of the methyl group affected the activity. Furthermore, violapyrones having an isomethyl group in the alkyl side chain showed better activity than others. Violapyrones (A–G) also showed quite similar tendency in their antibacterial activities [18].

Table 2

Growth Inhibition (GI50, μg/mL) of 1–4 against a Panel of Human Tumor Cell Lines.

Cell Lines	GI50a (μg/mL)
	1	2	3	4	ADR b
Cervical cancer: HeLa	25.05	5.54	18.12	9.91	0.09
Renal cancer: ACHN	1.79	5.42	1.18	1.55	0.04
Colon cancer: HCT-15	1.10	3.38	2.01	5.22	0.08
Colon cancer: HCT-116	8.99	18.08	15.83	26.12	0.09
Breast cancer: MDA-MB-231	1.51	6.29	1.80	4.94	0.99
Lung cancer: NCI-H23	1.24	3.47	1.90	3.24	0.04
Lung cancer: NCI-H460	4.45	21.04	6.37	10.80	0.07
Stomach cancer: NUGC-3	1.27	3.36	2.24	4.02	0.12
Liver cancer: Hep-G2	2.30	14.60	2.04	3.96	0.08
Prostate cancer: PC-3	1.37	5.44	1.40	2.06	0.06

a GI50 values are the concentration corresponding to 50% growth inhibition; b ADR: adriamycin as standard.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotation was measured on a JASCO DIP-1000 digital polarimeter (JASCO Corporation, Tokyo, Japan), with a 1 cm cell. UV spectra were obtained on a Shimadzu UV-1650PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were recorded on a JASCO FT/IR-4100 spectrophotometer, (JASCO Corporation, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra, including 1H–1H COSY, HSQC and HMBC experiments, were collected on a Varian Unity 500 spectrometer (Varian Inc., Palo Alto, CA, USA) operating at 500 MHz (1H) and 125 MHz (13C) with chemical shifts given in ppm (δ). High-resolution ESI mass spectroscopy was recorded on a hybrid ion-trap time-of-flight mass spectrometer (SYNAPT G2, Waters Corporation, Milford, CT, USA). High performance liquid chromatography (HPLC) was conducted with a PrimeLine pump (Analytical Scientific Instruments, Inc., El Sobrante, CA, USA) with RI-71 refractive index detector (Shodex, Shoko Scientific Co. Ltd., Yokohama, Japan). Open column chromatography was carried out over a Pyrex glass (300 mm × 50 mm). RP-C18 silica gel (YMC-Gel ODS-A, 12 nm S-75 μm) was used for column chromatography. All solvents used were either spectral grade of distilled prior to use. Continuous centrifugation was done on a centrifugal separator (Kansai Centrifugal Separator Manufacturing Co. Ltd., Osaka, Japan).

3.2. Isolation and Identification of the Strain 112CH148

The strain designated as 112CH148 was isolated from a crown-of-thorns starfish, Acanthaster planci, collected from Chuuk, Federated States of Micronesia in 2011. A portion of sample was rinsed with sterilized sea water under aseptic condition and then put on Bennett’s agar plates (1% dextrose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, 1.7% agar, salinity 32 g/L, pH 7.02 before sterilization). The plates were incubated for 12 days at 28 °C, and the resulting colony of the strain 112CH148 was isolated and maintained on Bennett’s agar plates. The strain was identified as Streptomyces sp. on the basis of 16S rRNA sequence analysis. The sequence was deposited in the GenBank under the accession number KJ419328. This strain is currently preserved in the Microbial Culture Collection, KIOST, with the name of Streptomyces sp. 112CH148 under the curatorship of Hee Jae Shin.

3.3. Seed and Mass Cultures of the Strain

The seed and mass culture were carried out in Bennett’s medium (1% dextrose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, salinity 32 g/L, pH 7.02 before sterilization). The 200 mL medium was dispensed in a 500 mL conical flask and sterilized. A single colony of the strain from the agar plate was inoculated aseptically into the flask and incubated at 28 °C for 2 days on a rotary shaker at 120 rpm. An aliquot (0.2% v/v) from the seed culture was inoculated aseptically into 2 L flasks (total 24 flasks) containing 1.3 L medium and a 20 L fermenter containing 18 L of sterilized culture medium, respectively. The production culture was incubated under the same conditions as the seed culture for 7 days and then harvested. The mass cultures were carried out three times.

3.4. Extraction and Isolation of Compounds

The culture broth (total 150 L) was harvested by high speed centrifugation (60,000 rpm) and then extracted with EtOAc (2 times). The EtOAc extract was evaporated to obtain crude extract (13.85 g). The crude extract was subjected to ODS open column chromatography followed by stepwise gradient elution with MeOH/H2O (v/v) (1:4, 2:3, 3:2, 4:1 and 100:0) as eluent. The subfraction eluted with MeOH/H2O (4:1) was again applied to an ODS open column chromatography with a MeOH/H2O solvent system (6:4, 7:3, 8:2 and 100:0). The fraction eluted with MeOH/H2O (8:2) was purified by a reversed-phase HPLC (YMC ODS-A column, 250 × 10 mm i.d, 5 μm; 70% MeOH in H2O; flow rate: 2.0 mL/min; detector: RI) to yield pure compounds 1 (1.9 mg, tR 32.5 min) and 4 (9.0 mg, tR 31.0 min). The subfraction eluted with MeOH/H2O (7:3) was also purified by a RP-HPLC (YMC ODS-A column, 250 × 10 mm i.d, 5 μm; 60% MeOH; flow rate: 2.0 mL/min; detector: RI) to get compounds 2 (2.7 mg, tR 30.5 min) and 3 (5.0 mg, tR 34.0 min).

