HETEROCYCLES

Paper | Special issue | Vol. 90, No. 1, 2015, pp. 425-431
Received, 9th June, 2014, Accepted, 24th June, 2014, Published online, 30th June, 2014.
DOI: 10.3987/COM-14-S(K)38

■ 2-Debromonagelamide U, 2-Debromomukanadin G, and 2-Debromonagelamide P from Marine Sponge Agelas sp.

Kenta Nakamura, Taishi Kusama, Naonobu Tanaka, Kanae Sakai, Tohru Gonoi, Jane Fromont, and Jun'ichi Kobayashi*

Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo, Hokkaido 060-0812, Japan

Abstract

Two new monomeric bromopyrrole alkaloids (1 and 2) and one new dimeric bromopyrrole alkaloid (3) were isolated from an Okinawan marine sponge Agelas sp. Spectroscopic analyses of these compounds revealed the structures of 1–3 to be 2-debromonagelamide U, 2-debromomukanadin G, and 2-debromonagelamide P, respectively. Antimicrobial activity of 1–3 was evaluated.

INTRODUCTION
Marine sponges have been recognized as a rich source of bioactive metabolites with unique chemical structures.1 Among them, bromopyrrole alkaloids are one of the most common metabolites contained in marine sponges. These alkaloids have attracted widespread interest due to their fascinating chemical structures with high N to C ratio (ca 1:2).2-4 During our search for new metabolites from Okinawan marine sponges, we have reported many bromopyrrole alkaloids with a variety of chemical structures from sponges Agelas spp.5-8 Recently, we isolated three new bromopyrrole alkaloids, 2-debromonagelamide U (1), 2-debromomukanadin G (2), and 2-debromonagelamide P (3) (Chart 1) from a sponge Agelas sp. (SS-156). In this article, we describe the isolation, structure elucidation, and antimicrobial activity of 1–3.

RESULTS AND DISCUSSION
The sponge Agelas sp. (SS-156, 6.3 kg, wet weight) collected at Kerama Islands, Okinawa, was extracted with MeOH to give the extract, a part of which was partitioned with n-hexane, n-BuOH, and water. Chromatographic separations of the n-BuOH-soluble materials gave 2-debromonagelamide U (1, 0.00003%, wet weight), 2-debromomukanadin G (2, 0.00017%), and 2-debromonagelamide P (3, 0.00010%), together with six known bromopyrrole alkaloids, mukanadin A,9 tauroacidin B,10 laughine,11 2-debromotaurodispacamide A,12 ageliferin,13 and 2,2’-didebromonagelamide B.7
2-Debromonagelamide U (1) was isolated as an optically active colorless amorphous solid. The pseudomolecular ion peaks at m/z 449 and 451 (1:1) in the ESIMS suggested the existence of one bromine atom in the molecule. The molecular formula of 1, C13H19N6O5BrS, was established by the HRESIMS (m/z 449.02591 [M–H]-, Δ+1.09 mmu). The presence of a pyrrole amide moiety, a common unit on bromopyrrole alkaloids, was disclosed by an IR absorption at 1684 cm-1 and a UV absorption at 268 nm. The 1H and 13C NMR spectra (Table 1) showed the signals from a 3-bromopyrrole amide moiety (N-1–N-7), a guanidine moiety (N-13–N-16), an ethanesulfonic acid moiety (C-2’ and C-3’), two sp3 methylenes, and two sp3 methines. The 1D NMR spectra resembled those of nagelamide U,6 except for the resonances due to the bromopyrrole amide moiety. From these observations, 1 was presumed to be a 2-debromo form of nagelamide U. The gross structure of 1 was confirmed by analysis of the 1H-1H COSY and HMBC spectra (Figure 1). The anti relationship for H-9/H-11 was implied by a ROESY correlation for H-8a/H-11. Thus, the structure of 2-debromonagelamide U (1) was assigned as shown in Chart 1.

