Novel Adociaquinone Derivatives from the Indonesian Sponge Xestospongia sp.

Seven new adociaquinone derivatives, xestoadociaquinones A (1a), B (1b), 14-carboxy-xestoquinol sulfate (2) and xestoadociaminals A–D (3a, 3c, 4a, 4c), together with seven known compounds (5–11) were isolated from an Indonesian marine sponge Xestospongia sp. Their structures were elucidated by extensive 1D and 2D NMR and mass spectrometric data. All the compounds were evaluated for their potential inhibitory activity against eight different protein kinases involved in cell proliferation, cancer, diabetes and neurodegenerative disorders as well as for their antioxidant and antibacterial activities.


Introduction
Marine sponges of the genus Xestospongia have proved to be an extremely rich source of secondary metabolites with unprecedented molecular structures and various bioactivities [1][2][3]. Adocia- [4], halena- [5] and xesto-quinone [6] are the three main quinone-type skeletons identified from sponges of the genus Xestospongia sp. Among the most significant compounds, adociaquinones A and B, first isolated from the sponge Adocia sp. and then from the Philippine sponge Xestospongia sp. revealed inhibition of topoisomerase II in catalytic DNA unwinding and decatenation assays as well as inhibition of enzyme in the potassium sodium dodecyl sulfate KSDS assay [7]. Previous investigations on the South Pacific Xestospongia sp. by our group led to the isolation of a series of halenaquinone-type compounds, including xestosaprol C methylacetal, 3-ketoadociaquinones A and B, tetrahydrohalenaquinones A and B, halenaquinol sulfate, halenaquinone and orhalquinone [8]. Orhalquinone demonstrated significant inhibitory activities against both human and yeast farnesyltransferase enzymes, with IC50 values of 0.4 µM. In our continuing search for bioactive compounds from marine sponges, we have chemically investigated the Indonesian sponge of Xestospongia sp. collected off North Sulawesi, the methanolic crude extract of which showed kinase inhibition, antimicrobial and antioxidant activities. Bio-guided fractionation of the extract led to the isolation of seven new adociaquinone derivatives 1a-4c, together with seven known compounds, adociaquinone A 5 and B 6 [4], secoadociaquinones A 7 and B 8, 15-chloro-14-hydroxyxestoquinone 9, 14-chloro-15-hydroxyxestoquinone 10 [7] and xestoquinol sulfate 11 [9] (Figure 1). The known compounds were identified by comparison of their spectroscopic data with those of the literature. In this article, we describe the isolation and structural elucidation of the new compounds as well as their biological activities.
In addition, correlations from the protons at δH 7.80 (H-18) and 3.84 (H2-21) to the carbon δC 173.0 (C-16) were observed in the HMBC spectrum of compound 1a, while correlations from the protons at δH 8.37 (H-11) and 3.84 (H2-21) to the carbon δC 172.8 (C-13) were found in compound 1b. Thus, the structures of compounds 1a and 1b were determined as presented in Figure 1 and named as xestoadociaquinone A (1a) and xestoadociaquinone B (1b).   Table 1) showed similarities with those of the xestoquinol skeleton of compound 11. The major difference between compounds 2 and 11 [8] was the presence in the 13 C NMR spectrum of one carboxyl carbon signal at δC 171.8. The position of a carboxyl group was determined thanks to the HMBC correlations between the proton at δH 7.75 (H-15) and the carbons at δC 132.2 (C-17), 162.1(C-13), and 171.8 (C-21) ( Figure 3). Another major difference is the lack of the ortho-coupled protons system between H14-H15, replaced by the singlet due to H15. Therefore, the structure of 2 was determined as being 14-carboxy-xestoquinol sulfate. The structure elucidation of xestoadociaminals A 3a and B 3c was performed on two fractions, each of which contained the two natural products, and their hemiaminal diastereomers, in differing proportions. Compound 3a was obtained as a yellowish solid after being recrystallized slowly from a methanolic solution of 3a and 3c. 1 H NMR rapidly established that the sample was comprised of 3a and 3c in a ratio of 1:0.14, in addition to two further minor compounds (hemiaminal diastereomers, see later) also being present, again in a relative ratio of 1:0.14 (see 1 H NMR spectrum in Supplementary Information). Particularly diagnostic of the presence of four compounds, were 1 H resonances at δH 7.84, 7.78, 7.86 and 7.81 (H-18, all singlets) and δH 8.66, 8.64, 8.65 and 8.62 (H-11, all singlets), with both sets of signals observed in relative ratios of 1:0.14:0.14:0.02, respectively. The molecular formula of 3a was assigned as C24H21NO7S based on its HRESIMS data (m/z 468.1100 [M + H] + ), indicating 17 degrees of unsaturation. The presence of hydroxyl and carbonyl functional groups was deduced from the bands at 3649, 1752, and 1717 cm −1 in the IR spectrum. The DEPT spectrum indicated 24 carbons, including one methyl, six methylenes, four methines, and thirteen quaternary carbons. The 1 H and 13 C NMR data (recorded in DMSO-d6, Table 1) of 3a showed some similarities with adociaquinone A [6,7]. In the HMBC spectrum, the correlations between the proton at δH 8.66 (H-11) and the carbons at δC 132.9 (C-17), 154.3 (C-19), 170.4 (C-9) and 173.6 (C-13), between the proton at δH 7.84 (H-18) and the carbons at δC 36.7 (C-6), 130.2 (C-12), 142.6 (C-10), as well as between the proton at δH 7.94 (H-1) and the carbons at δC 121.4 (C-2), 143.1 (C-8), and 147.3 (C-7), confirmed the partial fragment of adociaquinone skeleton [5,7]. The main differences were the lack of one carbonyl group and the presence of additional signals corresponding to one oxygenated quaternary carbon [δC 72.7 (C-16)], one oxymethine [δC 87.5 (C-23)/δH 5.61 (1H, m, H-23)] and one methylene [δC 44.1 (C-24)/δH 3.24 (2H, m, H-24)] signal in the 1 H and 13 C NMR spectra of 3a. From the 1 H-1 H COSY spectrum, we observed the correlations between the protons at δH 2.83-2.59 (H2-3) and the protons at δH 2.20-2.05 (H2-4), which in turn gave correlations with the protons at δH 2.67, 1.56 (H2-5), and between the proton at δH 3.93-3.90 (H2-22) and the protons at