Cytotoxic Aaptamines from Malaysian Aaptos aaptos

In a preliminary screen, Aaptos aaptos showed significant cytotoxic activity towards a panel of cell lines and was thus subjected to bioassay-guided isolation of the bioactive constituents. In addition to the known aaptamine, two new derivatives of the alkaloid were isolated from the bioactive chloroform fraction of the crude methanolic extract. Detailed analysis by NMR and mass spectroscopy enabled their identification to be 3-(phenethylamino)demethyl(oxy)aaptamine and 3-(isopentylamino)demethyl(oxy) aaptamine. The cytotoxic activities of the three alkaloids were further evaluated against CEM-SS cells.


Introduction
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][2][3][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 OPEN ACCESS 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.

Results and Discussion
The methanolic extract of Aaptos aaptos was cytotoxic against all the cell lines tested, with CD 50 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 C 20 H 17 N 3 O 2 (calc. 332.1321). In the 1 H 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 1 H 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 13 C 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 1 H-1 H 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/MS n 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 C 7 H 7 + . 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 1 H 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-CH 2 -CH 2 -which was also evident from the    [1,6]naphthyridine moiety, as well as the -NH-CH 2 -CH 2 -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 2 J and 3 J HMBC correlations between H 2 -3' and H 2 -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-CH 2 -CH 2spin system. Again, a 3 J correlation observed from H 2 -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.
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-9one (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][16][17] suggested that hydroxylation at C-9 is important for cytotoxicity, and parasubstituted 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.

General Exprimental Procedures
UV and IR spectra were recorded on CARY 100 Conc UV-Vis (Varian) and Perkin-Elmer RXI FTIR spectrometers, respectively. Mass spectra were recorded on Polaris Q Mass Spectrometers (Thermo Finnigan San Jose CA), with ionization being induced by electron impact at 70eV. HRESIMS were measured using Finnigan MAT95XL-T spectrometers. LCMS/MS n were performed on a ThermoFinnigan model LCQ Deca (San Jose, CA). 1

Animal material
Aaptos aaptos was collected from the coastal waters of Terengganu, on the eastern part of Peninsular Malaysia. A specimen (registry No. P03.015) has been deposited at the Department of Biological Science, Faculty of Science and Technology, Universiti Malaysia Terengganu.

Extraction and isolation
Fresh samples of Aaptos aaptos (250 g) were cut and macerated in a high-speed blender with methanol at room temperature. The methanolic extract (5.5 g) was filtered, evaporated in vacuo, and lyophilized before subjected to cytotoxity assay [18] against a panel of cancer cell lines. The crude methanolic extract was further partitioned into n-hexane (1 g), chloroform (0.6 g), ethyl acetate (1.1 g) and aqueous (2.3 g) fractions. All the fractions were assayed against CEM-SS, where the cytotoxity (CD 50 =2.4 μg/ml) was found to be concentrated in the chloroform fraction. Further isolation was thus focused on this bioactive fraction. The extract was chromatographed on a silica gel column eluting with chloroform with increasing amounts of MeOH as eluent. The fraction eluted with 20% MeOH yielded large amounts of aaptamine (50 mg). The fraction eluted with 10% MeOH was further subjected to silica gel column, eluted with dichloromethane with increasing amounts of acetone. The subfraction eluted with 30% acetone was further subjected to reversed-phase HPLC using MeOH/H 2 O 7:3 as eluent (isocratic, flow rate 3 ml/min, wavelength 366 nm) to yield 1.0 mg each of compound 1 and 2.

Evaluation of Cytotoxicity
Cytotoxic activity was measured against a panel of cell lines as described previously [18].