HETEROCYCLES

Paper | Special issue | Vol. 82, No. 1, 2010, pp. 333-338
Received, 5th January, 2010, Accepted, 9th February, 2010, Published online, 10th February, 2010.
DOI: 10.3987/COM-10-S(E)3

■ Amphidinolide C3, a New Cytotoxic 25-Membered Macrolide from Marine Dinoflagellate Amphidinium sp

Takaaki Kubota, Akiko Suzuki, Mika Yamada, Shigeyuki Baba, and Jun'ichi Kobayashi*

Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo, Hokkaido 060-0812, Japan

Abstract

A new cytotoxic 25-membered macrolide, amphidinolide C3 (1), has been isolated from marine dinoflagellate Amphidinium sp. (Y-56 strain), and the structure of 1 was elucidated on the basis of spectroscopic data and chemical means.

INTRODUCTION
Marine dinoflagellates have been recognized as a source of novel bioactive substances with unique structures.1-9 During our continuing search for structurally unique secondary metabolites from marine dinoflagellates Amphidinium sp, a series of cytotoxic macrolides, amphidinolides, as well as long chain polyhydroxy compounds have been isolated so far.1-4 Further investigation of extracts of dinoflagellates Amphidinium spp. resulted in isolation of a new cytotoxic 25-membered macrolide, amphidinolide C3 (1), from the Y-56 strain of the dinoflagellate Amphidinium sp., which was separated from the inside cells of the marine acoel flatworms Amphiscolops sp. In this paper we describe the isolation and structure elucidation of amphidinolide C3 (1).

RESULTS AND DISCUSSION
The dinoflagellate Amphidinium sp. (Y-56 strain) was obtained from an acoel flatworm Amphiscolops sp. collected off Zanpa, Okinawa. The harvested cells of the cultured dinoflagellate (304 g, wet weight) obtained from 580 L of culture were extracted with MeOH/toluene (3:1), and the extracts were partitioned between toluene and water. The toluene soluble materials were subjected to a silica gel column followed by a C18 column and C18 HPLC to afford amphidinolide C3 (1, 0.00006%) together with amphidinolide C (2, 0.0004%).10-13
The molecular formula of amphidinolide C3 (1) was revealed to be C41H60O10 by HRFABMS [m/z 735.41075 (M + Na)+, +2.33 mmu]. The IR spectrum indicated the presence of hydroxy (νmax 3470 cm-1) and carbonyl (νmax 1730 and 1710 cm-1) functionalities, while UV absorptions at 237 nm (39000) and 280 nm (3200) suggested the presence of conjugated system. The 1H and 13C NMR, and HMQC spectra of 1 disclosed the existence of three keto carbonyls, an ester carbonyl, four sp2 quaternary carbons, four sp2 methines, two sp2 methylenes, eleven sp3 methines, ten sp3 methylenes, and six methyls, which were similar to those of amphidinolide C (2) except for the presence of a keto carbonyl carbon in place of an oxymethine carbon of amphidinolide C (2).

The 1H-1H COSY and TOCSY spectra of 1 revealed connectivities of five partial structures, a (C-2 to C-8 and C-4 to C-35), b (C-12 to C-14 and C-38), c (C-16 to C17 and C-39), d (C-19 to C-27), and e (C-31 to C-34), as shown in Figure 1. 1H-1H COSY cross-peaks of H-36 to C-10 and H3-37 to C-10 due to allyl couplings, and HMBC correlations for H2-36/C-8, H3-37/C-11, H3-38/C-11, and H-13/C-11 suggested that C-8 and C-12 were connected through a diene moiety (C-9 to C-11, C-9 to C-36, and C-11 to C-37). Connectivities of C-14 and C-16 to C-15, and C-17 and C-19 to C-18 were implied by HMBC cross-peaks of H3-39 to C-15, H2-17 to C-18, and H2-19 to C-18. Two cross-peaks due to allyl coupling of H-27 to H3-40 and H2-31 to H-41 observed in the 1H-1H COSY spectrum, and HMBC cross-peaks of H3-40 to C-28, H3-40 to C-29, H-41 to C-29, and H2-31 to C-30 disclosed the linkages of C-27, C-29, and C-40 via C-28, and C-29, C-31, and C-41 via C-30. The presence of two tetrahydrofuran rings (C-3 to C-6 and C-20 to C-23) were deduced from HMBC cross-peaks of H-6 to C-3 and H-20 to C-23, respectively. The HMBC correlation for H-2 to an ester carbonyl carbon (C-1, δ 172.40) and the chemical shifts for H-24 (δ 5.45) and C-24 (δ 76.64) suggested that C-2 and C-24 were connected through an ester linkage. The NOESY correlation for H-36b (δ 5.00)/H3-37 indicated that the conjugated diene C-36-C-9-C-10-C-11 was s-cis form. E-geometry of the C-25-C-26 double bond was deduced from the 1H-1H coupling constant (3JH-25/H-26 = 15.6 Hz), while that of the C-27-C-28 double bond was assigned as E on the basis of a NOESY cross-peak of H-26/H3-40. Thus, the gross structure of amphidinolide C3 was elucidated to be 1 (Figure 1).

