VOLUME 67, NUMBER 19
SEPTEMBER 20, 2002
© Copyright 2002 by the American Chemical Society
Amphidinolides H2-H5, G2, and G3, New Cytotoxic 26- and 27-Membered Macrolides from Dinoflagellate Amphidinium sp. Jun’ichi Kobayashi,* Kazutaka Shimbo, Masaaki Sato, and Masashi Tsuda Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
[email protected] Received October 22, 2001
Six new macrolides, amphidinolides H2 (5), H3 (6), H4 (7), H5 (8), G2 (9), and G3 (10), have been isolated from a marine dinoflagellate Amphidinium sp. (strain Y-42). Cytotoxicity of five derivatives (11-15) of amphidinolide H (1) in addition to 10 amphidinolides (1-10) containing amphidinolides H (1), G (2), B (3), and D (4) was examined, and it was found that the presence of an allyl epoxide, an S-cis-diene moiety, and the ketone at C-20 was important for the cytotoxicity of amphidinolide H (1)-type macrolides. Introduction Amphidinolides are a series of unique cytotoxic macrolides isolated from dinoflagellates Amphidinium sp., which were separated from marine acoel flatworms Amphiscolops sp.1 Amphidinolides H (1), G (2), B (3), and D (4) are potent cytotoxic 26- or 27-membered macrolides possessing unique structural features such as an allyl epoxide and vicinally located one-carbon branches.2-4 The absolute stereochemistry of amphidinolide B (3) has been established by Shimizu’s4 and our groups.5 Recently, we have separated a strain (Y-72) of the genus Amphidinium producing a relatively large amount of amphidinolides H (1) and G (2) and determined the absolute stereochemistry of 1 and 2 on the basis of the X-ray diffraction analysis and synthesis of a degradation product.6 During our continuing search for bioactive and structurally unique metabolites from marine dinoflagellates,1,7,8 investigation of extracts from another strain (Y42) of the genus Amphidinium resulted in the isolation * To whom correspondence should be addressed. Phone and Fax: +81 11 706 4985. Fax: +81 11 706 4989. (1) Kobayashi, J.; Kubota, T.; Endo, T.; Tsuda, M. J. Org. Chem. 2001, 66, 134-142 and references therein. (2) Kobayashi, J.; Shigemori, H.; Ishibashi, M.; Yamasu, T.; Hirota, H.; Sasaki, T. J. Org. Chem. 1991, 56, 5221-5224. (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. (4) Bauer, I.; Maranda, L.; Shimizu, Y.; Peterson, R. W.; Cornell, L.; Steiner, J. R.; Clardy, J. J. Am. Chem. Soc. 1994, 116, 2657-2658. (5) Ishibashi, M.; Ishiyama, H.; Kobayashi, J. Tetrahedron Lett. 1994, 35, 8241-8242. (6) Kobayashi, J.; Shimbo, K.; Sato, M.; Shiro, M.; Tsuda, M. Org. Lett. 2000, 2, 2805-2807.
