Note pubs.acs.org/jnp
Curacin E from the Brittle Star Ophiocoma scolopendrina Reiko Ueoka,† Yuki Hitora,† Akihiro Ito,‡,§ Minoru Yoshida,‡,§ Shigeru Okada,† Kentaro Takada,† and Shigeki Matsunaga*,† †
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan ‡ Chemical Genomics Reserach Group, RIKEN Center for Sustainable Resource Science, Wako Saitama 351-0198, Japan § Chemical Genetics Laboratory, RIKEN, Wako Saitama 351-0198, Japan S Supporting Information *
ABSTRACT: Bioassay-guided fractionation of the extract of the brittle star Ophiocoma scolopendrina afforded curacin E (1), a congener of curacin A (2). Curacin A (2) is an antimitotic agent of cyanobacterial origin. The structure of curacin E was studied by interpretation of NMR data and the ECD spectrum. Curacin E has an ethylcarbonyl terminus in its side chain and inhibits the proliferation of P388 cells.
T
he brittle star Ophiocoma scolopendrina (Lamarck), Ophiocomidae, is widely distributed in shallow waters of tropical and subtropical latitudes of the Indo-Pacific.1 It is a microphagous suspension- and deposit-feeder. On the flooding tide it feeds on particles at the air−water interface, which constitutes its major diet.2 Secondary metabolites with apparent cyanobacterial or other algal origin have been isolated from O. scolopendrina and other ophiuroides.3 It is not known whether these metabolites were procured from the air−water interface or by direct feeding. We isolated ophiodilactones from O. scolopendrina collected at Amami-Oshima Island.4 Ophiodilactones are structurally related to maculatalactones, metabolites of a cyanobacterium.5 In our search for cytotoxic constituents from marine organisms, we found potent activity in the extract of O. scolopendrina collected at Kabira Reef of Ishigaki Island, Okinawa. We have isolated the major cytotoxic constituent and identified the compound as a homologue of curacin A (2), a cytotoxic constituent of cyanobacteria, with unprecedented functionalization at the terminus of the side chain. The organic extract of O. scolopendrina was subjected to solvent−solvent partitioning, ODS column chromatography, and silica gel column chromatography, followed by four rounds of reversed-phase HPLC to afford a minute amount of curacin E (1).
Curacin E had the molecular formula C23H35NO2S as assigned by HRESIMS. The 1H NMR signals of curacin E (1) were well dispersed and exhibited one doublet methyl, one triplet methyl, and six olefinic protons (Table 1). Interpretation of the COSY spectrum in conjunction with the HSQC spectrum gave partial structures a−c (Figure 1). Partial structure a was 2-substituted 1-methylcyclopropane, and partial structure b was an isolated ethyl group attached to an sp2 carbon. In partial structure c, there were a 1,4-disubstituted conjugated diene and a 1,2-disubstituted olefin, which were connected through two contiguous methylenes. The other end of the diene was connected to a linear C4-chain in which the third position was substituted by a heteroatom. The other side of the disubstituted olefin was connected to a methine carbon that was linked to a methylene carbon. The methine was substituted by a heteroatom. Because the presence of partial structures a and c was reminiscent of curacin D,6 we analyzed the HMBC data and found that curacin E (1) was a congener of curacin D whose terminal vinyl group was replaced by an ethylcarbonyl group. The configuration of the C-3 to C-4 double bond was assigned as Z on the basis of the coupling constant of 10.9 Hz between H-3 and H-4. Coupling constants between H-7 and H-8 and between H-9 and H-10 were not determined due to degenerate olefinic proton signals. The 13C chemical shift of 33.4 ppm for C-6 suggested the E-geometry of the C-7 to C-8 double bond.7 The chemical shift of 29.2 ppm for C-11 was an intermediate value for an allylic methylene carbon of an E-olefin (33 ppm) and Z-olefin (27 ppm). Considering the O-methyl substitution at C-13, which causes a deshielding effect of ca. 3 ppm by γgauche interaction to C-11,8 the value of 29.2 ppm was consistent with the allylic carbon of an E-olefin. The carbon chemical shift of 28.5 ppm was reported for the corresponding Received: July 29, 2016 Published: September 29, 2016
© 2016 American Chemical Society and American Society of Pharmacognosy
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DOI: 10.1021/acs.jnatprod.6b00701 J. Nat. Prod. 2016, 79, 2754−2757
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Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Curacin E (1) and Curacin A in CD3OD curacin A
1 position
δC, type
1
40.0, CH2
2 3 4 5 6
74.3, 130.9, 132.9, 28.8, 33.4,
CH CH CH CH2 CH2
7 8 9 10 11
132.3, 132.6, 132.1, 132.9, 29.2,
CH CH CH CH CH2
12
34.7, CH2
13 13-OMe 14
78.1, CH 57.3, CH3 47.7, CH2
