Since the first reported isolation of a diketopiperazine (DKP) from the sponge Dysidea herbacea [1], there have been several reports describing the isolation of this class of compound from other marine sponges [2–4]. DKPs are also reported from marine microbial sources [5–8], including the proteobacteria Alcaligenes faecalis, isolated from the sponge Stelletta tenuis [9]. The metabolites reported in these investigations are mostly the products of 4-hydroxy-proline [2,5,6,8] or proline [7] reacting with phenylalanine [2,5], arginine [4], leucine [5–7], isoleucine [7], norvaline [3] or alanine [8].

Sponges from the genus Stelletta are known to produce a number of other bioactive classes of compounds, including but not limited to steroids [10], alkaloids [11,12], isomalabaricane triterpenes [13], acetylenic acids [14] and lysophosphatidylcholines [15]. Initial interest in the methanol (MeOH) extract of the sponge Stelletta sp. was motivated by to its potent activity in the NCI 60 cell line screen and a unique COMPARE analysis profile (average GI₅₀ 0.5 μg/mL) [16]. This profile was different to that of the standard chemotherapeutic agents paclitaxel, cisplatin, gemcitadine, bryostatin 1, didemnin B, tamoxifen and vinblastine (data provided by NCI). Subsequent bioassay-guided investigations of this extract led to the isolation of a new DKP cyclo-(4-S-hydroxy-R-proline- R-isoleucine) (1), the previously reported bengamides; A (2) [17], F (3) [18], N (4) [19], Y (5) [20], and the previously reported bengazoles; Z (6) [20], C₄ (7) [21] and C₆ (8) [21]. Described in this publication is the isolation and structure elucidation of 1, together with the activity of compounds 1–8.

The DKP cyclo-(4-S-hydroxy-R-proline-R-isoleucine) 1 was isolated and the molecular formula C₁₁H₁₈N₂O₃, corresponding to four double-bond equivalents, was determined by (+)-ESI-FTMS accurate mass measurement. The ¹³C NMR data of 1 contained resonances consistent with the presence of two amide carbonyl groups (δ_C 170.5 (C-7), 165.4 (C-1)) as the only multiple bonds within the molecule, and a hydroxy methine (δ_C 66.8 (C-4); δ_H 4.28, 1H, dd, J = 4.6, 4.6 Hz) (Table 1). These functionalities accounted for all of the oxygen and nitrogen atoms and all of the multiple bonds in 1, indicating the molecule to be bicyclic. Analysis of the COSY NMR data of 1 showed an extended ¹H–¹H spin system from H-9 to H₃-12 via H-10 and H-11, as well as a vicinal COSY NMR correlation from H-10 to H₃-13. Observed gHMBC NMR correlations from δ_H 7.97 to the ¹³C NMR resonances at δ_C 59.1 (C-9), δ_C 56.7 (C-6), δ_C 34.8 (C-10) and C-1 positioned this hydrogen at N-8. Further gHMBC NMR correlations from δ_H 4.00 (H-9) to δ_C 23.9 (C-11) and δ_C 14.9 (C-13), as well as to C-1 and C-10, clearly positioned H-9 adjacent to the C-1 carbonyl and N-8, giving rise to an isoleucine moiety (1A). Additional gHMBC NMR correlations from 8-NH and H-9 to C-7 revealed it was attached to N-8. A further contiguous ¹H–¹H spin system from H-6 to H₂-3, in addition to gHMBC NMR correlations from the 8-NH and H_b-3 to C-6, and from H_b-3 to C-1 established the remaining nitrogen (N-2) to be attached to C-1, C-6 and C-3, giving rise to the two rings within 1. The planar structure of 1 is as shown (Scheme 1).

