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Chemical Constituents of the Marine Sponge Aaptos aaptos (Schmidt, 1864) and Their Cytotoxic Activity

Seven compounds (1-7) were isolated from the marine sponge Aaptos aaptos living in the Vietnamese sea. Their structures were determined as 2 hours, 5H,7H,9H-9S-hydroxy-imidazo[1,5-α]pyridine-1,3-dione (1), 3-([9-methylhexadecyl]oxy)propane-1,2-diol 2, 2,3-dihydro-2,3-dioxoaaptamine (3), indol-3-aldehyde (4), methyl indole-3-carboxylate (5) 4-hydroxy-5-(indole-3-yl)−5-oxo-pentan-2-one (6), and thymidine (7) by extensive analysis of HR-ESI-MS, 1D, and 2D NMR spectral data, as well as by comparison of the spectral data with those reported in the literature. In addition, the absolute configuration of 1 was determined from the experimental ECD spectrum and comparison of this with the theoretical ECD calculations using the TDDFT method. Compounds 1 and 2 were isolated from nature for the first time. Compound 3 induced cytotoxic activity against SK-LU-1, MCF-7, HepG2, and SK-Mel-2 cell lines with IC50 values of 41.27 ± 2.63, 40.70 ± 2.65, 34.31 ± 3.43, and 36.63 ± 1.40 µM, respectively.


Results and Discussion
As described in the Materials and Methods, after checking by TLC each fraction of the methanol extract of Aaptos aaptos, and using various chromatograph methods, compounds 1-7 were obtained. Compound 1 was isolated as a light yellow solid and the presence of OH and HN groups (3235.7 cm -1 , broad band) and C = O groups (1771.7 and 1724.0 cm -1 ) were indicated by its IR spectrum (Supplemental Figure S6b). Its molecular formula was deduced to be C 7 H 10 Figure S3) exhibited seven carbon signals, including four methylenes (δ C 37.2, 25.8, 19.7, and 32.8), two carbonyl groups (δ C 177.7 and 155.5), and one quaternary carbon bearing an oxygen atom at δ C 84.2. From the above evidence, with 4 degrees of unsaturation, compound 1 was suggested to have two rings. Analysis of the chemical shift values, the proton couplings, and HSQC and HMBC correlations from positions C-5 to C-9 suggested that carbon C-9 (δ C 84.2) linked to C-8 (δ C 32.8), the nitrogen atom linked to C-5 (δ C 37.2), and one ring was formed from C-9 to C-5 through a nitrogen atom (Supplemental Figure S4 and S5). Obviously, the other ring must be formed between two carbonyl carbons through NH group. From the HR-ESI-MS and NMR results, the chemical structure of 1 was suggested to form as a bicyclic skeleton, 2 hours, 5H,7H,9H-9-hydroxy-imidazo [1,5-α] pyridine-1,3-dione, as shown in Figure 1. These suggestions were further confirmed by the HMBC correlations. The HMBC interactions from H-5 (3.04/3.92) to carbons C-9 (84.2), C-6 (25.8), C-7 (19.7), and C-3 (155.5), as well as from H-8 (1.50/2.06) to C-1 (177.7), C-9 (84.2), and C-6 (25.8) confirmed that C-5 and C-9 were connected through a nitrogen atom, and the second ring was formed from C-1 to C-3 through the other nitrogen atom indicated from its molecular formula. As reported in previous papers, 2 hours, 5H,7H,9H-9-hydroxyimidazo[1,5-α]pyridine-1,3-dione (C) was believed to be an intermediate product produced in the conversion of α-keto-ɛ-carbamidocaproic acid to 2 hours,5H,7H-imidazo[1,5-α] pyridine-1,3-dione (A) by enzymatic transamination in vivo (Supplemental Scheme S1) 12 ; letter it was synthesized in 1983. 13 The NMR data of 1 were consistent with those previously reported. 13 The absolute configuration at C-9 of compound 1 was determined from the ECD spectrum. The experimental ECD spectrum of 1 was compared with the TDDFT calculated ECD spectra of the two enantiomers (9R and 9S). As shown in Supplemental Figure S6a, the experimental ECD of 1 appeared like that of the 9S-enantiomer, confirming the S configuration at C-9. From the above evidence, compound 1 was determined to be 2 hours, 5H,7H,9H-9(S)-hydroxyimidazo[1,5-α]pyridine-1,3-dione, a new compound from nature and named as aaptos VN1.
Compound 2 was obtained as a colorless oil. Its molecular formula was deduced to be C 20 H 42   long alkyl chain from δ H 1.00 to 1.60 ppm. The 13 C NMR and HSQC spectra of 2 indicated the presence of three oxygenated methylene carbons at δ C 64.2, 71.8, and 72.4, one oxygenated methine carbon at δ C 70.6, two methyl carbons at δ C 14.1 and 19.7, one methine carbon at δ C 32.8, and 12 methylene carbons from δ C 22.7 to 37.1. The above evidence suggested that compound 2 was a glyceryl ether derivative having a long alkyl chain. The NMR spectral data of 2 (measured in CDCl 3 : Supplemental Figures S8-11; measured in CD 3 OD + Py-d 5 , 1/1: Supplemental Figure S11b) were compared with the corresponding data of lysoplasmanglinositol (isolated from marine sponge Theonella swinhoei) 14 and found to match, except for the lower field signal of C-3 of 2 (δ C 64.2), indicating only one long alkyl chain linked to C-1 of the glyceryl molecules, which was further confirmed by HMBC correlations. However, the absolute configuration at C-2 of this compound has not yet been determined. Compound 2 was reported as a hydrolysis product of lysoplasmanglinositol, 14 but its NMR data have not been reported. Consequently, compound 2 was identified as 3-((9-methylhexadecyl)oxy)propane-1,2-diol. This is the first report of 2 from nature.

General Experimental Procedures
Optical rotation was measured on a Jasco P-2000 polarimeter, NMR spectra on a Bruker 500 MHz spectrometer, and HR-ESI-MS on an Agilent 6530 Accurate Mass Q-TOF LC/MS. Flash column chromatography was performed using either silica gel or reversed phase (RP-18) resins as adsorbent. Thin layer chromatography was carried out on pre-coated silica gel 60 F 254 and/or RP-18 F 254S plates. TLC plates were visualized under UV irradiation (254 and 365 nm) or by spraying with H 2 SO 4 solution (5%) followed by heating with a heat gun. HPLC was carried out using an AGILENT 1100 HPLC system.

Animal Material
The sponge samples were collected in Vanphong Bay, Nha Trang, Vietnam in May 2020 and identified as Aaptos aaptos (Schmidt, 1864) by Prof. Do Cong Thung, the Institute of Marine Environment and Resources, Vietnam Academy of Science and Technology (VAST). A voucher specimen (NCCT-B70) was deposited at the Institute of Marine Biochemistry, VAST.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.