of six new potent cytotoxic macrolides, amphidinolides H2 (5), H3 (6), H4 (7), H5 (8), G2 (9), and G3 (10). The stereochemistries of 5-9 were elucidated on the basis of JHH and JCH coupling constant data, distance geometry calculation, and chemical means. Five derivatives (1115) of amphidinolide H (1) were prepared, and cytotoxicity of 11-15 in addition to 10 amphidinolides (1-10) was examined. As a result, it was found that the presence of an allyl epoxide, an S-cis-diene moiety, and the ketone at C-20 was important for the cytotoxicity of amphidinolide H (1)-type macrolides. Here, we describe the isolation and structure elucidation of 5-9 and the structure-activity relationship of amphidinolide H-type macrolides. Results and Discussion Isolation and Structures of Amphidinolides H2 (5), H3 (6), H4 (7), H5 (8), G2 (9), and G3 (10). The dinoflagellate Amphidinium sp. (Y-42 strain) was isolated from an acoel flatworm Amphiscolops sp. collected off Sunabe, Okinawa, and mass cultured unialgally at 25 °C for 2 weeks in a seawater medium enriched with 1% Provasoli’s Erd-Schreiber (ES) supplement. The mass cultured algal cells (110 g, wet weight) obtained from 200 (7) Amphidinolides: (a) Tsuda, M.; Endo, T.; Kobayashi, J. Tetrahedron 1999, 55, 14565-14570. (b) Kubota, T.; Tsuda, M.; Kobayashi, J. Tetrahedron Lett. 2000, 41, 713-716. (c) Tsuda, M.; Endo, T.; Kobayashi, J. J. Org. Chem. 2000, 65, 1349-1352. (8) Colopsinols: (a) Kobayashi, J.; Kubota, T.; Takahashi, M.; Ishibashi, M.; Tsuda, M.; Naoki, H. J. Org. Chem. 1999, 64, 14781482. (b) Kubota, T.; Tsuda, M.; Takahashi, M.; Ishibashi, M.; Naoki, N.; Kobayashi, J. J. Chem. Soc., Perkin Trans. 1 1999, 3483-3488. (c) Kubota, T.; Tsuda, M.; Takahashi, M.; Ishibashi, M.; Oka, S.; Kobayashi, J. Chem. Pharm. Bull. 2000, 48, 1447-1451.
10.1021/jo016222c CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/16/2002
J. Org. Chem. 2002, 67, 6585-6592
6585
Kobayashi et al. CHART 1
L of culture were extracted with MeOH/toluene (3:1), and the extracts were partitioned between toluene and 1 M NaCl (aq). The toluene extracts were subjected to a silica gel column followed by SiO2 and/or C18 HPLC to afford amphidinolides H2 (5, 0.0003%, wet weight), H3 (6, 0.0004%), H4 (7, 0.0005%), H5 (8, 0.0002%), G2 (9, 0.0005%), and G3 (10, 0.0003%) together with known related macrolides, amphidinolides H (1, 0.0007%) and G (2, 0.0008%) (Chart 1).2 Amphidinolides H2 (5) and H3 (6) had the same molecular formula, C32H50O8, as that of amphidinolide H (1) as revealed by HRMS data [[m/z 668.4390 (M + diethanolamine (DEA) + H)+, ∆ +1.7 mmu] for 5 and [m/z 585.3419 (M + Na)+, ∆ +1.6 mmu] for 6]. 1H and 13C NMR data (Tables 1-3) of 5 and 6 were similar to those of 1. Detailed analyses of 2D NMR data including 1H1 H COSY, TOCSY, HSQC, and HMBC spectra indicated that 5 and 6 were stereoisomers of amphidinolide H (1). The relative stereochemistry at nine chiral centers of amphidinolides H2 (5) and H3 (6) was elucidated mainly on the basis of 1H-1H coupling constants and NOESY correlations. The geminal and vicinal 1H-1H coupling constants (Table 3) for 5 and 6 were mainly obtained by 6586 J. Org. Chem., Vol. 67, No. 19, 2002
resolution-enhanced spectra of decoupling difference experiments. Two- and three-bond 13C-1H coupling constants, which were obtained from hetero half-filtered TOCSY (HETLOC) spectra,9 were used for configurational assignments of portions with 1,3-chiral centers.10 The relative stereochemistry for the C-16-C-22 part of amphidinolide H2 (5) was elucidated by comparison of the coupling constants (Table 3) and NOESY correlations (Figures 1 and 2) with those of amphidinolide H (1). As shown in Figure 1a, the coupling constant between H-18 and H-19a in 5 was 2.6 Hz, whereas that between H-18 and H-19b was 9.8 Hz, indicating that H-18/H-19a and H-18/H-19b were gauche and anti relations, respectively. The anti relation between H-19a and OH-18 was deduced from the two-bond C-H coupling constants for C-18/H-19a (∼0 Hz) and C-18/H-19b (-5.3 Hz) of 5. The NOESY correlation for H-18/H-19a also supported the relative stereochemistry as shown in Figure 1a, which (9) (a) Otting, G.; Wu¨thrich, 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. (10) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866-876.
