Numerous marine organisms are known to produce secondary metabolites as defense mechanisms [1–4] which ultimately prove to have other interesting biological activities [5–7]. Many of these compounds are brightly coloured. Crinoids are known producers of brightly coloured, typically orange red pigments that are predominantly anthraquinoid in nature. The NMR data for these compounds are difficult to assign and as a result are often incorrectly assigned [8–10]. NMR techniques that are not routinely used need to be applied to resolve assignment issues for this class of compounds [11]. During a screening campaign to identify marine extracts with whole cell anticancer activity, a dichloromethane/methanol (DCM/MeOH) extract of a specimen of the crinoid Colobometra perspinosa, collected from south east of Richards Island (Bedara), Family Islands, Central Great Barrier Reef, Australia, was found to be active. Subsequent bioassay and ¹H-NMR guided fractionation of the MeOH soluble fraction led to the isolation of compounds 1, 3, 4 and 5. Outlined in this publication are details of the structure elucidation of 1, 3, 4 and 5, as well as a detailed discussion of the ¹³C-NMR data associated with 1 and 3, together with their cytotoxicity, as assessed in a series of anticancer cell based assays.

Rhodoptilometrin (1) was isolated as an orange-yellow microcrystalline solid with a molecular weight corresponding to the molecular formula C₁₇H₁₄O₆; the molecule had eleven double bond equivalents of unsaturation. Analysis of the NMR data for 1 (Table 1) showed it to have 6 × C=C, and 2 × C=O as the only multiple bonds within the molecule, so 1 was tricyclic. Other structural features of the molecule, gleaned from its spectroscopic data, were the presence of two pairs of meta-coupled protons and a 1-hydroxypropyl moiety. These deductions indicated 1 to be a tetra-substituted anthraquinone, where the substituents were three hydroxyl groups and a 1-hydroxypropyl moiety. From ¹³C- and ¹H-¹³C HMBC data (see Table 1) it was possible to establish the substitution pattern of the anthraquinone as shown in Figure 1. Comparison of the NMR data in Table 1 for 1 with those reported for rhodoptilometrin (see Table 1 and Table 2) [8–10], raised doubts about the structure, in particular the ¹³C-NMR resonances for C-4, 5, 6, 8a and 9a, associated with rings A and C of 1, and the ¹H-NMR data for H₃-3′. If the reported structure of 1 was incorrect, the only other structure that fits all of the available data is the regioisomer 2. In an attempt to resolve this regio-chemical issue and provide reliable NMR data, a 1,1-ADEQUATE spectrum [11] (see Figure 2 and Table 1), as well as ¹³C-NMR spectra at various concentrations (see Table 2), were recorded, and all of the NMR data re-examined. There was a slight influence on ¹³C-NMR chemical shifts with changing concentration (see Table 2), however, nothing to the extent that would account for the differences seen between resonances associated with C-4, 5, 6, 8a and 9a reported in the literature [10] and current values. The 1,1-ADEQUATE cross-peaks observed for 1 between H-4 and C-3, 4, and 4a, between H-2 and C-1 and 3, and between H-1′ and C-2′ and 3, (see Figure 2 and Table 1), coupled with the HMBC cross-peaks observed between H-4 and C-1, 2, 9 (weak), 9a and 10, defined ring C, the 1-hydroxypropyl moiety, and C-10, as shown in 1. Also 1,1-ADEQUATE cross-peaks were observed between H-5 and C-6 and 10a, and between H-7 and C-6, together with HMBC cross-peaks observed between H-5 and C-6, 7, 8, 8a, 9 (weak) and 10, and between H-7 and C-5, 6, 8, 8a and 9 (weak). This defined ring A and C-9, thus completing ring B of the molecule and confirming the planar structure. Based on this analysis, the compound isolated in this study was assigned as 1. The specific rotation of 1 measured at 589 nm was -22, which is in agreement with that reported for rhodoptilometrin by Lee at al. [10]. Careful consideration of the C-4, 5, 6, 8a and 9a ¹³C-NMR chemical shift differences observed between our 1 and that reported by Lee et al. [10], highlighted that, with the exception of C-6, these carbons would possibly be influenced by tautomeric effects. This raised the issue of pH and whether the compounds were in the same form prior to NMR analysis. In our case, all samples were acid and base free prior to measurement as were NMR solvents used for the measurement. Adding formic acid or sodium hydroxide to our NMR sample in CD₃OD did not alter the chemical shifts of these carbons. Compound 1 was also isolated from a more recent collection of Colobometra perspinosa collected from Coombe Island in the Family Islands, Central Great Barrier Reef, Australia in 2005. It seems likely that the compound isolated and characterized by Lee et al., [10] probably is rhodoptilometrin (1), but in the absence of more details concerning isolation and measurement conditions (e.g., temperature, concentration and pH) this cannot be stated with certainty.

