**1. Introduction**

The ocean has the characteristics of high salinity, high pressure, low temperature, low oxygen content, and oligotrophic environment, which enables microorganisms to have unique metabolic adaptation mechanisms and produce natural products with novel structures and diverse bioactivities [1]. Marine-derived fungi have been found to be a rich source of natural products due to their complex genetic background and abundant metabolites [2]. In recent years, a large number of novel secondary metabolites, such as polyketides, alkaloids, terpenes, steroids, peptides, etc., have been discovered from marine-derived *Aspergillus* species [3], and showed diverse bioactivities like antibacterial, antitumor, antioxidant, and anti-inflammatory activities [4]. More than 80% natural products were directly or indirectly related to small molecule drugs for the treatment of various diseases in the last 30 years, and many marine alkaloids with bioactivities have been comprehensively studied for drug development [5,6].

**Citation:** Li, P.; Zhang, M.; Li, H.; Wang, R.; Hou, H.; Li, X.; Liu, K.; Chen, H. New Prenylated Indole Homodimeric and Pteridine Alkaloids from the Marine-Derived Fungus *Aspergillus austroafricanus* Y32-2. *Mar. Drugs* **2021**, *19*, 98. https://doi.org/10.3390/md19020098

Academic Editor: Asunción Barbero

Received: 16 January 2021 Accepted: 5 February 2021 Published: 9 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In our previous study, a series of fungal secondary metabolites were isolated and characterized with antitumor or cardiovascular effects [7,8]. To discover more natural products with pharmacological activities from marine-derived fungi, the fungal strain *Aspergillus austroafricanus* Y32-2 has been isolated from a seawater sample collected from the Indian Ocean. Chemical investigation of the secondary metabolites of Y32-2 fermented on rice medium resulted in the isolation of fourteen compounds, including two new prenylated indole alkaloid homodimers and one new pteridine alkaloid, named di-6- hydroxydeoxybrevianamide E (**1**), dinotoamide J (**2**) and asperpteridinate A (**3**), along with eleven known compounds (**4**–**14**) [7,9–16] (Figure 1). Among them, compound **4** was isolated for the first time as a natural product.

**Figure 1.** Structures of Compounds **1**–**14**.

The prenylated indole alkaloids contain a bicyclo[2.2.2]diazaoctane or diketopiperazine ring, and has been reported to have antitumor, antibacterial, and insecticidal activities [17]. Here two new prenylated indole alkaloid homodimers and other isolated compounds were all tested for pro-angiogenic and anti-inflammatory effects in zebrafish models and cytotoxicity towards HepG2 human liver carcinoma cells. Compounds **2**, **4**, **5**, **7**, and **10** exhibited angiogenesis promoting activity in a dose-dependent manner. Compounds **7**, **8**, **10**, and **11** also displayed anti-inflammatory activity in a dose-dependent

manner. In addition, compound **6** showed cytotoxicity against HepG2 cells. In this paper, the isolation, structure elucidation, and bioactivity of all isolated compounds are reported.

