Iriomoteolide-1a, a Potent Cytotoxic 20-Membered Macrolide froma Benthic Dinoflagellate Amphidinium Species
Masashi Tsuda,*,†,‡ Keiko Oguchi,‡ Rie Iwamoto,‡ Yumiko Okamoto,‡ Jun’ichi Kobayashi,‡
Eri Fukushi,§ Jun Kawabata,§ Tomoko Ozawa,| Atsunori Masuda,| Yoshiaki Kitaya,⊥ andKenji Omasa#
Center for AdVanced Marine Core Research, Kochi UniVersity, Kochi 783-8502, Japan, Graduate Schoolof Pharmaceutical Sciences, Hokkaido UniVersity, Sapporo 060-0812, Japan, Graduate School of
Agriculture, Hokkaido UniVersity, Sapporo 060-8589, Japan, MARINE FARM, Yanmar Company Limited,
Oita 873-0421, Japan, Graduate School of Life and EnVironmental Sciences, Osaka Prefecture UniVersity,
Osaka 599-8531, Japan, and Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo,
Tokyo 113-8657, Japan
mtsuda@kochi-u.ac.jp
ReceiVed February 28, 2007

A potent cytotoxic 20-membered macrolide, iriomoteolide-1a (1), has been isolated from a benthic
dinoflagellate Amphidinium sp. (strain HYA024), and the structure was elucidated on the basis of detailed
analyses of 2-D NMR data. The relative and absolute stereochemistries were assigned by the combinationof conformational analyses using NMR data and modified Mosher’s method of 1.

Introduction
Dinoflagellates belonging in the genus Amphidinium are
known to produce unique polyketide metabolites with interestingbioactivities.1,2 Cytotoxic macrolides designated as amphidinolides or caribenolide have been isolated from symbiotic or freeswimming dinoflagellates, Amphidinium species.2 These macrolides have a variety of backbone skeletons and different sizesof macrocyclic lactone rings (12- ∼ 29-membered rings).
Some of the amphidinolides contain biosynthetically uniquepartial structures such as vicinally located C1 branches.2* To whom correspondence should be addressed. Phone: +81-88-864-6720.
Fax: +81-88-864-6713.
† Kochi University.
‡ Graduate School of Pharmaceutical Sciences, Hokkaido University.
§ Graduate School of Agriculture, Hokkaido University.
| Yanmar Co. Ltd.
⊥ Osaka Prefecture University.
# The University of Tokyo.

(1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Princep,
M. R. Nat. Prod. Rep. 2006, 23, 26-78 and its previous reviews.
(2) Kobayashi, J.; Tsuda, M. Nat. Prod. Rep. 2004, 21, 77-93 andreferences cited therein.

Furthermore, some of these macrolides (e.g., amphidinolides
B,3 G,4 H,4 and N5 and caribenolide-I6) exhibit potent cytotoxicity against tumor cell lines in vitro and antitumor activity invivo. Therefore, these macrolides have attracted great interest
as challenging targets for total synthesis7 and biosynthetic
studies.8(3) (a) Ishibashi, M.; Ohizumi, Y.; Hamashima, M.; Nakamura, H.;
Hirata, Y.; Sasaki, T.; Kobayashi, J. J. Chem. Soc., Chem. Commun. 1987,
1127-1129. (b) Kobayashi, J.; Ishibashi, M.; Nakamura, H.; Ohizumi, Y.;
Yamasu, T.; Hirata, Y.; Sasaki, T.; Ohta, T.; Nozoe, S. J. Nat. Prod. 1989,
52, 1036-1041. (c) Bauer, I.; Maranda, L.; Shimizu, Y.; Peterson, R. W.;
Cornell, L.; Steiner, J. R.; Clardy, J. J. Am. Chem. Soc. 1994, 116, 26572658.
(4) (a) Kobayashi, J.; Shigemori, H.; Ishibashi, M.; Yamasu, T.; Hirota,
H.; Sasaki, T. J. Org. Chem. 1991, 56, 5221-5224. (b) Kobayashi, J.;
Shimbo, K.; Sato, M.; Shiro, M.; Tsuda, M. Org. Lett. 2000, 2, 28052807. (c) Kobayashi, J.; Shimbo, K.; Sato, M.; Tsuda, M. J. Org. Chem.
2002, 67, 6585-6592.
(5) Ishibashi, M.; Yamaguchi, N.; Sasaki, T.; Kobayashi, J. J. Chem.
Soc., Chem. Commun. 1994, 1445-1446.
(6) Bauer, I.; Maranda, Y.; Young, K. A.; Shimizu, Y.; Fairchild, C.;
Cornell, L.; MacBeth, J.; Huang, S. J. Org. Chem. 1995, 60, 10841086.

