Bromopyrrole Alkaloids from Okinawan Marine Sponges Agelas spp.

Abstract

In our continuing study for structurally and biogenetically interesting natural products from marine organisms, Okinawan marine sponges Agelas spp. were investigated, resulting in the isolation of 18 unique alkaloids including five dimeric bromopyrrole alkaloids (1–5), ten monomeric bromopyrrole alkaloids (6–15), and three conjugates of monomeric bromopyrrole alkaloid and hydroxykynurenine (16–18). In this mini-review, the isolation, structure elucidation, and antimicrobial activities of these alkaloids are summarized.

1. Introduction

Bromopyrrole alkaloids are one of the most common secondary metabolites found in marine sponges. These alkaloids are argued to be taxon-specific of at least the Agelasida order and can be used as chemical markers of these phylogenetically related sponges.^1–3) It has been shown that oroidin and sceptrin, which are bromopyrrole alkaloids contained in sponges Agelas spp. (Agelasidae), are not associated with the symbiotic bacteria but with the sponge cells,⁴⁾ and that sponges Agelas spp. share bromopyrrole alkaloids as a common chemical defense against fish predators.^5,6) In addition to these chemotaxonomic and ecological roles, bromopyrrole alkaloids are interesting for their diverse biological activities⁷⁾ including antifeedent, antibiofilm, anticancer, antiinflammatory, antimicrobial, immunomodulatory, analgesic, antiserotonergic, and antihistamic activities.

Representative bromopyrrole alkaloids, oroidin^8,9) and hymenidin,¹⁰⁾ are composed of an aminoimidazole moiety and a brominated pyrrole-amide moiety linked through a C₃ unit (Fig. 1). Proline and lysine have been suggested to be biogenetic precursors of oroidin.¹¹⁾ Cyclization and/or dimerization of the monomeric bromopyrrole alkaloids yielded polycyclic and dimeric bromopyrrole alkaloids, which have attracted widespread interest as challenging targets for total synthesis by virtue of their fascinating functionalized structures with a high N : C ratio (1 : 2).^12,13) Though a number of bromopyrrole alkaloids have been isolated from marine sponges to date, stylissadines A and B¹⁴⁾ from Stylissa flabellata, are the only known examples of tetrameric bromopyrrole alkaloids.

Fig. 1. Structures of Oroidin and Hymenidin

In our continuing search for structurally and biogenetically unique natural products from marine organisms, we have reported the isolation of some bromopyrrole alkaloids from Okinawan marine sponges.¹⁵⁾ As part of that research project, bromopyrrole alkaloids in sponges Agelas spp. (SS-162, SS-307) were investigated, resulting in the isolation of 18 unique alkaloids including five dimeric bromopyrrole alkaloids (1–5), ten monomeric bromopyrrole alkaloids (6–15), and three conjugates of monomeric bromopyrrole alkaloid and hydroxykynurenine (16–18). In this mini-review, we describe the isolation, structure elucidation, and antimicrobial activities of these alkaloids.

2. Extraction and Isolation of New Bromopyrrole Alkaloids

The sponges Agelas spp. (SS-162, SS-307) were collected off Kerama Islands, Okinawa, and were individually extracted with MeOH. Repeated chromatographic separations of the extract from SS-162 using a silica gel column, an octadecylsilyl (ODS) column, a Sephadex LH-20 column, and a Toyopearl HW-40 column gave fractions containing bromopyrrole alkaloids. The fractions were purified by reversed-phase HPLC and/or hydrophilic interaction liquid chromatography (HILIC) HPLC to isolate new bromopyrrole alkaloids; agelamadins A (1), B (2), C (16), D (17), and E (18); nagelamides U (7), V (8), W (9), X (3), Y (4), and Z (5); tauroacidins C (10) and D (11); mukanadin G (13); 2-bromokeramadine (14); and 2-bromo-9,10-dihydrokeramadine (15). Similarly, the MeOH extract from SS-307 was separated to give agelamadin F (6) and tauroacidin E (12).