δH 3.38, 3.34 (H2-21), and particularly between the proton at δH 5.61 (H-23) and the protons at δH 3.24-2.12 (H2-24) and the exchangeable proton at δH 6.96 (OH-23). The presence of a pyrrolidine ring in compound 3a was deduced by HMBC correlations between the proton at δH 3.24-2.12 (H2-24) and the carbons at δC 72.7 (C-16), 87.5 (C-23), and 164.2 (C-15), and between the proton at δH 5.61 (H-23) and the carbon at δC 44.1 (C-24). Furthermore, the HMBC correlations observed from the exchangeable proton at δH  and the proton at δH 7.84 (H-18) to the carbon at δC 72.7 (C-16), and from the other exchangeable proton at δH  to the carbon at δC 87.5 (C-23) (Figure 4) confirmed the presence of two hydroxyl groups at positions 16 and 23. Therefore, it was concluded that the structure of 3a contained a pyrrolidine ring fused between the dioxothiazine and quinone rings of adociaquinone A. With two stereogenic centres being present in this new ring system (at C-16 and C-23), in addition to the chiral centre at C-6, the four compounds in the sample being studied were ascribed to being pairs of hemiaminal diastereomers (each present in a 1:0.14 relative ratio) associated with each of the two possible C-16 stereoisomers. Observation of a NOESY correlation between the hemiaminal proton at δH 5.61 (H-23) and the hydroxyl proton at δH 6.78 (OH-16), indicated the relative disposition between these two protons in the dominant diastereomer present in the sample, however a lack of other correlations prevented determination of configuration relative to the stereocentre at C-6. While many of the 1 H and 13 C resonances associated with the hemiaminal diastereomer of 3a (3b) were co-incident with those of 3a, discernible signals attributable to 3b were observed at δH 8.64/δC 124.9 (CH-11), δH 7.78/δC 123.3 (CH-18), δH 5.40/δC 88.3 (CH-23) and δC 74.7 (C-16). With NMR resonances assigned for each of 3a/3b, attention then turned to a second column fraction that also contained the same four sets of NMR signals. Integration of the 1 H NMR spectrum of this second fraction indicated it to be enriched in a second compound 3c, also present with its hemiaminal diastereomer 3d, with relative ratios of 1:0.15:1:0.15 (3a:3b:3c:3d). Differences between 1 H NMR data observed for 3c compared to 3a were associated with the resonance of the exchangeable proton at OH-16 (δH 6.78 for 3a and δH 6.88 for 3c), H-11 (8.66 vs. 8.65), H-18 (7.84 vs. 7.86) while 13 C NMR shift differences were observed for C-16 (72.7 vs. 72.6), C-23 (87.45 vs. 87.40), and C-18 (123.1 vs. 123.3). It was thus concluded that compound 3c was likely the C-16 epimer of 3a. It was named xestoadociaminal B. The fourth very minor constituent of the fraction mixtures was then presumed to be the hemiaminal diastereomer of 3c (Figure 4). Compound 4 was obtained as a 1:1:0.29:0.29 mixture of four diastereoisomers (see 1 H NMR spectrum in supplementary data), isolated as a yellowish amorphous solid. The molecular formula was also assigned as C24H21NO7S, being isomeric with 3a-d. The 1 H and 13 C NMR data (recorded in DMSO-d6, Table 1) were also similar to those observed for compounds 3a-d. The partial fragment of an adociaquinone skeleton was confirmed by the HMBC correlations between the proton at δH 8.19 (H-11) and the carbons at δC 133.9 (C-17), 150.9 (C-19) and 170.3 (C-9), between the proton at δH 8.18 (H-18) and the carbons at δC 36.5 (C-6), 135.2 (C-10), 137.8 (C-12) and 173. 5 (C-16), and between the proton at δH 7.95 (H-1) and the carbons at δC 122.8 (C-2), 143.3 (C-8) and 147.8 (C-7). However, in contrast to the case of compound 3a, the HMBC spectrum also showed key correlations between the proton at δH 8.18 (H-18) and the carbon at δC 173. 5 (C-16), and between the proton at δH 8.19 (H-11) and the carbon at δC 72.4 (C-13), between the exchangeable proton at δH  and the carbons at δC 44.1 (C-24), 72.4 (C-13), and 137. 8 (C-12) and between the other exchangeable proton at δH  to the carbons at δC 44.1 (C-24) and 87.5 (C-23) ( Figure 5). These correlations again identified the presence of a pyrrolidine ring fused between the dioxothiazine and quinone rings of an adociaquinone-type molecule, but in contrast to ring fusion to C-16 in 3a-d, fusion in compound 4 was at C-13. Therefore, it was concluded that the fraction contained all four diastereomers represented by structures 4a-d with the two major components 4a and 4c being C-13 epimers and the minor components 4b and 4d being their corresponding hemiaminal diastereomers. The major components were named xestoadociaminals C and D; their relative configurations remain unresolved due to overlapping signals. Biogenetically, the structures of 3a-d and 4a-d represent the addition of ethanal to adociaquinones A and B. However, it should be noted that ethanol was not used for the storage of the sponge nor in any chromatographic purification steps. All compounds were tested against eight different protein kinases relevant to cell proliferation, cancer, diabetes and neurodegenerative disorders along with the related compounds 12 and 13 (Figure 6), previously isolated from the marine sponge Xestospongia sp., [8] in order to establish structure-activity relationships. Compounds 6 and 8 revealed a modest but selective inhibitory activity towards CDK9/cyclin T (IC50: 3 µM) and CDK5/p25 (IC50: 6 µM), respectively. Compound 12, which is a sodium derivative and differs from 11 by the presence of the ketone group in the position 3, showed significant activity against most protein kinases ranging from 0.5 to 7.5 µM (Table 2), while compound 13, with a hydroxyl in position C-1 and a methoxyl at C-8, showed marginal activity against DYRK1A. This information suggests that the presence of a ketone group in position 3 and eventually a non-substituted furan ring are important for the activity. Therefore, adociaquinone derivatives could be of interest in the discovery of new potential kinase inhibitors.
All the compounds were also tested for potential antioxidant and antibacterial activities. Only compound 11 showed moderate antibacterial activity with an IC50 value of 125 μM against Staphylococcus aureus.

