Iriomoteolides-14a and 14b, New Cytotoxic 15-Membered Macrolides from Marine Dinoflagellate Amphidinium Species

Abstract

Two new macrolides, iriomoteolides-14a (1) and 14b (2) have been isolated from the marine dinoflagellate Amphidinium species (strain KCA09057). Compounds 1 and 2 are 15-membered macrolides, which are structural analogs of amphidinolides O (3) and P (4). The structures of 1 and 2 were assigned on the basis of detailed NMR analyses and chemical conversion studies. Compounds 1 and 2 showed moderate cytotoxic activity against human cervix adenocarcinoma HeLa cells.

Introduction

Marine dinoflagellates of the genus Amphidinium have been recognized as rich sources of biologically active polyketides possessing unique chemical structures.¹⁾ Macrolides are one of the representative secondary metabolites of the Amphidinium dinoflagellates.^2,3) During our investigation on bioactive Amphidinium metabolites,^4,5) we have recently reported a 15-membered Amphidinium macrolide, iriomoteolide-9a,⁶⁾ from the marine benthic dinoflagellate Amphidinium species (KCA09052 strain). Further investigation on the other Amphidinium dinoflagellate (KCA09057 strain) have led to the discovery of two 15-membered Amphidinium macrolides, iriomoteolides-14a (1) and 14b (2) (Fig. 1). In this paper, we describe the isolation and structural elucidation of 1 and 2.

Fig. 1. Structures of Iriomoteolides-14a (1) and -14b (2) and Amphidinolides O (3) and P (4)

Results and Discussion

The dinoflagellate Amphidinium species (strain KCA09057) was cultivated at 25°C for 2 weeks in seawater medium under illumination. The algal cells were extracted with methanol (MeOH)–toluene (3 : 1). The toluene-soluble materials of the extract were subjected to silica gel, octadecylsilyl (ODS), and amino silica gel column chromatography followed by reversed-phase HPLC to afford iriomoteolides-14a (1, 0.0015% yield from dry weight) and 14b (2, 0.0022% yield) (Fig. 1).

Iriomoteolide-14a (1) was obtained as an optically active and colorless amorphous solid. The molecular formula of 1 was determined to be C₂₂H₃₂O₇ by electrospray ionization (ESI)-MS (m/z 431.2031 [M + Na]⁺, Δ −0.89 mmu). The ESI-MS data (m/z 434.2222 [M + Na]⁺, C₂₂H₂₉D₃O₇Na, Δ −0.68 mmu) obtained in MeOH-d₄ suggested the presence of three hydroxy groups in 1. The ¹³C-NMR spectrum in chloroform-d (CDCl₃) (Table 1) showed 22 carbon resonances, and their chemical shifts and multiplicities assigned using the heteronuclear multiple quantum coherence (HMQC) spectrum indicated the presence of 1 ester carboxyl, 2 sp² and 2 sp³ quaternary carbons, 8 methines including 2 sp² ones, 6 methylenes including 2 sp² ones, and 3 methyl carbons. Three olefins and one carbonyl accounted for four out of the seven degrees of unsaturation, implying that compound 1 contains three rings.