3. Dimeric Bromopyrrole Alkaloids, Agelamadins A and B and Nagelamides X–Z

Agelastatins, which are monomeric bromopyrrole alkaloids, possess a unique 5/5/6/5 tetracyclic ring system. Among these, agelastatin A^16,17) (Fig. 2) is interesting for its potent cytotoxicity against various cancer cell lines and for its inhibitory activity against glycogen synthase kinase-3β (GSK-3β). Agelamadins A (1) and B (2)¹⁸⁾ have a common structure consisting of an agelastatin-like tetracyclic moiety and an oroidin-like linear moiety (Fig. 2). The high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) and the one dimensional (1D) NMR spectra suggested that 1 and 2 were dimeric bromopyrrole alkaloids, and their gross structures were elucidated by analyses of the ¹H–¹H correlation spectroscopy (COSY) and heteronuclear multiple bond coherence (HMBC) spectra. Rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) analysis indicated that the relative configurations of the agelastatin-like tetracyclic moieties for 1 and 2 were coincident with that of agelastatin A. The optical resolutions on chiral HPLC revealed both 1 and 2 to be racemates.

Fig. 2. Structures of Agelamadins A (1) and B (2) and Agelastatin A

The 1D NMR spectra implied that nagelamides X (3) and Y (4)¹⁹⁾ are dimeric bromopyrrole alkaloids with an N-ethanesulfonic acid moiety (Fig. 3). The novel tricyclic skeleton shared by 3 and 4, comprising spiro-bonded tetrahydrobenzaminoimidazole and aminoimidazolidine moieties, was elucidated by 2D-NMR analyses. Nagelamides X (3) and Y (4) seemed to have been derived from oroidin (Fig. 1) and taurodispacamide A²⁰⁾ (Fig. 3) by [4+2] cycloaddition. Nagelamide Z (5)¹⁹⁾ was the first example of a dimer of oroidin-like linear bromopyrrole alkaloid involving dimerization at C-8 (Fig. 3). The absolute configuration of C-8 for 5 remained unsolved.

4. Monomeric Bromopyrrole Alkaloids, Agelamadin F and Nagelamides U–W

Fig. 3. Structures of Nagelamides X–Z (3–5) and Taurodispacamide A

The 1D NMR spectra suggested that agelamadin F (6)²¹⁾ had an oroidin-like linear moiety and a 3-hydroxypyridinium moiety (Fig. 4). The connectivity between C-15 of the linear moiety and N-1′ of the 3-hydroxypyridinium moiety was revealed by HMBC and ROESY analyses. Agelamadin F (6) is the first example of a bromopyrrole alkaloid with a 3-hydroxypyridinium moiety. Nagelamide U (7)²²⁾ was a monomeric bromopyrrole alkaloid having a γ-lactam ring with an N-ethanesulfonic acid moiety and a guanidino moiety, while nagelamide V (8)²²⁾ was assigned as a stereoisomer of 7. The relationships between the substituents at C-9 and C-11 for 7 and 8 were assigned as anti and syn, respectively, by ROESY analysis. Though nagelamides U (7) and V (8) were optically active, their absolute configurations were not assigned. Interpretation of the 2D-NMR spectra revealed nagelamide W (9)²²⁾ to be a monomeric bromopyrrole alkaloid with two aminoimidazole moieties in the molecule (Fig. 4). Nagelamide W (9) might be biogenetically generated by the addition of guanidine to oroidin.

Fig. 4. Structures of Agelamadin F (6) and Nagelamides U–W (7–9)

Analyses of the NMR spectra implied that tauroacidins C–E (10–12)^21,23) were monomeric bromopyrrole alkaloids with an N-ethanesulfonic acid moiety (Fig. 5), and their structures were closely related to those of tauroacidin A,²⁴⁾ previously isolated by our group from the Okinawan marine sponge Hymeniacidon sp. Mukanadin G (13)²³⁾ had a tricyclic skeleton consisting of a fused tetrahydrobenzaminoimidazole moiety and a 2,5-dioxopyrrolidine moiety (Fig. 5). The relative stereochemistry of 13 was concluded as shown in Fig. 5 by ROESY analysis and a comparison of the experimental ¹H coupling constants with those calculated for the most stable stereoisomers of 13 obtained by conformational searches. This is the first example of the isolation of 13 from a natural source, whereas the synthesis of 13 as a Diels–Alder adduct of maleimide and oroidin has been reported.²⁵⁾ Chiral HPLC analyses disclosed tauroacidins C–E (10–12) and mukanadin G (13) to be racemates. The structures of 14 and 15 were assigned as 2-bromo and 2-bromo-9,10-dihydro analogues²³⁾ of keramadine,²⁶⁾ respectively (Fig. 5).

