HETEROCYCLES

Paper | Special issue | Vol. 82, No. 1, 2010, pp. 371-375
Received, 31st January, 2010, Accepted, 20th May, 2010, Published online, 21st May, 2010.
DOI: 10.3987/COM-10-S(E)7

■ Sunabedine, a Novel Toxic Bromotyrosine-derivative Alkaloid from Okinawan Sponge, Order Verongida

Norihito Maru, Tomoyuki Koyama, Osamu Ohno, Kaoru Yamada, and Daisuke Uemura*

Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Hiyoshi, Kohoku-ku Yokohama 223-8522, Japan

Abstract

Sunabedine (1), a new bromotyrosine-derivative alkaloid, was isolated from the Okinawan sponge, order Verongida. The structure of 1 was determined by spectroscopic analyses. 1 showed the cytotoxicity against B16 mouse melanoma cells and toxicity against brine shrimp.

Bromotyrosine-derivative alkaloids are characteristic secondly metabolites from marine sponges of the order Verongida. The structures of these metabolites are typically based on one or two spirocyclohexadienyl isoxazole moieties connected to diverse side chains, as seen in aerothinin1 and aerophobin-1.2 Various biological activities have been reported, such as cytotoxic,3 antibacterial,4 antihistaminic,5 and enzyme-inhibitory6,7 activities. We report here the isolation, structural determination and biological activities of the novel bromotyrosine-derivative alkaloid, sunabedine (1).

The ESIMS of 1 showed a [M+H]+ ion 1:4:6:4:1 quintet at m/z 894.9, 896.9, 898.9, 900.9 and 902.9, which indicated the presence of four bromine atoms in its structure. 1 has a molecular formula of C28H30Br4N6O8, as suggested by HRFABMS at m/z 896.8943 [M+H]+ (calcd for C28H3179Br381Br N6O8, 896.8917). The 1H and 13C NMR data are summarized in Table 1. The absorption bands in the IR spectrum showed amino and hydroxyl groups (3339 cm-1) and α-iminoamide functions (1662, 1597 and 1541 cm-1), and the UV spectrum showed cyclohexadienyl moieties (284 nm). The 1H NMR signals displayed the characteristic signals of two 2,4-dibromo-1-hydroxyl-3-methoxy-8-carbamoyl spirocyclohexadienyl isoxazole moieties at 3.06/3.07, 3.72, 3.74/3.75, 4.07 and 6.40/6.41, and corroborating 13C NMR signals at 40.2, 60.4, 75.5, 92.4, 114.1, 122.8, 132.3, and 149.3. The COSY spectral analysis allowed us to connect two spin systems of methylene chains of C10-C11 and C15-C17 (Figure 1). The HMBC correlations of H7/C9, H10/C9, H17/C18 and H20/C18 indicated the connectivity between C7-C10 and C17-C20 via an amide bond. The remaining proton signals at 6.96 and 7.59, and carbon signals at 117.7, 138.2 and 140.0 were thought to represent an aromatic moiety. The HMBC correlations of H10/C12, H11/C12, H11/C13, H13/C12, H13/C14, H14/C13, H15/C13 and H15/C14 revealed the structure of a 1,4-disubstituted imidazole ring that was linked to methylene chains.

The geometries of the vicinal oxygen atoms at C1 (C26) and C6 (C21) were established by comparison of the chemical shifts value with the synthetic trans and cis spiroisoxazoline derivertives.8 The 1H NMR signals of H1, H6 and H7 in trans compound at 4.08, 6.40, and 3.05/3.78 and cis compound at 4.40, 6.55, and 3.3-3.4 reveals trans relationship of these in 1. The absolute configurations of spirocyclohexadienyl isoxazole moieties were thought to be determined based on the CD spectrum by comparison with the reported spectrum of aerothionin, the configuration of which was established by X-ray analysis.9 However, 1 showed no absorption at 240-300 nm in its CD spectrum. These data, supported by the large active optical rotation {[α] –137.1 (c 0.30, CHCl3)}, suggest that 1 is not a racemic mixture and the absolute configurations of C1, C6, C21 and C26 might be 1R, 6S, 21R and 26S or 1S, 6R, 21S and 26R.
The biological activities of compound 1 were examined with regard to cytotoxicity against B16 mouse melanoma cells and toxicity against brine shrimp (genus Artemia). After incubation, 1 showed potency with an IC50 of 39 μM against B16 cells and a LD50 of 110 μM against brine shrimp. To the best of our knowledge, this is the first report of a bromotyrosine-derivative alkaloid that is toxic toward brine shrimp.

EXPERIMENTAL
General Experimental Procedures. The 1H, 13C, and 2D NMR spectra were recorded on a JEOL A-300 spectrometer, and the 1H and 13C chemical shifts were referenced to the solvent peaks [δH 3.30 and δC 49.0 in CD3OD and δH 1.93 in CD3CN]. The IR spectrum was measured using a JASCO FT/IR-6100 spectrometer, the UV spectrum was measured using a JASCO V-570 spectrometer, the optical rotation was measured on a JASCO DIP-1000 polarimeter, and the CD spectrum was obtained in MeOH using a JASCO J-720WN spectrophotometer. The low-resolution mass spectrum (ESIMS) was determined on a Bruker HCTultra mass spectrometer, and the high-resolution mass spectrum (HRFABMS) was determined on a JEOL JMS-700 mass spectrometer. Column chromatography was performed on Silicagel BW-820MH (Fuji Silysia Chemical). Preparative TLC was performed using Silicagel 60 F254 (Merck). All solvents used were reagent grade.
Extraction and Isolation. The sponge order Verongia (270 g wet wt.), collected at Okinawa Island, Okinawa Prefecture, Japan, was crushed and extracted with 80% aqueous EtOH (1 L) for 12 days. The extract was filtered, concentrated, and partitioned between EtOAc and water. The EtOAc-soluble material was further partitioned between 90% aqueous MeOH and hexane. The material obtained from the aqueous MeOH portion was subjected to fractionation using a silica gel (MeOH/CHCl3) and a preparative TLC (MeOH/CHCl3) to give 5 mg of 1 as a colorless solid.
Sunabedine (1): [α] –137.1 (c 0.30, CHCl3); IR (neat): 3339, 1662, 1597, 1541, 1437, 1308, 1268, 1219, 989 cm-1; UV (MeOH): λmax 227, 284 nm; 1H NMR (CD3OD, 300 MHz), see Table 1; 13C NMR (CD3OD, 75 MHz), see Table 1; HRFABMS: [m / z (M + H)+] found 896.8943, calcd for C28H3179Br381Br N6O8 (Δ +2.6 mmu).
Cytotoxic Activity. Cultured cells of B16 mouse melanoma were seeded at 4 x 104 cells/mL in each well of a 96-well plate, and samples dissolved in MeOH were added. The mixture was incubated at 37 °C for 96 h in a CO2 incubator with a humidified atmosphere containing 5% CO2. The cell number was counted by the MTT method.
Brine Shrimp Toxicity. Brine shrimp eggs ware hatched in artificial seawater with oxygenation by an aquarium pump at 24 ˚C for 24-48 h. Ten hatched brine shrimp in 100 μL seawater were placed in each well of a 96-well microplate, and samples dissolved in 100 μL of 2.5% MeOH/seawater were added. After 24 h, the numbers of dead and live brine shrimp were counted under a microscope.

ACKNOWLEDGEMENTS
This work was supported in part by JSPS via Grants-in-Aid for Scientific Research (16GS0206, 21221009, and 20611006) and the Global-COE program in Chemistry, Nagoya University.

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