Table 1. ¹H- and ¹³C-NMR Data of Iriomoteolides-14a (1) and 14b (2) in CDCl₃

Position	1				2
	13C		1H		13C		1H
1	172.6	C			173.8	C
2	44.0	CH2	2.70	d, 12.4	44.3	CH2	2.72	d, 12.5
			2.65	d, 12.4			2.67	d, 12.5
3	98.9	C			99.3	C
4	44.9	CH	2.28	m	44.8	CH	2.34	m
5	143.5	C			143.8	C
6	39.4	CH2	2.59	dd, 12.9, 3.0	39.4	CH2	2.57	dd, 13.0, 2.7
			2.26	dd, 12.9, 11.4			2.30	m
7	73.2	CH	3.33	ddd, 11.4, 8.0, 3.0	73.9	CH	3.33	ddd, 11.5, 8.5, 2.7
8	62.9	CH	2.67	dd, 8.0, 2.0	59.4	CH	2.94	dd, 8.5, 2.0
9	54.3	CH	2.82	dt, 9.5, 2.0	54.0	CH	2.65	m
10	39.6	CH2	2.34	dd, 13.9, 2.0	39.1	CH2	2.36	dd, 14.0, 2.0
			1.41	dd, 13.9, 9.5			1.45	dd, 14.0, 9.5
11	73.2	C			74.2	C
12	133.5	CH	5.96	d, 16.1	134.2	CH	5.96	d, 15.8
13	129.7	CH	5.88	dd, 16.1, 5.2	129.0	CH	5.80	dd, 15.8, 7.0
14	74.4	CH	5.32	dd, 7.6, 4.9	76.3	CH	5.43	t, 7.0
15	45.4	CH	2.49	m	44.6	CH	2.50	m
16	146.4	C			145.9	C
17	112.4	CH2	4.80	s	112.5	CH2	4.80	s
			4.75	s			4.77	s
18	12.0	CH3	1.11a)	d, 6.7	12.0	CH3	1.10a)	d, 6.7
19	110.2	CH2	4.98	s	110.1	CH2	4.97	s
			4.84	s			4.83	s
20	15.6	CH3	1.04a)	d, 6.7	15.7	CH3	1.04a)	d, 6.7
21	20.1	CH3	1.71a)	s	20.1	CH3	1.71a)	s
22	70.6	CH2	3.40b)	br s	69.0	CH2	3.52b)	br s

1: 3-OH: 4.49 (br s), 11-OH: 2.47 (br s), 20-OH: 1.87 (br s) 2: 3-OH: 4.42 (br s), 11-OH: 2.32 (br s), 20-OH: 1.78 (br s) a) 3H. b) 2H.

The planar structure of 1 was elucidated on the basis of detailed NMR studies, including ¹H–¹H correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) spectra recorded in CDCl₃. The ¹H–¹H COSY and TOCSY spectra revealed three ¹H–¹H networks from H-4 to H₃-18, from H₂-6 to H₂-10, and from H-12 to H₃-20 (Fig. 2). The disubstituted C-12–C-13 double bond was assigned to be E, as suggested by the J(H-12/H-13) value (16.1 Hz). The presence of the trans epoxide at C-8 was attributed to the ¹³C chemical shifts for C-8/C-9 (δ_C 62.9/54.3) and the specific J(H-8/H-9) value (2.0 Hz). HMBC correlations for H₂-2 (δ_H 2.70 and 2.65)/C-3 (δ_C 98.9), H₃-18 (δ_H 1.11)/C-3, and 3-OH (δ_H 4.49)/C-4 (δ_C 44.9) suggested that the hemiketal carbon at C-3 was attached to C-2 and C-4. The presence of two exomethylene groups at C-5 and C-16 was established by the HMBC correlations for H₂-6 (δ_H 2.59 and 2.26)/C-5 (δ_C 143.5), H₃-18/C-5, H₂-19 (δ_H 4.98 and 4.84)/C-6 (δ_C 39.4), H₂-17 (δ_H 4.80 and 4.75)/C-15 (δ_C 45.4), H₂-17/C-21 (δ_C 20.1), and H₃-20 (δ_H 1.04)/C-16 (δ_C 146.4). The NOESY correlations for H₂-10 (δ_H 2.34 and 1.41)/H₂-22 (δ_H 3.40, 2H) and H-12 (δ_H 5.96)/H₂-22 and HMBC correlations for H-10a/C-11 (δ_C 73.2) and H-12/C-11 indicated that the oxymethylene carbon at C-22 was connected to C-10 and C-12 via the oxygenated quaternary carbon at C-11. The HMBC correlation was observed from H₂-2 to C-1 (δ_C 172.6), indicating that an ester carbonyl was attached to C-2. The relative low-field chemical shift for H-14 (δ_H 5.32) suggested that C-14 was involved in an ester linkage with C-1. The third ring system in the molecule was revealed to be a six-membered ether ring at C-3–C-7 by the NOESY correlation for 3-OH/H-7 (δ_H 3.33). Therefore, the gross structure of 1 was concluded to possess a 15-membered lactone ring associated with a tetrahydropyran and an epoxide rings. Moreover, the structure of 1 corresponded to the 11,22-dihydroxy form of a known 15-membered macrolide, amphidinolide P (4).^7–12)

