Iriomoteolides-10a and 12a, Cytotoxic Macrolides from Marine Dinoflagellate Amphidinium Species

Abstract

Two new macrolides, iriomoteolides-10a (1) and -12a (2), have been isolated from a marine dinoflagellate Amphidinium sp. (KCA09053 strain), and their structures were elucidated on the basis of a detailed two dimensional (2D)-NMR analysis. Compound 1 is a novel 21-membered Amphidinium macrolide, which contains one tetrahydrofuran ring, two ketone carbonyls, two hydroxyl groups, and six one-carbon branches. Compound 2 is a new 12-membered macrolide related to amphidinolide Q. Compound 1 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa and murine hepatocellular carcinoma MH134 cells.

Marine dinoflagellates of the genus Amphidinium have been proven to produce unique polyketide-like metabolites with interesting biological activities.¹⁾ A series of macrolides, represented by amphidinolides, are well-known secondary metabolites of Amphidinium dinoflagellates^2,3) with various carbon skeletons forming 12–29-membered (including odd-numbered) macrolactone rings and biosynthetically unique partial structures such as vicinally located C₁ branches. During our investigation of bioactive Amphidinium metabolites,^4,5) we have recently reported the isolation of 22- and 23-membered Amphidinium macrolides, i.e., iriomoteolides-13a⁶⁾ and -2a,⁷⁾ respectively, from the marine benthic dinoflagellate Amphidinium species (KCA09053 and HYA024 strains, respectively). Further investigation of the cytotoxic fractions of KCA09053 strain led to the discovery of the first 21-membered Amphidinium macrolide, iriomoteolide-10a (1), together with a new 12-membered macrolide, iriomoteolide-12a (2) (Fig. 1). Herein, we describe the isolation and structural elucidation of 1 and 2.

Fig. 1. Structures of Iriomoteolides-10a (1) and -12a (2)

C-3, C-6, C-8, C-9 and C-11 and C-14, C-16, C-17, C-18, and C-20 of 1 were independently relative.

Results and Discussion

The dinoflagellate Amphidinium sp. (strain KCA09053) 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 cytotoxic assay-guided fractionation using a silica gel column with chloroform (CHCl₃)–MeOH. One of the cytotoxic fractions was separated with an octadecylsilyl (ODS) column followed by reversed-phase HPLC to afford iriomoteolide-10a (1, 0.069% from dry weight). On the other hand, iriomoteolide-12a (2) was obtained from the less polar fraction of the first silica gel column chromatography (CC) in 0.016% yield. Known macrolides, iriomoteolides-1a,⁸⁾ -3a,⁹⁾ and -13a,⁶⁾ have been separated by the first SiO₂ gel column from a more polar fraction than those containing 1 and 2.

Iriomoteolide-10a (1) was obtained as optically active and colorless amorphous solid, and the molecular formula of C₃₅H₆₀O₇ was revealed by electrospray ionization (ESI)-MS data (m/z 615.4315 [M+Na]⁺, Δ+0.1 mmu). The ¹³C-NMR spectrum in benzene-d₆ (C₆D₆) (Table 1) showed the 35 carbon resonances, and their chemical shifts and multiplicities assigned by heteronuclear multiple quantum coherence (HMQC) and CH₂-selected heteronuclear single quantum coherence (HSQC) spectra indicated the presence of 2 ketones, 1 ester carboxyl, 13 methines including two sp² ones, 12 methylenes, and 7 methyl carbons. One olefin and three carbonyl groups accounted for four out of the six degrees of unsaturation; thus, the remaining double bond equivalents were attributed to the presence of two rings in the molecule of 1.

