Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai Delfly B. Abdjul, Hiroyuki Yamazaki,* Syu-ichi Kanno, Ohgi Takahashi, Ryota Kirikoshi, Kazuyo Ukai, and Michio Namikoshi Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan *S Supporting Information ABSTRACT: Three new N-methyladenine-containing diterpenes, 2- oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G(10), and ageline B (11). The structures of 1-3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5-11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 μg/disc, respectively. All compounds were inactive (IC 50 > 10 μM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC 50 value of 15 μM. M arine organisms produce a wide variety of metabolites that display diverse biological activities and unique structural features. 1 Marine sponges of the genus Agelas have been identified as a rich source of diterpenes with polar functionalities in addition to their chemical markers and bromopyrrole alkaloids. 2-5 In the course of our search for bioactive and useful substances from marine organisms, we have reported cancer preventive compounds, protein tyrosine phosphatase 1B (PTP1B) inhibitors, and antimycobacterial substances. 6-8 Further investigations on extracts from marine invertebrates revealed that the EtOH extract of the Okinawan marine sponge Agelas nakamurai Hoshino exhibited antibacterial activity against Mycobacterium smegmatis with an inhibition zone of 22 mm at 50 μg/disc. Bioassay-guided isolation afforded three new diterpene alkaloids possessing an N-methyladenine unit, 2- oxoagelasine A (1), 2-oxoagelasine F (2), and 10-hydro-9- hydroxyagelasine F (3), together with eight known agelasine derivatives: 2-oxoagelasine B (4), 3a agelasines A (5), 3b B(6), 3b D(7), 3b E(8), 3c F(9), 3c and G (10), 3d and ageline B (11). 3e We herein described the isolation, structure elucidation including absolute configurations, and biological activities of compounds 1-11. ■ RESULTS AND DISCUSSION The marine sponge (159.3 g, wet weight) was extracted with EtOH. The obtained extract (4.1 g) inhibited the growth of M. smegmatis (22 mm at 50 μg/disc) and was separated by an octadecylsilane (ODS) column (100 g) into seven fractions. The bioactive fractions were further purified by repeated high- performance liquid chromatography (HPLC) using an ODS column and yielded compounds 1 (6.8 mg), 2 (1.5 mg), 3 (3.3 mg), 4 (4.6 mg), 5 (2.8 mg), 6 (2.6 mg), 7 (4.7 mg), 8 (2.3 mg), 9 (3.5 mg), 10 (4.4 mg), and 11 (1.4 mg). Compounds 4-11 were identified as the known agelasine derivatives, 2-oxoagelasine B, agelasines A, B, D, E, F, and G, and ageline B, respectively, by comparing their spectroscopic data with those for the reported values. 3 Various kinds of agelasines, including 4-11, have previously been reported from several marine sponges of the genus Agelas. 3-5 The 1 H and 13 C NMR spectra of 1 resembled those of 2- oxoagelasine B (4) and agelasine A (5). The molecular formula of 1 was deduced from HRFABMS and NMR data (Table 1) as C 26 H 38 N 5 O, which was the same as that of 4. Although information from COSY data was limited, HMBC data revealed that 1 had the same planar structure as 4 (Figure 1a). The NOESY correlations between H 2 -12 (δ H 2.00)/H-14 (δ H 5.48) and H 2 -15 (δ H 5.12)/H 3 -16 (δ H 1.80) showed the E-orientation of the double bond at C-13 (Figure 1b). The cis-configuration of the clerodane moiety was established from NOESY correlations between H-1a (δ H 2.73)/H 3 -19 (δ H 1.18), H-1b (δ H 2.44)/H 3 -20 (δ H 0.51), and H-10 (δ H 1.90)/H 3 -19. The NOESY correlations between H-7b (δ H 1.16)/H 3 -20 and H 3 -17 (δ H 0.75)/H 3 -20 revealed the configurations at the C-8 and C-9 positions. These NOESY correlations were similar to those of nakamurol C possessing a cis-clerodane moiety, which has also been isolated from the Okinawan marine sponge Agelas Received: April 29, 2015 Article pubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.5b00375 J. Nat. Prod. XXXX, XXX, XXX-XXX