Absolute stereochemistry of amphidinolide C.

Article abstract of DOI:10.1021/ol015741z

[structure in text] The absolute configurations at 12 chiral centers in amphidinolide C (1), a potent cytotoxic 25-membered macrolide isolated from a marine dinoflagellate Amphidinium sp., were determined to be 3S, 4R, 6R, 7R, 8R, 12R, 13S, 16S, 20R, 23R, 24R, and 29S by combination of NMR analyses, degradation experiments, and synthesis of the C-1-C-7 segment.

Full text of DOI:10.1021/ol015741z

ORGANIC
LETTERS
2001
Vol. 3, No. 9
1363-1366
Absolute Stereochemistry of
Amphidinolide C
Takaaki Kubota, Masashi Tsuda, and Jun’ichi Kobayashi*
Graduate School of Pharmaceutical Sciences, Hokkaido UniVersity,
Sapporo 060-0812, Japan
jkobay@pharm.hokudai.ac.jp
Received February 21, 2001
ABSTRACT
The absolute configurations at 12 chiral centers in amphidinolide C (1), a potent cytotoxic 25-membered macrolide isolated from a marine
dinoflagellate Amphidinium sp., were determined to be 3S, 4R, 6R, 7R, 8R, 12R, 13S, 16S, 20R, 23R, 24R, and 29S by combination of NMR
analyses, degradation experiments, and synthesis of the C-1−C-7 segment.
Amphidinolides are a series of cytotoxic macrolides pos-
sessing unique structural features isolated from laboratory-
cultured marine dinoflagellates Amphidinium sp.1 Amphid-
inolides C2 (1, Scheme 1)) and F,3 isolated from dinoflagellates
2D NMR data, and the relative stereochemistry of the C-1-
C-8 and C-20-C-23 portions has been assigned tentatively
by NOESY correlations of 1 and its 7,8-O-isopropylidene
derivative (2).4 During our search for bioactive metabolites
from marine dinoflagellates,5 relatively large amounts of
amphidinolide C (1) have been recently isolated from three
strains (Y-56,1d,e Y-59, and Y-71) of the genus Amphidinium,
which were separated from the inside cells of the marine
acoel flatworms Amphiscolops sp. This sample was utilized
to reinvestigate the relative stereochemistry and to determine
the absolute configurations at 12 chiral centers in 1.
Scheme 1
Investigation of Relative Stereochemistry. In our previ-
ous studies, the relative stereochemistry of H-3/H-4, H-3/
(1) (a) Ishibashi, M.; Kobayashi, J. Heterocycles 1997, 44, 543-572.
(b) Tsuda, M.; Endo, T.; Kobayashi, J. Tetrahedron 1999, 55, 14565-
14570. (c) Kubota, T.; Tsuda, M.; Kobayashi, J. Tetrahedron Lett. 2000,
41, 713-716. (d) Tsuda, M.; Endo, T.; Kobayashi, J. J. Org. Chem. 2000,
65, 1349-1352. (e) Kobayashi, J.; Kubota, T.; Endo, T.; Tsuda, M. J. Org.
Chem. 2001, 66, 134-142.
(2) Kobayashi, J.; Ishibashi, M.; Wa¨lchli, M. R.; Nakamura, H.; Hirata,
Y.; Sasaki, T.; Ohizumi, Y. J. Am. Chem. Soc. 1988, 110, 490-494.
(3) Kobayashi, J.; Tsuda, M.; Ishibashi, M.; Shigemori, H.; Yamasu, T.;
Hirota, H.; Sasaki, T. J. Antibiot. 1991, 44, 1259-1261.
(4) Ishiyama, H.; Ishibashi, M.; Kobayashi, J. Chem. Pharm. Bull. 1996,
44, 1819-1822.
Amphidinium sp. (Y-5 and Y-26 strains, respectively), are
unique 25-membered macrolides having two tetrahydrofuran
rings and vicinally located one-carbon branches. Particularly,
amphidinolide C (1) exhibited potent cytotoxicity against
tumor cells. The gross structure of 1 has been elucidated by
(5) Kobayashi, J.; Shimbo, K.; Sato, M.; Shiro, M.; Tsuda, M. Org. Lett.
2000, 2, 2805-2807.
10.1021/ol015741z CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/06/2001

