Nagelamide I and 2,2′-Didebromonagelamide B, New Dimeric Bromopyrrole–Imidazole Alkaloids from a Marine Sponge Agelas sp.

Abstract

New dimeric bromopyrrole–imidazole alkaloids, nagelamide I (1) and 2,2′-didebromonagelamide B (2), have been isolated from an Okinawan marine sponge Agelas species. The structures of 1 and 2 were elucidated based on analyses of the spectral data. Nagelamide I (1) was the first symmetric dimeric bromopyrrole–imidazole alkaloid consisting of two subunits connected with a single bond.

Marine sponges of the genus Agelas have been demonstrated to be a rich source of unique bioactive products.¹⁾ During our search for bioactive metabolites from marine organisms, a series of bromopyrrole–imidazole alkaloids²⁾ and diterpene derivatives with polar functionalities³⁾ have been isolated from marine sponges of the genus Agelas. Recently, we have isolated seven new diterpene alkaloids with a 9-N-methyladenine unit, agelasines O–U from the extract of a sponge Agelas sp. (NSS-19).³⁾ Further investigation of the extract resulted in isolation of two new dimeric bromopyrrole–imidazole alkaloids, nagelamide I (1) and 2,2′-didebromonagelamide B (2). Here we describe the isolation and structure elucidation of 1 and 2.

The sponge Agelas sp. (NSS-19) collected at Okinawa was extracted with MeOH, which was partitioned between organic solvents (n-hexane, CHCl₃, and n-BuOH) and H₂O. n-BuOH-soluble materials of the extract were fractionated by gel filtration on Sephadex LH-20, and a fraction was purified by C₁₈ column chromatography and C₁₈ HPLC to yield nagelamide I (1, 0.00015%, wet weight) and 2,2′-didebromonagelamide B (2, 0.00010%). Eighteen known alkaloids, agelasidines A–C,^4–7) agelasines E,⁸⁾ G,⁹⁾ H,¹⁰⁾ I,¹⁰⁾ and O–U,³⁾ 2-oxo-agelasine B,¹¹⁾ ageline B,¹²⁾ hymenidin (4),¹³⁾ and sceptrin,¹⁴⁾ have been isolated in the purification process of 1 and 2.

Nagelamide I (1) was obtained as brown amorphous solid. The electrospray ionization mass spectrometry (ESI-MS) spectrum of 1 showed the molecular ion peaks at m/z 617, 619, and 621 (1 : 2 : 1) implying the presence of two bromine atoms. The molecular formula of 1 was revealed to be C₂₂H₂₂N₁₀O₂⁷⁹Br₂ by high resolution (HR)-ESI-MS data [m/z 617.03772 (M+H)⁺, Δ+1.05 mmu]. The UV absorption [λ_max 273 (ε 12800) nm] was attributed to heteroaromatic rings, while IR absorptions (3335, 1682 cm⁻¹) indicated the existence of amino and carbonyl functionalities, respectively. The ¹³C-NMR spectrum of 1 (Table 1) showed one signal for amide carbonyl (δ_C 159.5), five signals for sp² quaternary carbons (δ_C 147.7, 126.6, 121.3, 119.1, 95.0), four signals for sp² methine (δ_C 126.7, 121.2, 115.7, 111.6), and one signal for sp³ methylene (δ_C 40.4). These data and the molecular formula of 1 clarified that 1 was a symmetric dimeric bromopyrrole–imidazole alkaloid. Among eleven carbon signals, two signals for sp² quaternary carbons (δ_C 126.6, 95.0) and two signals for sp² methine (δ_C 121.2, 111.6) were ascribed to two 5-monosubstituted 3-bromopyrrole rings (N-1–C-5 and N-1′–C-5′), while three signals for sp² quaternary carbons (δ_C 147.7, 121.3, 119.1) were assigned to a 4,5-disubstituted 2-aminoimidazole rings (C-11–C-15 and C-11′–C-15′).^13–17) The ¹H–¹H correlated spectroscopy (COSY) and total correlation spectroscopy (TOCSY) spectra of 1 disclosed the connection for N-7 to C-10 (N-7′ to C-10′). Heteronuclear multiple bond correlation (HMBC) correlations for H-2/C-3 (H-2′/C-3′), H-2/C-4 (H-2′/C-4′), and H-2/C-5 (H-2′/C-5′) supported the presence of 5-monosubstituted 3-bromopyrrole rings. Geometry of the double bond at C-9 (C-9′) was assigned as E from a coupling constant (15.9 Hz) for H-9/H-10 (H-9′/H-10′). Connectivity of C-5 and N-7 (C-5′ and N-7′) through an amide bond was inferred from an HMBC correlation for NH-7/C-6 (NH-7′/C-6′) and a rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlation for H-4/NH-7 (H-4′/NH-7′). An HMBC correlation for H-10/C-15 (H-10′/C-15′) suggested that a 2-aminoimidazole ring was attached to C-10 (C-10′). Considering that 1 was a symmetric molecule, two subunits were connected with a single bond between C-15 and C-15′. Thus, the structure of nagelamide I (1) was assigned as shown in Fig. 1.