Table 1. ¹H- and ¹³C-NMR Data for Iriomoteolide-10a (1) in C₆D₆

Position	13C	1H (mult, J=in Hz)	Position	13C	1H (mult, J=in Hz)
1	171.3 C		17	90.6 CH	3.77 dd, 7.0, 2.8
2	42.9 CH2	2.36 dd, 14.0, 4.7	18	34.5 CH	1.94 m
		2.21 dd, 14.0, 9.8	19	33.1 CH2	2.03 ddd, 15.0, 9.7, 3.3
3	34.3 CH	2.78 m			1.58 ddd, 15.0, 10.0, 3.1
4	133.7 CH	5.50 dd, 15.0, 7.2	20	75.3 CH	5.37 dd, 9.7, 3.1
5	135.4 CH	5.30 dd, 15.0, 8.6	21	210.0 C
6	34.1 CH	2.54 m	22	42.8 CH	2.61 m
7	38.7 CH2	1.43 m	23	32.5 CH2	1.95 m
		1.27 m			1.17 m
8	71.8 CH	3.74 ddd, 9.8, 3.5, 1.8	24	37.2 CH2	1.11a) m
9	37.1 CH	1.81 m	25	23.8 CH2	1.51 m
10	41.5 CH2	1.41 m			1.34 m
		0.96 m	26	41.7 CH2	1.92a) t, 7.0
11	31.3 CH	1.49 m	27	210.1 C
12	33.6 CH2	1.52 m	28	35.7 CH2	1.91a) q, 7.0
		1.20 m	29	7.9 CH3	0.94b) t, 7.0
13	34.5 CH2	1.57 m	30	20.5 CH3	0.98b) d, 7.0
		1.54 m	31	22.6 CH3	1.10b) d, 7.0
14	78.1 CH	4.17 m	32	15.2 CH3	0.99b) d, 7.0
15	41.8 CH2	1.84 m	33	20.6 CH3	0.92b) d, 7.0
		1.64 ddd, 14.5, 8.0, 6.6	34	16.5 CH3	1.15b) d, 7.0
16	74.0 CH	4.12 ddd, 7.0, 6.6, 3.5	35	17.9 CH3	1.22b) d, 7.0

a) 2H. b) 3H.

The planar structure of 1 was elucidated on the basis of detailed NMR studies, namely ¹H–¹H correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear multiple bond correlation (HMBC), and CH₂-selected HSQC-TOCSY spectra measured in benzene-d₆. ¹H–¹H COSY and TOCSY spectra revealed three ¹H–¹H networks from H₂-2 to H-20, H₃-30, H₃-31, H₃-32, H₃-33, and H₃-34, from H-22 to H₂-26 and H₃-35, and from H₂-28 to H₃-29 (Fig. 2). The E-geometry was assigned to the disubstituted C-4–C-5 double bond, as suggested by the J(H-4/H-5) value (15.0 Hz). The long-range ¹H–¹³C correlations for H₂-26 (δ_H 1.92)/C-24 (δ_C 37.2) and H₃-35 (δ_H 1.22)/C-24 observed in CH₂-selected HSQC-TOCSY spectrum supported an aliphatic substructure for the C-22–C-26 portion. HMBC correlations from H-20 (δ_H 5.37) and H₃-35 to a ketone carbonyl (C-21: δ_C 210.0) and from H₂-26 (δ_H 1.92) and H₃-29 (δ_H 0.94) to another ketone carbonyl (C-27: δ_C 210.1) suggested that the ketone groups connect C-20 to C-22 and C-26 to C-28. The HMBC correlation for H-17 (δ_H 3.77)/C-14 (δ_C 78.1) revealed the presence of a tetrahydrofuran ring at C-14–C-17. The ester linkage between C-1 and C-20 was established by the HMBC correlations for H₂-2 (δ_H 2.36 and 2.21)/C-1 (δ_C 171.3) and H-20/C-1. Thus, the gross structure of 1 was concluded to contain a 21-membered macrolactone ring, as shown in Fig. 1.

Fig. 2. Selected 2D-NMR Correlations for Iriomoteolide-10a (1)

The relative stereochemistry of 1 was investigated on the basis of nuclear Overhauser effect spectroscopy (NOESY) correlations and coupling constants. Magnitude of the ¹H–¹H couplings was estimated from the intensity of the ¹H–¹H COSY correlations, when signals overlapped with other signals or showed multiple couplings. The J-based configuration analysis¹⁰⁾ was applied to the investigation of C-8–C-9 and C-17–C-18 bonds. Long-range ¹³C–¹H coupling constants were obtained from analyses of the hetero half-filtered TOCSY (HETLOC) correlations or from the cross-peak intensities in the phase-sensitive HMBC spectrum.