10.1021/jo070414b CCC: $37.00 © 2007 American Chemical Society

Published on Web 05/15/2007

J. Org. Chem. 2007, 72, 4469-4474

4469

Tsuda et al.

In our continuing investigation for unique metabolites fromthe dinoflagellate Amphidinium sp., molecular phylogenetic
studies of 18S ribosomal DNA sequences have been examinedfor dinoflagellates Amphidinium species with or without amacrolide-producing ability. It was found that the 18S ribosomal
DNA sequences of the macrolide-producing Amphidinium
strains belonged in a different clade from those of the strains
without a macrolide-producing ability.9 On the basis of the DNA
sequence homology, a rapid searching strategy for macrolideproducing strains, using specific DNA sequences and one-cell
PCR protocol, was developed.9
Recently, 250 or more Amphidinium strains isolated frombenthic sources collected off the South-East Islands, Japan, were
tested by one-cell PCR. The extracts of dinoflagellates positivein the PCR studies were further evaluated by cytotoxic screening
and metabolomics analyses using 2-D NMR analyses.10 This
led to the discovery of a marine benthic Amphidinium strain
HYA024 that produced unknown macrolides. Mass cultivationof this strain and examination of the extract resulted in theisolation of a novel, potently cytotoxic, 20-membered macrolide,iriomoteolide-1a (1). Herein, we describe the isolation andstructure elucidation of 1.
Results and Discussion
The dinoflagellate Amphidinium sp. (HYA024 strain) was
monoclonally separated from benthic sea sand collected off
Iriomote Island, Japan, and mass cultured unialgally at 23 °C
for 2 weeks in a 2% Provasoli’s enriched seawater (PES)
medium enriched with 3 mM NaHCO3. The algal cells (15.3 g,
dry weight) obtained from 400 L of the medium were extractedwith MeOH/toluene (3:1), and the extracts were partitioned
between toluene and H2O. The toluene soluble materials were
subjected to a SiO2 gel column to obtain cytotoxic fractions
against human B lymphocyte DG-75 cells.
Metabolomics analyses of these cytotoxic fractions werecarried out using HMQC experiments.10 HMQC spectra showedcharacteristic correlations for δ ca. 70/5.4 ∼ 5.2 and ca. 110/
4.8 (F1/F2), the former of which was considered to be a marker
correlation for ester functionality, while the latter was a markerof the exomethylene functional group. One of the cytotoxic(7) Total synthesis of amphidinolides: (a) Amphidiolide, A: Trost, B.
M.; Harrington, P. E.; Chrisholm, J. D.; Wrobleski, S. T. J. Am. Chem.
Soc. 2005, 127, 13598-13610. (b) Trost, B. M.; Wrobleski, S. T.;
Chrisholm, J. D.; Harrington, P. E.; Jung, M. J. Am. Chem. Soc. 2005,
127, 13589-13597. (c) Trost, B. M.; Harrington, P. E. J. Am. Chem. Soc.
2004, 126, 5028-5029. (d) Maleczeka, R. E., Jr.; Terrell, L. R.; Gang, F.;
Ward, J. S., III. Org. Lett. 2002, 4, 2841-2844. (e) Lam, H. W.; Pattenden,
G. Angew. Chem., Int. Ed. 2002, 41, 508-811. (f) Trost, B. M.; Chisholm,
J. D.; Wrobleski, S. T.; Jung, M. J. Am. Chem. Soc. 2002, 124, 1242012421. (g) Amphidinolide E: Va, P.; Roush, W. R. J. Am. Chem. Soc.
2006, 128, 15960-15961. (h) Amphidinolide J.: Williams, D. R.; Kissel,
W. S. J. Am. Chem. Soc. 1998, 120, 11198-11199. (e) Amphidinolide K:
Williams, D. R.; Meyer, K. G. J. Am. Chem. Soc. 2001, 123, 765-766. (f)Amphidinolide P: Williams, D. R.; Myers, B. J. Org. Lett. 2000, 2, 945948. (g) Amphidinolide T1: Ghosh, A. K.; Liu, C. J. Am. Chem. Soc. 2003,
125, 2374-2375. (h) Amphidinolide T4: Fürstner, A.; Aı̈ssa, C.; Riveiros,R.; Ragot, J. Angew. Chem., Int. Ed. 2002, 41, 4763-4766.
(8) (a) Rein, K. S.; Borrone, J. Comp. Biochem. Physiol., Part B:
Biochem. Mol. Biol. 1999, 124, 117-131. (b) Snyder, R. V.; Gibbs, P. D.;
Palacio, A.; Abiy, L.; Dickey, R.; Lopez, J. V.; Rein, K. S. Mar. Biotechnol.
2003, 5, 1-12. (c) Kubota, T.; Iinuma, Y.; Kobayashi, J. Biol. Pharm. Bull.
2006, 29, 1314-1318.
(9) Iwamoto, R.; Kobayashi, J.; Horiguchi, T.; Tsuda, M. Phycologia
2005, 44, 104.
(10) (a) Fiehn, O. Plant Mol. Biol. 2002, 48, 155-171. (b) Reo, N. V.
Drug Chem. Toxicol. 2002, 25, 375-382. (c) Kikuchi, J.; Shinozaki, K.;
Hirayama, T. Plant Cell Physiol. 2004, 45, 1099-1104.