#### **2. Results and Discussion**

*2.1. Structure Elucidation*

Compound **1**, obtained as yellow amorphous powder, possessed a molecular formula of C42H48N6O6 by the negative HR-ESI-MS (*m/z* 731.3559 [M − H]<sup>−</sup>, calculated 731.3557), requiring 22 unsaturations. The HPLC chromatographic behavior of **1** was unusual and existed always as a 1:1 inseparable mixture. Many of the NMR signals also appeared in pairs, hinting towards structural distinctiveness and complexity. The 1H NMR spectrum (Table 1) in DMSO-*d*6 of **1** showed two pairs of mutually coupled aromatic protons at *δ*H 7.35, 7.37 (each H, d, *J* = 8.4 Hz) and 6.80, 6.81 (each H, d, *J* = 8.4 Hz), a set of vinyl proton signals at *δ*H 6.15, 6.17 (each H, dd, *J* = 17.5, 10.7 Hz), 5.06 (2H, br d, *J* = 17.5 Hz) and 5.01 (2H, br d, *J* = 10.7 Hz), four methyl singlets at *δ*H 1.42 (12H, s), as well as six active hydrogen signals at *δ*H 9.09 (2H, br s), 8.45, 8.58 (each H, s) and 6.21, 6.30 (each H, s). The 13C NMR spectrum (Table 1) showed four amidocarbonyl carbon signals at *δ*C 169.3, 169.4 (C-18, 18-) and 165.6 (C-12, 12-, overlapped), twenty aromatic or olefinic carbon signals, containing four vinyl carbons at *δ*C 146.4, 146.5 (C-21, 21-) and 111.29, 111.33 (C-20, 20-), and four nitrogen-bearing methine signals at *δ*C 58.5 (C-17, 17-, overlapped) and 55.0, 55.3 (C-11, 11-). The above NMR features were similar to those of 6-hydroxydeoxybrevianamide E [18], a cyclic dipeptide produced by *Aspergillus* and *Penicillium* species, and a careful and rigorous analysis of the 1H, 1H-COSY and HMBC correlations (Figure 2) also supported this inference. However, there were two obvious differences in their NMR signals: (1) different substitution patterns on the indole ring; (2) most of the NMR signals in **1** appeared in pairs. Based on the HSQC and HMBC correlation, the C-7, 7- (*δ*C 104.0, 104.1) were aromatic quaternary carbon signals that were different from 6-hydroxydeoxybrevianamide E, confirmed that the positions C-7, 7- of the indole ring were substituted. Considering its molecular formula, compound **1** was deduced as a dimer of 6-hydroxydeoxybrevianamide E via C-7 and C-7-. Due to a certain steric hindrance, the structure existed as a 1:1 mixture of inseparable rotamers. The relative configuration of the cyclic dipeptide moiety was determined by the NOESY correlation (Figure 2) of H-11 and H-17. Based on the relative configuration, two probable forms of its absolute configuration, 1a (11*S*, 17*S*, 11-*S*, 17-*S*) and 1b (11*R*, 17*R*, 11-*R*, 17-*R*), were respectively used for the ECD calculations, and the absolute configuration was assigned as 11*S*, 17*S*, 11-*S*, 17-*S* (Figure 3), which was in consistent with that of 6-hydroxydeoxybrevianamide E. Therefore, the structure of **1** was unequivocally established as shown in Figure 1 and named as di-6-hydroxydeoxybrevianamide E.




**Table 1.** *Cont.*

**Figure 2.** The 1H, 1H-correlation spectroscopy (1H, 1H-COSY), key heteronuclear multiple-bond correlation spectroscopy (HMBC) and nuclear overhauser effect spectroscopy (NOESY) correlations of compounds **1**, **2** (only half showed) and **3**.

**Figure 3.** Measured CD and calculated equivalent circulating density (ECD) curves of compounds **1** (**A**), **2** (**B**) and **3** (**C**).