H-6, and H-20/H-23 on the two tetrahydrofuran rings was
proposed to be all anti from NOESY data of amphidinolide
C (1).6 An erythro relationship for the 7,8-diol was deduced
from analysis of the NOESY spectrum of the 7,8-O-
3
isopropylidene derivative (2) of 1. The J(H-12,H-13) (8.8
Hz) was a typical value for an anti relationship7 (Figure 1a).
Figure 2. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
(S)- and (R)-MTPA esters (3a and 3b, respectively) of the 7,8-O-
isopropylidene derivative (2) of amphidinolide C (1).
positive ∆δ values were observed for H-7, H-8, H-10, H-12,
H-23, H-24, H-25, H-26, H-27, H2-36, H3-37, H3-38, and
H3-40, thus indicating that C-13 and C-29 both had S-
configurations.
Figure 1. Rotation models for (a) C-12-C-13 and (b) C-23-C-
24 bonds of amphidinolide C (1). NOESY correlations are illustrated
by solid arrows.
Absolute Configurations at C-3, C-4, and C-6. To
investigate the absolute stereochemistry at C-3, C-4, and C-6,
the oxidative degradation reaction for the 7,8-diol unit in
amphidinolide C (1) was performed as follows. Reduction
of 1 with DIBAL, oxidative cleavage of the 7,8-diol unit
with NaIO4, reduction with NaBH4, esterification with (R)-
(-)-MTPACl, and then HPLC separation furnished the bis-
(S)-MTPA ester (4a) of the C-1-C-7 segment (Scheme 2),
The values for 2J(C-13,H-12) (-6.3 Hz), 3J(C-14,H-12) (3.1
Hz), and 3J(C-38,H-13) (3.5 Hz), which were obtained from
the hetero half-filtered TOCSY (HETLOC)8 spectrum,
indicated that H-12 was gauche to 13-OH, while H-12 and
H-13 were gauche to C-14 and C-38, respectively. The
gauche relation between the C-13-C-14 and C-12-C-38
bonds was deduced from the intense NOESY correlation for
H-14 (δH 2.53)/H3-38. Thus, the erythro relation for the
C-12-C-13 bond was established. On the other hand, an anti
relationship for H-23 and H-24 (Figure 1b) was inferred from
3
Scheme 2
the J(H-23,H-24) value (7.7 Hz). The NOESY correlation
for H-22 (δH 1.60)/H-24 as well as the J(C,H) values for
C-23/H-24 (-4.4 Hz) and C-22/H-24 (1.4 Hz) indicated that
C-22 and 23-O were both gauche to H-24. The NOESY
cross-peak for H-22 (δH 1.87)/H-25 was suggestive of the
gauche relation between C-22-C-23 and C-24-C-25 bonds,
3
and the J(C-25,H-23) value (1.0 Hz) was a typical one for
a gauche relation, thus indicating that the relative configu-
ration of C-23-C-24 was threo.
of which the structure was elucidated by analysis of 1H-1H
COSY and NOESY spectra. On the other hand, both bis-
(S)- and -(R)-MTPA esters (4a and 4b, respectively) of the
C-1-C-7 segment were prepared from the (4R,6R)-6-
hydroxymethyl-4-methyl-γ-butyrolactone (7), which was
derived from D-glutamic acid10 (Scheme 3). Two-carbon
Absolute Configurations at C-13 and C-29. Determi-
nation of the absolute configurations of two oxymethine
carbons at C-13 and C-29 was accomplished by a modified
Mosher method.9 The 7,8-O-isopropylidene derivative (2)
of amphidinolide C (1) was treated with (R)-(-)- and (S)-
(+)-2-methoxy-2-trifluoromethyl-2-phenylacetyl chloride (MT-
PACl) to afford the bis-(S)- and bis-(R)-MTPA esters (3a
and 3b, respectively), respectively. ∆δ values (δS - δR) are
shown in Figure 2. The ∆δ values for H2-14, H2-17, H2-31,
H2-32, H2-33, H3-34, H3-39, and H2-41 were negative, while
Scheme 3
(6) Kobayashi, J.; Ishibashi, M. Chem. ReV. 1993, 93, 1753-1769.
(7) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana,
K. J. Org. Chem. 1999, 64, 866-876.
(8) (a) Otting, G.; Wu¨thrich, K. Q. ReV. Biophys. 1990, 23, 39-96. (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.
(9) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092-4095.
1364
Org. Lett., Vol. 3, No. 9, 2001

