Tunicates are a group of marine invertebrates particularly abundant along coastal regions, often found as the dominant organisms in sessile benthic communities [1]. In Brazil, as in many other regions, information regarding these animals is restricted to faunal inventories and, in many cases, is inaccurate or incomplete [2]. In regards to Brazilian tropical waters, the deficiency in species inventories is quite evident, as very few references are available [3,4,5].

The faunal list for the state of Ceará, on the northeastern coast of Brazil, indicated that 22 species of ascidians were registered [5,6], including novel species and many cases of regional endemism. This coastline has been scarcely explored as a source of natural products. In 2003, a pioneering study investigated the cytotoxicity of aqueous methanol extracts of the ten most abundant ascidians of Ceará. The results of these assays suggested that Eudistoma vannamei Millar, 1977 (Figure 1 a) might be of interest due to its marked bioactivity, especially the inhibition of growth in tumor cell lines [7]. Evaluation of the cytotoxicity data from these extracts suggested the presence of amino acid-derived compounds in fractions of intermediate polarity. Treatment of leukemia cells with the amino acid-derived compounds resulted in apoptosis [8].

The genus Eudistoma is the most diverse among the Polycitoridae, with most of its species living in tropical regions [9]. On the Brazilian coast, only one species of Eudistoma was found in temperate waters [10], while in the warmer Northeast region, there are at least seven species of this genus. E. vannamei is endemic to the Northeast coast of Brazil where it forms large colonies on the underside of ledges of the beach rocks and is characterized by its conspicuous orange or yellow bulbus heads [3,5]. E. vannamei’s sessile habit and apparent lack of mechanical defenses would make it an easy target for predators. However, taking into account its dominance on the intertidal reefs, the species most probably relies on chemical deterrents for protection.

Several cytotoxic substances have been isolated from Eudistoma ascidians, including the alkaloids eudistomins A–T, eudistomidins A–F and eudistalbin [11,12,13,14,15,16,17,18,19] and the macrolides iejimalides A–D [20,21]. The present study reports the bioactivity-guided isolation of two new members of the staurosporine group of alkaloids from the extract of E. vannamei.

The aqueous methanol extract from E. vannamei was submitted to cytotoxicity-guided fractionation (flowchart provided in the supplementary material), resulting in the isolation of 5 mg of an active fraction, which appeared as a single spot by TLC and a single peak via LC-MS analysis.

This material was submitted to NMR and HRMS analysis. The ESI-HRMS spectrum showed one signal at m/z 497.1830 (error value: 2.21 ppm), suggesting the molecular formula C₂₈H₂₄N₄O₅. The initial analysis of the ¹³C NMR contained more carbons than expected, and appeared as two sets of peaks with similar chemical shifts. Detailed analysis of the MS and MS/MS data along with evaluation of the ¹³C NMR data suggested that the material was a mixture of two staurosporine derivatives, compounds 1 and 2.

A detailed set of NMR spectral data was then collected including ¹H, ¹³C, COSY, HSQC and HMBC spectra to identify the structures of 1 and 2. Each of the protons in 1 and 2 were assigned using a combination of chemical shift data and coupling constants from the ¹H NMR spectrum (Table 1) and correlations from a COSY spectrum (Figure 2). Analysis of the chemical shift and coupling constant data indicated that the glycosidic unit matched that in staurosporine [22,23,24,25]. An HSQC experiment enabled the assignments of each methine, methylene and methyl carbon (Table 1). The COSY spectrum revealed the scalar coupling correlations for the most deshielded proton attached to sp³ carbons H-6′ (δ_H 6.62 for 1 and 6.69 for 2) with the heterotopic methylene protons 2H-5′ (δ_H 2.36/2.72 for 1 and δ_H 2.46/2.74 for 2) which, in turn, showed geminal coupling. The oxymethine protons at δ_H 3.95 (H-3′ for 1) and 3.97 (H-3′ for 2) showed correlations with the azomethine protons at δ_H 3.23 and 3.26 (H-4′) for 1 and 2, respectively. In the aromatic region, the most deshielded proton at δ_H 9.94 (H-8 for both 1 and 2) coupled to one at δ_H 7.46 (H-9 for both 1 and 2), which coupled with one at δH 7.61 (H-10 for both 1 and 2) and that, in turn, coupled with the ones at δ_H 8.15 (for 1) and 8.17 (for 2). On the hydroxyl substituted benzene ring, the most deshielded doublet (J = 8.4 Hz) at δ_H 9.63 (H-4 for 1) correlated with the one at δ_H 7.40 (H-3 of 1), while the correspondent H-4 of 2 at δ_H 9.58 (br, d) coupled weakly with the proton at δ_H 7.61 that, in turn, coupled with the one at δ_H 7.54 (d, J = 8.7 Hz, H-1 of 2).

