Bioactive Secondary Metabolites of Marine Fungi

Edited by Hee Jae Shin

mdpi.com/journal/marinedrugs

## **Bioactive Secondary Metabolites of Marine Fungi**

## **Bioactive Secondary Metabolites of Marine Fungi**

Editor

**Hee Jae Shin**

Basel • Beijing • Wuhan • Barcelona • Belgrade • Novi Sad • Cluj • Manchester

*Editor* Hee Jae Shin Korea Institute of Ocean Science and Technology (KIOST) Busan, Republic of Korea

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Marine Drugs* (ISSN 1660-3397) (available at: https://www.mdpi.com/journal/marinedrugs/ special issues/MarineFungi2022).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Lastname, A.A.; Lastname, B.B. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-9086-8 (Hbk) ISBN 978-3-0365-9087-5 (PDF) doi.org/10.3390/books978-3-0365-9087-5**

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) license.

## **Contents**


Reprinted from: *Mar. Drugs* **2023**, *21*, 293, doi:10.3390/md21050293 ................. **163**

### **Olesya I. Zhuravleva, Ekaterina A. Chingizova, Galina K. Oleinikova, Sofya S. Starnovskaya, Alexandr S. Antonov, Natalia N. Kirichuk, et al.**


### **About the Editor**

#### **Hee Jae Shin**

Hee Jae Shin is a principal research Scientist at the Department of Marine Biotechnology and Bioresource Research, Korea Institute of Ocean Science and Technology (KIOST) and a professor at the University of Science and Technology. He received his PhD from the University of Tokyo (1997), where he studied the isolation and structure determination of protease inhibitors from cyanobacteria. He undertook two post-doctoral positions at the Marine Biotechnology Institute, Japan (1997–1999) and the center for marine biotechnology and biomedicine, Scripps Institution of Oceanography with Professor William Fenical (1999–2000). He spent 3 years working in the pharmaceutical industry (2000–2003) and then returned to the Korea Ocean Research and Development Institute, which is now KIOST. His research interest is on the isolation and structure determination of bioactive marine natural products from marine microorganisms including fungi, actinomycetes, deep-sea and symbiotic microorganisms, and discovery and development of drug candidates.

### **Preface**

Marine fungi can be isolated from marine animals, plants, sediments, and seawater. Due to the complex marine environments, marine fungal metabolites have novel structures and diverse activities. Over 1,500 species of marine fungi, including about 530 species of obligate marine fungi, are known. Marine fungi are important sources of biologically active natural products due to their ability to produce secondary metabolites with novel structures and pharmacological activities. Over recent decades, pharmaceutical and medical applications of marine fungi have been explored, and new drugs from relatively underexplored sources are essential. Halimide (phenylahistin), a naturally occurring fungal natural product with a diketopiperazine structure isolated from *Aspergillus ustus*, is being studied in a Phase 3 clinical trial for the treatment of non-small-cell lung cancer (NSCLC). Its synthetic analog, plinabulin (NPI 2358), is being developed by BeyondSpring Pharmaceuticals, and a New Drug Application (NDA) has been submitted in the United States and China for its use in the treatment of NSCLC and chemotherapy-induced neutropenia (CIN).

This book provides details about the isolation, structure determination, and bioactivities of marine fungal natural products. Hence, bioactive secondary metabolites from marine fungi are important for academic research, pharmaceutical, nutraceutical, and biomedical industries. I would like to acknowledge *Marine Drugs* for their encouragement and suggestions to get this wonderful compilation related to the special issue "Bioactive Secondary Metabolites of Marine Fungi". I would also like to sincerely thank all the contributors for their high-quality manuscripts, support, and advice.

> **Hee Jae Shin** *Editor*

### *Review* **Natural Products from Chilean and Antarctic Marine Fungi and Their Biomedical Relevance**

**Dioni Arrieche 1,†, Jaime R. Cabrera-Pardo 2,†, Aurelio San-Martin 3, Héctor Carrasco 4,\* and Lautaro Taborga 1,\***


**Abstract:** Fungi are a prolific source of bioactive molecules. During the past few decades, many bioactive natural products have been isolated from marine fungi. Chile is a country with 6435 Km of coastline along the Pacific Ocean and houses a unique fungal biodiversity. This review summarizes the field of fungal natural products isolated from Antarctic and Chilean marine environments and their biological activities.

**Keywords:** marine natural products; marine fungi; Chilean marine fungi; biological activities

#### **1. Introduction**

Natural products (NPs) represent a rich and vast biologically relevant chemical space that remains extremely difficult to access with the current arsenal of tools in chemical synthesis [1]. NPs are characterized by enormous scaffold diversity and structural complexity. Nature, via evolution, has optimized secondary metabolites to serve pivotal biological functions, including endogenous defense mechanisms as well as interaction with other organisms [2]. Natural products-based medicines can be traced back thousands of years and still contribute to many approved drugs. Indeed, natural products and their derivatives represented 27% of all therapeutics approved by the FDA between 1981 and 2019 [3,4]. In recent years, this proportion has increased, illustrating the continued importance of NPs. Research programs focused on unveiling new NPs from understudied microorganisms, such as fungi isolated from Chilean marine environments, are crucial to the future drug development pipeline.