elongation of the γ-butyrolactone (7) using a Wittig reaction
gave a E-olefin 8 in 90% yield in two steps. The unsaturated
ester 8 was converted into a tetrahydrofuran (94%) by
treatment with tert-butylammonium fluolide (TBAF) in THF
through diastereoselective Michael reaction, and then the
ester carbonyl group was reduced by DIBAL to give
compound 9 in 91% yield. Relative configurations of H-3/
H-4 and H-3/H-6 in 9 were both assigned as anti by NOESY
correlations for H2-2/H-6 and H-3/H3-35. Deprotection of
the benzyl group in 9 was achieved by hydrogenation using
palladium-charcoal in EtOH, and then esterification with
(R)-(-)- and (S)-(+)-MTPACl afforded the bis-(S)- and (R)-
MTPA esters (4a and 4b, respectively) of the C-1-C-7
segment.
derived from a natural specimen were identical with those
of the synthetic bis-(S)-MTPA ester (4a). Therefore, the
absolute configurations at C-3, C-4, and C-6 were determined
to be S, R, and R, respectively.
Absolute Configurations at C-7, C-8, and C-24. The
absolute configuration at C-24 was elucidated by application
of a modified Mosher method8 to the linear methyl esters of
amphidinolide C (1). Treatment of amphidinolide C (1) with
K2CO3 in MeOH yielded a mixture of four linear methyl
esters generated by epimerization of C-16 and C-20.11 One
of the four methyl esters purified by C18 HPLC was treated
with (R)-(-)- and (S)-(+)-MTPACl to afford the pentakis-
(S)-(-)- and -(R)-(+)-MTPA esters (5a and 5b, respectively).
1
∆δ values obtained from the H chemical shifts of 5a and
The 1H NMR spectrum of the bis-(S)-MTPA ester (4a) of
the C-1-C-7 segment obtained from natural amphidinolide
C (1) was compared with those of synthetic bis-(S)- and -(R)-
MTPA esters (4a and 4b) of the C-1-C-7 segment (Figure
3). Though 4a and 4b showed very similar NMR profiles,
5b are shown in Figure 4. The ∆δ values of the protons
from C-19 to C-22 are negative in sign, while those of H-25,
H-26, and H-27 are positive, suggesting a 24R configuration.
The absolute stereochemistry of C-7 and C-8 was eluci-
dated on the basis of the application of Mosher’s method
for erythro-glycol proposed by Kusumi et al.12 Negative ∆δ
values for H2-2, H-3, H-4, H2-5, and H-6 and positive ones
for H-10, H2-36, and H3-37 obtained from 5a and 5b
indicated that 5a and 5b had the 7S and 8R configurations.
Therefore, the absolute configurations at C-7 and C-8 were
concluded to be both R.
Absolute Configuration at C-16. To determine the
absolute configuration at C-16 of amphidinolide C (1),
Scheme 4
Figure 3. 1H NMR spectra (partial) of (a) bis-(S)-MTPA ester (4a)
of the C-1-C-7 segment derived from amphidinolide C (1) and
(b) synthetic bis-(S)- and (c) bis-(R)-MTPA esters (4a and 4b,
respectively) of the C-1-C-7 segment.
significant differences were observed for signals due to the
methylene proton at C-7 (4a, δH 4.06 and 3.98; 4b, δH 4.13
Baeyer-Villiger oxidation using trifluoroperacetic acid13
(TFPA) was applied to obtain the segment including the
1
and 3.79). H NMR data of the bis-(S)-MTPA ester (4a)
Figure 4. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the pentakis-(S)- and -(R)-MTPA esters (5a and 5b, respectively) of the linear
methyl ester of amphidinolide C (1).
Org. Lett., Vol. 3, No. 9, 2001
1365