Fig. 2. Selected 2D-NMR Correlations for Iriomoteolide-14a (1)

Elucidation of the stereochemistry of eight stereocenters in 1 was achieved by chemical conversion studies and spectroscopic analysis. Treatment of 1 with sodium periodate (NaIO₄) afforded a ketone compound, of which the ¹H-NMR spectrum and the optical rotation were identical to those of amphidinolide O (3).^7,11) Thus, seven of eight stereocenters in 1 (except C-11) were established to have the same configurations as those of amphidinolides O (3) and P (4). The stereochemistry at C-11 was elucidated by the conformational analysis of 1, as shown in Fig. 3. NOESY correlations for H-8 (δ_H 2.67)/H-10b, H-8/H₂-22, and H-10b/H₂-22 suggested that the C-22 hydroxymethyl group adopted β-orientation. Therefore, the structure of iriomoteolide-14a was concluded to be 1 as shown in Fig. 1.

Fig. 3. Stereostructure for C-3 − C-11 Portion in Iriomoteolide-14a (1)

“a” and “b” for geminal proton pairs denoted low-and high-field resonances, respectively. Vicinal ¹H–¹H coupling constants (Hz) (H/H): 3.0 (6a/7), 11.4 (6b/7), 8.0 (7/8), 2.0 (8/9), 2.0 (9/10a), 9.5 (9/10b).

Iriomoteolide-14b (2) was obtained as an optically active and colorless amorphous solid. The molecular formula, C₂₂H₃₂O₇, of 2 (m/z 481.2034 [M + Na]⁺, Δ －0.62 mmu) was identical to that of 1. The ¹H- and ¹³C-NMR data of 2 were closely similar to those of 1. Detailed analysis of the spectroscopic data showed that 2 possessed the same gross structure as that of 1. The structure of 2 including its absolute stereochemistry was elucidated to be the 11R-isomer of 1 upon conversion of 2 to amphidininolide O (3) through NaIO₄ oxidation and stereochemical analysis as shown in Fig. 4. The NOESY correlation for H-9/H₂-22 certainly indicated that compound 2 possessed α-orientation for the C-22 hydroxymethyl group.

Fig. 4. Stereostructure for C-3 − C-11 Portion in Iriomoteolide-14b (2)

Vicinal ¹H–¹H coupling constants (Hz) (H/H): 2.7 (6a/7), 11.5 (6b/7), 8.5 (7/8), 2.0 (8/9), 2.0 (9/10a), 9.5 (9/10b).

Iriomoteolide-14a (1) and 14b (2) are new 15-membered macrolides related to amphidinolides O (3) and P (4). The presence of 1 and 2 may indicate that 3 is generated from 4 through epoxidation of the exomethylene group at C-11, hydration of the oxirane, and cleavage of the resulting diol (Fig. 5), though compounds 3 and 4 have not been detected in this algal cell extract so far. Compounds 1 and 2 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa cells (IC₅₀: 10 µM for both 1 and 2), which were almost equivalent to those of 3 and 4 (IC₅₀: 8 and 12 µM, respectively).