The absolute stereochemistry of amphidinolide C3 (1) was elucidated by chemical correlation with amphidinolide C (2) as follows (Figure 2). Amphidinolide C (2) was treated with 2,2-dimethoxypropane (DMP) and pyridinium p-toluenesulfonate (PPTS) to afford 7,8-O-isopropylidene derivative 3 of amphidinolide C (2). Selective oxidation of the allylic alcohol at C-29 in 7,8-O-isopropylidene derivative 3 with manganese dioxide gave 7,8-O-isopropylidene 29-keto derivative 4 of amphidinolide C (2). Deprotection of acetonide group at C-7 and C-8 of 7,8-O-isopropylidene 29-keto derivative 4 afforded 29-keto derivative of amphidinolide C (2), whose NMR data and [α]D value {[α]D22 -32.7 (c 0.20, CHCl3)} were coincident with those of natural amphidinolide C3 (1). Thus, the absolute stereochemistry of amphidinolide C3 was concluded to be 1.
Amphidinolide C3 (1) exhibited cytotoxicity against murine lymphoma P388 and L1210, and human epidermoid carcinoma KB cells (IC50 2.9, 7.6, and 10.0 µg/mL, respectively) in vitro, which were less potent than those of amphidinolides C (2) and C2.14

EXPERIMENTAL
General Experimental Procedures.
Optical rotation was recorded on a JASCO P-1030 polarimeter. IR and UV spectra were recorded on JASCO FT/IR-230 and Shimadzu UV-1600PC spectrophotometer, respectively. 1H and 13C NMR spectra were recorded on a Bruker AMX-500 or AMX-600 spectrometer using 2.5 mm micro cells (Shigemi Co., Ltd.) for C6D6. The 7.20 and 128.0 ppm resonances of residual C6D6 were used as internal references for 1H and 13C NMR spectra, respectively. ESI mass spectra were obtained on a JEOL JMS-700TZ spectrometer.

Cultivation and Isolation.
The dinoflagellate Amphidinium sp. (strain number Y-56) was isolated from the inside cells of a marine acoel flatworm Amphiscolops sp. collected off Zanpa, Okinawa. The dinoflagellate was unialgally cultured at 25 °C for two weeks in a seawater medium enriched with 1% ES supplement. The harvested cells of the cultured dinoflagellate (304 g wet weight, from 580 L of culture) were extracted with MeOH/toluene (3:1, 600 mL x 3). After addition of 1 M NaCl aq. (500 mL), the mixture was extracted with toluene (500 mL x 3). The toluene soluble fractions were evaporated under reduced pressure to give a residue (2.55 g), which was subjected to a silica gel column (Wakogel C-300, Wako Pure Chemical Industries, Ltd., CHCl3/MeOH) and a C18 column (Cosmosil 140C18-PREP, nacalai tesque, MeOH/H2O) followed by C18 HPLC [YMC Pack Pro C18, 5 mm, YMC Co., Ltd., 10 x 250 mm; eluent, CH3CN/H2O (70:30); flow rate, 3.0 mL/min; UV detection at 210 nm] to afford amphidinolide C3 (1, 0.2 mg, 0.00006%, tR 38.0 min) together with amphidinolide C (2, 1.3 mg, 0.0004%, tR 25.0 min).

Amphidinolide C3 (1): colorless amorphous solid; [α]D22 -31.5 (c 0.20, CHCl3); UV (MeOH) λmax 204 (ε 82000), 237 (39000), and 280 nm (3200); IR νmax 3470, 1730, 1710 and 1630 cm-1; ESIMS (pos.) m/z 735 [M + Na]+; HRESIMS (pos.) m/z 735.41075 [(M + Na)+, calcd for C41H60O10Na, 735.40842].

Amphidinolide C3 (1) derived from amphidinolide C (2). Amphidinolide C (2, 1.0 mg) was dissolved in acetone (30 μL) and treated with 2,2-dimethoxypropane (10 μL) and PPTS (0.24 mg) at room temperature for 1 h. After addition of triethylamine (0.24 μL), the reaction mixture was concentrated and purified by a silica gel column (n-hexane/acetone, 3:1) to afford 7,8-O-isopropylidene derivative (3, 0.9 mg) of amphidinolide C (2). To a solution of 7,8-O-isopropylidene derivative (3, 0.9 mg) of amphidinolide C (2) in CH2Cl2 (300 µL) was added MnO2 (1.8 mg). The resultant mixture was stirred at room temperature for 48 h. The mixture was filtered through a cotton plug, and the filtrate was concentrated to afford 7,8-O-isopropylidene 29-keto derivative (4, 0.7 mg) of amphidinolide C (2). The 7,8-O-isopropylidene 29-keto derivative (4, 0.6 mg) of amphidinolide C (2) was dissolved in MeOH (300 mL) and treated with PPTS (0.23 mg) at 55 °C for 3 h. After addition of triethylamine (0.23 μL), the reaction mixture was concentrated and purified by a silica gel column (n-hexane/acetone, 3:1 to 3:2) to afford amphidinolide C3 (1, 0.6 mg,).

ACKNOWLEDGMENTS
We thank Dr. E. Fukushi, Graduate School of Agriculture, Hokkaido University, for measurement of HMBC spectrum of 1, and Ms. S. Oka, Equipment Management Center, Hokkaido University, for measurements of ESIMS. This work was supported by Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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