Note pubs.acs.org/jnp
Sacchathridine A, a Prostaglandin Release Inhibitor from Saccharothrix sp. Koichi Nakae,* Ikuko Kurata, Fukiko Kojima, Masayuki Igarashi, Masaki Hatano, Ryuichi Sawa, Yumiko Kubota, Hayamitsu Adachi, and Akio Nomoto Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan S Supporting Information *
ABSTRACT: Sacchathridine A (1) was isolated from the fermentation broth of strain Saccharothrix sp. MI559-46F5. The structure was determined as a new naphthoquinone derivative with an acetylhydrazino moiety by a combination of NMR, MS spectral analyses, and chemical degradation. Compound 1 showed inhibitory activity of prostaglandin E2 release in a concentration-dependent manner from human synovial sarcoma cells, SW982, with an IC50 value of 1.0 μM, but had no effect on cell growth up to 30 μM.
P
rostanoids are bioactive lipid mediators composed of unsaturated twenty-carbon fatty acids. The biosynthesis of prostanoids involves oxidation and subsequent isomerization of arachidonic acid, which is released from the cell membrane by phospholipase A2 enzymes.1 Arachidonic acid is enzymatically converted to various kinds of prostanoid via cyclooxygenase and prostanoid synthase.2 The released prostanoid binds to its specific G protein-coupled receptor and activates second messengers.3 Prostaglandin E2 (PGE2) is known to play a prominent role in several diseases such as inflammatory disorders, sensory neuron related disorders, and cancer, as well as in maintaining physiological homeostasis.4−7 To discover effective molecules that could be beneficial against these disorders, we conducted a screening program for inhibitors of PGE2 release from natural sources and identified an inhibitory sample from the culture medium of a Saccharothrix sp. In this study, we report the isolation, physicochemical properties, structural determination, and biological activities of sacchathridine A found from this microorganism. The strain Saccharothrix sp. MI559-46F5 was cultured in a 500 mL Erlenmeyer flask containing 110 mL of a seed medium. The inoculated medium was incubated at 27 °C for 48 h on a rotary shaker. This seed culture was used to inoculate into 110 mL of the same medium in a 500 mL Erlenmeyer flask, which was then incubated at 27 °C for 5 days on a rotary shaker. From multiple flasks, 15 L of culture broth was loaded onto DIAION HP-20 and eluted with MeOH. The extract was concentrated, and the resulting aqueous solution was extracted with EtOAc. The organic phase was evaporated to give a solid, which was subjected to silica gel column chromatography followed by gel filtration and reverse phase HPLC to give the red powder of 1. 1 H and 13C signals for 1 were assigned by 1H, 13C, DEPT135, and HMQC spectral analysis. Compound 1 has four methyls, five sp3 methines, two sp2 methines, nine quartnary carbons, © 2013 American Chemical Society and American Society of Pharmacognosy
and two exchangeable protons from these spectra. The analysis of the 1H−1H COSY spectrum of 1 revealed the connectivity from an anomeric proton of a sugar at δ 5.41 (H-1′) to an sp3 methine proton at δ 3.94 (H-2′), 3.75 (H-3′), 3.30 (H-4′), and 3.37 (H-5′) and a methyl at δ 1.10 (H-6′) and from sp2 methine protons at δ 7.50 (H-5) to 7.37 (H-6) (Figure 1). The connectivities from H-5′ to δ 99.0 (C-1′), from H-1′ to δ 69.9
Figure 1. Key correlations observed in the 2D NMR spectra of sacchathridine A by COSY, HMBC, and SIMBA. Received: September 13, 2012 Published: April 12, 2013 720
dx.doi.org/10.1021/np3006327 | J. Nat. Prod. 2013, 76, 720−722
Journal of Natural Products
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(C-5′), and from an sp2 carbon at δ 151.0 (C-7) revealed the presence of a sugar moiety that was attached to an olefinic or an aromatic moiety. In addition to 1H−13C long-range correlations from H-5 to δ 184.1 (C-4), C-7, and δ 115.8 (C-8a), from H-6 to δ 125.6 (C-4a) and 142.0 (C-8), from methyl protons at δ 1.94 (H-13) to δ 158.5 (C-2), δ 130.3 (C-3), and C-4, and from a methoxy group at δ 3.93 (H-14) to C-2, the long-range correlation at δ 182.8 (C-1) was obtained by the analysis of a selective inverse multiple-bond analysis (SIMBA)8 experiment, which revealed four-bond correlations. In particular, irradiation at the C-1 carbon by SIMBA revealed the correlation at the methyl proton of H-13 and the aromatic proton of H-5. These observations indicated that 1 has a 2-methoxy-3-methylnaphthalene-1,4-dione skeleton. Remaining exchangeable protons (δ 9.89 and 10.03) were assigned to be from the two NH groups by 1H−15N-HMQC. Finally, long-range coupling from the methyl proton at δ 1.83 (H-12) to the carbonyl carbon at δ 168.2 (C-11), from NH-10 (δ 9.89) to C-8 and C-11, and from NH-9 (δ 10.03) to C-7 and C-8a established that the acetylhydrazino moiety was attached to C-8 and the sugar moiety was attached to C-7 of the 2-methoxy-3-methylnaphthalene-1,4-dione skeleton. The planar structure of 1 was elucidated as shown in Figure 1.
