Antiproliferative bistramides from Trididemnum cyclops from Madagascar

In our continuing search for biologically active natural products from Madagascar as part of an International Cooperative Biodiversity Group (ICBG) program,1 an extract was obtained from the tunicate Trididemnum cyclops Michaelsen 1921 (Didemnidae). The marine invertebrate Trididemnum has been the source of a number of potently bioactive compounds, of which the most frequently reported in the literature and subsequently found to be the most bioactive are a class of cyclic depsipeptides known as didemnins. Rinehart et al. reported the first occurrence of these amino acid macrocycles from Trididemnum solidum.2 One of the isolates was didemnin B, the first marine-derived compound to enter Phase I and Phase II clinical trials by the National Cancer Institute as a potential antitumor agent. In the current study, an extract of T. cyclops showed significant antiproliferative activity against the A2780 ovarian cancer cell line, and it was thus selected for study. Fractionation yielded one new and two known bistramides. Bistramides are a class of polyether amides with methylated tetrahydropyran and spiroketal subunits. The first of its kind, bistramide A (2), was discovered in 1988 by Gouiffes et al. from the ascidian Lissoclinum bistratum, also of the family Didemnidae.3 The bistramides have been shown to display diverse bioactivities, including immunomodulating and weak antimalarial activity,4,5 but their most notable trait is their antitumor properties. Statsuk et al. reported that actin, a protein essential for cellular motility and division, is the primary cellular receptor of bistramide A.6 In a series of cell-based and in vitro studies in conjunction with affinity-based protein isolation, it was determined that bistramide A (2) aggravates the actin cytoskeleton by depolymerizing filamentous F-actin and binding to monomeric G-actin. An actin-bistramide A complex was later crystallized, allowing for detailed analysis of the binding interactions of this marine metabolite with its monomeric G-actin counterpart.7 Recently, Rizvi et al. described the mechanism of action of bistramide A, namely, the “severing of actin filaments and covalent sequestration of monomeric actin in the cell.”8 They postulated the C-1-C-4-enone subunit to be crucial for covalent protein modification, an event that helps trigger actin depolymerization. Since the identification of bistramide A there has been only one report of the isolation of additional bistramides as natural products (analogs B-D and K, also from L. bistratum),9 in comparison with nearly 40 journal articles focusing on their synthesis and bioactivity. To date, the only natural source of bistramides has been from L. bistratum, and the first report of these potential antitumor compounds from another source is described herein. Bioassay-guided fractionation of an extract from a Trididemnum cyclops afforded 300 µg of the new compound 1, together with the known bistramides A (2) and D (3). Compound 1 was obtained as an amorphous solid (0.3 mg). HRFABMS (positive-ion mode) analysis indicated a molecular formula of C40H68N2O8. Its 1H NMR spectrum exhibited large solvent peaks and was noisy because of the limited sample size, and it was not possible to obtain a 13C NMR spectrum, but the 13C NMR shifts could be inferred from the HMBC and g-HSQC spectra of 1. Analysis of data from these spectra indicated the presence of two amide carbonyls (δC 173.2, C-13; 177.3, C-18), one ketone carbonyl (δC 202.5, C-39), and six sp2 olefinic carbons (δC 126.8, C-2; 134.7, C-3; 128.7, C-6; 132.1, C-7; 151.4, C-36; 137.1, C-37). There was also evidence of six sp3 oxygenated carbons (δC 73.4, C-4; 72.3, C-11; 73.1, C-15; 75.6, C-22; 96.7, C-27; 70.0, C-31), and two sp3 nitrogenated carbons (δC 44.1, C-14; 40.2, C-19) (Table 1). The 1H NMR resonances of 1 in CD3OD showed evidence for five unsaturated methines (δH 5.61, dq, J = 15.1, 6.4 Hz, H-2; 5.45, m, H-3; 5.45, m, H-6; 5.45, m, H-7; 6.53, d, J = 10.3 Hz, H-36) and seven methyl groups (δH 1.67, br d, J = 6.4 Hz, H3-1; 0.90, d, J = 6.8 Hz, H3-10; 1.13, d, J = 7.0 Hz, H3-17; 0.83, d, J = 6.5 Hz, H3-24; 1.06, d, J = 6.6 Hz, H3-35; 1.75, s, H3-38; 2.31, s, H3-40) (Table 1). There were eight degrees of unsaturation for the proposed molecular formula of 1, which corresponded to three double bonds, three carbonyl groups, and two rings. Table 1 NMR Data of Compound 1a The aforementioned components of 1 were pieced together using both g-COSY and g-HMBC data, as well as comparing 1H and 13C NMR chemical shift values with those of bistramide K,9 although noticeable differences in chemical shifts suggested the occurrence of slightly different functionalization. Most