Fig. 5. Plausible Biogenetic Scheme from Amphidinolide P (4) to Amphidinolide O (3) via Iriomoteolides-14 (1 or 2)

Experimental

General Experimental Procedures

The optical rotations and IR data were measured on a JASCO P-2300 polarimeter and a JASCO FT/IR-4100 spectrophotometer, respectively. The NMR data for 1 and 2 were recorded on an NMR400WB spectrometer (Agilent Technologies, U.S.A.) using 5.0 mm microcells (Shigemi Co., Ltd., Japan). The ¹H-NMR spectra of the products treated with NaIO₄ were measured on the same spectrometer equipped with a PFG-HX nanoprobe. All chemical shifts were reported in ppm relative to the residual proton and carbon signals of CDCl₃ (δ_H 7.26 and δ_C 77.16, respectively) and benzene-d₆ (C₆D₆, δ_H 7.20 and δ_C 128.0, respectively). ESI-MS was conducted on an LTQ Orbitrap XL spectrometer (ThermoFisher Scientific Inc., U.S.A.) The cytotoxic assay and the cultivation/harvest protocol was carried out using a literature procedure.⁶⁾

Materials

The dinoflagellate Amphidinium species (strain KCA09057) was monoclonally separated from benthic sea sands collected off Iriomote Island, Japan in May 2009.⁶⁾ The voucher specimen was deposited at the Center for Advanced Marine Core Research, Kochi University.

Extraction and Isolation

The dried algal cells (53.54 g) obtained from 2000 L of medium were extracted with MeOH–toluene (3 : 1), and partitioned between toluene and water (H₂O). The toluene-soluble materials (2.94 g) of the extract were subjected to silica gel CC (Wakogel^® C-300, FUJIFILM Wako Pure Chemical Corporation, Japan) using a gradient elution of 0–5% MeOH in CHCl₃. The fraction eluted with CHCl₃–MeOH (95 : 5) was then chromatographed using an ODS [Wakogel^® 50C18, FUJIFILM Wako Pure Chemical Corporation; eluent: acetonitrile (CH₃CN)–H₂O, 7 : 3] and amino silica gel [Wakogel^® 50NH2, FUJIFILM Wako Pure Chemical Corporation; eluent: hexane–ethyl acetate (EtOAc), 1 : 1–1 : 4] columns. The fraction eluted with hexane–EtOAc (1 : 4) was separated by reversed-phase HPLC [YMC-Pack Pro C₁₈, 5 µm, YMC Co., Ltd., 10×250 mm; eluent, CH₃CN–H₂O (70 : 30); flow rate, 2 mL/min; UV detection at 230 nm] to afford 1 [0.8 mg, 0.0015%, retention time (t_R) 5.3 min] and 2 (1.2 mg, 0.0022%, t_R 4.5 min).

Iriomoteolide-14a (1)

Colorless amorphous solid; [α]_D²⁰ + 20 (c = 0.25, CHCl₃); IR (neat) cm⁻¹: 3452 (broad), 2926, 1710; ¹H- and ¹³C-NMR (400 MHz, CDCl₃): Table 1; ESI-MS (MeOH) m/z: 431.2031 [M + Na]⁺ (Calcd for C₂₂H₃₂O₇Na, 431.2040); ESI-MS (MeOH-d₄) m/z 434.2222 [M + Na]⁺ (Calcd for C₂₂H₂₉²H₃O₇Na, 434.2229).

Iriomoteolide-14b (2)

Colorless amorphous solid; [α]_D²⁰ + 11 (c = 0.20, CHCl₃); IR (neat) cm⁻¹: 3430 (broad), 2953, 1715; ¹H- and ¹³C-NMR (400 MHz, CDCl₃): Table 1; ESI-MS m/z: 431.2034 [M + Na]⁺ (Calcd for C₂₂H₃₂O₇Na, 431.2040); ESI-MS (MeOH-d₄) m/z: 434.2222 [M + Na]⁺ (Calcd for C₂₂H₂₉²H₃O₇Na, 434.2229).