Table 1. ¹H- and ¹³C-NMR Data of Nagelamide I (1) and 2,2′-Didebromonagelamide B (2) in DMSO-d₆

Position	1			2
	δHa)	Multi (J in Hz)	δCb)	δHa)	Multi (J in Hz)	δCb)
1	11.82	br s	—	11.82	br s	—
2	6.98	s	121.2	6.98	s	121.3
3	—		95.0	—		95.0
4	6.87	s	111.6	6.86d)	s	111.6
5	—		126.6	—		126.7
6	—		159.5	—		159.4
7	8.41	t (5.5)	—	8.18	t (5.2)	—
8	3.95c)	t (5.3)	40.4	3.38	nde)	43.6
				2.90	m
9	6.03	dt (15.9, 5.8)	126.7	4.16	nde)	69.3
9-OH	—		—	5.83	br s	—
10	6.40	d (15.9)	115.7	4.16	nde)	35.0
11	—		119.1	—		124.9
12	12.83	br s	—	—	—
13	—		147.7	—		147.0
13-NH2	7.71c)	s	—	7.48c)	m	—
14	—		—	12.15	br s	—
15	—		121.3	6.77	s	110.6
1′	11.82	br s	—	11.82	br s	—
2′	6.98	s	121.2	6.98	s	121.3
3′	—		95.0	—		95.0
4′	6.87	s	111.6	6.87d)	s	111.6
5′	—		126.6	—		126.7
6′	—		159.5	—		159.9
7′	8.41	t (5.5)	—	8.38	t (5.6)	—
8′	3.95c)	t (5.3)	40.4	3.92c)	m	40.6
9′	6.03	dt (15.9, 5.8)	126.7	6.04	dt (16.1, 6.3)	126.6
10′	6.40	d (15.9)	115.7	6.42	d (16.1)	116.2
11′	—		119.1	—		122.2
12′	12.83	br s	—	12.78	br s	—
13′	—		147.7	—		147.7
13′-NH2	7.71c)	s	—	7.48c)	m	—
14′	—		—	—		—
15′	—		121.3	—		111.8

a) 600 MHz. b) 150 MHz. c) 2H. d) Interchangeable. e) J-Values were not determined because of overlapping with other signals.

Fig. 1. Structures of Nagelamide I (1), 2,2′-Didebromonagelamide B (2), Nagelamide B (3), Hymenidin (4), and Oroidin (5)