Fig. 5. Structures of Tauroacidins C–E (10–12), Mukanadin G (13), 2-Bromokeramadine (14), 2-Bromo-9,10-dihydrokeramadine (15), and Keramadine

5. Conjugates of Monomeric Bromopyrrole Alkaloid and Hydroxykynurenine, Agelamadins C–E

Agelamadins C–E (16–18)²⁷⁾ possessed a unique hybrid structure of oroidin and 3-hydroxykynurenine connected through a dihydro-1,4-oxazine moiety (Fig. 6). Interpretations of the 1D and 2D-NMR spectra indicated that 16–18 had the same planar structure. To assign the absolute configurations for the α-carbons (C-9′) of 16–18, a phenylglycine methyl ester (PGME) method was applied,^27,28) suggesting the configuration to be S in each case. ROESY analysis indicated the H-9/H-10 anti relationship for 16 and 17, whereas the H-9/H-10 syn relationship was assigned for 18. The absolute configurations at C-9 and C-10 of 16–18 were elucidated by comparison of the electronic circular dichroism (ECD) spectra with the time-dependent density functional theory (TDDFT) calculated spectra as shown in Fig. 6.

Fig. 6. Structures of Agelamadins C–E (16–18)

6. Antimicrobial Activities of Isolated Bromopyrrole Alkaloids

In the course of our search for antimicrobial marine natural products,^29–34) we evaluated the antimicrobial activities of isolated bromopyrrole alkaloids against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger (IFM62678), Trichophyton mentagrophytes (IFM62679), Candida albicans (IFM62680), and Cryptococcus neoformans (IFM62681) (Table 1). Among tested alkaloids, nagelamide Z (5) exhibited significant antimicrobial activity against C. albicans (MIC 0.25 µg/mL) and moderate activities against several microorganisms, while agelamides A (1) and B (2), nagelamides X (3), Y (4), U (7), and W (9) also showed moderate antimicrobial activities.

Table 1. Antimicrobial Activities of 1–5, 7, 9, 10, and 13–18

Strain	1	2	3	4	5	7	9
Escherichia colia)	>32	32	>32	>32	>32	>32	>32
Staphylococcus aureusa)	32	32	8.0	>32	16	>32	>32
Bacillus subtilisa)	16	16	>32	>32	>32	>32	>32
Micrococcus luteusa)	4.0	8.0	8.0	>32	8.0	>32	>32
Aspergillus nigerb)	>32	>32	32	>32	4.0	>32	>32
Trichophyton mentagrophytesb)	>32	>32	16	>32	4.0	>32	>32
Candida albicansb)	>32	>32	2.0	2.0	0.25	4.0	4.0
Cryptococcus neoformansb)	8.0	4.0	>32	>32	2.0	>32	8.0
Strain	10	13	14	15	16	17	18
Escherichia colia)	>32	>32	>32	>32	>32	>32	>32
Staphylococcus aureusa)	>32	>32	32	>32	>32	>32	>32
Bacillus subtilisa)	>32	>32	>32	>32	>32	>32	>32
Micrococcus luteusa)	>32	>32	>32	>32	>32	>32	>32
Aspergillus nigerb)	>32	32	>32	>32	>32	>32	>32
Trichophyton mentagrophytesb)	>32	>32	>32	32	>32	>32	>32
Candida albicansb)	>32	16	>32	32	>32	>32	>32
Cryptococcus neoformansb)	>32	8.0	>32	>32	32	>32	32

a) MIC value (µg/mL). b) IC₅₀ value (µg/mL).

Acknowledgments

We thank Prof. T. Gonoi, Dr. A. Takahashi-Nakaguchi, and Dr. K. Sakai, Mycology Research Center, Chiba University, for evaluation of antimicrobial activity, and Mr. Z. Nagahama for help with sponge collection. 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.

Conflict of Interest

The authors declare no conflict of interest.

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