4470 J. Org. Chem., Vol. 72, No. 12, 2007

CHART 1

fractions was purified with C18 and NH2-SiO2 column chromatographies followed by C18 HPLC to afford iriomoteolide1a (1, 0.028% from dry weight) (Chart 1) as one of cytotoxic
components. No macrolides, such as amphidinolides or caribenolides, have been isolated previously from strain HYA024.
Iriomoteolide-1a {1, [R]D20 +31° (c 0.35, CHCl3)} showeda pseudo-molecular ion peak at m/z 529 (M + Na)+ in the
ESIMS spectrum, and the molecular formula of C29H46O7 wasrevealed by HRESIMS data [m/z 529.3148 (M + Na)+, ∆+0.7 mmu]. 1H and 13C NMR data (Table 1) in CDCl3 assigned
by using HMQC and CH and CH2 selected E-HSQC spectra11
revealed the presence of a total of 29 carbon atoms due to anester carbonyl, two quaternary sp2 carbons, five sp2 methines,
an sp2 methylene, two quaternary sp3 carbons, one of which isa hemiketal carbon, seven sp3 methines including four oxygenated ones, five sp3 methylenes, and six methyls. Since five outof seven degrees of unsaturation were accounted for, iriomoteolide-1a (1) was inferred to possess two rings. The 1H NMR
spectrum in CDCl3 included a sharp singlet D2O-exchangeable
proton (δH 3.52, OH-13), indicating the presence of intramolecular hydrogen bonding.
The planar structure of 1 was mainly elucidated on the basisof 2-D NMR data measured in CDCl3. Analyses of 1H-1H
COSY and TOCSY spectra disclosed three proton-proton
networks from H-2 to H3-24, from H-4 to H2-10 and H3-25,
and from H-15 to H3-23, H3-28, and H3-29 (Figure 1). The
presence of two disubstituted E-double bonds at C-6-C-7 andC-15-C-16 were implied by JHH values [H-6 (δH 5.57)/H-7(δH 5.68): 15.7 Hz, H-15 (δH 5.68)/H-16 (δH 5.76): 15.5 Hz],while a trisubstituted double bond at C-2-C-3 was indicatedto possess Z-geometry by the chemical shift (δH 2.12, δC 23.8)
of C-24 and the ROESY correlation for H-2 (δH 6.02)/H3-24(δH 2.10) in C6D6. The double bond at C-2-C-3 was shown tobe adjacent to C-4 by HMBC correlations for H-2 (δH 5.72)/
C-3 (δC 162.0), H3-24/C-4 (δC 47.9), and H3-25 (δH 1.24)/C-3
in CDCl3. HMBC correlations for H2-26 (δH 4.82 and 4.81)/
C-10 (δC 40.7), H2-26/C-11 (δC 141.7), and H2-26/C-12 (δC
36.88) suggested that C-10 was attached to C-12 through an
exomethylene unit (C-11-C-26). The connection between C-12
and C-15 via two sp3 oxygenated quaternary carbons (C-13 andC-14) was revealed by HMBC correlations for H2-12 (δH 2.40
and 2.26)/C-13 (δC 99.7), 13-OH (δH 3.52)/C-12, 13-OH/C13, 13-OH/C-14 (δC 77.2), H3-27 (δH 1.25)/C-13, H3-27/C-14,
and H3-27/C-15 (δC 134.9). The existence of a six-memberedhemiacetal ring was deduced from the ROESY correlation for
H-9 (δH 3.81)/13-OH. The relatively low-field resonance of
H-19 (δH 5.11) suggested that C-19 was involved in an ester(11) (a) Davis, D. G. J. Magn. Reson. 1991, 91, 665. (b) Fukushi, E.;
Tanabe, S.; Watanabe, M. Kawabata, J. Magn. Reson. Chem.1998, 36, 741746.

Iriomoteolide-1a, Potent Cytotoxic 20-Membered Macrolide
TABLE 1.