importantly, an additional ketone carbonyl signal (C-39) was observed instead of a hydroxylated carbon signal at C-39. The molecular weight of 1 was found to be two mass units less than that of bistramide K. The absence of one sp3-oxygenated hydrogen resonance in the 1H NMR spectrum (H-39, bistramide K), in addition to the deshielded nature of H-40 (δH 2.31, s) and a downfield shift of the C-36 olefinic carbon (δC 151.4) signal as the result of becoming integrated into an α,β-unsaturated ketone system, signified that the sole structural difference between 1 and bistramide K is the oxidation of the C-39 hydroxyl moiety to a ketone functional group. Some key HMBC correlations are listed in Figure 1. Notably, HMBC correlations of H3-40 and H3-38 to the carbonyl at C-39 and the α-quaternary olefinic carbon (C-37), in addition to correlations of H3-40 and H3-38 to C-36, assisted in making the assignments at the tail end of the polyketide chain of 1. Figure 1 Key HMBC (arrows) and ROESY (dashed curves) correlations for compound 1 Because of the small amount of compound isolated, spectroscopic methods were relied on to establish the relative configuration of 1. The planar structures of 1 and 2 are identical from C-12 to C-37, and superimposing their 1H NMR spectra showed no significant differences between chemical shifts and coupling patterns, indicating that the two compounds share the same relative configuration at these positions. A correlation in the ROESY spectrum (axial H-22 to equatorial H3-24) also supported this hypothesis, and similarities were observed between the 1H NMR chemical shifts and coupling constants (JH-9–H-11) of 1 with those found in the literature for bistramide K.9 Despite overlap of the H-6 and H-7 olefinic resonances in the 1H NMR spectrum, the regiochemistry of the double bond was assigned as E due to similar chemical shifts of these two positions to those of bistramide K and the observance of ROESY correlations of H-6 with H4/H-8, and H-7 with H-5/H-9. Finally, although the small amount of sample available precluded the running of a quantitative CD spectrum of 1, the qualitative CD spectra of 1 and 2 were similar, with each showing a broad negative Cotton effect between 210 and 240 nm. These data suggested that bistramide K and compound 1 have the same absolute and relative configurations. The stereochemistry of the new chiral center at C-4 could not be determined from the available data. Bistramides A (2) and D (3) were identified on the basis of comparison of MS and one and two-dimensional NMR spectroscopic data to those reported by Biard et al.9 The IC50 values for the antiproliferative activities of compounds 1 and 2 against the A2780 human ovarian cancer cell line were 0.34 and 0.26 µM, respectively. Compared with IC50 values against other cell lines reported in the literature (45, 20, and 22 nM against the KB, P388, and normal endothelial cells, respectively),10 the values against the A2780 cell line were about ten-fold larger. In addition to bistramides 1–3, 1H NMR spectroscopic analysis of other minor peaks from the HPLC separation indicated the presence of several other bistramide analogs or bistramide-derived secondary metabolites; we estimate that this extract may contain as many as ten additional bistramides. Since only five known naturally occurring bistramides have been isolated over the past 20 years, this suggests that this structural class is more diverse than previously known. Given their nanomolar potency against selected cell lines and their well-defined mechanism of action against cancer, the isolation of additional bistramide derivatives will increase the chances of discovering analogs that exhibit similar activity with less toxicity. The current work has proven a Trididemnum cyclops to be a source of small quantities of structurally unique bistramides. Since Trididemnum spp. are also sources of the didemnins, this genus is a versatile producer of bioactive metabolites. This raises the question of the true origin of the bistramides, which were originally isolated from the ascidian Lissoclinum bistratum.4,9 It has been reported that other species of Lissoclinum, as well as a few species of Trididemnum, contain obligate Prochloron spp. bacterial symbionts,10 and the collection used for this work also had a crust of a Prochloron sp. These cyanobacteria act as important nutrient sources for their host ascidians (particularly of the family Didemnidae) via photosynthesis and nitrogen fixation.11 They have been implicated in the production of cyclic peptides,12,13 a class of compounds that includes both bistratamides (Lissoclinum) and didemnins (Trididemnum). It is thus very possible that the bistramides are in fact products of microbial origin.