The molecular formula of 2-debromomukanadin G (2) was assigned by the HRESIMS (m/z 407.04590 [M]+, Δ–0.28 mmu). The 1H and 13C NMR data for 2 (Table 1) were similar to those for mukanadin G,8 a bromopyrrole alkaloid with the tricyclic skeleton consisting of a tetrahydrobenzaminoimidazole and a 2,5-dioxopyrrolidine moieties, and the resonances due to a 3-bromopyrrole amide moiety (N-1–N-7) in 2 were discerned in place of the signals of a dibromopyrrole amide moiety in mukanadin G. The relative configurations of C-9, C-16, and C-20 were deduced to be the same as those of mukanadin G by resemblance of their 1H coupling constants. Given correlations observed in the 2D NMR spectra (Figure 2), 2 was assigned as 2-debromomukanadin G.

2-Debromonagelamide P (3) was obtained as a colorless amorphous solid. The ESIMS showed the pseudomolecular ion peaks at m/z 633, 635, and 637 (1:2:1), indicating the existence of two bromine atoms in the molecule. The HRESIMS suggested the molecular formula of 3 to be C22H23N10O3Br2 (m/z 633.03191 [M]+, Δ+0.32 mmu). Comparison of the 1D NMR data for 3 (Table 2) with those for bromopyrrole alklaoids from sponges of Agelas spp. implied 3 to be a 2-debromo derivative of nagelamide P.14 This was confirmed based on analysis of the 2D NMR spectra (Figure 2).
2-Debromomukanadin G (2) and 2-debromonagelamide P (3) were both optically inactive, implying that they are racemates, while mukanadin G8 and nagelamide P14 were also assigned as racemates by analysis of the chiral HPLC and the CD spectrum, respectively.
Antimicrobial activities of 1–3 against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, Trichophyton mentagrophytes, Candida albicans, and Cryptococcus neoformans were evaluated. As a result, 2-debromonagelamide U (1) and 2-debromonagelamide P (3) showed antimicrobial activity against Trichophyton mentagrophytes (IC50 16 and 32 µg/mL, respectively), while 1 and 2-debromomukanadin G (2) exhibited activity against Cryptococcus neoformans (IC50 32 µg/mL each). 1–3 showed no cytotoxicity against human epidermoid carcinoma KB and murine lymphoma L1210 cells (IC50 >10 µg/mL) in vitro.

CONCLUSION
The investigation of the extract from an Okinawan marine sponge Agelas sp. (SS-156) resulted in the isolation of two new monomeric bromopyrrole alkaloids (1 and 2) and one new dimeric bromopyrrole alkaloid (3). The structures of 1–3 were assigned as 2-debromonagelamide U, 2-debromomukanadin G, and 2-debromonagelamide P, respectively, on the basis of spectroscopic analyses.

EXPERIMENTAL
General procedures: Optical rotations were recorded on a JASCO P-1030 digital polarimeter. IR and UV spectra were recorded on a JASCO FT/IR-230 and a Shimadzu UV-1600PC spectrophotometers, respectively. NMR spectra were measured by a Bruker Avance 600 NMR spectrometer. The resonances of CHD2SOCD3 (δH 2.49) and DMSO-d6 (δC 39.5) were used as internal references for 1H and 13C NMR spectra, respectively. Mass spectra were recorded on a Thermo Scientific Exactive spectrometer.

Sponge description: The sponge (SS-156: Order Agelasida, Family Agelasidae, Agelas sp.) with smooth, finely conulose surface collected at Kerama Islands, Okinawa, was kept frozen until used. Mesohyl is compact with large internal canals. Sponges are firm, springy and compressible. Skeleton is dense, reticulate fibre skeleton, with grainy texture over fibres, echinated by verticillate spined acanthostyles. Fibres sparsely cored by spicules, more coring of spicules towards surface of sponge. Spicules are verticillate, regularly spined acanthostyles, 170x15 µm.