1H and 13C NMR Data for Iriomoteolide-1a (1)1H

position
1
2
3
4
5
6
7
8a
8b
9
10a
10b
11
12a
12b
13
14
15
16
17a
17b
18
19
20a
20b
21
22
23
24
25
26a
26b
27
28
29

13C

CDCl3

C6D6

CDCl3

5.72

brs

6.02

brs

2.46
4.28
5.57
5.68
2.18a

dq, 2.9, 7.3
mdd, 4.1, 15.7
m
m

3.81
2.21b
1.90b

brt, 11.5
brd, 12.7
brt, 12.3

1.90
3.89
5.25
5.80
2.19
2.00
4.00
2.15
1.90

m
m
dd, 4.6, 15.3
ddd, 4,6, 10.0, 14.6m
m
tt, 2.0, 11,9m
dd, 11.9, 13.0

2.40
2.26
3.52c

d, 13.6
brd, 13.6
brd, 1.9

2.73
2.45
3.82c

d, 13.3
brd, 13.3
brs

5.68
5.76
2.15
1.96
1.82
5.11
1.80
1.15
1.40
3.58
1.11d
2.12d
1.24d
4.82a

brd, 15.5
ddd, 3.1, 10.8, 15.5m
dt, 14.1, 11.6mm
ddd, 4.4, 8.7, 13.8
ddd, 4.4, 8.8, 13.8m
quint, 6.3
d, 6.3sd, 7.3
brs

1.25d
0.99d
0.91d

s
d, 6.8d, 6.7

6.07
5.97
2.08
2.14
1.63
5.38
1.94
1.14
1.40
3.41
0.98d
2.10d
1.08d
5.01
4.97
1.54d
0.94d
0.97d

d, 14.6
ddd, 4.6, 10.0, 14.6mmm
ddd, 2.7, 4.6, 8.0
ddd, 4.0, 8.0, 13.9
ddd, 4.6, 8.6, 13.9mdq, 6.0, 6.6
d, 6.6sd, 6.6brs
brs
sd, 6.8d, 6.7

167.4
115.8
162.0
47.9
72.3
132.0
126.8
39.5

C
CH
C
CH
CH
CH
CH
CH2

71.8
40.7

CH
CH2

141.7
36.88

C
CH2

99.7
77.2
134.9
128.8
38.2

C
C
CH
CH
CH2

36.94
70.8
36.5

CH
CH
CH2

36.5
72.2
19.8
23.8
15.6
110.6

CH
CH
CH3
CH3
CH3
CH2

23.1
14.2
15.5

CH3
CH3
CH3

a 2H. b Coupling constants for H-10a are small and large values for H-9 and H-10b, respectively. On the other hand, coupling constants for H-10b areboth large values for H-9 and H-10a. c 13-OH. d 3H.

FIGURE 1. Selected 2-D NMR correlations for iriomoteolide-1a (1).

linkage with C-1, which was also supported by HMBC
correlations for H-2/C-1 (δC 167.4) and H-19/C-1 and ROESY
correlation for H-2/H-19. Thus, the planar structure of iriomoteolide-1a was concluded to be 1.
The relative stereochemistry of 1 was deduced from detailed
analyses of bond rotations12 based on 1H-1H and 13C-1H
coupling constants and ROESY data in CDCl3 and C6D6. 1H1H coupling constants were obtained from the resolution(12) (a) Tsuda, M.; Nozawa, K.; Shimbo, K.; Ishiyama, H.; Fukushi, E.;
Kawabata, J.; Kobayashi, J. Tetrahedron Lett. 2003, 44, 1395-1399. (b)Nozawa, K.; Tsuda, M.; Ishiyama, H.; Sasaki, T.; Tsuruo, T.; Kobayashi,
J. Bioorg. Med. Chem. 2006, 14, 1063-1067.

enhanced 1H NMR spectrum and differential homonuclear
decoupling experiments, while long-range 13C-1H coupling
constants were obtained by analyses of HETLOC13 and JIMPEACH-MBC14 spectra. Coupling constants and ROESY
correlations in CDCl3 and C6D6 were close to each other, thus
suggesting that the solution conformations of 1 in both solventswere similar to each other. For typical vicinally located oxythinemethylated methine bonds such as C-4-C-5, C-18-C-19, andC-21-C-22, the relative configurations were concluded to beerythro, erythro, and threo, respectively, by J-based configuration analysis15 as described next (Figure 2).
For the C-1-C-14 portion (Figure 3), a gauche configurationfor H-4-H-5 was suggested by the J(H-4/H-5) value (2.9 Hz),while relatively small J(C-5/H3-25) and J(C-5/H-4) values16 (<3and 3.0 Hz, respectively) indicated gauche alignment for C-25(13) (a) Otting, G.; Wüthrich, K. Quart. ReV. Biophys. 1990, 23, 3996. (b) Wollborn, U.; Leibfritz, D. J. Magn. Reson. 1992, 98, 142-146.
(c) Kurz, M.; Schmieder, P.; Kessler, H. Angew. Chem., Int. Ed. Engl. 1991,
30, 1329-1331.
(14) Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Martin, G. E.;
Krishnamurthy, V. V. Magn. Reson. Chem. 2001, 39, 127-132.
(15) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana,K. J. Org. Chem. 1999, 64, 866-876.
(16) When the dihedral angles between 13C-attached oxygen and theproton are 180 and 60°, typical 2JCH values were 0 to approximately -2
and -5 to approximately -7 Hz, respectively. On the other hand, when
the dihedral angles between carbon and proton through three bonds are
180 and 60°, typical 3JCH values were 6 to ∼8 and 1 to ∼3 Hz, respectively.
For review, see: Hansen, P. E. Prog. NMR Spectrosc. 1981, 14, 175-296.

