Agesasines A and B, Bromopyrrole Alkaloids from Marine Sponges Agelas spp

Exploration for specialized metabolites of Okinawan marine sponges Agelas spp. resulted in the isolation of five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9-hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5). Their structures were elucidated on the basis of spectroscopic analyses. Agesasines A (1) and B (2) were assigned as rare bromopyrrole alkaloids lacking an aminoimidazole moiety, while 3–5 were elucidated to be linear bromopyrrole alkaloids with either aminoimidazolone, aminoimidazole, or N-methylated aminoimidazole moieties.


Introduction
A number of structurally unique bioactive specialized metabolites have been isolated from marine sources including sponges, algae, cnidarians, and marine microorganisms, etc. [1]. To date, more than 8000 species of marine sponges (phylum Porifera) have been found under the sea throughout tropical, temperate, and polar area [2]. Marine sponges utilize some of their specialized metabolites as chemical defenses against predator attacks, microbial infections, biofouling, and overgrowth of other sessile organisms [3,4]. On the other hand, natural products isolated from marine sponges are recognized as an attractive source of leads for therapeutic agents due to a diversity of their chemical structures and biological activities.
Marine sponges belonging to the genus Agelas are known to be a rich source of bromopyrrole alkaloids and diterpene alkaloids that have been used as a taxonomically characteristic maker [5]. In our search for structurally unique marine natural products [6][7][8], we have recently reported the isolation of diterpene alkaloids from the extracts of a marine sponge Agelas spp. [9]. As part of this research project, we have investigated another specimen of Agelas marine sponges, which resulted in the isolation of five new bromopyrrole alkaloids (1)(2)(3)(4)(5). Among others, agesasines A (1) and B (2) are rare bromopyrrole alkaloids lacking an aminoimidazole moiety, from the point of view that typical bromopyrrole alkaloids consist of a brominated pyrrolecarboxamide moiety and an aminoimidazole moiety linked through a C 3 unit. Herein, we describe the isolation and structure elucidation of 1-5.