Isolation of 2-debromonagelamide U (1), 2-debromomukanadin G (2), and 2-debromonagelamide P (3): The sponge Agelas sp. (6.3 kg, wet weight) was extracted with MeOH (6 L x 3) to give the extract (424.0 g). A part of the extract (205.6 g) was partitioned successively with n-hexane (800 mL x 3), n-BuOH (800 mL x 3), and water (800 mL). The n-BuOH-soluble materials (77.1 g) were subjected to a silica gel column (CHCl3/MeOH/AcOH, 80:20:2→0:100:2) to give six fractions (frs. 1–6). Separation of fr. 3 on a TOYOPEARL HW-40 column (MeOH/H2O/TFA, 30:70:0.1→100:0:0.1) afforded six fractions (frs. 3.1–6), and fr. 3.4 was separated by a C18 column (MeOH/H2O/TFA, 20:80:0.1→100:0:0.1) to give seven fractions (frs. 3.4.1–7). Fr. 3.4.2 was purified using C18 HPLC (YMC ODS-AQ, 20 x 250 mm; flow rate 5.0 mL/min; UV detection at 254 nm; eluent MeCN/H2O/TFA, 25:75:0.1) to isolate 2-debromomukanadin G (2, 10.9 mg). Fr. 3.4.5 was loaded on a Sephadex LH-20 column (MeOH/H2O/TFA, 20:80:0.1→100:0:0.1) chromatography to give five fractions (frs. 3.4.5.1–5). Fr. 3.4.5.4 was purified by C18 HPLC (YMC ODS-AQ, 20 x 250 mm; 5.0 mL/min; 254 nm; MeCN/H2O/TFA, 30:70:0.1, and then YMC Hydrosphere C18, 10 x 250 mm; 2.5 mL/min; 254 nm; MeCN/H2O/TFA, 23:77:0.1) to afford 2-debromonagelamide P (3, 6.7 mg). Separation of fr. 4 using a TOYOPEARL HW-40 column (MeOH/H2O/TFA, 10:90:0.1→100:0:0.1) and a Sephadex LH-20 column (MeOH/H2O/TFA, 10:90:0.1→100:0:0.1) gave a fraction containing bromopyrrole alkaloids. The fraction was purified by C18 HPLC (YMC ODS-AQ, 20 × 250 mm, 5.0 mL/min, 254 nm, MeCN/H2O/TFA, 15:85:0.1) and HILIC HPLC (Cosmosil HILIC, 10 × 250 mm, 3.0 mL/min, 254 nm, MeCN/H2O, 87:13) to isolate 2-debromonagelamide U (1, 1.7 mg).
2-Debromonagelamide U (1). Colorless amorphous solid; [α]21D +14.3 (c 0.025, MeOH); UV (MeOH) νmax 209 (ε 17200 sh) and 268 (4200) nm; IR (KBr) λmax 3444, 1684, and 1673 cm-1; ESIMS m/z 449 and 451 (1:1) [M–H]-; HRESIMS: m/z 449.02591 [M–H]- (calcd for C13H18N6O579BrS, 449.02482; 1H and 13C NMR (Table 1).
2-Debromomukanadin G (2). Colorless amorphous solid; [α]21D ≈0 (c 0.25, MeOH); UV (MeOH) νmax 221 (ε 7100 sh) and 268 (6900) nm; IR (KBr) λmax 3181, 1717, and 1683 cm-1; ESIMS m/z 407 and 409 (1:1) [M]+; HRESIMS: m/z 407.04590 [M]+ (calcd for C15H16N6O379Br, 407.04618); 1H and 13C NMR (Table 1).
2-Debromonagelamide P (3). Colorless amorphous solid; [α]21D ≈0 (c 0.25, MeOH); UV (MeOH) νmax 221 (ε 12200 sh) and 271 (13800) nm; IR (KBr) λmax 3181, 1690, and 1637 cm-1; ESIMS m/z 633, 635, and 637 (1:2:1) [M]+; HRESIMS: m/z 633.03191 [M]+ (calcd for C22H23N10O379Br2, 633.03159; 1H and 13C NMR (Table 2).

ACKNOWLEDGMENTS
We thank Mr. Z. Nagahama for his help with sponge collection, Ms. S. Oka and Ms. A. Tokumitsu, Equipment Management Center, Hokkaido University, for measurements of mass spectra. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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