The antiperiplanar relationship for H-3–H-4 and H-5–H-6 was deduced from NOESY correlations for H-3/H-5 and H-4/H-6 and from the relatively large J(H-3/H-4) (7.2 Hz) and J(H-5/H-6) values (8.6 Hz), thus suggesting an equatorial orientation for both C-30 and C-31 methyl groups (Fig. 3a). The 1,3-syn relation for C-31 and 8-OH was indicated by the coupling constants for H-6/H-7a (>8 Hz), H-6/H-7b (<4 Hz), H-7a/H-8 (9.8 Hz), and H-7b/H-8 (3.5 Hz), and by NOESY correlations for H-5/H-7b and H-5/H-8. Considering the coupling constants for H-8/H-9 (1.8 Hz), C-8/H-9 (−1 Hz), C-10/H-8 (<3 Hz), and C-32/H-8 (<6 Hz), the C-8–C-9 bond was suggested to have the threo configuration (Fig. 3b). The NOESY correlation observed for H-8/H-11 indicated that the methine proton of C-8 was oriented toward the inside of the macrocyclic ring. The relatively large J(H-9/H-10a) and J(H-10a/H-11) values (both >8 Hz) were deduced to be an antiperiplanar relationship between these protons and an equatorial orientation for both C-32 and C-33 methyl groups, which was corroborated by the NOESY correlation for H-9/H-11.

Fig. 3. Stereostructures for (a) C-2–C-8, (b) C-8–C-11, and (c) C-14–C-20 Portions in Iriomoteolide-10a (1)

“a” and “b” for germinal proton pairs denoted low-and high-field resonances, respectively. ¹H–¹H coupling constants (Hz) (H/H): 4.7 (2a/3), 9.8 (2b/3), 7.2 (3/4), 7.0 (3/30), 15.0 (4/5), 8.6 (5/6), >8 (6/7a), <4 (6/7b), 7.0 (6/31), 9.8 (7a/8), 3.5 (7b/8), 1.8 (8/9), >8 (9/10a), <4 (9/10b), 7.0 (9/32), >8 (10a/11), <4 (10b/11), 7.0 (11/33), <4 (14/15a), 8.0 (14/15b), 3.5 (15a/16), 6.6 (15b/16), 7.0 (16/17), 2.8 (17/18), 3.3 (18/19a), 10.0 (18/19b), 7.0 (18/34), 9.7 (19a/20), 3.1 (19b/20). ¹³C–¹H coupling constants (Hz) (C/H): −1 (8/9), <3 (10/8), ca. 0 (17/18), 6 (19/17), <6 (32/8), 2 (34/17).

For the tetrahydrofuran portion at C-14–C-17 (Fig. 3c), NOESY correlations for H-14/H-17 and H-16/H-18 revealed syn and anti configurations for H-14–H-17 and H-16–H-17, respectively. The erythro configuration of the C-17–C-18 bond was elucidated from the coupling constants of H-17/H-18 (2.8 Hz), C-17/H-18 (ca. 0 Hz), C-19/H-17 (6 Hz), and C-34/H-17 (2 Hz). The anti relations for H-18–H-19b and H-19a–H-20 were implied by their relatively large coupling constants (10.0, 9.7 Hz, respectively). Moreover, a 1,3-syn configuration for the C-18–C-20 portion was suggested by NOESY correlations for H-16/H-18, H-16/H-19a, H-17/H-19b, H-18/H-20, and H-20/H₃-34. On the other hand, the stereochemical relationship between C-20 and C-22 through the C-21 ketone could not be determined, although NOESY correlations were found for H-20/H-22, H-20/H-23b, and H-20/H₃-35. In addition, because of the overlap between H-12a and H₂-13, the relative stereochemistry between C-11 and C-14 could not be unambiguously assigned. In order to determine the absolute configuration at C-8 and C-16, the modified Mosher’s method was applied using a small amount of 1; however, 1 decomposed in the reaction with α-methoxy-α-trifluormethyphenylacetyl chloride (MTPACl).

