Amphidinolide C2, New Macrolide from Marine Dinoflagellate Amphidinium Species

Abstract

A new cytotoxic 25-membered macrolide, amphidinolide C2 (1), has been isolated from marine dinoflagellate Amphidinium sp. (Y-71 strain), and the structure 1 was elucidated on the basis of spectroscopic data and chemical means.

Introduction

Amphidinolides are a series of unique cytotoxic macrolides isolated from marine dinoflagellates Amphidinium species, which were separated from acoel flatworms Amphiscolops species [1,2]. Amphidinolide C (2) isolated previously from the Y-5 strain of Amphidinium sp. is a unique 25-membered macrolide having two tetrahydrofuran rings and vicinally-located one-carbon branches. [3] Recently, relatively large amounts of 2 have been isolated from three strains (Y-56, Y-59, and Y-71) of the genus Amphidinium, which were separated from the inside cells of the marine acoel flatworms Amphiscolops species. This allowed us to elucidate the absolute configurations at twelve chiral centers in 2. [4] The biosynthetic origins of amphidinolide C (2) were investigated on the basis of ¹³C NMR data of ¹³C enriched samples obtained by feeding experiments with [1-¹³C], [2-¹³C], and [1,2-¹³C₂] sodium acetates in cultures of a dinoflagellate Amphidinium species. [5] Amphidinolides F [6] and U [7] are considered to belong to the same group as amphidinolide C (2) in the structural diversity of amphidinolides. In this paper we describe the isolation and structure elucidation of a new 25-membered macrolide, amphidinolide C2 (1), isolated from the Y-71 strain of the symbiotic dinoflagellate Amphidinium species.

Results and Discussion

The dinoflagellate Amphidinium sp. (Y-71 strain) was obtained from an acoel flatworm Amphiscolops sp. collected off Sunabe, Okinawa. The mass cultured algal cells (1200 g, wet weight) obtained from 1300 L of culture were extracted with MeOH/toluene (3:1), and the extracts were partitioned between toluene and water. The toluene extracts were subjected to silica gel and then successive C₁₈ column chromatographies followed by C₁₈ HPLC to afford amphidinolide C2 (1, 0.00015 %) together with known macrolides, amphidinolides B, [8–10] C (2), [3] T1, [11–13] and T2. [12]

Amphidinolide C2 (1) had the molecular formula of C₄₃H₆₄O₁₁ as revealed by HRFABMS [m/z 779.4323, (M+Na)⁺, +2.3 mmu]. The IR spectrum indicated the presence of hydroxyl(s) (λ_max 3298 cm⁻¹) and carbonyl group(s) (λ_max 1738 and 1705 cm⁻¹). The UV spectrum showed the absorption at 230 nm (ɛ 20000) due to a conjugated diene chromophore. ¹H and ¹³C NMR data disclosed the existence of two ketones, two ester carbonyls, four sp² quaternary carbons, four sp² methines, two sp² methylenes, twelve sp³ methines (nine of them bearing an oxygen atom), ten sp³ methylenes, and seven methyls (two of them attached to olefins), which were similar to those of amphidinolide C (2) except for the presence of additional methyl and ester carbonyl carbons. Interpretation of the ¹H-¹H COSY and TOCSY spectra revealed proton connectivities of the following partial structures: from H₂-2 to H₂-37, from H₃-38 to H₂-14, from H₃-39 to H₂-17, from H₂-19 to H₃-40, and from H-29 to H₃-34. Connections among five units were deduced from the following HMBC correlations; H₃-37/C-12, H₂-14/C-15, H₃-39/C-15, H₂-17/C-18, H₂-19/C-18, and H₃-40/C-29. Geometries of three internal olefins at C-10–C-11, C-25–C-26, and C-27–C-28 were assigned as all E on the basis of NOESY cross-peaks for H-10/H-12, H-24/H-26, H-25/H-27, H-26/H₃-40, H-27/H-29, and H-36b/H₃-37. HMBC cross-peaks from H-6/C-3 and H-20/H-23 suggested that two tetrahydrofuran rings were formed between C-3 and C-6 and between C-20 and C-23, and their relative stereochemistries were implied by analysis of NOESY correlations. The existence of the 25-membered macrocylcic ring was implied by HMBC correlations from H₂-2 and H-24 to C-1. HMBC correlations from the low-field oxymethine proton (δ_H 5.89, s) at C-29 and a single methyl proton (δ_H 1.74, s, C-43) to an ester carbonyl carbon (δ_C 168.91, C-42), suggesting that an acetoxy group was attached to C-29. Thus the gross structure of amphidinolide C2 (1) was elucidated to be the 29-O-acetyl form of amphidinolide C (2).

Amphidinolide C2 (1) was converted into its 7,8,13-O-trisacetate (5), while the tetrakisacetate of amphidinolide C (2) was also prepared (Scheme 1). The spectral data of 3 derived from 1 were identical to those at the tetrakisacetate prepared from 2. Therefore, the absolute configurations of twelve chiral centers in 1 must be 3S, 4R, 6R, 7R, 8R, 12R, 13S, 16S, 20R, 23R, 24R, and 29S, the same as those previously determined for amphidinolide C (2).

Amphidinolide C2 (1) exhibited cytotoxicity against murine lymphoma L1210 and human epidermoid carcinoma KB cells (IC₅₀ 0.8 and 3 μg/mL, respectively) in vitro, which were less potent than those of amphidinolide C (2).