identical to those of standard methyl-α-L-rhamnoside (Wako Pure Chemical Industries, Osaka, Japan) (Figures S3 and S4). Finally, the structure of 2 was determined to be methyl-α-Lrhamnoside by comparison of the optical rotation (2, [α]21D = −57.9 (c 0.15 H2O); standard, [α]21D = −59.1 (c 0.15 H2O)). Combined with this result and the 1JCH (C-1′) values (171.6 Hz) of compound 1, the sugar moiety was proven to be α-Lrhamnose. On the basis of all of these results, structure 1 was proposed for sacchathridine A as N′-(7-methoxy-6-methyl-5,8dioxo-2-(α- L -rhamnosyloxy)-5,8-dihydronaphthalen-1-yl)acetohydrazide. The effect of compound 1 on PGE2 release from human synovial sarcoma SW982 cells was investigated. SW982 cells are known to produce prostaglandins and cytokines.9,10 We monitored bradykinin-induced PGE2 release from SW982 cells in a concentration-dependent manner (Figure 2a). As shown in Figure 2b, compound 1 inhibited PGE2 release in a concentration-dependent manner from SW982 cells with an IC50 value of 1.0 μM. We also examined the effect of cell growth on 1, and the results indicated that there was no cell growth inhibition up to 30 μM (Figure 2c). In conclusion, we isolated sacchathridine A (1) from the culture broth of Saccharothrix sp. MI559-46F5. The structure of 1 was elucidated to be a new naphthoquinone derivative with an acetylhydrazino moiety. Sacchathridine A inhibited PGE2 release from SW982 cells without affecting cell growth and would be a useful tool for the analysis of the mechanism of PGE2 release as a bioprobe.
Table 1. 1H and 13C NMR Spectroscopic Data for Sacchathridine A (DMSO-d6) position 1 2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 14 1′ 2′ 3′ 4′ 5′ 6′
δ
C
type
182.8 158.5 130.3 184.1 125.6 120.1 118.6 151.0 142.0 115.8
C C C C C CH CH C C C NH NH C CH3 CH3 CH3 CH CH CH CH CH CH3
168.2 20.5 9.0 60.7 99.0 69.7 70.3 71.8 69.9 17.9
δ
H
multiplicity, J Hz
HMBC
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SIMBA
General Experimental Procedures. The UV spectrum was obtained on a U-2800 spectrophotometer (Hitachi High-Technologies, Tokyo, Japan). The IR spectrum was obtained on an FT-210 Fourier transform infrared spectrometer (Horiba, Kyoto, Japan). Optical rotations were measured on a P-1030 polarimeter (JASCO, Tokyo, Japan). NMR spectra were recorded using a JNM-ECA 600 (JEOL, Tokyo, Japan) with TMS as an internal standard. The HRESIMS spectrum was measured using an LTQ Orbitrap mass spectrometer (Thermo Fischer Scientific, San Jose, CA, USA). Microorganism. The strain MI559-46F5 was isolated from a soil sample collected at Higashikagawa, Kagawa Prefecture, Japan. The substrate hyphae were branched and were pale yellow to pale brown. The aerial mycelia were straight or flexuous and were fragmented into cylindrical and ellipsoidal spores (0.5 to 0.7 by 0.9 to 1.3 μm). The surface of the spore was smooth and was white to brownish-white. These characteristics were observed on oatmeal agar (ISP medium No.3) and inorganic salt-starch agar (ISP medium No.4). Whole-cell hydrolysates contained meso-A2 pm, rhamnose, and galactose as the characteristic whole-cell sugars. A polar lipid analysis showed phosphatidylethanolamine as the diagnostic phospholipid. The predominant menaquinone was MK-9(H4), and a trace amount of MK-10(H4) was contained. The cell-wall acyl type was acetyl type. The partial 16S rRNA gene sequence (1446 bp) of the strain showed high identity with those of the genus Saccharothrix such as Saccharothrix texasensis (NRRL B-16107T, T: type strain, 1429/1444 bp, 98.9%) and S. xinjiangensis (NBRC 101911T, 1418/1441 bp, 98.4%). