Marine sponges have received a lot of research attention in the last decade and have been shown to be a prolific source of novel chemicals with promising therapeutic potentials [1–4]. The genus Aaptos in particular have yielded a group of 1H-benzo[d,e][1,6]-naphthyridine alkaloids, known collectively as aaptamines. Aaptamine [5,6] the first isolated alkaloid of this series, is a useful chemotaxonomic marker for sponges of the order Hadromerida [7] although it has also been found to occur in a sponge of the order Haplosclerida [8]. Several members of this class of compounds have been reported to show antitumour properties [9,10], antiHIV [11], mycobacterial [11], cardiac activity [12], and more recently, sortase A inhibitory [13] and antidepressant activities [14].

In our search for pharmacologically active substances from marine organisms, we screened a number of marine sponges for cytotoxic activity against a panel of cell lines consisting of HL-60 (promyelocytic leukemia), CEM-SS (T-lymphoblastic leukemia), MCF-7 (breast cancer), HeLa (cervical cancer), HT-29 (colon cancer) and L929 (murine fibrosarcoma from mouse). In our screen, the methanolic extract of Aaptos aaptos showed significant cytotoxic activity towards all the cell lines. Bioassay guided fractionation of the extract led us to the isolation of two new 9H-benzo[d,e][1,6]-naphthyridine alkaloids, 1 and 2, in addition to the known aaptamine (3). Structural elucidation of these alkaloids and their cytotoxic activity against CEM-SS cell line are described herein.

The methanolic extract of Aaptos aaptos was cytotoxic against all the cell lines tested, with CD₅₀ values ranging from 3.2 to 24.1 μg/ml (Table 1). Bioassay guided solvent fractionation of the crude methanolic extract of A. aaptos retained a maximum of activity in the chloroform soluble fraction. Chromatographic separation of the active fraction using gel filtration, normal phase chromatography and semi-preparative reverse phase HPLC, yielded small amounts of the two new aaptaminoids, 1 and 2, in addition to aaptamine [5], 3, as the major constituent. The structures of the two alkaloids were elucidated based on NMR and mass spectroscopic methods.

The ESI-MS (positive ion mode) of 1 exhibited [M+Na]⁺ and [M+H]⁺ pseudomolecular ion peaks at m/z 354.20 and 332.07, respectively. The molecular formula was determined by HR-ESIMS data (m/z 332.1291), to be C₂₀H₁₇N₃O₂ (calc. 332.1321). In the ¹H NMR spectra, the signals observed at δ_H 8.76 (d, J 4.5), 7.47 (d, J 4.5), 6.63 (s) and 8.42 (s) were characteristic of the coupled protons H-5 and H-6, the lone proton H-7, and H-2 of the aaptamines [8], suggesting a benzo[de][1,6]naphthyridine skeleton. Unlike the other aaptaminoids which have been thus far reported, H-2 appeared as a singlet, thus indicating that C-3 was substituted. The ¹H NMR spectra also exhibited a signal for only one methoxyl group, observed at δ 3.99 (s), which could be assigned to either position 8 or 9. However, the ¹³C NMR also exhibited a carbonyl signal at δ_c 176.3 which is consistent with the characteristic C-9 carbonyl observed in demethyl(oxy)aaptamine (4), previously isolated from the Okinawan Aaptos aaptos [6]. Careful analysis of the ¹H-¹H COSY, HSQC and HMBC correlations, as summarized in Table 2 further confirmed the placement of the carbonyl carbon on position 9 and substitution on C-3.

