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

Abstract

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) [2–7], 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.

Results and Discussion

The dinoflagellate Amphidinium sp. (strain Y-100) was isolated from a marine acoel flatworm Amphiscolops sp. collected off Ma’eda Cape, Okinawa, and mass cultured unialgally at 25 °C for 2 weeks in a seawater medium enriched with 1% Provasoli’s Erd-Schreiber (ES) supplement and 1% NaH¹³CO₃. The mass cultured algal cells (60.5 g, wet weight) obtained from 30 L of culture were extracted with MeOH/toluene (3:1), and the extracts were partitioned between toluene and 1M NaCl aq. The toluene soluble materials were subjected to a silica gel column followed by SiO₂ and/or C₁₈ HPLC to afford amphidinolides B4 (1, 0.0008 %, wet weight) and B5 (2, 0.0002 %) together with known related macrolides, amphidinolides B [2–4], (3, 0.0016 %), C [9,10] (0.0025 %), and T1 [11–13] (0.0028 %).

Amphidinolide B4 {1, [α]_D²³ −13° (c 0.2, CHCl₃)} showed the pseudomolecular ion peak at m/z 569.5 (M+Na)⁺ in the ESIMS spectrum, and the ¹³C-enrichment was estimated as 32% by the pattern of the pseudomolecular ion peak. The molecular formula of C₃₂H₅₀O₈ was revealed on the basis of HRESIMS data [m/z 569.3467 (M+Na)⁺, Δ +1.3 mmu]. The ¹H NMR spectrum of 1 was similar to that of amphidinolide B (3). The ¹³C NMR data (

Table 1. ¹³C NMR Data of Amphidinolides B4 (1), B5 (2), B (3), H (4), H2 (5), and H3 (6) in CDCl₃.

positn	1	2	3	4	5	6
1	167.8	167.8	167.7	168.7	168.9	168.8
2	128.5	128.1	128.3	127.9	127.6	128.1
3	139.8	139.4	139.9	141.0	140.8	140.7
4	26.8	26.9	26.8	27.0	26.9	26.8
5	31.2	31.0	30.8	30.9	31.0	30.7
6	135.9	136.1	135.4	135.7	136.1	136.1
7	128.7	129.7	128.5	128.6	129.7	127.9
8	60.6	60.1	60.0	60.3	60.1	60.5
9	59.5	59.9	59.3	59.5	59.8	59.7
10	39.8	40.4	39.4	39.8	40.4	39.6
11	29.4	29.9	29.1	29.1	29.8	29.3
12	47.1	46.7	46.7	47.1	46.6	47.1
13	144.2	144.3	144.4	144.1	144.3	143.9
14	126.5	126.6	124.3	126.1	126.7	125.8
15	141.7	140.6	143.1	141.7	140.6	141.5
16	40.9	40.9	75.9	40.7	40.8	40.9
17	40.8	40.2	45.2	40.9	40.2	40.8
18	67.4	65.9	66.5	67.5	65.9	67.1
19	45.1	43.9	45.9	45.2	43.8	46.6
20	212.9	212.0	212.4	212.2	211.3	215.1
21	77.9	78.8	77.7	77.7	78.4	77.0
22	75.8	76.3	75.5	75.4	76.2	77.2
23	33.2	32.9	33.2	33.0	32.3	30.1
24	39.2	39.4	39.3	33.5	33.9	30.2
25	68.3	73.4	68.3	73.4	73.3	73.7
26	21.2	21.2	21.0	66.1	66.6	66.4
27	12.4	12.5	12.4	12.6	12.7	12.6
28	18.0	19.5	18.2	18.0	19.5	18.2
29	114.8	114.9	114.8	114.7	115.0	114.8
30	13.1	12.3	15.6	13.2	12.3	12.8
31	20.5	20.2	28.3	20.3	20.4	20.8
32	15.8	15.2	15.0	15.6	15.1	16.2

) of 1 revealed total 32 carbon signals due to a ketone, an ester carbonyl, three sp² quaternary carbons, four sp² methines, an sp² methylene, nine sp³ methines consisting of six oxygenated ones, seven sp³ methylenes, and six methyls. Detailed analyses of the HMQC and INADEQUATE spectra of 1 established the carbon chain from C-1 to C-26 and six C₁ branches including five methyls (C-27, C-28, C-30, C-3, and C-32) and an exomethylene (C-29). The relatively lower-field resonance of H-25 (δ_H 5.08) indicated that C-25 was involved in an ester linkage with C-1. This was supported by HMBC correlations for H-3/C-1, H₃-27/C-1, and H-25/C-1. Thus, the gross structure of amphidinolide B4 was assigned as 1.