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of MI559-46F5 is AB795585. These phenotypic and genotypic analyses suggested that the strain MI559-46F5 belonged to the genus Saccharothrix. Therefore, the strain was tentatively designated as Saccharothrix sp. MI559-46F5. Fermentation and Isolation of Sacchathridine A. A slant culture of MI559-46F5 was used for inoculation in 500 mL Erlenmeyer flasks. Each flask contained 110 mL of seed medium consisting of galactose 2%, dextrin 2%, glycerol 1%, soy peptone 1%, corn steep liquor 0.5%, (NH4)2SO4 0.2% and CaCO3 0.2% in deionized water
5, 13 7.50 7.37
10.03 9.89 1.83 1.94 3.93 5.41 3.94 3.75 3.30 3.37 1.10
d (8.5) d (8.5)
4, 6, 7, 8a 4a, 5, 8, 7
br br
7, 8a 8, 11
(s) (s) (s) brd (1.4) m dd (3.4, 9.3) t (9.3) dq (6.1, 9.3) d (6.1)
11 2, 3, 4 2 5′, 7 3′, 4′ 4′ 2′, 3′, 6′ 1′, 3′, 4′ 4′, 5′
EXPERIMENTAL SECTION
In regard to the analysis of the sugar constituent of 1, the vicinal coupling constant 3JHH values (3J1′,2′ = 1.4 Hz, 3J2′,3′ = 3.4 Hz, 3J3′,4′ = 9.3 Hz, 3J4′,5′ = 9.3 Hz, and J5′,6′ = 6.1 Hz) suggested that the sugar moiety was rhamnose. The absolute configuration of rhamnose was determined by chemical degradation. Sacchathridine A (15.4 mg) was dissolved in 1 mL of 5−10% HCl−methanol and stirred at 60 °C for 24 h. The reaction mixture was diluted by EtOAc, and the aqueous phase was evaporated. The residue was purified by preparative TLC (CHCl3−MeOH−H2O, 10:5:1) to give pure methylrhamnoside (2) (2.6 mg). The 1H and 13C NMR data of 2 were 721
dx.doi.org/10.1021/np3006327 | J. Nat. Prod. 2013, 76, 720−722
Journal of Natural Products
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Figure 2. Biological activity of 1. (a) Concentration−response of PGE2 release from SW982 cells induced by bradykinin. (b) Effect of sacchathridine A on PGE2 release from SW982 cells. (c) Effect of sacchathridine A on cell growth of SW982 cells. Data represent mean ± SD of 3 or 4 independent experiments. 1 was initially dissolved in DMSO and diluted by assay buffer. The final concentration of DMSO was 0.1%.
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and adjusted to pH 7.0 before sterilization. The flasks were shaken on a rotary shaker at 200 rpm at 37 °C for 2 days. An aliquot of 2 mL of the seed culture was inoculated into 110 mL of the culture medium consisting of glycerol 2%, dextrin 2%, soy peptone 1%, yeast extract 0.3%, (NH4)2SO4 0.2% and CaCO3 0.2% in deionized water (pH 7.4) in a 500 mL Erlenmeyer flask and incubated at 27 °C for 5 days on a rotary shaker (180 rpm). The cultured broth (15 L) was centrifuged to separate the supernatant and a mycelial cake. The supernatant was loaded on DIAION HP-20 (1.5 L) (Mitsubishi Chemical Co., Tokyo, Japan), washed with 50% MeOH, and then eluted with MeOH. The MeOH fraction was concentrated, and the resulting aqueous solution was extracted with EtOAc (4.5 L). The extract was dried over anhydrous Na2SO4 and evaporated to give a red oil (1.13 g). The oil was subjected to silica gel column chromatography (60 g) and eluted with 900 mL of CHCl3−MeOH (10:1) followed by 1200 mL of CHCl3−MeOH (50:1). The active fractions (127 mg) were combined and evaporated to give a solid, which was further subjected to column chromatography using TOYOPEARL HW-40F (Tosoh Co., Tokyo, Japan) eluted with MeOH to yield a red residue (44 mg). Finally, active fractions were separated via preparative HPLC (CAPCELL PAK C18 UG column, 5 μM 20 i.d. × 250 mm (Shiseido, Tokyo, Japan) with photodiode