Amphidinolides H2-H5 TABLE 1.
1H NMR Chemical Shifts of Amphidinolides H (1), H2 (5), H3 (6), H4 (7), and H5 (8) in CDCl3
TABLE 2.
13C NMR Data of Amphidinolides H (1), H2 (5), H3 (6), H4 (7), and H5 (8) in CDCl3
position
1
5
6
7
8
position
1
5
6
7
8
3 4a 4b 5a 5b 6 (6a) 6b 7 (7a) 7b 8 9 10a 10b 11 12a 12b 14 16 17a 17b 18 19a 19b 21 22 23 24a 24b 25 26a 26b 27 28 29a 29b 30 31 32
6.82 2.46 2.22 2.38 2.11 5.90
6.80 2.48 2.23 2.41 2.15 5.87
6.82 2.43 2.24 2.34 2.16 5.93
5.15
5.24
5.18
3.14 2.96 1.52 1.18 1.61 2.14 1.88 5.58 2.26 1.81 1.45 3.94 2.75 2.62 4.32 3.76 1.90 2.04 1.29 5.08 3.74 3.68 1.84b 0.86b 4.98 4.81 1.73b 1.07b 1.02b
3.08 2.97 1.72 0.94 1.57 2.28 1.66 5.53 2.24 1.82 1.43 4.07 3.06 2.53 4.23 3.61 1.91 2.39 1.21 5.09 3.71a
3.14 2.98 1.56 1.11 1.56 2.07 1.92 5.53 2.22 1.82 1.48 4.00 2.92 2.74 4.15 3.56 2.06 1.96 1.40 5.09 3.76 3.67 1.84b 0.89b 5.00 4.82 1.73b 1.08b 1.01b
6.97 2.31 2.18 1.59 1.44 1.66 1.46 1.90 1.18 2.75 2.90 1.55 1.17 1.63 2.16 1.80 5.52 2.21 1.81 1.42 3.98 2.72 2.54 4.44 3.74 1.95 2.01 1.28 5.07 3.76 3.68 1.83b 0.88b 5.00 4.83 1.75b 1.07b 1.06b
6.81 2.32 2.22 1.65 1.54 1.60 1.52 1.84 1.21 2.83 2.97 1.52 1.00 1.52 2.16 1.84 5.56 2.22 1.84 1.42 4.03 2.89 2.56 4.27 3.57 1.92 2.42 1.17 5.15 3.76 3.72 1.83b 0.84b 4.81 4.97 1.75b 1.07b 0.97b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
168.7 127.9 141.0 27.0 30.9 135.7 128.6 60.3 59.5 39.8 29.1 47.1 144.1 126.1 141.7 40.7 40.9 67.5 45.2 212.2 77.7 75.4 33.0 33.5 73.4 66.1 13.2 18.0 114.7 12.6 20.3 15.6
168.7 127.6 140.8 26.9 31.0 136.1 129.7 59.8 60.1 40.4 29.8 46.6 144.3 126.7 140.6 40.8 40.2 65.9 43.8 211.3 78.4 76.2 32.3 33.9 73.3 66.6 12.7 19.5 115.0 12.3 20.4 15.1
168.8 128.1 140.7 26.8 30.7 136.1 127.9 60.5 59.7 39.6 29.3 47.1 143.9 125.8 141.5 40.9 40.8 67.1 46.6 215.1 77.0 77.2 30.1 30.2 73.7 66.4 12.6 18.2 114.8 12.8 20.8 16.2
168.7 127.4 143.1 28.1 25.2 27.9 32.1 59.9 58.2 40.5 29.0 47.2 144.1 126.3 141.4 41.1 40.5 67.5 44.9 210.8 77.5 75.0 32.4 34.2 73.4 66.2 12.3 17.6 114.9 12.7 20.3 15.6
168.6 127.2 143.6 27.1 25.0 27.9 31.6 59.1 58.9 40.1 28.8 47.0 144.0 126.0 141.0 41.1 41.0 65.6 44.0 210.2 78.8 76.3 32.7 34.1 73.2 66.6 12.1 17.6 114.9 12.5 20.5 15.3
a
1.83b 0.89b 4.96 4.80 1.71b 1.07b 0.96b
2H. b 3H.