J. Org. Chem, Vol. 72, No. 12, 2007 4471

Tsuda et al.

FIGURE 4. ROESY correlations and relative stereochemistry for C-8FIGURE 2. Rotation models for (a) C-4dC-5, (b) C-18-C-19, and(c) C-21-C-22 bonds of iriomoteolide-1a (1).

FIGURE 3. ROESY correlations and relative stereochemistry for C-1C-14 portion in iriomoteolide-1a (1).

H-5 and anti for H-4-OH-5. Thus, the C-4-C-5 bond was
revealed to be in the erythro configuration (Figure 2a). ROESY
correlations for H-4 (δH 1.90)/H3-24 (δH 2.10), H-4/H-6 (δH
5.25), H-6/H-8b (δH 2.00), and H-6/H3-24 in C6D6 indicatedthat H-4, H-6, H-8b, and C-24 were oriented to the same sideof the macrocyclic ring. Gauche and anti relations for H-7H-8a and H-7-H-8b, respectively, were deduced from J(H-7/
H-8a) and J(H-7/ H-8b) values (4.6 and 10.0 Hz, respectively).
J(H-8a/H-9) and J(H-8b/H-9) values were relatively large andsmall (11.9 and 2.0 Hz, respectively), indicating that relationships for H-8a-H-9 and H-8b-H-9 were anti and gauche,
respectively. On the other hand, gauche and anti relations for
H-9-H-10a and H-9-H-10b, respectively, were implied by J(H9/H-10a) and J(H-9/H-10b) values (2.0 and 11.9 Hz, respectively). From the ROESY correlation for H-8b/H-10a, theorientations of H-8b and H-10a were shown to be the sameside of the macrocyclic ring. ROESY correlations were observedfor H-10a (δH 2.21)/H-26b (δH 4.97), H-10b (δH 1.90)/H-12b(δH 2.45), and H-12a (δH 2.73)/H-26a (δH 5.01), suggesting that
H-10a and H-12a were both equatorial configurations, while
H-10b and H-12b were axial. The syn configuration for H-913-OH in the tetrahydropyran ring was deduced from ROESY
correlations for H-9 (δH 3.81)/13-OH (δH 3.52) observed in
CDCl3.
4472 J. Org. Chem., Vol. 72, No. 12, 2007

C-20 portion in iriomoteolide-1a (1). The rotation of C-21-C-22 bondrepresented as one of two rotamers shown in Figure 2c.

For the C-13-C-20 portion (Figure 4), the integrated valueof the ROESY correlation for H-12b (δH 2.45)/H3-27 (δH 1.54)
in C6D6 was larger than that of H-12a (δH 2.73)/H3-27, and the
ROESY correlations were observed for H-16 (δH 5.97)/H3-27(in C6D6) and 13-OH/H-15 (δH 5.68) (in CDCl3). These
indicated that H-12b, H-16, and H3-27 were oriented to the sameside of the macrocylic ring. Both anti relations to H-16-H17b and H-17b-H-18 were elucidated by relatively large J(H16/H-17b) and J(H-17b/H-18) values (10.0 and 11.2 Hz,
respectively) and ROESY correlations for H-15/H-17b (δH 2.14)
and H-16/H-18 (δH 1.63) in C6D6. The J(H-18/H-19) (2.0 Hz)
and |J(C-19/H-18)| values (6 Hz)17 suggested both gauche
relations for H-18-H-19 and H-18-19-O. Considering the
ROESY correlations for H-16/H-19 (δH 5.38) and H2-20 (δH
1.94 and 1.14)/H3-28 (δH 0.94), the C-18-C-19 bond was
revealed to be in the erythro configuration (Figure 2b), thus
suggesting both equatorial orientations for the methyl group (C28) on C-18 and the side chain (C-20 to ∼C-23) on C-19.
Antiperiplanar relations for H-19-H-20a and H-20b-H-21 werededuced from the following coupling constants: J(H-19/H-20a)
8.0 Hz, J(H-19/H-20b) 4.6 Hz, J(H-20a/H-21) 4.0 Hz, J(H-20b/
H-21) 8.6 Hz and ROESY correlations: H-18/H-20a, H-19/H21 (δH 1.40).
For the C-21-C-22 bond, J(H-21/H-22) and J(C-21/H3-23)
were typical medium values (6.1 and 4.8 Hz, respectively) for3J3
HH and JCH, suggesting that this bond underwent a conformational change between anti and gauche. On the other hand,
the gauche configuration for H-22-C-29 was deduced from therelatively small J(C-22/H3-29) value (<3 Hz). Of the six
possible rotamers arising from erythro and threo, one pair of
the threo relations shown in Figure 2c satisfied the ROESY
correlations for H2-20/H-22 (δH 3.41), H-21/H3-23 (δH 0.98),
and H-22/H3-29 (δH 0.97). Therefore, the total relative configurations of nine chiral centers were concluded as shown in
Figures 3 and 4.
(17) Although the modulus of J(C-19/H-18) was obtained from the
J-IMPEACH MBC spectrum, the sign was not determined.

