Amphidinolides B4 and B5, Potent Cytotoxic 26-Membered Macrolides from Dinoflagellate Amphidinium Species

Two new cytotoxic 26-membered macrolides, amphidinolides B4 (1) and B5 (2), have been isolated from a marine dinoflagellate Amphidinium sp. (strain Y-100), and the structures were elucidated on the basis of detailed analyses of 2D NMR data including 13C–13C correlations.


Introduction
Amphidinolides are a series of unique cytotoxic macrolides isolated from dinoflagellates Amphidinium species, which were separated from marine acoel flatworms Amphiscolops species [1]. The 26-or 27-membered macrolides, which were represented by amphidinolides B (3) and H (4) [2][3][4][5][6][7], possess unique structural features such as an allyl epoxide and vicinally located one-carbon branches, and exhibit potent cytotoxicity against tumor cell lines. From our previous studies, the presence of an allyl epoxide, an S-cis-diene moiety, and the ketone at C-20 in 3 and 4 was indicated to be important for the cytotoxicity of amphidinolide H-type macrolides [7]. More recently, it was indicated that one of the mechanism of action for the potent cytotoxicity of 4 was due to bind to actin covalently [8].
During our continuing search for bioactive metabolites from marine dinoflagellates, two new amphidinolide B-type macrolides, amphidinolides B4 (1) and B5 (2), have been isolated from a marine dinoflagellate Amphidinium species (strain Y-100). In this paper, we describe the isolation, structure elucidation, and cytotoxicity of 1 and 2.
The relative stereochemistry of 1 was deduced from detailed comparison of the 13 C NMR data ( Table 1) of 1 with those of amphidinolides B (3), H (4), H2 (5), and H3 (6). The 13 C chemical shifts for C-1 ~ C-13 part with two methyls (C-27 and C-28), and an exomethlene (C-29) of 1 corresponded well to those of 3 ~ 5, and the 13 C chemical shifts for C-20 ~ C-26 and C-32 of 1 were also close to those of 4 and 5, suggesting that the relative stereochemistry for these parts of 1 might be common to that of amphidinolides B (3), H (4), and H2 (5). On the other hand, the 13 C NMR data of the corresponding portion of amphidinolide H3 (6) were different from those of 1, while the 13 C NMR data for C-14 ~ C-23 and C-28, C-29, C-31 of 1 were similar to those for the corresponding parts of amphidinolide H (4) rather than those of amphidinolide H2 (5) as shown in Figure 1 (Table 1) spectra of 2 were reminiscent of those of 1. The gross structure of 2 was elucidated to be the same as that of 1 from analyses of the HMQC, HMBC, and INADEQUATE spectra. The relative stereochemistry of 2 was deduced from comparison of the carbon chemical shifts with those of 3, 4, and 5 as follows. The 13 C chemical shifts for C-1 ~ C-13 and C-27 ~C-29 of 2 were similar to those of 3 ~ 5. The 13 C NMR data for C-1 ~ C-23 and C-27 ~ C-32 of 2 were closer to those of 5 than those of 4 ( Figure 2), indicating that the relative stereochemistry of this part in 2 was the same as that of the corresponding part in 5. The chemical shifts for C-18 (δ C 65.9), C-19 (δ C 43.9), and C-30 (δ C 12.3) of 2 corresponded well to those of 5 (C-18: δ C 65.9, C-19: δ C 43.8, and C-30: δ C 12.3), while those of 2 were slightly different from those of 4.
Hokkaido University, for ESIMS measurements. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

General
The IR and UV spectra were taken on a FT/IR-5300 and a UV-1600PC spectrophotometers, respectively. NMR spectra were recorded on a Bruker AMX-600 spectrometer. Positive-mode ESI mass spectra were obtained on a JEOL JMS-700TZ spectrmeter.

Cultivation and Isolation
The dinoflagellate Amphidinium species (strain number Y-100) was separated from inside cells of a marine acoel flatworm Amphiscolops species, which was collected off Ma'eda Cape, Okinawa. The dinoflagellate was unialgally cultured at 25 °C for two weeks in seawater medium enriched with 1% ES supplement, 16 h light and 8 h dark. The harvested cells (60.5 g, wet weight, from 30 L of culture) were extracted with MeOH/toluene (3:1, 200 mL x 3). After addition of 1 M NaCl aq. (100 L), the mixture was extracted with toluene (100 mL x 3). The toluene-soluble fractions (730 mg) were subjected to a silica gel column (CHCl 3 /MeOH, 98:2) to afford a macrolide-containing fraction, which was separated with a Sep-Pak cartridge C 18 (MeOH/H