2,2′-Didebromonagelamide B (2) was obtained as brown amorphous solid. The presence of two bromine atoms were inferred from the molecular ion peaks at m/z 635, 637, and 639 (1 : 2 : 1) observed on the ESI-MS spectrum of 2. The molecular formula of 2 was established as C₂₂H₂₄N₁₀O₃⁷⁹Br₂ by HR-ESI-MS data [m/z 635.04839 (M+H)⁺, Δ+1.15 mmu]. Analyses of the heteronuclear multiple quantum coherence (HMQC) spectrum with ¹H- and ¹³C-NMR data of 2 (Table 1) uncovered the existence of twenty-two carbons consisting of two amide carbonyls (δ_C 159.9, 159.4), nine sp² quaternary carbons [δ_C 147.7, 147.0, 126.7 (2C), 124.9, 122.2, 111.8, 95.0 (2C)], seven sp² methines [δ_C 126.6, 121.3 (2C), 116.2, 111.6 (2C), 110.6], two sp³ methines (δ_C 69.3, 35.0), and two sp³ methylenes (δ_C 43.6, 40.6). Among twenty-two carbons, four sp² quaternary carbons [δ_C 126.7 (2C), 95.0 (2C)] and four sp² methine [δ_C 121.3 (2C), 111.6 (2C)] were ascribed to 5-monosubstituted 3-bromopyrrole rings (N-1–C-5 and N-1′–C-5′).^13–17) Two sp² quaternary carbons (δ_C 147.0, 124.9) and one sp² methine (δ_C 110.6) were attributed to a 4-monosubstituted 2-aminoimidazole ring (C-11–C-15), while three sp² quaternary carbons (δ_C 147.7, 122.2, 111.8) were assigned to a 4,5-disubstituted 2-aminoimidazole ring (C-11′–C-15′).^13–17) The ¹H–¹H COSY and TOCSY spectra of 2 disclosed connections for N-7 to C-10, C-9 to 9-OH, and N-7′ to C-10′. The configuration of a double bond at C-9′ was assigned as E from a coupling constant (16.1 Hz) for H-9′/H-10′. HMBC correlations for H-9/C-11 and H-9/C-15′ suggested that two 2-aminoimidazole rings were attached to C-10, while the connection of C-10′ and C-11′ in the 4,5-disubstituted 2-aminoimidazole ring was implied from HMBC correlations for H-9′/C-11′ and H-10′/C-11′. ROESY correlations for H-4/NH-7 and H-4′/NH-7′ indicated that a 5-monosubstituted 3-bromopyrrole ring was connected to each of N-7 and N-7′ through an amide carbonyl carbon (δ_C 159.4, 159.9, respectively). Thus, the gross structure of 2 was elucidated to be 2,2′-didebromo form of nagelamide B (3)¹⁵⁾ as shown in Fig. 1.

The threo configuration for C-9–C-10 was deduced from a relatively small J value for H-9 and H-10 (³J_H-9/H-10 <5.0 Hz)¹⁸⁾ as well as ROESY correlations for H₂-8/H-10 and H-9/H-15 (Fig. 4).

Fig. 2. Selected 2D-NMR Correlations for Nagelamide I (1)

Fig. 3. Selected 2D-NMR Correlations for 2,2′-Didebromonagelamide B (2)

Fig. 4. Rotation Model for the C-9/C-10 Bond of 2,2′-Didebromonagelamide B (2)

A series of dimeric bromopyrrole–imidazole alkaloids, which might be biosynthesized from two monomeric bromopyrrole–imidazole alkaloid subunits such as hymenidin (4)¹³⁾ and oroidin (5),^16,17) are attracting great interests from biosynthetic and biological points of view.¹⁹⁾ Nagelamide I (1) might be generated by dimerization of hymenidin (4), and was the first example of symmetric dimeric bromopyrrole–imidazole alkaloid consisting of two subunits connected with a single bond. Nagelamide I (1) and 2,2′-didebromonagelamide B (2) did not show cytotoxicity (IC₅₀>10 µg/mL) against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro.

Experimental

General Methods

Optical rotation was recorded on a JASCO P-1030 polarimeter. IR and UV spectra were recorded on a JASCO FT/IR-230 and a Shimadzu UV-1600PC spectrophotometer, respectively. ¹H- and ¹³C-NMR spectra were recorded on a Bruker AMX-600 spectrometer using 2.5 mm micro cells (Shigemi Co., Ltd.) for DMSO-d₆. The 2.49 and 39.5 ppm resonances of residual DMSO-d₅ and DMSO-d₆ were used as internal references for ¹H- and ¹³C-NMR spectra, respectively. MS spectra were recorded on a Thermo Scientific Exactive and a Thermo Scientific LTQ-OrbitrapXL spectrometers.

