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

**Sanghoon Lee 1,2, Naonobu Tanaka 1,\*, Sakura Takahashi 1, Daisuke Tsuji 1, Sang-Yong Kim 3, Mareshige Kojoma 3, Kohji Itoh 1, Jun'ichi Kobayashi <sup>4</sup> and Yoshiki Kashiwada 1,\***


Received: 29 June 2020; Accepted: 27 August 2020; Published: 30 August 2020

**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 9*E*-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.

**Keywords:** agesasines; bromopyrrole alkaloid; marine sponge; *Agelas*

#### **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 [6–8], 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 9*E*-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].

**Figure 1.** Structures of agesasines A (**1**) and B (**2**), 9-hydroxydihydrodispacamide (**3**), 9-hydroxydihydrooroidin (**4**), and 9*E*-keramadine (**5**).

#### *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]+, <sup>Δ</sup> <sup>−</sup> 0.05 mmu). The 1H and 13C NMR spectra (Table 1) 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.


**Table 1.** One-dimensional (1D) NMR data for agesasines A (**1**) and B (**2**) in DMSO-*d*6.

**Figure 2.** Key two-dimensional (2D) NMR correlations for agesasines A (**1**) and B (**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 *<sup>m</sup>*/*<sup>z</sup>* 443.92824 ([M <sup>−</sup> <sup>H</sup> <sup>+</sup> Na]+, <sup>Δ</sup> <sup>−</sup> 0.04 mmu), suggesting the molecular formula of C11H14N5O3Br2. The 1H and 13C 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 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**.

**Figure 3.** Selected 2D NMR correlations for 9-hydroxydihydrodispacamide (**3**).


**Table 2.** 1D NMR data for 9-hydroxydihydrodispacamide (**3**), 9-hydroxydihydrooroidin (**4**), and 9*E*-keramadine (**5**) in DMSO-*d*6.

nd: Not detected.

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<sup>H</sup> 3.76, and δ<sup>C</sup> 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*/*<sup>z</sup>* 405.9510 [M]+, <sup>Δ</sup> <sup>−</sup> 0.4 mmu), **<sup>4</sup>** 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.

**Figure 4.** Selected 2D NMR correlations for 9-hydroxydihydrooroidin (**4**) and 9*E*-keramadine (**5**).

9*E*-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 <sup>3</sup>*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 9*E*-keramadine from a natural source, although the synthesis of 9*E*-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 9*E*-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**
