**1. Introduction**

Pyrrole-imidazole alkaloids (PIAs) and simple pyrrole alkaloids represent a specific structural class of compounds isolated from sponges including those from the genus *Agelas*, *Axinella*, *Hymeniacidon*, *Phakellia* and *Stylissa* [1–5]. PIAs can be divided into monomeric and polymeric groups. Like sceptrin1, palau'amine [6], ageliferin [7], and stylissadine [8] represent members of polymeric PIAs. Biosynthesis of mono-PIAs originates from proline and lysine [9], evolving to form several skeletons such as oroidin [10], phakellin [11], ugibohlin [12], and spongiacidin types [13], which have 5/5 bicyclic, 5/6/5/5 tetracyclic, 5/6/5 tricyclic, and 5/7/5 tricyclic systems, respectively. Currently, although hundreds of PIAs have been discovered from sponges, the structural diversity of this alkaloid family, especially for the monomeric ones, is relatively conservative.

We collected the *Stylissa massa* sponge from the Xisha Islands (Paracel island) in the South China Sea. Targeted isolation for methanol extraction yielded twelve new and four known compounds. Five biosynthetic-related PIA skeletons including the 5/7 imidazoleacyclic compound **1**, 5/7/5 spongiacidins (**2**–**5**, and **13**–**15**), 5/6/5/5 phakellins (**6** and **16**), 5/7 bicyclic (**<sup>7</sup>**–**9**), and pyrrole single ring alkaloids (**10**–**12**) were obtained from *Stylissa massa*. Compound **1** is the first identified precursor metabolite of the classic 5/7/5 tricyclic skeleton with unesterified guanidine and carboxyl groups. Compounds **2a** and **2b** are a scalemic mixture with a hydroxyl group positioned at C-9. Compounds **3a** and **3b** are also scalemic mixture compounds with the *E*-configuration of Δ10,11 versus the

**Citation:** Wang, Q.; Gao, C.; Wei, Z.; Tang, X.; Ji, L.; Luo, X.; Peng, X.; Li, G.; Lou, H. A Series of New Pyrrole Alkaloids with ALR2 Inhibitory Activities from the Sponge *Stylissa massa*. *Mar. Drugs* **2022**, *20*, 454. https://doi.org/10.3390/ md20070454

Academic Editor: Asunción Barbero

Received: 20 June 2022 Accepted: 8 July 2022 Published: 12 July 2022

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*Z*-configuration of **2**. Compound **4** has a hydroxide at C-9 with a double bond Δ9,10 at a different position, and the new compound **5** has an extra methyl group at 13-NH.

Guanidine compounds often exhibit effective diabetes related activities [14]. ALR2 is a key limiting enzyme of the glucose polyol metabolic pathway, which is the key targe<sup>t</sup> for the treatment of diabetes complications [15]. In the ALR2 enzyme activity assay in vitro, spongiacidin-type PIAs (compounds **2**–**5**, and **13**–**15**) presented superior inhibitory activities than the other skeletons at 20 μM. The IC50 values ranged from 8.6 to 13.6 μM, respectively (Figure S23). The SPR experiments verified the interaction between ALR2 and compound **14**, with the KD value of 6.9 μM. In summary, spongiacidin-PIAs are efficient ALR2-targeted inhibitors with 5/7/5 tricyclic skeletons. Thus, the structural elucidation and the ALR2 inhibitory activities of these PIAs are concluded below (Figure 1).

**Figure 1.** The structures of compounds **1**–**16**.