FIGURE 1. Rotation models for (a) C-18-C-19, (b) C-17-C18, and (c) C-16-C-17 bonds of amphidinolide H2 (5). 1H-1H coupling constants are given in hertz. NOESY correlations are illustrated by solid arrows.
was different from that of amphidinolide H (1).11 In the C-17-C-18 bond (Figure 1b), the vicinal coupling constants for H-17a/H-18 (3.5 Hz) and H-17b/H-18 (10.6 Hz) suggested that H-17a/H-18 and H-17b/H-18 were gauche and anti relations, respectively. The 3J(H-16/H-17a) (12.2 Hz) and 3J(H-16/H-17b) (4.0 Hz) values were typical for (11) Amphidinolide H (1) had anti and gauche relations for H-18/ H-19a and H-18/H-19b, respectively, as indicated by the 3J(H-18/H19a) and 3J(H-18/H-19b) values (8.5 and 1.8 Hz, respectively) and 2J(C-18/H-19a) and 3J(C-18/H-19b) values (-6.7 Hz and -2.6 Hz, respectively).
FIGURE 2. NOESY correlations and 1H-1H coupling constants for C-16-C-22 portion in amphidinolide H2 (5). Key NOEs are illustrated by solid arrows, and vicinal 1H-1H coupling constants are given in hertz.
anti and gauche relations, respectively (Figure 1c). NOESY correlations observed for H-17a/H3-31 and H-17b/ H3-31 indicated that H-17a/C-31 and H-17b/C-31 both had gauche relations, which was also supported by relatively small 3JC-H values for C-31/H-17a (3.2 Hz) and C-31/H-17b (2.8 Hz). NOESY correlations for H-16/H-19a and H-17b/H-19b (Figure 2) indicated a 1,3-anti relation of C-31/18-OH, while those for H-19b/H-21 and H-19a/ H-22 of 5 indicated that H-19b and H-21 had the same orientations. Thus, the relative configurations at C-16, C-18, C-21, and C-22 of amphidinolide H2 (5) were elucidated as shown in Figure 2. J. Org. Chem, Vol. 67, No. 19, 2002 6587
Kobayashi et al. TABLE 3.
1H-1H Coupling Constants of Amphidinolides H (1), H2 (5), H3 (6), H4 (7), and H5 (8) in CDCl3
position
1
5
6
7
8
6/7 7/8 8/9 9/10a 9/10b 10a/10b 10a/11 10b/11 11/12a 11/12b 11/28 12a/12b 16/17a 16/17b 16/31 17a/18 17b/18 17a/17b 18/19a 18/19b 19a/19b 21/22 22/23 23/24a 23/24b 24a/24b 23/32 24a/25 24b/25 25/26a 25/26b 26a/26b
15.4 8.5 2.4 2.4 9.4 14.5 10.0 2.9 5.8 9.8 6.4 13.7 11.7 4.4 6.9 4.5 9.6 13.8 8.5 1.8 16.2 1.8 12.2 2.3 11.0 13.9 6.6 10.6 2.4 6.2 6.6 11.5
15.4 8.8 2.5 2.2 9.0 14.0 9.8 2.8 4.5 11.1 6.5 13.5 12.2 4.0 6.8 3.5 10.6 13.8 2.6 9.8 17.7 1.8 11.0 2.6 11.4 14.3 6.6 11.3 2.6 3.3 6.2 12.0
15.5 8.4 2.4 2.0 9.9 12.5 10.2
