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

Abstract

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.

1. 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 (1–5). 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 C₃ unit. Herein, we describe the isolation and structure elucidation of 1–5.

2. Results and Discussion

2.1. Isolation of 1–5 from Marine Sponges Agelas spp.

Two specimens of the marine sponge Agelas spp. (SS-516 and SS-1302) were separately extracted with MeOH to give extracts, each of which was partitioned between n-hexane and 90% MeOH aq. Repeated chromatographic separations of the 90% MeOH aq.-soluble materials from SS-516 gave two new bromopyrrole alkaloids, agesasines A (1, 2.5 mg) and B (2, 2.2 mg) (Figure 1) together with two known bromopyrrole alkaloids, tauroacidin A [10] and taurodispacamide A [11]. In contrast, the 90% MeOH aq.-soluble materials of SS-1302 were further partitioned with n-BuOH and water. The n-BuOH-soluble materials were separated by column chromatographies to give three new bromopyrrole alkaloids, 9-hydroxydihydrodispacamide (3, 5.0 mg), 9-hydroxydihydrooroidin (4, 2.1 mg), and 9E-keramadine (5, 3.1 mg) (Figure 1), together with four known alkaloids, oroidin [12,13], keramadine [14], 2-bromo-9,10-dihydrokeramadine [15], and nagelamide L [16].

2.2. 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, C₉H₁₀N₂O₄Br₂, was determined by the high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 390.89045 [M + Na]⁺, Δ − 0.05 mmu). The ¹H and ¹³C NMR spectra (

Table 1. One-dimensional (1D) NMR data for agesasines A (1) and B (2) in DMSO-d₆.

Position	1		2
	13C	1H (J in Hz)	13C	1H (J in Hz)
1	–	12.67 (brs)	–	12.65 (brs)
2	104.8	–	104.7	–
3	98.0	–	98.0	–
4	113.1	6.93 (brs)	113.0	6.93 (d, 2,7)
5	128.1	–	128.3	–
6	159.3	–	159.3	–
7	–	8.20 (t, 5.8)	–	8.12 (t, 5.5)
8	42.7	3.46, 3.36 (each 1 H, m)	44.9	3.20 (2 H, m)
9	69.3	4.17 (q, 6.1)	66.6	3.99 (m)
10	173.1	–	40.6	2.49 (m), 2.27 (dd, 15.2, 8.8)
11	–	–	171.8	–
9-OH	–	5.71 (d, 5.9)	–	nd
OMe	51.8	3.61 (3 H, brs)	51.4	3.56 (3 H, brs)

nd: Not detected.

) displayed the signals of one sp³ methine, one sp³ 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 ¹H-¹H 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 H₂-8 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]⁻, Δ + 0.2 mmu), corresponding to the molecular formula of C₁₀H₁₂N₂O₄Br₂. The 1D NMR spectra of 2 (Table 1) were closely correlated to those of 1, except for the presence of an additional sp³ methylene signal (CH₂-10) in 2. The methylene protons (H₂-10) showed a ¹H-¹H 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 ¹H and ¹³C NMR spectra of 3 (

Table 2. 1D NMR data for 9-hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5) in DMSO-d₆.

Position	3		4		5
	13C	1H (J in Hz)	13C	1H (J in Hz)	13C	1H (J in Hz)
1	–	12.66 (brs)	–	12.66 (brs)	–	11.83 (brs)
2	104.7	–	104.8	–	121.6	6.98 (dd, 2.9, 1.6)
3	98.0	–	98.2	–	95.2	–
4	113.1	6.94 (d, 2.8)	113.2	6.86 (s)	111.8	6.92 (s)
5	128.3	–	128.4	–	126.9	–
6	159.3	–	159.4	–	159.7	–
7	–	8.15 (t, 5.9)	–	8.19 (t, 5.6)	–	8.40 (t, 5.5)
8	45.3	3.18 (2 H, m)	44.8	3.23 (m), 3.16 (m)	40.4	3.99 (2 H, t, 5.5)
9	66.3	3.79 (m)	68.4	3.76 (m)	130.8	6.19 (dt, 16.1, 5.5)
10	34.8	1.96 (ddd, 14.4, 5.5, 2.6) 1.71 (ddd, 14.4, 10.9, 5.5)	30.1	2.57 (dd, 15.2, 4.2) 2.40 (dd, 15.2, 7.8)	115.3	6.30 (d, 16.1)
11	56.8	4.34 (t, 5.5)	124.3	–	126.6	–
12	–	9.47 (brs)	–	11.95 (brs)
13	158.2	–	147.1	–	146.9	–
14	–	nd	–	11.87 (brs)	–	12.35 (brs)
15	175.6	–	110.1	6.58 (brs)	109.4	7.14 (brs)
N-Me					29.8	3.38 (3 H, s)
13-NH2	–	nd	–	7.35 (2 H, brs)	–	7.71 (2 H, brs)

nd: Not detected.

