Amphidinolides are a series of cytotoxic macrolides isolated from laboratory-cultured marine dinoflagellates Amphidinium sp.1 Amphidinolide N (1),2 isolated from Amphidinium sp. (Y-5 strain), is a 26-membered macrolide having an allyl epoxide and a tetrahydropyran with 13 chiral centers. Among all amphidinolides isolated so far, amphidinolide N (1) is the most potent cytotoxic macrolide with IC50 values against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro being 0.00005 and 0.00006 μg ml−1, respectively. Owing to the complex structure and the extraordinarily potent cytotoxicity, amphidinolide N (1) has been a challenging target for total synthesis.3, 4, 5, 6 In our previous studies,2 gross structure 1b was proposed based on 2D NMR and mass spectral data, and the configuration of epoxide and the relative stereochemistry of the C-14-C-19 portions were assigned from J-values and NOESY data. However, the stereochemistry of the remaining part has not yet been elucidated. Here we describe a revision of the gross structure from 1b to 1a (Figure 1) and full assignment of the relative stereochemistry of amphidinolide N (Figure 5).

Figure 1
figure 1

Revised (1a) and proposed (1b) gross structures of amphidinolide N.

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The molecular formula of amphidinolide N was established as C33H52O11 from ESIMS (m/z 647 (M+Na)+ (pos.), m/z 623 (M-H)− (neg.)) (in our previous studies,2 pseudo molecular ion peaks of amphidinolide N, m/z 730 (M+DEA+H)+ and m/z 748 (M+DEA+H3O)+, were misinterpreted as m/z 730 (M+DEA+H-H2O)+ and m/z 748 (M+DEA+H)+, respectively). Deuterium-induced shifts in 13C NMR resonances for amphidinolide N were measure by using C6D6/CD3OH (3:1) and C6D6/CD3OD (3:1) as solvents. Of 11 signals observed for oxygenated sp3 carbons, six oxymethines did not show deuterium-induced upfield shifts (Table 1). Of the unchanged oxymethines, two high-field methine carbons at δ 54.92 (C-5) and 62.98 (C-4) in C6D6/CD3OH (3:1) were attributed to those of an epoxide ring. Two carbons at δ 98.08 (which did show the upfield shift) and 66.73 were assigned as a hemiacetal carbon at C-15 and an oxymethine carbon at C-19, respectively, whereas an oximethine carbon (δ 75.66) was elucidated to be that forming a lactone linkage between C-1 and C-25. The remaining two unchanged oxygenated carbons at δ 75.61 and 80.22 were assigned as C-21 and C-24, respectively, suggesting that C-21 and C-24 were not connected to hydroxy groups. The cross-peak of H-24 to C-21 observed in the HMBC spectrum of 1a indicated that C-21 and C-24 were connected to each other through an ether linkage. Thus, the gross structure of amphidinolide N was revised from 1b to 1a (Figure 1).

Table 1 13C NMR data of amphidinolide N (1)
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In our previous studies,2 the configuration of the 4,5-epoxide was deduced to be trans from the coupling constant (3JH-4/H-5=1.9 Hz), and the relative stereochemistry of the C-14-C-19 portions was elucidated from J-values and NOESY data. In this study, the relative stereochemistry of the C-2-C-4 segment was analyzed by the J-based configuration analysis method.7 The values for 3JH-2/H-3 (7.2 Hz), 3JC-4/H-2 (3.3 Hz), 3JC-1/H-3 (3.4 Hz) and 2JC-3/H-2 (−4.7 Hz), which were obtained from the 1H NMR and J-HMBC8, 9, 10, 11, 12 spectra, indicated that C-1 was gauche to C-4, H-2 was gauche to 3-OH and H-3 was gauche to C-30. The gauche relation between the H-3 and C-30 was deduced from the intense NOESY correlation for H-3/H3-30 as well, suggesting the threo relation for the C-2-C-3 bond (Figure 2a). The values for 3JH-3/H-4 (4.6 Hz), 3JC-5/H-3 (4.6 Hz), 3JC-2/H-4 (3.3 Hz) and 2JC-4/H-3 (−3.2 Hz), and the intense NOESY correlation for H-2/H-5 indicated that C-2 was gauche to C-5, H-3 was gauche to O-4 and H-4 was gauche to 3-OH, suggesting assignment of the relative configuration of C-3-C-4 as threo (Figure 2b).

Figure 2
figure 2

Rotation models for (a) C-2-C-3 and (b) C-3-C-4 bonds of amphidinolide N.

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The relative stereochemistry of the C-5-C-10 segment was deduced from J-values and NOESY data. The values for 3JH-7/H-8a (10.4 Hz) and 3JH-7/H-8b (2.3 Hz) indicated an anti relationship for H-7 and H-8a, while NOESY correlations of H-5/H-8b and H-8b/H-10 showed proximity of H-5, H-8b and H-10, reading to the relative configurations shown in Figure 3.

Figure 3
figure 3

Selected NOESY correlations and relative stereochemistry for amphidinolide N (C-4-C-10 part).

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Proximity of H-18, H-21 and H-25 was indicated by NOESY correlations of H-18/H-21 and H-21/H-25, suggesting that the relative stereochemistry of H-21 and H-24 in the tetrahydrofuran ring was anti. The proximity of H-24 and H-2 was deduced from the NOESY correlation of H-24/H-2. The relative configurations from C-15 through C-2 as shown in Figure 4 were suggested by these NOESY correlations. Thus, the relative stereochemistry of amphidinolide N (1) was elucidated as shown in Figure 5.

Figure 4
figure 4

Selected NOESY correlations and relative stereochemistry for amphidinolide N (C-15-C-26 and C-1-C-2 parts).

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Figure 5
figure 5

Relative stereochemistry of amphidinolide N (1).

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