The molecular formula, C₂₅H₄₀O₅, of iriomotrolide-12a (2) was established by ESI-MS (m/z 443.2768 [M+Na]⁺, Δ+0.0 mmu). The ¹³C-NMR spectrum in chloroform-d₁ (CDCl₃) disclosed the presence of a total of 25 carbon signals due to 2 ketones, 1 ester carbonyl, 2 sp² quaternary carbons, 2 sp² methines, 6 sp³ methines including 2 oxygenated ones, 5 sp³ methylenes, and 7 methyl groups. The ¹H-NMR spectrum showed the presence of two singlet sp² methines [δ 5.99 (H-17), 5.89 (H-2)] and three singlet methyl groups [δ 2.12 (H₃-19), 2.07 (H₃-20), and 2.06 (H₃-25)], all presumably adjacent to quaternary carbons. ¹H–¹H COSY and TOCSY spectra revealed two ¹H–¹H networks from H-4 to H₂-5 and from H-7 to H₂-15, H₃-21, H₃-22, H₃-23, and H₃-24 (Fig. 4). HMBC correlations for H-2/C-3 (δ_C 154.9), H₃-20/C-3, and H₃-20/C-4 (δ_C 73.1) indicated that C-2 and C-20 were connected to C-4 through the sp² quaternary carbon C-3. The presence of a ketone carbonyl at C-6 was implied by HMBC correlations for H₂-5 (δ_H 3.02, 2.48)/C-6 (δ_C 217.0) and H₃-21 (δ_H 1.03)/C-6. Attachment of a pent-2-en-3-one terminus at C-15 was deduced from HMBC correlations for H₂-15 (δ_H 2.07, 1.84)/C-16 (δ_C 157.9), H-17/C-18 (δ_C 198.7), H₃-19/C-18, H₃-25/C-16, and H₃-25/C-17. The E-geometries for the two trisubstituted double bonds at C-2–C-3 and C-16–C-17 were inferred from the ¹³C chemical shifts of the C-20 and C-25 methyl groups (δ_C 16.8, 14.7, respectively). A four-bond HMBC correlation was observed from H₃-20 to C-1 (δ_C 167.7), thus indicating that an ester carbonyl was attached to C-2. The relatively low-field resonance for the oxymethine H-11 (δ_H 5.06) suggested that C-11 was involved in an ester linkage with C-1. Thus, the planar structure of 2 was found to consist of a 12-membered macrolide associated with two α,β-unsaturated carbonyls, a saturated ketone, six C₁ branches, and a hydroxyl group. Moreover, the gross structure of the macrocyclic portion of 2 corresponded to that of amphidinolide Q^11–13) (3), a known 12-membered macrolide.

Fig. 4. Selected 2D-NMR Correlations for Iriomoteolide-12a (2)

In order to elucidate the stereochemistry of 2, the ¹³C chemical shifts of 2 in CDCl₃ were compared with the data reported¹²⁾ for 3. Figure 5 shows the absolute values of chemical shift differences [Δδ (in ppm)=δ (2 in CDCl₃)−δ (3 in CDCl₃)] for C-1–C-13 and four branched methyl groups at C-3, C-7, C-9, and C-13. The absolute Δδ values for C-1–C-11 and methyls at C-3, C-7, and C-9 were extremely small (≤0.2 ppm), strongly suggesting that the relative stereochemistries for C-4, C-7, C-9, and C-11 in the macrolactone ring of 2 were the same as those of 3. On the other hand, because of side-chain structural differences between 2 and 3, the Δδ values for C-12, C-13, and 13-Me were high. C-11 and C-13 of 2 were assumed to take a syn configuration, as that in 3, because the strongly coupled signal for H₂-12 [δ_H 1.42 (2H)] of 2 was reminiscent of the signal pattern of 3.¹²⁾ Moreover, the optical rotation ([α]_D) of 2 showed the same sign and similar magnitude as that reported for 3, presumably indicating that the absolute stereochemistries for C-4, C-7, C-9, C-11, and C-13 of 2 corresponded to those of 3. The relatively small coupling constant (≤4 Hz) for H-13–H-14 suggested a gauche relation between H-13 and H-14. Nevertheless, the relative stereochemistry of the C-13–C-14 bond could not be assigned, because no information on long-range ¹³C–¹H coupling around the C-13–C-14 bond was obtained from the HETLOC spectrum.