) 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 ¹H-¹H COSY cross-peaks 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.

9-Hydroxydihydrooroidin (4) was obtained as a pale yellow amorphous solid. Although the ¹H and ¹³C 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 ¹H-¹H 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 ¹H and ¹³C 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 ³J (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–S45).

In conclusion, five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9-hydroxydihydrodispacamide (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 have been isolated from marine sponges Agelas spp. collected off the South China Sea [21,22].

3. Materials and Methods

3.1. General Procedures

Optical rotations were obtained on a JASCO P-2200 digital polarimeter (JASCO Co., Tokyo, Japan). UV spectra were recorded on a Hitachi U-3900H spectrophotometer (Hitachi, Ltd., Tokyo, Japan). NMR spectra were measured by a Bruker AVANCE-500 instrument (Bruker, Billerica, MA, USA) using tetramethylsilane as an internal standard. HRESIMS were recorded on a Waters LCT PREMIER 2695 (Waters Co., Milford, MA, USA) and a JEOL JMS-T100LP (JEOL, Ltd., Tokyo, Japan). Column chromatography was performed with silica gel 60 N (Kanto Kagaku, Tokyo, Japan) and Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan). Medium pressure liquid chromatography (MPLC) was carried out on Toyopearl HW-40F (TOSOH Co., Tokyo, Japan), MCI gel CHP20P (Mitsubishi Chemical, Tokyo, Japan), and Biotage SNAP Cartridge KP-C18-HS (Biotage, Uppsala, Sweden).

3.2. 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.

3.3. 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.-soluble materials of SS-516 were separated by column chromatography on Diaion HP-20 (MeOH/H₂O, 0:100–100:0) to give six fractions (frs. 1–6). Fr. 3 was subjected to silica gel column chromatography (CHCl₃/MeOH/TFA, 95:5:0.1–80:20:0.1) to yield 12 fractions (frs. 3.1–3.12). Fr. 3.7 was applied to ODS MPLC (MeCN/H₂O/TFA, 5:95:0.1–80:20:0.1), and then purified by ODS HPLC (YMC Hydrosphere C18, ϕ20 × 250 mm, MeCN/H₂O/TFA, 35:65:0.1) to furnish agesasines A (1, 2.5 mg) and B (2, 2.2 mg). Separation of fr. 3.11 by ODS MPLC (MeCN/H₂O/TFA, 5:95:0.1–80:20:0.1) gave five fractions (frs. 3.11.1–3.11.5). Tauroacidin A (124.1 mg) and taurodispacamide A (34.5 mg) were purified from fr. 3.11.3 by ODS MPLC (MeCN/H₂O/TFA, 20:80:0.1).

The 90% MeOH aq.-soluble materials of SS-1302 were further partitioned between n-BuOH and water. The n-BuOH-soluble materials (58.0 g) were applied to silica gel column chromatography (CHCl₃/MeOH/TFA, 9:1:0.1–5:5:0.1) to give six fractions (frs. 1′–6′) including oroidin (17.1 g, fr. 3′). Fr. 4′ was subjected to MPLC on a Toyopearl HW-40F column (MeOH/H₂O/TFA, 10:90:0.1–90:10:0.1), an MCI gel CHP 20P column (MeOH/H₂O/TFA, 10:90:0.1–90:10:0.1) to yield seven fractions (frs. 4′.4.1–4′.4.7). Fr. 4′.4.3 was loaded to MPLC on an ODS column (MeCN/H₂O/TFA, 10:90:0.1–60:40:0.1) to give six fractions (frs. 4′.4.3.1–4′.4.3.6), and then fr. 4′.4.3.3 was purified by ODS HPLC (COSMOSIL 5C₁₈-MS-Ⅱ, ϕ 20 × 250 mm, MeCN/H₂O/TFA, 17:83:0.1). Further purification of fr. 4′.4.3.3.2 on ODS HPLC (YMC Hydrosphere C18, ϕ 10 × 250 mm, MeCN/H₂O/TFA, 13:87:0.1) afforded 9-hydroxydihydrodispacamide (3, 5.0 mg), 9E-keramadine (5, 3.1 mg), and keramadine (6.7 mg). 9-Hydroxydihydrooroidin (4, 2.1 mg) was isolated from fr. 4′4.3.3.3 by ODS HPLC (YMC Hydrosphere C18, ϕ 10 × 250 mm, MeCN/H₂O/TFA, 13:87:0.1). Fr. 4′.4.4 was subjected to ODS MPLC (MeCN/H₂O/TFA, 10:90:0.1–50:50:0.1) and then ODS HPLC (YMC Hydrosphere C18, ϕ 10 × 250 mm, MeCN/H₂O/TFA, 15:85:0.1) to furnish 2-bromo-9,10-dihydrokeramadine (2.1 mg). Fr. 5′ was applied to MPLC on a Toyopearl HW-40F column (MeOH/H₂O/TFA, 10:90:0.1–90:10:0.1) to give eight fractions (frs. 5′.1–5′.8). Fr. 5′ was passed through an MCI gel CHP 20P column (MeOH/H₂O/TFA, 10:90:0.1–100:0:0.1) and an ODS column (MeOH/H₂O/TFA, 10:90:0.1–0:10:0.1) to afford nagelamide L (187.5 mg). Tauroacidin A and nagelamide L did not show optical rotations.