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 FAB mass spectra were obtained on a JEOL JMS HX-110 using p-nitrobenzyl alcohol as a matrix.

Cultivation and Isolation

The dinoflagellate Amphidinium sp. (strain number Y-71) was isolated from the inside cells of the marine acoel flatworm Amphiscolops sp. collected off Sunabe, 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 (1200 g wet weight, from 1300 L of culture) were extracted with MeOH/toluene (3:1, 600 mL × 5). After addition of 1 M NaCl aq. (500 mL), the mixture was extracted with toluene (500 mL × 3). Parts (3.30 g) of the toluene-soluble materials (6.63 g) were subjected to silica gel (CHCl₃/MeOH, 98:2) and C₁₈ column chromatographies (MeOH/H₂O, 8:2) to give a fraction (31.5 mg) containing some macrolides. The fraction was separated by C₁₈ HPLC (Develosil ODS-HG-5, Nomura Chemical Co. Ltd., 10 × 250 mm; eluent, CH₃CN/H₂O (65:35); flow rate, 2.5 mL/min; UV detection at 210 nm) to afford amphidinolides C2 (1, 1.8 mg, 0.00015 %, wet weight, t_R 23.0 min), B (0.0017 %), C (2, 0.0012 %), T1 (0.00092 %), and T2 (0.00013 %).

Spectral Data

Amphidinolide C2 (1): colorless oil; UV (MeOH) λ_max 230 (ɛ 20000); IR (KBr) ν_max 3298, 1738, 1705 cm^{−1; 1}H-NMR (600 MHz, benzene-d₆) δ 0.72 (3H, d, J = 6.4 Hz, H₃-35), 0.86 (3H, t, J = 7.4 Hz, H₃-34), 1.00 (6H, d, J =7.0 Hz, H₃-38/H₃-39), 1.13 (1H, m, H-21), 1.24 (2H, m, H₂-33), 1.36 (1H, m, H-22), 1.45 (1H, m, H-5), 1.53 (1H, m, H-4), 1.56 (3H, m, H-22/H₂-32), 1.72 (3H, br s, H₃-40), 1.74 (3H, s, H₃-43), 1.75 (1H, m, H-21), 1.77 (3H, br s, H₃-37), 1.86 (1H, m, H-5), 1.95 (1H, m, H-31), 1.99 (1H, m, H-31), 2.17 (2H, m, H-2/H-17), 2.29 (1H, m, H-12), 2.35 (2H, m, H-14/H-19), 2.50 (1H, dd, J = 8.2, 14.9 Hz, H-2), 2.59 (1H, dd, J = 9.5, 15.7 Hz, H-14), 2.75 (1H, dd, J = 9.5, 15.7 Hz, H-19), 3.00 (1H, dd, J = 8.5, 17.6 Hz, H-2), 3.14 (1H, m, H-16), 3.62 (1H, br t, J = 4.5 Hz, H-7), 3.89 (1H, dt, J = 2.5, 9.4 Hz, H-3), 4.00 (1H,m, H-23), 4.09 (1H, m, H-6), 4.10 (1H, m, H-13), 4.30 (1H, m, H-20), 4.32 (1H, d, J = 4.5 Hz, H-8), 4.97 (1H, s, H-41), 5.00 (1H, s, H-36), 5.19 (2H, s, H-36/H-41), 5.47 (1H, t, J = 8.0 Hz, H-24), 5.64 (1H, dd, J = 8.0, 15.1 Hz, H-25), 5.89 (1H, s, H-29), 6.30 (1H, s, H-10),6.30 (1H, d, J = 10.9 Hz, H-27), 6.74 (1H, dd, J = 10.9, 15.1 Hz, H-26); ¹³C NMR (150 MHz, benzene-d₆) δ 13.06 (C-40), 14.07 (C-34), 15.09 (C-37), 15.39 (C-38), 15.71 (C-35), 16.27 (C-39), 20.59 (C-43), 22.64 (C-33), 28.40 (C-22), 30.10 (C-32), 32.18 (C-21), 32.38 (C-31), 37.06 (C-5), 39.13 (C-14), 40.12 (C-4), 42.57 (C-16), 45.83 (C-19), 46.25 (C-17), 48.72 (C-2), 49.38 (C-12), 71.03 (C-13), 75.60 (C-20), 76.64 (C-7), 77.13 (C-24), 77.67 (C-8), 79.28 (C-6), 80.02 (C-23), 80.34 (C-29), 81.73 (C-3), 111.36 (C-41), 115.27 (C-36), 125.42 (C-10), 127.46 (C-27), 129.44 (C-26), 130.46 (C-25), 136.67 (C-28), 140.35 (C-9), 146.07 (C-11), 146.15 (C-30), 168.91 (C-42), 171.13 (C-1), 207.38 (C-18), 213.27 (C-15); (+)-FABMS m/z 779 (M+Na)⁺; (+)-HRFABMS m/z 779.4323 (C₄₃H₆₄O₁₁Na requies (M+Na)⁺, 779.4300).

7,8,13-O-Trisacetate (3) of Amphidinolide C2 (1): Amphidinolide C2 (1, 0.2 mg) was treated with acetic anhydride and pyridine (both 100 μL) at room temperature for 17 h. After evaporation of the solvent, the residue was subjected to a silica gel column (hexane/acetone, 2:1) to afford 7,8,13-O-triacetate (3, 0.2 mg) of amphidinolide C2. The spectroscopic data for compound 3 were identical to those of reported 7,8,13,29-O-tetrakisacetate of amphidinolide C (2). [3]