The configuration at C-4, C-6 and C-9 of 1 was established through analysis of ¹H–¹H coupling constants, optical rotation measurement, molecular minimisations and comparison with literature compounds [3,6,7]. The magnitude of the coupling constants associated with H-6 (dd, J = 11.0, 6.1 Hz) and the observed COSY NMR correlations between H-6 and H_a/b-5 established it to have a pseudo-axial orientation, similar to that of cyclo[l-(4-hydroxyprolinyl)-l-leucine)] (H-6, dd, J = 11.1, 6.1 Hz) [6]. An apparent zero coupling between H-4 (J = 4.6, 4.6 Hz) and H_b-3 or H_a-5 as evident by lack of observed COSY NMR correlations, and observed couplings to H_a-3 (J = 12.5, 4.6 Hz) and H_b-5 (J = 12.9, 11.0, 4.6 Hz), was indicative of H-4 being orientated at approximately 90° to both H_b-3 and H_a-5. The observed weak COSY NMR correlation between 8-NH and H-9, and the broad singlet for H-9 (similar to that observed in cyclo-[S-proline-S-isoleucine)] [3]), revealed H-9 to be axial. Molecular modelling studies showed that the observed coupling constants were in agreement with either R,R (Figure 1) or S,S configuration at C-6/C-9 but definitely not R,S or S,R (Supplementary Data S6-S13 and Table S1). Based on optical rotation trends of DKPs from the literature [3,7], the overall positive [α]²¹_D = +12° indicated the absolute configuration at C-6 should be R, therefore supporting the R,R configuration. The magnitude of the optical rotation is also in agreement with other C-4 hydroxylated DKPs [5,7]. The molecular model shown in Figure 1, with calculated dihedral angles for H_a-5–C-5–C-6–H-6 (Φ = 41°), H_b-5–C-5–C-6–H-6 (Φ = 163°), H-4–C-4–C-5–H_a-5 (Φ = 79°), H-4–C-4–C-5–H_b-5 (Φ = −42°), H_a-3–C-3–C-4–H-4 (Φ = 29°), H_b-3–C-3–C-4–H-4 (Φ = −93°) and 8-NH–N-8–C-9–H-9 (Φ = 91°), best explained the observed COSY NMR correlations, ¹H–¹H coupling constants and the positive sign of [α]²¹_D confirmed the absolute configuration at C-3, C-6 and C-8 to be as shown. It is likely that this DKP was produced by an enzymatically controlled condensation reaction between d-isoleucine and 4-S-hydroxy-d-proline (Scheme 1) [22].

The cytotoxicity of 1–8 was investigated against the human tumour cell lines H460, SF-268, MCF-7, HT-29 and a normal mammalian cell line CHO-K1. The DKP 1 exhibited minimal activity towards MCF-7, H460 and HT-29 cells and no activity towards SF-268 or CHO-K1 cells at the highest dose (Table 2). In contrast, the GI₅₀ values (μM) for bengamides A (2), F (3), N (4), Y(5), and bengazoles Z (6), C₄ (7) and C₆ (8) were comparable to those reported in previous studies [19,20], and accounted for the activity observed in the original MeOH extract.

The DKP cyclo-(4-S-hydroxy-R-proline-R-isoleucine) (1), together with the known bengamides; A (2), F (3), N (4), Y (5), and bengazoles; Z (6), C₄ (7) and C₆ (8), was isolated from the Australian marine sponge Stelletta sp. Interestingly, this is the first report of bengamides or bengazoles from the genus Stelletta, however, it should be noted that they have previously been reported from species of Dorypleres splendens [24], which has since been reclassified as Jaspis splendens, and from Jaspis sp. [24], both of which belong to the Ancorinidae family of sponges. The cyclo-(4-S-hydroxy-R-proline-R-isoleucine) (1) was not cytotoxic against the cell lines MCF-7, H460, HT-29, SF-268 or CHO-K1. The DKP class of compounds has recently gained interest in drug discovery [25] due to their chiral, rigid and functionalised structures. These features enable them to bind to a large variety of receptors with high affinity giving rise to a broad range of biological activities, including cytotoxicity, quorum sensing, antibacterial, antifungal, antiviral, antiprion, antitumor, and immunosuppressive functions, even plant-growth regulators [7,26–28]. Our report adds to the vast knowledge of these potentially therapeutic molecules.