The remaining aromatic protons at δ_H 7.39 (t, 2H) and 7.31 (d, 3H, J 7.5) were those of a monosubstituted benzene ring, which must thus be part of the substituent on C-3. This was supported by LC-MS/MSⁿ experiments, where the MS/MS fragmentation of both the [M+Na]⁺ and the [M+H]⁺ ions, respectively, gave daughter ions at m/z 263 and 241 for the loss of 91 amu, indicative of the loss of a tropilium ion C₇H₇+. Another spin system, made up of a deshielded 1H broad triplet at δ 7.03, a 2H quartet at δ 3.85 and a 2H triplet at δ 3.15, was also evident from the ¹H NMR spectra. The latter two signals were due to a pair of methylene groups, adjacent to each other and directly attached to the carbons at δ_c 44.3 and 35.5, respectively, based on an HSQC experiment. However, the 1H triplet at δ 7.03 was not directly attached to any carbon and based on its highly deshielded nature, the proton was deduced to be an NH proton, giving the spin system –NH-CH₂-CH₂- which was also evident from the ¹H-¹H COSY spectrum. From the COSY correlations the protons could be assigned to H-1’ (δ 7.03) correlated to H₂-2’ (δ 3.85) which was in turn correlated to H₂-3’ (δ 3.15). Careful analysis of the correlations observed in the HSQC and HMBC spectra connected the ethylamino spin system to the monosubstituted benzene ring. A ³J correlation from H₂-2’ to the quaternary carbon at δ_c 144.1 (C-3) connected the 2-phenethylamino substituent to the main benzo[de][1,6]naphthyridine moiety. Based on these data, the structure of compound 1 was assigned as 3-(phenethylamino)demethyl(oxy)aaptamine.

The ESI-MS (positive ion mode) of 2 exhibited an [M+Na]⁺ and [M+H]⁺ pseudomolecular ion peaks at m/z 320.13 and 298.20, respectively. Again, HRESIMS (m/z 298.1445) gave the molecular formula C₁₇H₁₉N₃O₂ (calc. 298.1477) for 2. Meanwhile, the 1H NMR spectra of 2, closely resembled that of 1, showing the same C-3 substituted benzo[de][1,6]naphthyridine moiety, as well as the –NH-CH₂-CH₂- spin system of the C-3 substituent (Table 2). The difference was the absence of the phenyl protons of the C-3 substituent, which were replaced by an isopropyl group as deduced from the two equivalent methyl doublets at δ 1.05 (6H, J = 7.0 Hz, δ_c 22.6) and a methine proton at δ 1.87. The ²J and ³J HMBC correlations between H₂-3’ and H₂-2’, respectively, to this methine carbon (δ_c 26.0), assigned as C-4’, were the key correlations that connected the isopropyl group to the –NH-CH₂-CH₂- spin system. Again, a ³J correlation observed from H₂-2’ to the quaternary carbon C-3 (δ_c 144.4) connected the isopentylamino substituent to the main benzo[de][1,6]naphthyridine moiety, leading us to assign 2 as 3-(isopentylamino)demethyl(oxy)aaptamine. All the three isolated alkaloids exhibited significant cytotoxic activity against CEM-SS cells with CD₅₀ values of 5.3 (1), 6.7 (2) and 15.0 (aaptamine) μg/ml, respectively.

We have isolated two new aaptamines, 8-methoxy-2-(phenethylamino)-9H-benzo- [de][1,6]naphthyridin-9-one (1) and 2-(isopentylamino)-8-methoxy-9H-benzo[de][1,6]naphthyridin-9-one (2) from the tropical marine sponge Aaptos aaptos. To the best of our knowledge, this is the first report of naturally occurring C-3 substituted aaptamines. SAR studies carried out on analogs of aaptamine and isoaaptamine on several cell lines including against murine P-388 lymphocytic leukemia [9, 15–17] suggested that hydroxylation at C-9 is important for cytotoxicity, and para-substituted phenyl substituents on one or both of the nitrogens are important for increased activity. It is interesting to note that in this study, the cytotoxicity of the C-3 substituted aaptamines, 1 and 2, on CEM-SS human T-lymphoblastic leukaemia cells were also observably higher than that shown by aaptamine. This suggests that C-3 substitution may also influence the cytotoxity of this class of compounds, towards this type of cell line.