15 16
212.6, C 33.7, CH2
17 18 19 20
7.9, 174.8, 20.7, 14.5,
21 22
CH3 C CH CH2
16.2, CH 12.9, CH3
δH (J in Hz) 3.45, 2.95, 5.11, 5.53, 5.59, 2.25, 2.19, 2.15, 5.58, 6.03, 6.02, 5.55, 2.10,
δH (J in Hz)
HMBC
dd (11.0, 8.3) dd (11.0, 10.0) ddd (10.0, 9.2, 8.3) dd (10.9, 9.2) m m m m m m m m q (7.4)
18 2, 3 3, 18 5
4, 5, 7, 8 4, 5, 7, 8 8, 9
8, 9 9, 10, 12, 13
3.44, 2.95, 5.11, 5.53, 5.57, 2.26, 2.22, 2.18, 5.56, 6.25, 5.76,
dd (11.0, 8.3) dd (11.0, 10.0) ddd (10.0, 9.2, 8.3) dd (10.9, 9.2) m m m m m dd (15.1, 10.7) d (10.3)
1.59, m 1.53, m 3.68, tt (6.6, 5.3) 3.28 s 2.67, dd (15.7, 7.4) 2.52, dd (15.7, 5.0)
11, 13, 14 11, 13, 14 OMe 13 13, 15 12, 13, 15
2.49, q (7.3)
15, 17
1.00, t (7.3)
15, 16
2.10, m 2.05, m 1.57, m 1.57, m 3.21, dq (6.4, 5.5) 3.33 s 2.26, m 2.26, m 5.79, ddt (17.1, 10.2, 7.1) 5.05, dq (17.1, 1.7) 5.04, dd (10.2, 2.0) 1.71, s
18, 19, 21, 22 18, 22 19, 20, 21
1.88, 1.04, 0.88, 1.31, 1.08,
1.88, 1.04, 0.87, 1.31, 1.08,
dt (5.7, 8.5) dt (5.7, 8.5) q (5.7) m d (6.3)
dt (5.7, 8.5) dt (4.8, 8.2) q (5.7) m d (6.3)
a13
C chemical shifts were determined by HSQC and HMBC data.
the relative configuration of curacin A (2).11 This assignment was confirmed by an independent degradation study.12 Coupling constants of J1a,2 = 10.0 Hz and J1b,2 = 8.3 Hz in 1 suggested the 2R*,19S*,21R*-relative configuration, which was identical with that in the curacins of cyanobacterial origin.12 Due to the paucity of the sample, we were not able to measure the optical rotation of curacin E (1). Instead we measured the ECD spectrum and observed a positive Cotton effect at 230 nm, which was also observed for curacin A (2), suggesting that the absolute configuration of the two compounds around the thiazoline ring was identical.11 The absolute configuration at C13 of curacin E (1) was not determined. Curacin E (1) exhibited cytotoxicity against P388 murine leukemia cells at concentrations of 20 nM or higher.13 We examined the histone deacetylase (HDAC) inhibitory activity of 1, because the terminal ethylcarbonyl group was considered as a pharmacophore of potent inhibitors of HDAC, e.g., apicidins.14,15 Contrary to our expectations, curacin E inhibits neither HDAC1, HDAC6, nor SIRT1 at concentrations of 50 μM or lower. The biosynthesis of curacin A has been studied by precursor feeding experiments and analysis of its biosynthetic gene cluster.16 These studies demonstrated that curacin A was biosynthesized by a hybrid of NRPS and PKS pathways, and the terminal olefin was produced by decarboxylation with concomitant elimination of a sulfoxyl group.17 The ethylcarbonyl terminal group in curacin E is likely formed by
Figure 1. Partial structures (a−c) of compound 1.
carbon in curacin D, whose 9E-configuration was determined on the basis of the 1H−1H coupling constant. The relative configuration around the cyclopropane ring was assigned by analysis of 1H−1H coupling constants. The value of 8.8 Hz between H-19 and H-21 suggested that they were cis to each other. 9,10 Synthesis of two diastereomers, which correspond to the C-5 to the right portion of curacin A (2), differing in the configuration between the thiazoline moiety and the cyclopropane moiety and their 1H NMR study demonstrated that the two compounds were distinguishable on the basis of the 1H−1H coupling constants between H2-1 and H2.11 The compound with 2S,19S,21R-configuration exhibited J1a,2 = 6.7 Hz and J1b,2 = 8.4 Hz, whereas the one with 2R,19S.21R-configuration exhibited J1a,2 = 9.8 Hz and J1b,2 = 8.3 Hz; the values of the latter compound matched well with the values of the natural product, culminating in the assignment of 2755
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96 h. After addition of 50 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) saline solution (1 mg/mL) to each well, the plate was incubated for 3 h under the same condition to stain live cells. After the incubation, the plate was centrifuged, the supernatants were removed, and the cells were dissolved by addition of 150 μL of DMSO. The absorption at 570 nm was measured with a microplate reader. HDAC Assay. Enzyme inhibitory assays were conducted as described before.21
spontaneous or enzymatic decarboxylation of an α-methyl-βketocarboxylate intermediate (Scheme 1).18 Structure−activity Scheme 1. Proposed Formation of Curacin E (1) by Decarboxylation of a Biosynthetic Intermediate
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00701. NMR and ECD spectra for 1 (PDF)
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relationships of curacin A have been studied by using a large number of synthetic congeners.19,20 It is interesting to point out that none of these synthetic congeners had the ethylcarbonyl terminus in the side chain, emphasizing the richness of the natural product chemical scaffolds.