Agesasine A (1): Pale yellow amorphous solid; [α]${}_{\mathrm{D}}^{28}$ 0 (c 0.10, MeOH); UV (MeOH) λ_max 275 (ε 4900) nm; ¹H and ¹³C NMR data (Table 1); ESIMS: m/z 391, 393, and 395 (1:2:1), [M + Na]⁺; HRESIMS: m/z 390.89045 [M + Na]⁺ (calcd for C₉H₁₀N₂O₄Na⁷⁹Br₂, 390.89050).

Agesasine B (2): Pale yellow amorphous solid; [α]${}_{\mathrm{D}}^{28}$ 0 (c 0.10, MeOH); UV (MeOH) λ_max 274 (ε 3100) nm; ¹H and ¹³C NMR data (Table 1); ESIMS: m/z 381, 383, and 385 (1:2:1), [M − H]⁻; HRESIMS: m/z 380.9088 [M − H]⁻ (calcd for C₁₀H₁₁N₂O₄⁷⁹Br₂, 380.9086).

9-Hydroxydihydrodispacamide (3): Pale yellow amorphous solid; [α]${}_{\mathrm{D}}^{27}$ 0 (c 0.10, MeOH); UV (MeOH) λ_max 223 (ε 3900) and 275 (3400) nm; ¹H and ¹³C NMR data (Table 2); ESIMS: m/z 444, 446, and 448 (1:2:1), [M − H + Na]⁺; HRESIMS: m/z 443.92824 [M − H + Na]⁺ (calcd for C₁₁H₁₃N₅O₃Na⁷⁹Br₂, 443.92828).

9-Hydroxydihydrooroidin (4): Pale yellow amorphous solid; [α]${}_{\mathrm{D}}^{27}$ 0 (c 0.10, MeOH); UV (MeOH) λ_max 276 (ε 3900) nm; ¹H and ¹³C NMR data (Table 2); ESIMS: m/z 406, 408, and 410 (1:2:1), [M]⁺; HRESIMS: m/z 405.9510 [M]⁺ (calcd for C₁₁H₁₄N₅O₂⁷⁹Br₂, 405.9514).

9E-Keramadine (5): Pale yellow amorphous solid; UV (MeOH) λ_max 271 (ε 3300) nm; ¹H and ¹³C NMR data (Table 2); ESIMS: m/z 324 and 326 (1:1), [M]⁺; HRESIMS: m/z 324.04592 [M]⁺ (calcd for C₁₂H₁₅N₅O⁷⁹Br, 324.04600).

3.4. Optical Resolutions of 1–3

Optical resolutions of agesasine A (1) and 9-hydroxydihydrodispacamide (3), were performed on chiral HPLC (Chiral ART Cellulose-SB, YMC, ϕ 4.6 × 250 mm, flow rate 0.5 mL/min, UV detection 254 nm) at 35 °C with elution of MeOH/MeCN/H₂O/H₃PO₄ (30:10:60:0.1 for 1; 8:2:90:0.1 for 3) to give enantiomers in the integral ratio of ca. 1:1 (t_R 27.5 and 29.0 min for 1; t_R 12.5 and 14.3 min for 3) in each case. The separations of enantiomers were confirmed by MS analyses. Separated peaks for enantiomers of agesasine B (2) could not be obtained in any condition in this study.

3.5. 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% CO₂–95% air. Cells were seeded at 1 × 10⁴ 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 IC₅₀ values against HeLa, A549, and MCF7 cells were 11.7, 7.2, and 52.4 mM, respectively.