Together with rhodoptilometrin (1), 3-propyl-1,6,8-trihydroxy-9,10-anthraquinone (3), or dehydroxyrhodoptilometrin, was also isolated and characterized spectroscopically. From comparison of the ¹H- and ¹³C-NMR data for 1 with that obtained for 3, together with the accurate mass data, it was evident that 3 was also a tetra-substituted anthraquinone with three hydroxyl groups and an aliphatic moiety at C-3. The differences in the ¹H- and ¹³C-NMR chemical shifts of the aliphatic substituent between 1 and 3 clearly revealed the presence of a propyl moiety at C-3. For 3, complete assignment of ¹H- and ¹³C-NMR data are provided for the first time; see Experimental Section and Table 1.

The conjugate of phenylacetic acid and taurine, phenylacetyltaurine, or 2-[(phenylacetyl)amino]ethanesulfonic acid (4) was also isolated from the MeOH extract. The structural elucidation of this molecule was relatively straightforward once the elemental composition was determined from accurate mass measurement (C₁₀H₁₂NO₄S). It was possible to deduce two molecular fragments from the ¹H- and ¹³C-NMR data of 4 (see Experimental Section); the phenylacetic acid (amide) moiety, an ethyl chain with a terminal nitrogen at C-2, and a heteroatom, unlikely to be oxygen or nitrogen, at C-1. With the accurate MS measurement in hand it was evident that the group at C-1 was a sulfonic acid and that 4 was the conjugate from the reaction between phenylacetic acid and taurine. Surprisingly, this appears to be the first report of this entity as a natural product from the marine environment, even though it is encountered fairly often in metabolic studies involving the use of phenylacetic acid [12,13].

The final compound to be identified in this study was 4-hydroxybutanoic acid or gamma-hydroxybutyric acid (GHB, 5). The structure elucidation of this molecule was routine. From the ¹H- and ¹³C-NMR data of 5 (see Experimental Section), it was evident that the molecule was composed of a butyl chain terminating at one end with an OH (C-4, δ_C 68.5 ppm); and at the other with a carboxylic acid or amide (C-1, δ_C 177.8 ppm); these deductions were confirmed by routine low resolution MS of 5, and showed it to be the free acid and not the amide. The discovery of 5 in a marine organism is in itself not surprising, as the molecule is a simple one and commonly found in low concentrations in mammals [14]. What does make the discovery interesting is the colourful history associated with 5. It is a naturally occurring substance, probably synthesized from gamma-aminobutyric acid, found in the central nervous system (CNS) of mammals and thought to act as a neurotransmitter. It is also found in other organs such as the liver, kidneys, and heart as well as in bones. As a drug it is most commonly used in salt form [sodium gamma-hydroxybutyrate (Na-GHB, sodium oxybate) or potassium gamma-hydroxybutyrate (K-GHB)], and is used in the treatment of insomnia, depression [15], alcoholism [16], historically as an anesthetic, and for sleep disorders [17]. Its real notoriety comes from it being represented as a date rape drug, often being referred to as liquid ecstasy despite its unrelated effects [18].

Compounds 1, 3–5 were screened for their in vitro anti-cancer activity against three human tumour cell lines [MCF-7 (breast-pleural effusion adenocarcinoma), SF-268 (CNS-glioblastoma), and H460 (lung-large cell carcinoma)], the results of which are shown in Table 3. From the bioassay data it can be seen that 1, 3 and 5 all demonstrate some non-selective activity towards the three cell lines used for the testing, and that functionality in the side chain of 1, compared to 3, appears not to influence this activity. The activity observed for 3 is consistent with previous findings for its sulfate derivative [10]. Compound 4 appears to be weakly active in only one of the three cell lines (MCF-7).