Sponge Description

The sponge (NSS-19) Agelas sp. (order, Agelasida, family Agelasiidae) was collected off Kerama Islands, Okinawa, in July 1984, and kept frozen until used. The sponge was a large piece with a smooth, faintly patterned surface. Mesohyl is compact with numerous pores. Sponges are firm and compressible. Skeleton is reticulate with fibre development, with primary fibres echinated by verticillate spined acanthostyles. Meshes are small and compact. Spicules are verticillate spined acanthostyles, thick, slightly curved 230×12 µm, some thin forms occur. The voucher specimen was deposited at Graduate School of Pharmaceutical Sciences, Hokkaido University.

Extraction and Separation

The sponge (NSS-19, 1.6 kg, wet weight) was extracted with MeOH (2 L×3) to obtain the extract (76.0 g), which was partitioned stepwise between organic solvents [n-hexane (300 mL×3), CHCl₃ (300 mL×6), and n-BuOH (300 mL×3)] and H₂O (300 mL) to give n-hexane-soluble materials (2.1 g), CHCl₃-soluble materials (18.6 g), and BuOH-soluble materials (7.0 g). The n-BuOH soluble materials (7.0 g) were fractionated by gel filtration (Sephadex LH-20, GE Healthcare; eluent, MeOH–H₂O, 90 : 10), and a fraction was separated by C₁₈ column chromatography (Cosmosil 140 C₁₈ OPN, Nacalai Tesque Inc.; eluent, MeOH–H₂O–trifluoroacetic acid (TFA), 50 : 50 : 0.1 to 80 : 20 : 0.1) and C₁₈ HPLC (CAPCELL PAK C18 MG, 10×250 mm, SHISEIDO; eluent, MeCN–H₂O–TFA, 30 : 70 : 0.1; flow rate, 2.5 mL/min; UV detection at 254 nm) to yield two fractions including nagelamide I (1) and 2,2′-didebromonagelamide B (2), respectively. Nagelamide I (1, 2.43 mg, 0.00015%, wet weight) was purified by C₁₈ HPLC (Luna Phenyl-Hexyl, 10×250 mm, Phenomenex; eluent, MeCN–H₂O–TFA, 28 : 72 : 0.1; flow rate, 2.5 mL/min; UV detection at 254 nm). 2,2′-Dibromonagelamide B (2, 1.63 mg, 0.00010%) was purified by C₁₈ HPLC (COSMOSIL cholester, 10×250 mm, Nacalai Tesque Inc.; eluent, MeCN–H₂O–TFA, 25 : 75 : 0.1; flow rate, 2.5 mL/min; UV detection at 254 nm).

Nagelamide I (1): Brown amorphous solid; UV (MeOH) λ_max (ε) nm: 203 (11700), 273 (12800); IR (film) ν_max cm⁻¹: 3335, 2928, 1682, 1524, 1435, 1204, 1136; ¹H- and ¹³C-NMR see Table 1; ESI-MS m/z 617, 619, 621 [(M+H)⁺, 1 : 2 : 1]; HR-ESI-MS m/z 617.03772 [(M+H)⁺, Δ+1.05 mmu], Calcd for C₂₂H₂₃N₁₀O₂⁷⁹Br₂.

2,2′-Didebromonagelamide B (2): Brown amorphous solid; [α]_D²⁷ −2.5 (c=0.5, MeOH); UV (MeOH) λ_max (ε) nm: 205 (7800), 270 (6900), 377 (3700); IR (film) ν_max cm⁻¹: 3420, 2923, 1683, 1540, 1456, 1205, 1136; ¹H- and ¹³C-NMR see Table 1; ESI-MS m/z 635, 637, 639 [(M+H)⁺, 1 : 2 : 1]; HR-ESI-MS m/z 635.04839 [(M+H)⁺, Δ+1.15 mmu], Calcd for C₂₂H₂₅N₁₀O₃⁷⁹Br₂.

Acknowledgment

We thank Mr. Z. Nagahama, Okinawa, for his help with sponge collection, and Ms. S. Oka, Instrumental Analysis Division, Equipment Management Center, Creative Research Institution, Hokkaido University, for measurements of ESI-MS. This work was supported by the Naito Foundation and Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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