Fungi represent one of the largest groups of organisms. They are widely distributed across both mild and extreme ecosystems on our planet [5]. They have developed a unique metabolic plasticity, allowing them to rapidly adapt and survive through the biosynthesis of an array of fascinating natural products [6]. A recent analysis of fungal genomes has revealed many secondary metabolite pathways that can be tuned or modified, producing novel and valuable chemical scaffolds [7]. Fungal-derived natural products are pharmaceutically abundant, with several important biological applications ranging from highly potent toxins to approved drugs [8]. Since the discovery of penicillin, an antibiotic of fungal origin, many efforts around the globe have been devoted to searching for fungal-derived bioactive products. Fungi are a vast yet untapped source to search for pharmaceutically relevant molecules displaying a range of bioactivity, including anticancer, antioxidant, hepatoprotective, antibacterial, antidiabetic, and anti-inflammatory capabilities.

**Citation:** Arrieche, D.;

Cabrera-Pardo, J.R.; San-Martin, A.; Carrasco, H.; Taborga, L. Natural Products from Chilean and Antarctic Marine Fungi and Their Biomedical Relevance. *Mar. Drugs* **2023**, *21*, 98. https://doi.org/10.3390/ md21020098

Academic Editor: Hee Jae Shin

Received: 16 December 2022 Revised: 23 January 2023 Accepted: 26 January 2023 Published: 29 January 2023

**Copyright:** © 2023 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/).

Oceans are the source of a wide variety of natural products with unique structures mainly produced by marine macro-organisms, such as invertebrates (e.g., sponges, soft corals, tunicates) and algae. Additionally, many marine natural products have proved to be pharmacologically relevant [9–11].

Secondary metabolites obtained from marine fungi have been particularly interesting, mainly because of their unique chemical structures and biomedical applications [8,12]. In 1949, cephalosporin C was discovered from a culture of *Cephalosporium* fungus species obtained from the Sardinian coast [13]. Since then, extensive efforts over decades of work have revealed the vast chemical and biological potential of marine fungal natural products. Strains of marine fungi have been obtained from practically every possible marine habitat, including inorganic, marine microbial communities, marine plants, and marine vertebrates [10]. While the number of cultivable marine fungi is extremely low (1% or less) compared to their global biodiversity [8–10], the number of natural products that have been isolated and characterized from marine fungi exceeds 1000 molecules [14]. These include alkaloids, lipids, peptides, polyketides, prenylated polyketides, and terpenoids [14–17].

Chile has 6435 km of coastline and exercises exclusive rights over its maritime space called the Chilean Sea. This comprises four zones: the Territorial Sea (120,827 km2), the contiguous zone (131,669 km2), the exclusive economic zone (3,681,989 km2), and that corresponding to the Continental shelf (161,338 km2) [18].

The Chilean maritime territory, in the Pacific Ocean, consists of highly structured geographic sections displaying unique features that arise from the interactions of water masses with the seabed, emerged relief, air masses and centers of atmospheric action [19]. These phenomena lead to an environment suitable for a rich biodiversity ranging from microscopic organisms that swarm the waters in incredible numbers to large fish and other organisms [20]. Along the lengthy coastline, Chilean waters also differ in terms of important characteristics, e.g., mineral and saline composition [19].

The cold waters associated with both the Humboldt and Antarctic currents are characterized by a high gas and nitrogen content, unlike in temperate and warm waters. Consequently, phytoplankton is abundant in the Chilean sea and supports the growth of various marine organisms, specifically fungi. Therefore, Chile´s coastline provides a distinct environment for fungal biodiversity to flourish [21].

Despite its importance, there are not many reports about secondary metabolites from marine fungi or marine-derived fungi in Chile. Reports included in this review cover the period from 1996 until present. In this work, we have made a comprehensive review of compounds that have been isolated and chemically characterized during this time. Their biological activities are also reported.

#### **2. Secondary Metabolites Isolated from Chilean Marine Fungi in Continental Coasts**

Studies carried out on cultures of *Cladosporium cladosporioides*, a fungus isolated from the marine sponge *Cliona* sp. collected in Region IV of Chile in 2004, led to the identification of *p*-methylbenzoic acid (**1**) and peroxyergosterol (**2**) (Figure 1). This was the first time that **1** had been isolated as a natural product. It was reported that peroxyergosterol from *Inonotus obliquus* inhibited the growth of cancer cells and showed cytotoxic effects on the same cell lines. Additionally, peroxyergosterol displayed potent inhibition of lipid peroxidation and higher antioxidant activity than well-known antioxidants, such as αtocopherol and thiourea. A recent study also revealed inhibitory effects of peroxyergosterol on inflammation and tumor promotion in mouse skin [22]. In addition, compounds **1** and **2** did not show antimicrobial activity against Gram-positive (*Staphylococcus aureus, S. epidermidis*) or Gram-negative bacteria (*Escherichia coli, Proteus mirabilis*, *Enterococcus faecalis*) in the agar plate diffusion assay. Both compounds were inactive against *Artemia salina* [23].