Subsequent HMBC data facilitated the assignment of the remaining carbons (Table 1) with the exception of C-4c, C-5, C-7 and C-7a, which were interpreted by comparison with the literature [22,23,24,25]. Further long range correlations observed in the HMBC spectrum (Figure 2) provided definitive proof for the connectivity and thereby confirmed the structural assignment of 1 and 2 as 2-hydroxy-7-oxostaurosporine and 3-hydroxy-7-oxostaurosporine, respectively.

Staurosporines are a group of highly cytotoxic indole-carbazole alkaloids of which the parental molecule was isolated in 1977 from the fermentation broth of the soil actinomycete Streptomyces staurosporeus in a screening protocol directed to identify PKC (protein kinase C) inhibitors [26]. Currently, the staurosporines are a group of over 50 structurally related substances of natural and synthetic origins and have been found frequently in extracts from Eudistoma tunicates. 11-Hydroxystaurosporine and 3,11-dihydroxystaurosporine were isolated from Eudistoma sp. collected on a Micronesian island [27], while the staurosporine aglicone (K252-c) was obtained from another Eudistoma species found on the west coast of Africa [28]. Between 1999 and 2002, Schupp and co-workers published three articles covering the isolation and nM-range cytotoxicity of five new (3-hydroxy-4′-N-methylstaurosporine, 3-hydroxy-4′-N-demethylstaurosporine, 3′-demethoxy-3′-hydroxy-4′-N-demethylestaurosporine, 3-hydroxy-3′-demethoxy-3′-hydroxystaurosporine and 11-hydroxy-4′-N-demethylstaurosporine) and seven previously known staurosporines found in the Micronesian ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. [24,29,30].

The mixture of compounds 1–2 presented strong cytotoxic activity against a panel of seven human tumor cell lines (HL-60, Molt-4, Jurkat, K562, HCT-8, SF-295 and MDA-MB-435) and normal proliferating lymphocytes (PBMC), with an IC₅₀ ranging from 10.33 nM in Jurkat leukemia cells to 687.08 nM in normal PBMC cells (Table 2). Standard staurosporine (3), used as a positive control, was generally less potent than compounds 1–2 against tumor cells. Against normal proliferating lymphocytes, both 3 and 1–2 presented similar activity, suggesting a better selectivity index for 1–2 (Table 2). For example, 1–2 was 26.46 times more active against HL-60 cells than against PBMC, whereas STP was 2.00 times more active against HL-60 cells when compared with normal cells.

Structure-activity relationship studies involving staurosporine and structurally related compounds have shown that slight changes in structure lead to drastic changes in functionality or, essentially, selectivity towards a target [31,32,33,34]. A preliminary investigation on the mode of action of compounds 1 and 2 was conducted using HL-60 leukemia cells as a model. The mixture of 1 and 2 (80 nM) induced a notable and sustained cytostatic effect against HL-60 cells throughout the 72 h analysis. This observation was accompanied by an increase in the accumulation of cells in G2/M and a decrease of those in G0/G1 and S phases. The toxicity of STP (430 nM), however, was kinetically less consistent. Moreover, after 24 h, almost the entire STP-treated culture was arrested in G2/M, which was followed by an increase in polyploid cells at further time points, showing a less strict blockage. When the concentration of the mixture was increased to 160 nM, extensive DNA damage occurred, leading to cellular apoptosis (results not shown). However, in order to access the contribution of each isomer in the observed antiproliferative activity, it will be necessary to isolate the compounds and to access the mode of action of each one individually.

In the present study, two new 7-oxostaurosporine derivatives were obtained from the Brazilian endemic tunicate Eudistoma vannamei. The mixture of these compounds presented a strong antiproliferative effect against tumor cell lines, inducing a distinguished and persistent G2 arrest in sub-toxic concentrations, while at toxic concentrations, the treated cells underwent apoptosis.