Fig. 5. Chemical Shift Differences (Δδ) between Iriomoteolide-12a (2) and Amphidinolide Q (3)

Iriomoteolide-10a (1) is the first 21-membered Amphidinium macrolide, although several macrolides with odd-numbered, e.g. 13-, 15-, 17-, 19-, 23-, 25-, 27-, and 29-membered, lactone rings were isolated from the marine dinoflagellates Amphidinium species.³⁾ Isoapoptolidin^14,15) has been reported as the first example of 21-membered macrolide from natural sources, and corresponds to a ring-expanded isomer of the 20-membered macrolide, apoptolidin.^16,17) The interconversion between apoptolidin and isoapoptolidin was described to occur in dilute aqueous solution. However, such isomerization was not observed for 1, in alcohol or water. Furthermore, linear fatty acid precursors for 1 and 2 have not been observed in the extract. Irimoteolide-12a (2) is a new congener of amphidinolide Q (3), containing a 12-membered lactone ring. Compound 1 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa, human B lymphocyte DG-75, and murine hepatocellular carcinoma MH134 cells (IC₅₀: 1.5, 1.2, 3.3 µM, respectively). Compound 2 showed moderate cytotoxicity against DG-75 cells (IC₅₀: 50 µM).

Experimental

General Experimental Procedures

Optical rotation and IR data were measured on a JASCO DIP-370 polarimeter and a JASCO FT/IR-5300 spectrophotometer, respectively. NMR data were recorded using 2.5 mm microcells (Shigemi Co., Ltd., Japan). NMR spectra were measured on a Bruker AMX-500 spectrometer or a Varian-NMR500 spectrometer equipped with a triple resonance PFG Cold Probe or CH “Xsens” PFG Cold Probe. Chemical shifts were reported in ppm with reference to the residual proton and carbon signals of C₆D₆ (δ_H 7.20 and δ_C 128.0, respectively) and CDCl₃ (δ_H 7.26 and δ_C 77.0, respectively). ESI-MS spectra were recorded on a JEOL JMS-T100LC spectrometer.

Materials

A dinoflagellate Amphidinium species (strain KCA09053) 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

For the cultivation of the dinoflagellate, deep seawater pumped up from −344 m, offshore of Muroto, Japan, was sterilized by filtration performed using an ultrafiltration membrane, and was then utilized. Cultivation was performed using a 50 L fermenter (Earth Co., Japan) composed of polycarbonate resin. The culture was maintained in seawater supplemented with 1% Provasoli’s enriched seawater at 25–30°C with mechanical stirring at 180 rpm stirring using a MAZELA Z-1300 mechanical stirrer (Tokyo Rikakikai Co., Ltd., Japan) under illumination of approximately 30 µmol photons·m⁻²·s⁻¹ by fluorescent lights with a 16 h : 8 h light/dark cycle. The cultured algal cells were filtered and concentrated by using a MOLSEP® Fiber FS03-FC-FUS1582 ultrafiltration membrane (Daicen Membrane-Systems Ltd., Japan). The concentrated cells (ca. 2 L from 50 L culture) were centrifuged at 3000 rpm at 4°C for 10 min by using a Hitachi CR 22GIII high speed refrigerated centrifuge (Hitachi Koki Co., Ltd., Japan) and then lyophilized using an EYELA FD-511 freeze dryer (Tokyo Kasei Co., Ltd., Japan). The dried algal cells (30.3 g) obtained from 350 L of medium were extracted with MeOH–toluene (3 : 1, 300 mL×3), and partitioned between toluene (500 mL×3) and water (H₂O, 500 mL). The toluene-soluble materials (7.0 g) of the extract were subjected to silica gel CC using a gradient elution of 0–2% MeOH in CHCl₃. The fraction eluted with CHCl₃–MeOH (98 : 2) was then chromatographed using an ODS column [Cosmosil 140C₁₈-PREP; eluent: acetonitrile (CH₃CN)–H₂O, 7 : 3] to give a cytotoxic fraction with >80% inhibition of HeLa cells proliferation. The cytotoxic fraction was separated by reversed-phase HPLC [YMC-Pack Pro C₁₈, 5 µm, YMC Co., Ltd. (Japan) 10 mm×250 mm; eluent, CH₃CN–H₂O (70 : 30); flow rate, 2 mL/min; UV detection at 210 nm] to afford 1 [1.4 mg, 0.069%, retention time (t_R) 24 min]. The less polar fraction of the first silica column, eluted with CHCl₃ and CHCl₃–MeOH (98 : 2), was separated by ODS CC using a gradient of CH₃CN in H₂O (70 to 100%). The fraction eluted with 70% aqueous CH₃CN was purified by reversed-phase HPLC, using the conditions described above, to afford 2 (0.32 mg, 0.016%, t_R 17 min).