Violapyrone H (1): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 290 (0.52) nm; IR (MeOH) νmax 3341 (br), 2935, 1674 cm−1; 1H and 13C NMR data (CD3OD), Table 1; HR-ESIMS m/z 261.1466 [M + Na]+.

Violapyrone I (2): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 289 (0.70) nm; IR (MeOH) νmax 3345 (br), 2926, 1670 cm−1; 1H and 13C NMR data (CD3OD), Table 1; HR-ESIMS m/z 247.1313 [M + Na]+.

Violapyrone B (3): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 286.5 (1.34) nm; IR (MeOH) νmax 3347 (br), 2943, 1674 cm−1; 1H NMR (CD3OD) δH 5.97 (1H, s, H-5), 2.45 (2H, t, J = 7.5 Hz, H-7), 1.85 (3H, s, Me-3), 1.62 (2H, m, H-8), 1.54 (1H, m, H-11), 1.35 (2H, m, H-9), 1.23 (2H, m, H-10), 0.88 (6H, d, J = 7.0 Hz, H-12); 13C NMR (CD3OD) δC 169.4 (C-2), 168.8 (C-4), 164.9 (C-6), 101.5 (C-5), 98.9 (C-3), 39.9 (C-10), 34.4 (C-7), 29.2 (C-11), 28.3 (C-8), 27.9 (C-9), 23.1 (C-12), 8.4 (Me-3); HR-ESIMS m/z 225.1485 [M + H]+.

Violapyrone C (4): Yellowish amorphous solid; [α]D27 +50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 288.0 (0.91) nm; IR (MeOH) νmax 3343 (br), 2925, 1674 cm−1; 1H NMR (CD3OD) δH; 5.98 (1H, s, H-5), 2.47 (2H, t, J = 7.5 Hz, H-7), 1.85 (3H, s, Me-3), 1.62 (2H, m, H-8), 1.36 (2H, H-9), 1.34 (1H, Ha-12), 1.13 (1H, Hb-12), 1.33 (1H, Ha-10), 1.17 (1H, Hb-10), 1.32 (1H, H-11), 0.88 (3H, t, J = 6.5 Hz, H-13), 0.87 (3H, d, J = 6.0 Hz, Me-11); 13C NMR (CD3OD) δC; 169.5 (C-4), 169.2 (C-2), 164.9 (C-6), 101.8 (C-5), 98.8 (C-3), 37.5 (C-12), 35.7 (C-11), 34.4 (C-7), 30.7 (C-10), 28.4 (C-8), 27.6 (C-9), 19.7 (Me-11), 11.9 (C-13), 8.4 (Me-3); HR-ESIMS m/z 261.1461 [M + Na]+.

3.5. Cytotoxicity Test by SRB Assay

Human cancer cell lines, HeLa (cervix), ACHN (renal), HCT-15 (colon), HCT-116 (colon), MDA-MB-231 (breast), NCI-H23 (lung), NCI-H460 (lung), NUGC-3 (stomach), Hep-G2 (liver) and PC-3 (prostate), were purchased from American Type Culture Collection (Manassas, VA). The cell lines were cultured RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Cell cultures were maintained at 37 °C under a humidified atmosphere of 5% CO2. The growth inhibition assay against human cancer cell lines was carried out according to a sulforhodamine B (SRB) assay [20]. In brief, 8000 cells/well were seeded in a 96-well plate. Next day, the cells were treated with violapyrones H (1), I (2), B (3) and C (4) including vehicle control (0.1% DMSO) and positive control (adriamycin). After being incubated for 48 hours, cultures were fixed with 50% trichloroactetic acid (50 μg/mL) and stained with 0.4% sulforhodamine B in 1% acetic acid. Unbound dye was removed by washing with 1% acetic acid, and protein-bound dye was extracted with 10 mM Tris base (pH 10.5) for determination of optical density. The absorbance at 540 nm was determined using a VersaMax microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). GI50 values were calculated using GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA, USA).

4. Conclusions

As a result, we isolated two new 3,4,6-trisubstituted α-pyrone derivatives, violapyrones H (1) and I (2) and two known B (3) and C (4), from the culture broth of Streptomyces sp. 112CH148. These violapyrones exhibited the cytotoxicity against 10 human cancer cell lines (HeLa, ACHN, HCT-15, HCT-116, MDA-MB-231, NCI-H23, NCI-H460, NUGC-3, Hep-G2 and PC-3). Consequentially, these compounds could be new frontiers for the development of anticancer agents. Further studies are needed to clearly elucidate the mechanism of structure-activity relationship.