Iriomoteolide-1a, Potent Cytotoxic 20-Membered Macrolide

being peloruside A21 reported from the sponge Mycale hentscheli. Recently, the unique mechanism of action for peloruside A
has been revealed by chemical biological methods22 and in silico
studies.23 The mechanism of action for the target molecule for
1 is currently under investigation.
Experimental Section

FIGURE 5. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
5,22-bis-(S)- and (R)-MTPA esters (2a and 2b, respectively) of
iriomoteolide-1a (1).

To determine the absolute stereochemistry of iriomoteolide1a (1), a modified Mosher’s method18 was applied for twohydroxyl groups at C-5 and C-22. The 1H NMR data for 5,22bis-(S)- and (R)-MTPA esters (2a and 2b, respectively) of 1
were assigned by analysis of the TOCSY spectra. ∆δ values(∆δ ) δS - δR) obtained from 1H NMR data of 2a and 2b
showed positive signs for H-2 (+0.06), H-4 (+0.01), H-21(+0.08), H3-24 (+0.02), H3-25 (+0.11), and H3-29 (+0.04) andnegative signs for H-6 (-0.14), H-7 (-0.06), and H3-23 (-0.16)(Figure 5), thus suggesting that C-5 and C-22 possessed R- andS-configurations, respectively. Therefore, the absolute configurations of 1 were assigned as 4R, 5R, 9S, 13S, 14R, 18S, 19R,
21S, and 22S.
Iriomoteolide-1a (1) is a 20-membered macrolide having anovel carbon skeleton with four hydroxyl groups, five methyls,
an exomethylene branch, three endogenous double bonds, anda tetrahydropyran ring. Vicinally locating the C1 branches (C24 and C-25) on a C-4-C-5 portion were characteristic for themacrolides from the dinoflagellate Amphidinium species.19
Furthermore, iriomoteolide-1a (1) is the first macrolide with aZ-olefin and the first with an overall chain length composed of
23 carbons, in the series of macrolides isolated from theAmphidinium dinoflagellates thus far.
Iriomoteolide-1a (1) exhibited potent cytotoxicity against
human B lymphocyte DG-75 cells (IC50: 0.002 µg/mL), which
was 20 times more potent than that of doxorubicin (IC50:
0.04 µg/mL). The cytotoxic activity of 1 was almost equal tothat of amphidinolide H4,20 (IC50: 0.001 µg/mL), which was
one of the most potent cytotoxic macrolides from the Amphidinium dinoflagellates. Iriomoteolide-1a (1) also showed potent
cytotoxicity against Epstein-Barr virus (EBV)-infected human
B lymphocyte Raji cells (IC50: 0.003 µg/mL). This cytotoxic
activity against EBV-infected cells may be due not to an antiviral effect but rather to a general toxicity effect. In marineorganisms, potent cytotoxicity has been associated with macrolides containing a six-membered hemiketal ring, an example(18) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092-4095.
(19) In our work on acetate incorporation patterns of amphidinolides,
all of the C1 branches were found to be derived from C-2 of acetate.
Vicinally located C1 branches were also derived from C-2 of acetate: (a)Kobayashi, J.; Takahashi, M.; Ishibashi, M. J. Chem. Soc., Chem. Commun.
1995, 1639-1640. (b) Sato, M.; Shimbo, K.; Tsuda, M.; Kobayashi, J.
Tetrahedron Lett. 2000, 41, 503-506. (c) Kubota, T.; Tsuda, M.; Kobayashi,
J. Tetrahedron 2001, 57, 5975-5977. (d) Tsuda, M.; Kubota, T.; Sakuma,
Y.; Kobayashi, J. Chem. Pharm. Bull. 2001, 49, 1366-1367.
(20) Kobayashi, J.; Shimbo, K.; Sato, M.; Tsuda, M. J. Org. Chem. 2002,
67, 6585-6592.