The relative stereochemistry of 1 was deduced from detailed comparison of the ¹³C NMR data (Table 1) of 1 with those of amphidinolides B (3), H (4), H2 (5), and H3 (6). The ¹³C 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 ¹³C 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 ¹³C NMR data of the corresponding portion of amphidinolide H3 (6) were different from those of 1, while the ¹³C 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. The chemical shifts for C-18 (δ_C 67.4), C-19 (δ_C 45.1), and C-30 (δ_C 13.1) of 1; those of 4 (C-18: δ_C 67.5, C-19: δ_C 45.2, and C-30: δ_C 13.2); those of 5 (C-18: δ_C 65.9, C-19: δ_C 43.8, and C-30: δ_C 12.3). The ¹H-¹H coupling constants for H-18/H-19a (8.2 Hz) and H-18/H-19b (small) of 1 resembled those of 4 (H-18/H-19b: 8.5 Hz and H-18/H-19b: 1.8 Hz), while those for H-18/H-19a (2.6 Hz) and H-18/H-19b (9.8 Hz) of 5 were different from those of 1. These observations indicated the possibility that the relative stereochemistry of nine chiral centers in 1 might be the same as that in 4. The CD spectrum [λ_ext 262 (Δɛ +0.1) and 235 nm (−0.22)] of 1 was similar to those of 4 [λ_ext 261 (Δɛ +0.1) and 235 nm (−0.15)]. Considering the biosynthetical relationship among 1 and amphidinolides B (3) and H (4), the absolute configurations of 1 were elucidated to be the same as those of 3 and 4. Thus, amphidinolide B4 (1) was assigned as 16-deoxy form of amphidinolide B (3).

Amphidinolide B5 {2, [α]_D²³ −25° (c 0.2, CHCl₃)} was elucidated to have the same molecular formula, C₃₂H₅₀O₈, as 1. Profiles of the ¹H and ¹³C NMR (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 ¹³C chemical shifts for C-1 ~ C-13 and C-27 ~C-29 of 2 were similar to those of 3 ~ 5. The ¹³C 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. The absolute configurations of 2 were elucidated to be the same as those of 5, since the CD spectrum of 2 [λ_ext 262 (Δɛ +0.16) and 220 nm (0)] was quite similar to that of 5 [λ_ext 262 (Δɛ +0.17) and 221 nm (0)]. Thus, amphidinolide B5 (2) was assigned as 26-deoxy form of amphidinolide H2 (5) or 16-deoxy-16,18-epi form of amphidinolide B (3).

Amphidinolides B4 (1) and B5 (2) exhibited potent cytotoxicity against murine lymphoma L1210 (IC₅₀: 0.00012 and 0.0014 μg/mL, respectively) and human epidermoid carcinoma KB cells (IC₅₀: 0.001 and 0.004 μg/mL, respectively).

Experimental

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 × 3). After addition of 1 M NaCl aq. (100 L), the mixture was extracted with toluene (100 mL × 3). The toluene-soluble fractions (730 mg) were subjected to a silica gel column (CHCl₃/MeOH, 98:2) to afford a macrolide-containing fraction, which was separated with a Sep-Pak cartridge C₁₈ (MeOH/H₂O, 8:2) followed by C₁₈ HPLC [YMC-Pack Pro C₁₈, 5 μm, YMC Co., Ltd., 10 × 250 mm; eluent, CH₃CN/H₂O (75:25); flow rate, 3 mL/min; UV detection at 210 nm] to give amphidinolides B4 (1, 0.42 mg, 0.0008 %, wet weight, t_R 23.9 min) and B5 (2, 0.10 mg, 0.0002 %, t_R 27 min) together with amphidinolide B (3, 0.23 mg, 0.0005 mg). Amphidinolides C (0.0025 %) and T1 (0.0028 %) were isolated from another fraction.