Compound **2** was obtained as a yellow amorphous powder. The molecular formula was determined to be C42H48N6O8 by the negative HRESIMS (*m/z* 763.3440 [M − H]<sup>−</sup>, calculated 763.3456), indicating 22 degrees of unsaturation. The NMR spectra (Table 1) of **2** revealed 24 proton and 21 C-atom signals, suggesting **2** to be a symmetrical homodimer. The 1H NMR spectrum for **2** showed two aromatic proton signals at *δ*H 7.11 (1H, d, *J* = 8.2 Hz) and 6.39 (1H, d, *J* = 8.2 Hz), three vinyl proton signals at *δ*H 4.94 (1H, br d, *J* = 17.5 Hz), 5.00 (1H, br d, *J* = 10.9 Hz) and 6.15 (1H, dd, *J* = 17.5, 10.9 Hz), two methyl singlets at *δ*H 0.96 (3H, s), 0.98 (3H, s), as well as three active hydrogen signals at *δ*H 7.57 (1H, s), 9.28 (1H, br s), and 9.31 (1H, s). The 13C NMR data (Table 1) revealed the presence of three carbonyl carbon signals at *δ*C 180.1 (C-2), 169.8 (C-18) and 165.6 (C-12), eight aromatic or olefinic carbon signals containing two vinyl carbons at *δ*C 143.9 (C-21) and 112.6 (C-20), and two nitrogen-bearing methines at *δ*C 58.3 (C-17) and 52.6 (C-11). Extensive comparison of the above NMR spectra with those of notoamide J [17] revealed that both structures were very similar, except for the substitution patterns on the C-7 position of indole ring. Considering its molecular formula, compound **2** was also identified as a homodimer of notoamide J via C-7 and C-7-. Due to one single signal set in the NMR spectrum, one single peak in the chiral column chromatography and less steric hindrance in the structure than compound **1**, it was deduced to be a freely rotating homologous dimer. With the aid of the 1H, 1H-COSY, HSQC and HMBC correlations, the planar structure of **2** was established as shown (Figure 1). The relative configuration of the cyclic dipeptide moiety was deduced by a NOESY correlation between H-11 and H-17, suggested that both protons had the same co-facial orientation. Because of the similar NMR data between **2** and notoamide J, the relative configuration of the positions C-3, C-11 and C-17 were determined to be similar to that of notoamide J [17]. By comparison of the experimental and calculated ECD spectra of **2**, the absolute configuration was tentatively assigned as 3*R*, 11*S*, 17*S*, 3-*R*, 11-*S*, and

17- *S* (Figure 3), which was also probably verified by the identical CD spectrum between **2** and notoamide J. So, the structure of **2** was tentatively assigned as shown in Figure 1 and named as dinotoamide J.

Asperpteridinate A was obtained as a yellow amorphous powder. The molecular formula C20 H18 N4O8 was assigned on the basis of the HRESIMS peak at *m*/*z* 465.1018 [M + Na]+ (calcd. 465.1023), requiring 14 degrees of unsaturation. The 1H NMR spectrum of **3** showed the signals for a 1,2,4-trisubstituted benzene ring system at *δ*H 7.49 (1H, d, *J* = 1.4 Hz), 7.10 (1H, d, *J* = 8.3 Hz) and 7.65 (1H, dd, *J* = 8.3, 1.4 Hz), one vinyl proton at *δ*H 8.94 (1H, s), three O-methyl or N-methyl at *δ*H 3.74 (3H, s), 3.53 (3H, s), 3.31 (3H, s), one O-methylene at *δ*H 5.49 (2H, s), one methyl at *δ*H 1.90 (3H, s). The 13C NMR data (Table 2) revealed the presence of four carbonyl at *δ*C 150.6 (C-2), 159.7 (C-4), 164.8 (C-7-), 166.4 (C-3-'), ten aromatic or olefinic carbons, containing four vinyl carbons at *δ*C 145.8 (C-6), 147.2 (C-7), 147.7 (C-9), 127.2 (C-10), one O-methyl at *δ*C 53.5 (O-CH3), one O-methylene at *δ*C 64.7 (O-CH2-). 1H and 13C NMR (Table 2) spectra analysis revealed that some signals of **3** was similar to that of compound **4** [9] and 2, 2-dimethyl-1, 3-dioxa-benzo[d]pentane-6- carboxylic acid [19]. With the aid of the 1H, 1H-COSY, HSQC, and HMBC correlations, the structure of **3** was established as shown (Figures 1 and 2). The absolute configuration of **3** at C-2" was also determined as 2" *R* by ECD calculations (Figure 3).


**Table 2.** 400 MHz 1H and 150 MHz 13C NMR data of compound **3** in DMSO-*d*6.