Cytotoxic Assay

A cytotoxicity assay involving HeLa, DG-75, and MH134 cells was performed at 37°C in 5% carbon dioxide, at a density of 5000 cells per well in 96-well plates, using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. After 72 h, the number of viable cells was counted using Cell Counting Kit 8 (Dojindo Co., Kumamoto, Japan) according to the manufacturer’s instructions. The assay reagent was a tetrazolium compound (WST-8) that is reduced by live cells into a colored formazan product; the absorbance was measured at 450 nm using a microplate reader (Bio-Rad, U.S.A.). The experiments were repeated in triplicate wells. The viability of the treated groups was estimated as a percentage of that of the control groups. The cytotoxicity is expressed as IC₅₀. 5-Fluorouracil was used as an authentic sample, and IC₅₀ values against HeLa, DG-75, and MH134 cells were 30, 9, and 0.6 µM, respectively.

Iriomoteolide-10a (1)

Colorless amorphous solid; [α]_D²³ −20 (c=0.25, CHCl₃); IR (KBr) cm⁻¹: 3443 (broad), 2920, 1714; ¹H- and ¹³C-NMR (500 MHz, C₆D₆): Table 1; ESI-MS m/z: 615.4315 [M+Na]⁺ (Calcd for C₃₃H₆₀O₁₀Na, 615.4316).

Iriomoteolide-12a (2)

Colorless amorphous solid; [α]_D²³ +37 (c=0.16, MeOH); UV λ_max (MeOH) nm (ε): 223 (21300); IR (KBr) cm⁻¹: 3438 (broad), 2920, 1700; ¹H-NMR (500 MHz, CDCl₃) δ: 0.68 (3H, d, J=7.0 Hz, H₃-24), 0.76 (3H, d, J=7.0 Hz, H₃-23), 0.90 (3H, d, J=7.0 Hz, H₃-22), 0.90 (1H, m, H-8b), 0.91 (1H, m, H-9), 1.03 (3H, d, J=7.0 Hz, H₃-21), 1.27 (1H, br d, J=14.0 Hz, H-10b), 1.41 (1H, m, H-10a), 1.42 (2H, t J=7.0 Hz, H₂-12), 1.51 (1H, m, H-13), 1.84 (1H, dd, J 15.0, 8.4 Hz, H-15b), 1.91 (1H, m, H-14), 2.05 (1H, m, H-8a), 2.06 (3H, s, H₃-25), 2.07 (1H, m, H-15a), 2.07 (3H, s, H₃-20), 2.12 (1H, m, H-7), 2.12 (3H, s, H₃-19), 2.48 (1H, dd, J=12.6, 5.4 Hz, H-5b), 3.02 (1H, dd, J=12.6, 2.9 Hz, H-5a), 4.42 (1H, m, H-4), 5.06 (1H, m, H-11), 5.89 (1H, s, H-2), 5.99 (1H, s, H-17); ¹³C-NMR (500 MHz, CDCl₃) δ: 13.4 (CH₃, C-24), 14.7 (CH₃, C-25), 16.8 (CH₃, C-20), 17.9 (CH₃, C-21), 19.2 (CH₃, C-25), 23.2 (CH₃, C-22), 31.8 (CH₃, C-19), 33.18 (CH, C-9), 33.23 (CH, C-13), 33,4 (CH, C-14), 39.8 (CH₂, C-8), 40.4 (CH₂, C-12), 44.6 (CH₂, C-5), 44.9 (CH₂, C-10), 46.9 (CH₂, C-15), 50.7 (CH, C-7), 73.1 (CH, C-4), 74.2 (CH, C-11), 116.9 (CH, C-2), 124.9 (CH, C-17), 154.9 (C, C-3), 157.9 (C, C-16), 167.7 (C, C-1), 198.7 (C, C-18), 217.0 (C, C-6); ESI-MS m/z: 443.2768 [M+Na]⁺ (Calcd for C₂₅H₄₀O₅Na, 443.2768).

Acknowledgments

We thank Satoru Ibuki, Keita Ikebe, and Takahiro Tsushima, Kochi Prefectural Deep Seawater Laboratory, for the supply of deep seawater and assistance with dinoflagellate cultivation. This study was partially supported by a Grant-in-Aid for Scientific Research (No. 16K08295 to M.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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 are available as supplementary materials.

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