Amphidinolide B4 (1): colorless oil; [α]²³_D −13° (c 0.2, CHCl₃); UV (EtOH) λ_max 209 nm (ɛ 6800); IR (neat) ν_max 3436, 2956 and 1704 cm⁻¹; CD (EtOH) λ_ext 262 (Δɛ +0.1) and 235 nm (−0.22); ¹H NMR (CDCl₃) δ 0.85 (3H, d, J = 6.5 Hz, H₃-28), 0.98 (3H, d, J = 6.7 Hz, H₃-32), 1.06 (3H, d, J = 6.7 Hz, H₃-31), 1.15 (1H, m, H-10), 1.20 (1H, m, H-24), 1.24 (3H, d, J = 6.7 Hz, H₃-26), 1.42 (1H, m, H-17), 1.48 (1H, m, H-10), 1.56 (1H, m, H-11), 1.71 (3H, s, H₃-30), 1.82 (3H, s, H₃-27), 1.84 ~ 1.92 (3H, m, H-12, H-17, and H-23), 2.05 ~ 2.16 (3H, m, H-5, H-12, and H-24), 2.18 ~ 2.28 (2H, m, H-4 and H-16), 2.34 (1H, m, H-5), 2.42 (1H, m, H-4), 2.61 (1H, brd, J = 16.0 Hz, H-19), 2.74 (1H, dd, J = 8.9 and 16.0 Hz, H-19), 2.94 (1H, brd, J = 9.7 Hz, H-9), 3.14 (1H, brd, J = 8.0 Hz, H-8), 3.27 (1H, brs, OH), 3.55 (1H, brs, OH), 3.71 (1H, m, H-22), 3.86 (1H, brs, OH), 3.92 (1H, m, H-18), 4.31 (1H, brs, H-21), 4.81 (1H, s, H-29), 4.97 (1H, s, H-29), 5.08 (1H, m, H-25), 5.16 (1H, dd, J = 15.6 and 8.2 Hz, H-7), 5.56 (1H, s, H-14), 5.89 (1H, m, H-6), and 6.73 (1H, m, H-3); ¹³C NMR (Table 1); (+)-ESIMS m/z 569 ([M+Na]⁺, 6%), 570 (7%), 571 (11%), 572 (19%), 573 (32%), 574 (48%), 575 (64%), 576 (77%), 577 (87%), 578 (100%), 579 (96%), 580 (92%), 581 (86%), 582 (76%), 583 (64%), 584 (51%), 585 (41%), 586 (31%), 587 (25%), 588 (18%), 589 (14%), 590 (12%), 591 (13%), and 592 (12%); (+)-HRESIMS m/z 569.3467 (C₃₂H₅₀O₇Na require (M+Na)⁺, 569.3454).

Amphidinolide B5 (2): colorless oil; [α]_D −25° (c 0.2, CHCl₃); UV (EtOH) λ_max 209 nm (ɛ 6800); IR (neat) ν_max 3740, 2923, and 1706 cm⁻¹; CD (EtOH) λ_ext 262 (Δɛ +0.16) and 220 nm (0); ¹H NMR (CDCl₃) δ 0.86 (3H, d, J = 6.5 Hz, H₃-28), 0.92 (3H, d, J = 6.7 Hz, H₃-32), 0.98 (1H, m, H-10), 1.05 (3H, d, J = 6.7 Hz, H₃-31), 1.28 (1H, m, H-24), 1.28 (3H, d, J = 6.7 Hz, H₃-26), 1.45 (1H, m, H-17), 1.59 (1H, m, H-11), 1.64 (1H, m, H-11), 1.70 (1H, m, H-10), 1.71 (3H, s, H₃-30), 1.82 (3H, s, H₃-27), 1.82 (1H, m, H-17), 1.91 (1H, m, H-19), 2.05 (1H, m, H-24), 2.12 (1H, m, H-5), .2.22 ~ 2.40 (4H, m, H-4, H-5, H-12, and H-16), 2.45 (1H, m, H-4), 2.53 (1H, dd, J = 9.8 and 16.0 Hz, H-19), 2.97 (1H, brd, J = 9.7 Hz, H-9), 3.06 (1H, brd, J = 8.0 Hz, H-8), 3.09 (1H, d, J = 16.0 Hz, H-19), 3.57 (1H, m, H-22), 3.60 ~ 3.75 (3H, brs, OH × 3), 4.09 (1H, m, H-18), 4.22 (1H, brs, H-21), 4.80 (1H, s, H-29), 4.96 (1H, s, H-29), 5.09 (1H, m, H-25), 5.22 (1H, dd, J = 15.6 and 8.2 Hz, H-7), 5.56 (1H, s, H-14), 5.87 (1H, m, H-6), and 6.73 (1H, m, H-3); ¹³C NMR (Table 1); (+)-ESIMS m/z 569 ([M+Na]⁺, 6%), 570 (7%), 571 (11%), 572 (19%), 573 (32%), 574 (48%), 575 (64%), 576 (77%), 577 (87%), 578 (100%), 579 (96%), 580 (92%), 581 (86%), 582 (76%), 583 (64%), 584 (51%), 585 (41%), 586 (31%), 587 (25%), 588 (18%), 589 (14%), 590 (12%), 591 (13%), and 592 (12%); (+)-HRESIMS m/z 569.3448 (C₃₂H₅₀O₇Na require (M+Na)⁺, 569.3454).