NMR Measurement. CH and CH2 selected HSQC spectra were
measured with spectral widths of 5434 Hz for the F2 dimension
and 4545 Hz (ca. 50 to ∼20 ppm) for the F1 dimension, and 16
scans with two dummy scans were accumulated into 1000 data
points for each 64 t1 increments. The repetition delay, BIRD delay,and constant time for JCH evaluation were 2 s, 0.3 s, and 3.7 ms,
respectively. For CH selection, the editing flip angle and delay τwere set to π and 3.7 ms, respectively, while those for CH2 selection
were π/2 and 1.35 ms. The measuring times were both ca. 1 h.
The HETLOC spectrum was obtained with the pulse sequence
proposed by Woolborn and Leibfritz13b with composite pulses24 forbroadband constant rotations (broadwidth (0.60). The durations
for the trim pulse, the delay in the BIRD pulse, and the constanttime for JCH evaluation were 2.5, 300, and 3.57 ms, respectively.
The MLEV-17 spin-lock period was set to 30 ms for 2,3JCH. For
256 t1 increments, 384 transients with 16 dummy scans wereaccumulated in 4000 data points. Zero-filling to 1000 for F1 andmultiplication with squared cosine-bell windows shifted in bothdimensions were performed prior to 2-D Fourier transformation.
The measuring time was ca. 64 h.
The J-IMPEACH MBC experiment was carried out with spectral
widths of 5434 Hz for the F2 dimension and 27 777 Hz for the F1
dimension, and 512 scans with two dummy scans were accumulated
into 1000 data points for each 256 t1 increments. The scaling factor
and constant time variable delay were set to 20 and 175 ms,
respectively. The measuring time was ca. 72 h.
Cultivation and Isolation. To benthic sea sands collected off
Iriomote Island, Japan, was added seawater medium containing a
1% ES supplement, and the mixture was incubated for several
weeks. The dinoflagellate (strain number HYA024) was separated
monoclonally by micropippetting. The voucher specimen was
deposited at the Center for Advanced Marine Core Research, Kochi
University.
The dinoflagellate was unialgally cultured at 23 °C for 2 weeks
in seawater medium enriched with 2% PES supplement, 16 h lightand 8 h dark. The harvested cells (15.3 g, from 400 L of culture)
were extracted with MeOH/toluene (3:1, 500 mL × 3), and theextract was partitioned between toluene (500 mL × 3) and water(500 mL). The toluene-soluble fractions (2 g) were subjected to
SiO2 column chromatography (40 × 200 mm) using a stepwise
elution of CHCl3 (200 mL) and CHCl3/MeOH (98:2, 200 mL). The
fraction eluted with (CHCl3/MeOH, 98:2) was chromatographedsuccessively by using a C18 (CH3CN/H2O, 7:3) and then a NH2SiO2 column (hexane/EtOAc, 2:1). A macrolide containing fraction
was separated by C18 HPLC [YMC-Pack Pro C18, 5 µm, YMC Co.,
Ltd., 10 mm × 250 mm; eluent, CH3CN/H2O (60:40); flow rate, 2(21) West, L. M.; Northcote, P. T.; Battershill, C. N. J. Org. Chem. 2000,
65, 445-449.
(22) (a) Hood, K. A.; West, L. M.; Rouwe, B.; Northcote, P. T.; Berridge,
M. V.; Qakefield, S. J.; Miller, J. H. Cancer Res. 2002, 62, 3356-3360.
(b) Gaitanos, T. N.; Buey, R. M.; Diaz, J. F.; Northcote, P. T.; TeesdaleSpittle, P.; Andreu, J. M.; Miller, J. H. Cancer Res. 2004, 64, 5063-5067.
(c) Miller, J. H.; Rouwe, B.; Gaitanos, T. N.; Hood, K. A.; Crume, K. P.;
Backstrom, B. T.; La Flamme, A. C.; Berridge, M. V.; Northcote, P. T.
Apoptosis 2004, 9, 785-796.
(23) (a) Pineda, O.; Farras, J.; Maccari, L.; Manetti, F.; Botta, M.;
Vilarrasa, J. Bioorg. Med. Chem. Lett. 2004, 14, 4825-4829. (b) Hamel,
E.; Day, B. W.; Miller, J. H.; Jung, M. K.; Northcote, P. T.; Ghosh, A. K.;
Curran, D. P.; Cushman, M.; Nicolaou, K. C.; Paterson, I.; Sorensen, E. J.
Mol. Pharmcol. 2006, 70, 1555-1564.
(24) Shaka, A. J.; Pines, A. J. Magn. Reson. 1987, 71, 495-503.