NaIO₄ Degradation of Iriomoteolide-14a (1)

To a solution of iriomoteolide-14a (1, 0.2 mg) in a 1 : 1 mixture (50 µL) of CH₃CN/phosphate buffer saline (PBS, pH 7.0) added NaIO₄ (0.5 mg), and the stirring was continued for 1 h at room temperature. The mixture was extracted with CHCl₃, and concentrated in vacuo. The residue was subjected to C₁₈ HPLC [YMC-Pack Pro C₁₈, 5 µm, 10 × 250 mm; eluent, CH₃CN/H₂O (70 : 30); flow rate, 2 mL/min; UV detection at 230 nm] to afford amphidinolide O (3, 0.10 mg, t_R 10.5 min).

Amphidinolide O (3)

[α]_D²⁰ +100 (c = 0.02, benzene); ¹H-NMR (C₆D₆) δ: 0.93 (3H, d, J = 7.0 Hz), 1.01 (3H, d, J = 6.8 Hz), 1.59 (3H, s), 1.85 (1H, dd, J = 16.1 and 10.1 Hz), 1.98 (1H, m), 2.08 (1H, brt, J = 10.4 Hz), 2.21 (1H, dt, J = 11.0 and 2.5 Hz), 2.35 (1H, m), 2.36 (1H, d, J = 12.8 Hz), 2.37 (1H, m), 2.40 (1H, d, J = 12.8 Hz), 2.51 (1H, dd, J = 13.0 and 2.8 Hz), 2.97 (1H, dd, J = 16.0 and 2.4 Hz), 3.47 (1H, ddd, J = 11.4, 8.2, and 2.8 Hz), 4.80 (1H, d, J = 2.0 Hz), 4.81 (1H, br s), 4.84 (1H, br s), 4.87 (1H, br s), 4.89 (1H, br s), 5.53 (1H, ddd, J = 7.4, 2.6, and 1.6 Hz), 7.00 (1H, dd, J = 15.5 and 1.6 Hz), and 7.07 (1H, ddd, J = 15.5 and 2.6 Hz); ESI-MS m/z: 399.1776 [M + Na]⁺ (Calcd for C₂₁H₂₈O₆Na, 399.1784).

NaIO₄ Degradation of Iriomoteolide-14b (2)

Iriomoteolide-14b (2, 0.2 mg) was treated under the same conditions as described above to afford amphidinolide O (3, 0.16 mg, t_R 10.5 min).

Amphidinolide O (3)

[α]_D²⁰ +110 (c = 0.05, benzene) ; ¹H-NMR (C₆D₆) δ: 0.93 (3H, d, J = 7.0 Hz), 1.01 (3H, d, J = 6.8 Hz), 1.59 (3H, s), 1.85 (1H, dd, J = 16.1 and 10.1 Hz), 1.98 (1H, m), 2.08 (1H, brt, J = 10.9 Hz), 2.22 (1H, dt, J = 11.0 and 2.5 Hz), 2.35 (1H, m), 2.36 (1H, d, J = 12.8 Hz), 2.37 (1H, m), 2.40 (1H, d, J = 12.8 Hz), 2.51 (1H, dd, J = 13.0 and 2.7 Hz), 2.97 (1H, dd, J = 16.0 and 2.4 Hz), 3.47 (1H, ddd, J = 11.3, 8.2, and 2.7 Hz), 4.79 (1H, d, J = 1.9 Hz), 4.81 (1H, br s), 4.84 (1H, br s), 4.87 (1H, br s), 4.89 (1H, br s), 5.53 (1H, ddd, J = 7.4, 2.5, and 1.6 Hz), 7.00 (1H, dd, J = 15.4 and 1.6 Hz), and 7.07 (1H, ddd, J = 15.4 and 2.5 Hz) ; ESI-MS m/z: 399.1774 [M + Na]⁺ (Calcd for C₂₁H₂₈O₆Na, 399.1784).

Acknowledgments

We thank Tsuyoshi Matsuzaki, The Institute of Scientific and Industrial Research, Osaka University, for measurement of ESI-MS spectra.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials. Spectral data of compounds 1 and 2 and NaIO₄ degradation products of 1 and 2 are available as supplementary material.

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