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AUTHOR INFORMATION
Corresponding Author
*Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166. Email:
[email protected]. Notes
EXPERIMENTAL SECTION
The authors declare no competing financial interest.
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General Experimental Procedures. UV spectra were measured on a Shimadzu BioSpec-1600 spectrophotometer. ECD spectra were recorded in MeOH on a JASCO J-720 spectropolarimeter. NMR spectra were measured on a JEOL alpha 600 NMR spectrometer and referenced to the solvent peak: δH 3.30 and δC 49.0 for CD3OD. ESI mass spectra were recorded on a JEOL JMS-T100LC mass spectrometer. HPLC was carried out on a Shimadzu LC 20AT with an SCL-10Avp controller and an SPD-10Avp detector. Animal Material. The ophiuroid Ophiocoma scolopendrina was collected by hand at Kabira Reef on Ishigaki Island, Okinawa Prefecture, Japan, in 2009, frozen after collection, and kept at −20 °C until used. A voucher specimen was deposited at the Laboratory of Aquatic Natural Products Chemistry with a reference number E09001. Extraction and Isolation. The sample (1.6 kg) was extracted with MeOH (2.5 L × 2) and CHCl3 (2.5 L × 1), and the extracts were combined and concentrated in vacuo. The residue was suspended in H2O (500 mL) and extracted with CHCl3 (500 mL × 3) and n-BuOH (500 mL × 2). The CHCl3 fraction was partitioned between 90% MeOH (400 mL) and n-hexane (500 mL × 2). The 90% MeOH layer was diluted with H2O to yield a 60% MeOH solution and then extracted with CHCl3 (500 mL × 2). The CHCl3 layer (2.0 g) was concentrated and separated by ODS flash chromatography (eluents: 50% MeOH, 70% MeOH, 70% MeCN, 85% MeCN, 100% MeOH, and CHCl3/MeOH/H2O (7:3:0.5)) to give six fractions (A−F). The activity was concentrated in fraction D (85% MeCN fraction) (450 mg), which was separated by silica gel open column chromatography eluting with CHCl3 with increasing amount of MeOH. A fraction that eluted with CHCl3/MeOH (99:1) was subjected to four rounds of reversed-phase HPLC (1, Cosmosil AR-II, 20 × 250 mm with 85% MeOH; 2, Phenomenex Phenylhexyl, 10 × 250 mm with 85% MeOH; 3, Phenomenex Phenylhexyl, 10 × 250 mm with 80% MeCN; 4, Cosmosil π-nap, 4.6 × 250 mm with 60% MeCN) to give 0.2 mg of 1. Curacin E (1): yellow powder; UV (CHCl3) λmax (log ε) 245 (3.5); ECD (0.1 mg/mL, MeOH), λmax (Δε) 230 (0.5) nm, Figure S7; 1H NMR (CD3OD) and 13C NMR (CD3OD) data, Table 1; HRESIMS m/z 390.2466 (calcd for C23H36NO2S, 390.2467). Cell Culture and MTT Assay. P388 murine leukemia cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 μg/mL of kanamycin, and 10 μg/mL of 2-hydroxyethyl disulfide at 37 °C under an atmosphere of 5% CO2. To each well of the 96-well microplate containing 100 μL of tumor cell suspension (1 × 104 cells/ mL) was added 100 μL of test solution dissolved in RPMI-1640 medium, and the plate was incubated in a CO2 incubator at 37 °C for
ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” (23102007) and JSPS KAKENHI Grant Numbers 25252037, 25712024, 25660163, 15K14800, 16H04980, and 15K14799 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Dr. E. Glukhov and Prof. W. Gerwick, University of California, San Diego, Scripps Institution of Oceanography, for an authentic sample of curacin A. We also thank Prof. W. Gerwick for valuable discussions. We are grateful to Ms. A. Nakata, RIKEN, for conducting the enzyme inhibitory assay.
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REFERENCES
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