J. Org. Chem, Vol. 72, No. 12, 2007 4473

Tsuda et al.
mL/min; UV detection at 210 nm] to afford iriomoteolide-1a (1,4.3 mg, 0.028%).
Iriomoteolide-1a (1). Colorless amorphous; [R]D20 +31° (c 0.35,
CHCl3); IR (neat) νmax 3446 (broad), 2920 1686, 1214, and 1008
cm-1; 1H and 13C NMR data (Table 1); ESIMS (pos) m/z 529(M + Na)+; HRESIMS m/z 529.3148 [calcd for C29H46O7Na,(M + Na)+: 529.3141].
5,22-Bis-(S)-MTPA Ester (2a) of Iriomoteolide-1a (1). To asolution of iriomoteolide-1a (1, 0.2 mg) in 1% DMAP solution in
CH2Cl2 (20 µL) were added Et3N (2 µL) and (R)-(-)-MTPACl(1 µL), and the mixture was stirred at 4 °C for 15 h. After additionof N,N-dimethyl-1,3-propanediamine (2 µL), the solvent wasevaporated in vacuo. The residue was passed through a silica gelcolumn (hexane/acetone, 4:1) to afford 5,22-bis-(S)-MTPA ester(2a) of 1 (0.12 mg): 1H NMR (CDCl3); δ 0.92 (3H, d, J ) 6.6 Hz,
H3-28), 0.97 (3H, d, J ) 6.5 Hz, H3-29), 1.09 (3H, d, J ) 6.6 Hz,
H3-23), 1.13 (1H, m), 1.18 (3H, d, J ) 6.6 Hz, H3-25), 1.25 (3H,s, H3-27), 1.60 to ∼1.68 (2H, m), 1.70 (1H, m, H-21), 1.76 (1H,m), 1.87 (1H, m), 1.92 to ∼2.07 (2H, m), 2.07 (3H, s, H3-24), 2.08
to ∼2.27 (3H, m), 2.38 (1H, d, J ) 13.7 Hz, H-12), 2.49 (1H, m,
H-4), 3.48 (1H, m, H-9), 3.51 (3H, s), 3.60 (3H, s), 5.82 (2H, s,
H2-26), 5.02 (1H, m, H-22), 5.09 (1H, m, H-19), 5.25 (1H, m, H-5),
5.47 (1H, m, H-6), 5.50 (2H, m, H-15 and H-16), 5.68 (1H, m,
H-7), 5.78 (1H, s, H-2), 7.34 (6H, m), and 7.52 (4H, m); ESIMS(pos) m/z 961.5 (M + Na)+; HRESIMS m/z 961.3915 [calcd for
C49H60O11F6Na, (M + Na)+: 961.3927].
5,22-Bis-(R)-MTPA Ester (2b) of Iriomoteolide-1a (1). Iriomoteolide-1a (1, 0.2 mg) was treated with 4-dimethylaminopyridine

4474 J. Org. Chem., Vol. 72, No. 12, 2007

(50 µg), triethylamine (5 µL), and (S)-(+)-MTPACl (1.5 µL) by
the same procedure as described previously to afford 5,22-bis-(R)MTPA ester (2b) of 1 (0.08 mg): 1H NMR (CDCl3); δ 0.88 (3H,d, J ) 6.6 Hz, H3-28), 0.94 (3H, d, J ) 6.5 Hz, H3-29), 1.07 (3H,d, J ) 6.6 Hz, H3-25), 1.11 (1H, m), 1.24 (3H, s, H3-27), 1.25
(3H, d, J ) 6.6 Hz, H3-23), 1.62 (1H, m, H-21), 1.63 to ∼1.70(3H, m), 1.81 (1H, m), 2.02 (1H, m), 2.05 (3H, s, H3-24), 2.06
(1H, m), 2.08 to ∼2.30 (4H, m), 2.48 (1H, m, H-4), 3.49 (1H, m,
H-9), 3.51 (3H, s), 3.53 (3H, s), 4.85 (2H, s, H2-26), 5.09 (1H, m,
H-22), 5.10 (1H, m, H-19), 5.55 (1H, m, H-5), 5.61 (1H, m, H-6),
5.70 (2H, m, H-15 and H-16), 5.72 (1H, s, H-2), 5.74 (1H, m, H-7),
7.39 (6H, m), and 7.51 (4H, m); ESIMS (pos) m/z 961.5 (M +Na)+; HRESIMS m/z 961.3944 [calcd for C49H60O11F6Na, (M +Na)+: 961.3927].

Acknowledgment. We thank Y. Endo, Y. Nagakita, and Y.
Fukuda for help of dinoflagellate cultivation, Dr. M. Fujimuro,
Graduate School of Pharmaceutical Sciences, Hokkaido University, for offer of lymphoma cells and S. Oka, Center for
Instrumental Analysis, Hokkaido University, for ESIMS measurements. This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
Supporting Information Available: Spectral data for 1. This
material is available free charge via the Internet at http:// pub.acs.org.
JO070414B