Update of Spectroscopic Data for 4-Hydroxydictyolactone and Dictyol E Isolated from a Halimeda stuposa — Dictyota sp. Assemblage †

The methanol extract of an assemblage of Halimeda stuposa and a Dictyota sp., yielded three natural products characteristic of Dictyota sp., and one of Halimeda sp. These included the xenicane diterpene 4-hydroxydictyolactone (1), and the diterpenes dictyol E (2), 8α,11-dihydroxypachydictyol A (3) and indole-3-carboxaldehyde (4). A minor revision of 1 and new spectroscopic data for 1 and 2 are provided, along with associated anti-cancer activities of compounds 1–4.

While investigating marine derived extracts for their anti-cancer activity, the ethanol (EtOH) extract of the green-brown alga assemblage of Halimeda stuposa and Dictyota sp. was found to have significant activity and an unusual profile in the NCI 60 cell line COMPARE analysis [14] The methanol (MeOH) extract of a large scale recollection was subjected to bioassay-guided fractionation, using C 18 flash vacuum liquid chromatography and preparative C 18 HPLC, to yield the xenicane lactone 4-hydroxydictyolactone (1) [15], as well as the known diterpenes dictyol E (2) [16], 8,11-dihydroxypachydictyol A (3) [17], and indole-3-carboxaldehyde (4) [18] (Figure 1). Described below are a minor revision of 1, as well as CD data and molecular modelling studies, in accordance with the absolute configuration previously reported [19], and NMR evidence confirming the presence of the minor cis conformer of 1 [20]. Also presented are the complete 1 H-NMR data for 2, as well as the biological activities of 1-4 against a panel of human tumour and normal mammalian cell lines.   (1) was isolated from the MeOH extract with a HRESIMS molecular  weight indicative of the molecular formula C 20 H 30 O 3 and corresponding to six double bond  equivalents. 1 H-and 13 C-NMR resonances (Supporting information Table S1) were identical to those first reported for the naturally occurring [15] and the synthetic 4-hydroxy-dictyolactone (1) [19], except for the C-7 and C-13 resonances. HSQC correlations (Supporting information Figure S4) were observed from  H 5.32 (H-7) to  C 125.3 and from  H 5.02 (H-13) to  C 123.9, indicating that the original assignments of these carbons were reversed. The C-1-C-9 double bond was assigned an E-configuration owing to the large coupling constants exhibited due to the axial-axial orientation of H-1 (δ 5.32, dd, 11.4, 4.2 Hz) and H a -2 (δ 3.20, dddd, 17.5, 11.4, 2.2, 2.2) [21] whilst 13 C-NMR data for C-20 ( C 20.0) confirmed the E geometry of C-6-C-7 [22]. All other spectroscopic data matched that reported [15], however, as previously noted by Williams et al. [19], a differing optical rotation for the naturally occurring 1 {[α] 21 D −87° (c 0.25, CHCl 3 )}was observed. Guella et al. [20] showed that 1 undergoes a slow conformation medium-ring flipping between the predominant trans-(C-20 trans to H-3) and the minor cis-conformer (C-20 cis to H-3). Further inspection of the 1 H and COSY NMR data confirmed the presence of the minor cis-conformer (Supporting Information Table S1), the ratio of which may influence the optical rotation. Closer inspection of the 1 H-NMR of the 50% MeOH flash column fraction revealed the presence of both conformers, however, only the trans-conformer was detected in the MeOH extract due to overlapping signals and concentration. Molecular modeling studies, where the geometry of both the double bonds (C-2-C-9 and C-6-C-7) in the carbocycle was constrained to E, were performed to determine which of the 16  The naturally occurring [15,20] and synthetic studies [19] report measurement of optical rotation, but no CD data. The absolute configuration at C-2 of 1 was corroborated by CD measurement. The CD spectrum of 1 showed a large negative Cotton effect at 226 nm ( = −38.44, *), and a small positive Cotton effect at 258 nm ( = 5.42, n*). Applying the quadrant rule [23]; viewing the ring along the O-C-19-C-1 axis, resulted in C-3 extending into the negative upper right quadrant. This finding is reconcilable with an S configuration at C-2 (Figure 2c,d) and in agreement with the naturally occurring [15] and the synthetic 4-hydroxydictyolactone (1) [19], trans-2S,3S,4R,10R, where 10R has previously been determined by x-ray crystallography [24] and synthetic studies [20].
The configuration of the C-3-C-4 double bond must be Z in order to form the five-membered ring [26]. The relative stereochemical assignment was confirmed as 1R,5S,6R,7S by the positive optical rotation ([] 21 D +21 CHCl 3 ; c 0.11) [27] and comparison with literature values [16].
Outlined in Table 2 are the cytotoxic activities of 1-4 against a panel of human and mammalian cell lines. From these data there appears to be no obvious SAR, with 1-3 having approximately the same activities against the three human tumour cell lines SF-268, MCF-7 and H460. However, the response of compounds 1-3 against HT-29, a human colon tumour cell line, and CHO-K1, a Chinese hamster ovary non-tumour cell line, were between two and four-fold less active as compared to those for the three cancer cell lines mentioned above, suggesting some selectivity. Indole-3-carboxaldehyde (4) was not active against any of the cell lines.

General Procedures
General experimental procedures are as described previously [28]. CD spectra were collected on a JASCO J-715 spectropolarimeter with a 0.1 dm cell.

Plant Material
The green/brown algal assemblage of Halimeda stuposa (Udoteaceae, Caulerpales) and Dictyota sp., (Dictyotaceae, Dictyotales) was collected from the passage between Shaw and Maher Islands, Queensland, at a depth of 7 m, in October 1987. Collection of this material was conducted under the Queensland Fish or Marine Products Permit no. 1780 and the GBRMPA Permit no. 87/293. A voucher specimen (Accession number AQ642006) has been lodged with the Queensland Herbarium.

Bioassay
Cellular bioassays were undertaken as described previously [28].