Sponge Material
Specimens of Xestospongia sp. were collected in the North Sulawesi (Bunaken and other islands/reefs near Manado), Indonesia and were identified by Professor Van Soest, University of Amsterdam, the Netherlands.

Extraction and Isolation
Sponge specimens (500 g) were immediately immersed in MeOH after collection. The MeOH solution was evaporated and the aqueous residue was extracted and partitioned first with 1 L CH2Cl2 to give the CH2Cl2 extract (6.5 g). The aqueous phase was extracted with 1 L EtOAc and partitioned to afford to the EtOAc extract (1.7 g). Finally, the aqueous residue was extracted with 1 L BuOH and partitioned to obtain the BuOH extract (8.8 g).

Antibacterial Assay
The antibacterial activity assay was conducted on the bacterial strain Gram-positive Staphylococcus aureus ATCC 6538 and Gram-negative Escherichia coli ATCC 8739 evaluated in vitro by determining the IC50. A pre-culture of 5 mL LB (Luria Bertoni) medium was prepared by inoculating a colony of the bacterial strain and was incubated at 37 °C with stirring overnight. The concentration of the pre-culture was assessed by measuring the optical density OD at 620 nm and adjusted by dilution to obtain a suspension of 0.03 OD. The IC90 was determined by a liquid test in 96-well-plates. A quantity of 200 µL of the bacterial suspension was distributed in each well and 10 µL of the extracts, fractions or pure compounds solutions in DMSO (10, 5 and 2 mg/mL, respectively) were added in triplicate. The 96 well-plates were incubated at 30 °C for 16 to 18 h with shaking (450 rpm). The optical density of the wells was measured at 620 nm and the results were interpreted by calculating the percentage of growth inhibition in each well using the formula: % inhibition = 100 − (DOS − DOB)/(DOT − DOB) ×100 where T = bacterial suspension without test sample, B = culture medium without bacteria and S = bacterial suspension test sample. Ampicillin and chloramphenicol were used as positive control against S. aureus and E. coli, respectively.

Antioxidant Assay: Qualitative DPPH Screening
The potential antioxidant activity of sponge crude extract and fractions from different chromatography procedures was evaluated using the scavenging activity of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals. Active fractions were visualized by spraying a purple DPPH solution (2 mg/mL in MeOH) on a TLC, where extracts or fractions have been deposited. Immediate discoloration of DPPH around tested samples reveals their antioxidant activity. The well-known antioxidant ascorbic acid was used as positive control.