Structure Elucidation of 1-5
Agesasine A (1) displayed ion peaks at m/z 391, 393, and 395 (1:2:1), suggesting the presence of two bromine atoms in 1. The molecular formula of 1, C 9 H 10 N 2 O 4 Br 2 , was determined by the high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 390.89045 [M + Na] + , ∆ − 0.05 mmu). The 1 H and 13 C NMR spectra (Table 1) displayed the signals of one sp 3 methine, one sp 3 methylene, one methoxy group, and one carboxy carbon as well as resonances assignable to a 2,3-dibromopyrrole carboxamide moiety (N-1~N-7). Analysis of the 1 H-1 H correlation spectroscopy (COSY) spectrum revealed the connectivities from 7-NH to 9-OH ( Figure 2), while heteronuclear multiple bond coherence (HMBC) correlations for methoxy protons and H 2 -8 with C-10 suggested the presence of a methoxy carbonyl group at C-9. Thus, the planar structure of agesasine A (1) was elucidated as shown in Figure 2. Agesasine B (2) showed an ion peak at m/z 380.9088 ([M − H] − , ∆ + 0.2 mmu), corresponding to the molecular formula of C 10 H 12 N 2 O 4 Br 2 . The 1D NMR spectra of 2 (Table 1) were closely correlated to those of 1, except for the presence of an additional sp 3 methylene signal (CH 2 -10) in 2. The methylene protons (H 2 -10) showed a 1 H-1 H COSY cross-peak with H-9 and an HMBC correlation with a methoxy carbonyl carbon (C-11), suggesting the planar structure of 2 as shown in Figure 2.  The racemic nature of agesasines A (1) and B (2) indicated by their specific rotation values being nearly zero prompted us to perform the optical resolutions of 1 and 2. The analysis of 1 using the reversed phase chiral high performance liquid chromatography (HPLC) gave a pair of peaks in the integral ratio of ca 1:1, indicating agesasine A (1) to be a racemate. Agesasine B (2) was also deduced to be a racemate, although the optical resolution could not be achieved in spite of attempts being made at various separation conditions. 9-Hydroxydihydrodispacamide (3) was obtained as a pale yellow amorphous solid. The HRESIMS showed an ion peak at m/z 443.92824 ([M -H + Na] + , Δ − 0.04 mmu), suggesting the molecular formula of C11H14N5O3Br2. The 1 H and 13 C NMR spectra of 3 (Table 2) were similar to those of a known linear bromopyrrole alkaloid, dihydrodispacamide [17], except for the presence of an oxygenated methine signal (CH-9) in 3. Therefore, 3 was deduced to be a hydroxylated derivative of dihydrodispacamide. The presence of the hydroxy group at C-9 was confirmed by 1 H-1 H COSY crosspeaks of H2-8/H-9 and H-9/H2-10 ( Figure 3). The relative configuration of 3 was not assigned in this study, while the racemic nature of 3 was confirmed by HPLC analysis with chiral column with a similar manner as for 1.  The racemic nature of agesasines A (1) and B (2) indicated by their specific rotation values being nearly zero prompted us to perform the optical resolutions of 1 and 2. The analysis of 1 using the reversed phase chiral high performance liquid chromatography (HPLC) gave a pair of peaks in the integral ratio of ca. 1:1, indicating agesasine A (1) to be a racemate. Agesasine B (2) was also deduced to be a racemate, although the optical resolution could not be achieved in spite of attempts being made at various separation conditions. 9-Hydroxydihydrodispacamide (3) was obtained as a pale yellow amorphous solid. The HRESIMS showed an ion peak at m/z 443.92824 ([M − H + Na] + , ∆ − 0.04 mmu), suggesting the molecular formula of C 11 H 14 N 5 O 3 Br 2 . The 1 H and 13 C NMR spectra of 3 (Table 2) were similar to those of a known linear bromopyrrole alkaloid, dihydrodispacamide [17], except for the presence of an oxygenated methine signal (CH-9) in 3. Therefore, 3 was deduced to be a hydroxylated derivative of dihydrodispacamide. The presence of the hydroxy group at C-9 was confirmed by 1 H-1 H COSY cross-peaks of H 2 -8/H-9 and H-9/H 2 -10 ( Figure 3). The relative configuration of 3 was not assigned in this study, while the racemic nature of 3 was confirmed by HPLC analysis with chiral column with a similar manner as for 1.  9-Hydroxydihydrooroidin (4) was obtained as a pale yellow amorphous solid. Although the 1 H and 13 C NMR spectra (Table 2) implied that 4 was a bromopyrrole alkaloid related to dihydrooroidin [17], the signals of an oxygenated methine (CH-9, δH 3.76, and δC 68.4) were observed in 4. In the 1 H-1 H COSY spectrum, the oxygenated methine proton (H-9) showed cross-peaks with H2-8 and H2-10 ( Figure 4). Based on the above findings and the molecular formula of 4, C11H14N5O2Br2, obtained by the HRESIMS (m/z 405.9510 [M] + , Δ − 0.4 mmu), 4 was assigned as 9-hydroxydihydrooroidin ( Figure  1). A nearly zero value of the specific rotation indicated 4 to be a racemate, which was supported by the fact that 4 showed no cotton effect in the electronic circular dichroism (ECD) spectrum.   9-Hydroxydihydrooroidin (4) was obtained as a pale yellow amorphous solid. Although the 1 H and 13 C NMR spectra (Table 2) implied that 4 was a bromopyrrole alkaloid related to dihydrooroidin [17], the signals of an oxygenated methine (CH-9, δ H 3.76, and δ C 68.4) were observed in 4. In the 1 H-1 H COSY spectrum, the oxygenated methine proton (H-9) showed cross-peaks with H 2 -8 and  9-Hydroxydihydrooroidin (4) was obtained as a pale yellow amorphous solid. Although the 1 H and 13 C NMR spectra (Table 2) implied that 4 was a bromopyrrole alkaloid related to dihydrooroidin [17], the signals of an oxygenated methine (CH-9, δH 3.76, and δC 68.4) were observed in 4. In the 1 H-1 H COSY spectrum, the oxygenated methine proton (H-9) showed cross-peaks with H2-8 and H2-10 ( Figure 4). Based on the above findings and the molecular formula of 4, C11H14N5O2Br2, obtained by the HRESIMS (m/z 405.9510 [M] + , Δ − 0.4 mmu), 4 was assigned as 9-hydroxydihydrooroidin ( Figure  1). A nearly zero value of the specific rotation indicated 4 to be a racemate, which was supported by the fact that 4 showed no cotton effect in the electronic circular dichroism (ECD) spectrum. 9E-Keramadine (5) displayed the 1 H and 13 C NMR spectra ( Table 2) similar to those of a known bromopyrrole alkaloid possessing a 3-bromopyrrolecarboxamide moiety, keramadine [14]. The HRESIMS revealed the molecular formula of 5 to be C12H15N5OBr, which was identical to that of keramadine. However, the 3 J (H-9/H-10) value (J = 16.1 Hz) in 5 indicated the geometry of the double bond to be E, whereas keramadine has the Z-olefin. The E-geometry was further underpinned by rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations for H-9/H-15 and H2-8/H-10 ( Figure 4). This is the first report of 9E-keramadine from a natural source, although the synthesis of 9E-keramadine has been reported to date [18].
Bromopyrrole alkaloids isolated from marine sponges have attracted the interest of researchers due to their diverse chemical structures. Various intriguing biological activities of bromopyrrole alkaloids leading drug discovery such as cytotoxic, antibacterial (antibiofilm), and protein kinase C modulating activities have been reported [19,20]. We have also reported the isolation of antimicrobial bromopyrrole alkaloids to date [6]. In this study, the antiproliferative activity of 1-5 against human cancer cell lines (HeLa, A549, and MCF7) were evaluated, showing no cytotoxicity against all cell lines (IC50 > 100 µM) (Figures S43-45).
In conclusion, five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5) were isolated from Okinawan marine sponges Agelas spp. Typical bromopyrrole alkaloids such as oroidin [12,13] and keramadine [14] consist of a mono or dibrominated pyrrolecarboxamide moiety and an aminoimidazole moiety linked through a C3 unit. In contrast, agesasines A (1) and B (2) are rare bromopyrrole alkaloids lacking an aminoimidazole moiety, whereas 1 and 2 might be artifacts during the extraction and isolation process with acidic condition. Few alkaloids with such structural feature 9E-Keramadine (5) displayed the 1 H and 13 C NMR spectra ( Table 2) similar to those of a known bromopyrrole alkaloid possessing a 3-bromopyrrolecarboxamide moiety, keramadine [14]. The HRESIMS revealed the molecular formula of 5 to be C 12 H 15 N 5 OBr, which was identical to that of keramadine. However, the 3 J (H-9/H-10) value (J = 16.1 Hz) in 5 indicated the geometry of the double bond to be E, whereas keramadine has the Z-olefin. The E-geometry was further underpinned by rotating frame nuclear Overhauser effect spectroscopy (ROESY) correlations for H-9/H-15 and H 2 -8/H-10 ( Figure 4). This is the first report of 9E-keramadine from a natural source, although the synthesis of 9E-keramadine has been reported to date [18].
Bromopyrrole alkaloids isolated from marine sponges have attracted the interest of researchers due to their diverse chemical structures. Various intriguing biological activities of bromopyrrole alkaloids leading drug discovery such as cytotoxic, antibacterial (antibiofilm), and protein kinase C modulating activities have been reported [19,20]. We have also reported the isolation of antimicrobial bromopyrrole alkaloids to date [6]. In this study, the antiproliferative activity of 1-5 against human cancer cell lines (HeLa, A549, and MCF7) were evaluated, showing no cytotoxicity against all cell lines (IC 50 > 100 µM) (Figures S43-S45).