array detector) with CH3CN−H2O (20:80) containing 0.01% TFA at a flow rate of 8 mL/min. The fraction with a retention time of 12.9 min yielded compound 1 (31.8 mg). Physicochemical Properties of 1. The physicochemical properties of 1 are as follows: red powder; [α]23D −358 (c 0.0036 in MeOH); UV λmax (MeOH) 228, 263, 470 nm (ε 17 254, 9908, 2739); IR spectrum νmax (KBr) 3400, 1681, 1643, 1139, 953 cm−1. The molecular formula of sacchathridine A was determined to be C20H24N2O9 by HRESIMS (positive ion mode, m/z 459.1371 (M + Na)+ Δ −0.3 mDa as C20H24N2O9+Na). Measurement of PGE2. SW982 cells were incubated for 24 h in a 96-well plate (10 000 cells/well) at 37 °C and then washed with assay buffer (Hank’s balanced salt solution, 17 mM HEPES pH 7.4, 0.1% BSA). Cells were treated with 1 nM bradykinin (Sigma) at 37 °C. To detect PGE2, cells were incubated for 30 min. The release of PGE2 into the assay buffer was quantified using a fluorescence resonance energy transfer-based assay kit11 (Cisbio, Codolet, France) by an Envision microplate reader (PerkinElmer Inc., Waltham, MA, USA). Under this condition, the IC50 value of SC-58125, selective cyclooxygenase-2 inhibitor, was 33 nM. Cell Proliferation Assay. SW982 cells were grown in DMEM/ F12 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin, MEM-nonessential amino acids, and 1 mM sodium pyruvate at 37 °C. SW982 cells were seeded in a 96-well plate (3000 cells/well). Sacchathridine A was added, and the plates were incubated for 48 h. Cell counting kit-8 solution was added, and the cells were incubated for 90 min before measuring the absorbance at 450 nm using an ARVO microplate reader (PerkinElmer Inc.).
ASSOCIATED CONTENT
S Supporting Information *
1
H NMR, 13C NMR, and 1H−15N-HMQC spectra in DMSO-d6 for sacchathridine A (1) and 1H NMR and 13C NMR spectra of methylrhamnoside (2) and methyl-α-L-rhamnoside in CD3OD are available free of charge via the Internet at http://pubs.acs. org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-3441-4173. Fax: +81-3-3441-7589. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by a grant from JSPS KAKENHI (grant no. 22710227). The authors would like to thank M. Kawada, S. Ohba, and T. Masuda for providing technical assistance and helpful discussions.
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REFERENCES
(1) Murakami, M.; Kudo, I. J. Biochem. 2002, 131, 285−292. (2) Bos, C. L.; Richel, D. J.; Ritsema, T.; Peppelenbosch, M. P.; Versteeg, H. H. Int. J. Biochem. Cell Biol. 2004, 36, 1187−1205. (3) Breyer, R. M.; Bagdassarian, C. K.; Myers, S. A.; Breyer, M. D. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 661−690. (4) Funk, C. D. Science 2001, 294, 1871−1875. (5) Furuyashiki, T.; Narumiya, S. Curr. Opin. Pharmacol. 2009, 9, 31−38. (6) Ushikubi, F.; Segi, E.; Sugimoto, Y.; Murata, T.; Matsuoka, T.; Kobayashi, T.; Hizaki, H.; Tuboi, K.; Katsuyama, M.; Ichikawa, A.; Tanaka, T.; Yoshida, N.; Narumiya, S. Nature 1998, 395, 281−284. (7) Cathcart, M. C.; O’Byrne, K. J.; Reynolds, J. V.; O’Sullivan, J.; Pidgeon, G. P. Biochim. Biophys. Acta 2012, 1825, 49−63. (8) Crouch, R.; Martin, G. J. Magn. Reson. 1991, 92, 189−194. (9) Yamazaki, T.; Yokoyama, T.; Akatsu, H.; Tukiyama, T.; Tokiwa, T. In Vitro Cell. Dev. Biol. Anim. 2003, 39, 337−339. (10) Wada, Y.; Shimada, K.; Kimura, T.; Ushiyama, S. Immunol. Lett. 2005, 101, 50−59. (11) Goedken, E. R.; Gagnon, A. I.; Overmeyer, G. T.; Liu, J.; Petrillo, R. A.; Burchat, A. F.; Tomlinson, M. J. J. Biomol. Screen. 2008, 13, 619−625.
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