methine carbon at C-16. Amphidinolide C (1) was treated
with TFPA followed by reduction with LiAlH4, esterification
with (S)-(+)-MTPACl, and HPLC separation to afford a bis-
(R)-MTPA ester (6a) of 1,3-butanediol corresponding to the
C-16-C-18 segment of 1 (Scheme 4). On the other hand,
the two authentic bis-(R)-MTPA esters of (S)-(+)- and (R)-
(-)-1,3-butanediols (6a and 6b, respectively) were prepared.
1H NMR data of compounds 6a from a natural specimen
were identical with those of synthetic 16S-isomer (Figure
5), indicating that the absolute configuration at C-16 of
C (1) were elucidated to be 3S, 4R, 6R, 7R, 8R, 12R, 13S,
16S, 20R, 23R, 24R, and 29S.
Acknowledgment. This work was partly supported by a
Grant-in-aid from the Japan Securities Scholarship Founda-
tion, a Grant-in-aid from Uehara Memorial Foundation, and
a Grant-in-aid for Scientific Research form the Ministry of
Education, Sciences, Sports, and Culture of Japan.
Supporting Information Available: Experimental pro-
cedures, spectral data of 1, 2, 3a, 3b, 4a (natural and
synthetic), 4b, 5a, 5b, 6a (natural and synthetic), and 6b,
and Tables S1 and S2. This material is available via the
Internet at http://pubs.acs.org.
OL015741Z
(10) (a) Tomioka, K.; Cho, Y.-S.; Sato, F.; Koga, K. J. Org. Chem. 1988,
53, 4094-4098. (b) Nishida, Y.; Konno, M.; Fukushima, Y.; Ohrui, H.;
Meguro, H. Agric. Biol. Chem. 1986, 50, 191-193.
(11) Two of the four linear methyl esters were suggested to have a syn
relationship for H-20-H-23 by NOESY correlation for H-20/H-23, while
the relative stereochemistry of H-20-H-23 of the two other diastereomers
was anti. Epimerization at C-20 is explained by inversion of C-20, generated
through retro-Michael-type cleavage between the ether oxygen and C-20
followed by Michael-type re-formation of the ether linkage. Although such
a retro-Michael-Michael reaction for another tetrahydrofuran ring (C-3-
C-6) might occur, the anti relationship of H-3/H-4 in the tetrahydrofuran
ring was reported to be generally more kinetically and thermodynamically
stable than a syn relationship. Methanolysis of 1 with K2CO3 in MeOH-d4
1
afforded four linear methyl esters labeled with deuterium. In the H NMR
data of four linear methyl esters, the signals of R-protons (H2-2, H2-14,
H-16, H2-17, and H2-19) of three carbonyl groups at C-1, C-15, and C-18
disappeared. This indicated that one of the two 20,23-syn linear compounds
and one of two 20,23-anti compounds were products due to epimerization
at C-16. Nevertheless, the stereochemisry at C-16 of each compound was
not determined. The diastereomer using the modified Mosher method
possessed a 20,23-anti relationship.
Figure 5. 1H NMR spectra (partial) of (a) bis-(R)-MTPA ester
(6a) of the C-16-C-18 segment derived from amphidinolide C (1),
(b) the bis-(R)-MTPA ester (6a) of (S)-1,3-butanediol, and (c) the
bis-(R)-MTPA ester (6b) of (R)-1,3-butanediol.
(12) Ichikawa, A.; Takahashi, H.; Ooi, T.; Kusumi, T. Biosci. Biotechnol.
Biochem. 1997, 61, 881-883.
(13) Bailey, A. S.; Gilpin, M. L. Jones, E. R. H. J. Chem. Soc., Perkin
Trans. 1 1977, 265-270.
amphidinolide C (1) was determined to be S. Therefore, the
absolute configurations of 12 chiral centers in amphidinolide
1366
Org. Lett., Vol. 3, No. 9, 2001