Materials
The marine sponges Agelas spp. were collected off Kerama Islands, Okinawa, and identified by one of the authors (N.T.). The voucher specimens (SS-516 and SS-1302) were deposited in the Graduate School of Pharmaceutical Sciences, Tokushima University.

Extraction and Isolation
The marine sponges Agelas spp. SS-516 (5.22 kg, wet weight) and SS-1302 (3.42 kg, wet weight) were separately extracted with MeOH to give the extracts (197.1 and 376.3 g, respectively), each of which was partitioned with n-hexane and 90% MeOH aq. The 90% MeOH aq.

Evaluation for Antiproliferative Activity of 1-5
New bromopyrrole alkaloids 1-5 were evaluated for their antiproliferative activity against human cancer cell lines (HeLa, A549, and MCF7) according to the following procedure. The human cancer cell lines were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS). All cells were incubated at 37 • C in a humidified atmosphere with 5% CO 2 -95% air. Cells were seeded at 1 × 10 4 cells/well in a 96-well plate and preincubated for 24 h. Test samples were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 1%). After removal of the preincubated culture medium, 100 µL of medium containing various concentrations of test compound was added and further incubated for 48 h. A cell proliferation assay was performed with the Cell Counting Kit-8 (WST-8; Dojindo, Japan) according to the manufacturer's instruction. Briefly, the WST-8 reagent solution (10 µL) was added to each well of a 96-well microplate containing 100 µL of cells in the culture medium at various densities, and the plate was incubated for 2 h at 37 • C. Absorbance was measured at 450 nm using a microplate reader. Cisplatin was used as a positive control, whose IC 50 values against HeLa, A549, and MCF7 cells were 11.7, 7.2, and 52.4 mM, respectively.