**Figure 1.** Secondary metabolites isolated from *Cladosporium cladosporioides*.

Four previously reported metabolites (**3**–**6**, Figure 2) were isolated from *Penicillium brevicompactun*, collected in Quintay, Chile (region V). The mycelium and broth were extracted with ethyl acetate, and the solvent was evaporated to provide a crude extract that showed in vitro antibacterial activity against both Gram-positive (*Staphylococcus aureus*, *S. epidermidis*) and Gram-negative bacteria (*Escherichia coli*, *Proteus mirabilis*, *Enterococcus faecalis* and *Pseudomonas aeruginosa*) [24].

**Figure 2.** Secondary metabolites isolated from *Penicillium brevicompactun*.

Four steroids (**2**, **7**–**9**) (Figures 1 and 3) were isolated from cultures of *Geotrichum* sp., a fungus obtained from marine sediment collected in Concepción Bay, Chile (Region VIII). Compound **7** is commonly found in fungal extracts since it plays a structural role in the cytoplasmic membrane. Similarly, **2** is a ubiquitous NP present in a variety of lichens, fungi, sponges, and marine organisms. Compound **8** has been isolated from *Lampteromyces japonicus* and a luminous bacterium. Additionally, **8** has been found in non-luminous basidiomycete fungi, including *Fomes officinalis* and *Scleroderma polyrhizum*. It has also been isolated from a marine sponge *Dictyonella incisa* [25]. This is the first time this compound has been identified in a facultative marine fungus [26].

**Figure 3.** Secondary metabolites isolated from *Geotrichum* sp.

Compound **10** is the first indole derivative isolated from a marine fungus (*Cladosporium cladosorioides)*. The crystal structure of N-methyl-1H-indole-2-carboxamide (**10**) (Figure 4) was determined by single-crystal X-ray diffraction [27].

**Figure 4.** Secondary metabolite isolated from *Cladosporium cladosorioides*.

Two dibenzylbutyrolactones (**11**,**12**) (Figure 5) and two sesterterpenoids (**13**,**14**) (Figure 5) were obtained from *Aspergillus* sp. (2P-22) isolated from the marine sponge, *Cliona chilensis* collected in Los Molles, Chile (Region IV) [28]. Spectroscopic data highlighted compound **11** as a novel compound named butylrolactone-VI. All four compounds were then tested for antibacterial activity against both Gram-positive (*Clavibacter michiganensis* 807) and Gramnegative bacteria (*Pseudomonas syringae pv syringae, Xanthomonas arboricola pv juglandis* 833, *Erwinia carotovora, Agrobacterium tumefaciens* A348), vasorelaxant effects, and antitumor bioactivities employing a broth culture of *A. tumefaciens* [28].

**Figure 5.** Secondary metabolites isolated from *Aspergillus* sp.

#### **3. Secondary Metabolites Isolated from Antarctic Marine Fungi**

The Antarctic continent represents one of the most extreme environments on earth for life to exist [29]. This ecosystem is characterized by high-stress conditions, including low temperatures, scarce availability of nutrients, high acidity, and high levels of ultraviolet radiation [30]. In order to survive under these highly demanding conditions, fungi living in the Antarctic have had to adapt their biochemical machinery and have done so through modifications in gene expression as well as the biosynthesis of secondary metabolites. Thus, Antarctic fungi represent a unique, biologically relevant chemical space with tremendous potential to contribute to the development of effective therapeutics [31]. Indeed, a number of efforts have reported unique NPs isolated from fungi living in Antarctic environments [31] and this emerging field promises a vast capacity for expansion.

In cold marine ecosystems, the presence of fungi has been associated with macroalgae and invertebrates, although some species have also been recorded in seawater and sediments [32,33]. Five new asterric acid derivatives were identified and isolated from the fermentation of the Antarctic ascomycete *Geomyces* sp.: ethyl asterrate (**15**) (Figure 6), n-butyl asterrate (**16**) (Figure 6), and geomycins A–C (**17**–**19**) (Figure 6). These compounds were evaluated for antifungal and antibacterial properties. Geomycin B (**18**) showed significant activity against *Aspergillus fumigatus* ATCC 10894, with IC50/MIC values of 0.86/29.5 μM, indicating much higher antifungal activity than the positive control fluconazole, which showed IC50/MIC values of 7.35/163.4 μM [31,34].

**Figure 6.** Secondary metabolites isolated from *Geomyces* sp.

Six new peptaibols (linear or cyclic peptides), named asperelines A–F (**20**–**25**) (Figure 7), were characterized from the fermentation of the marine-derived fungus *Tridocherma asperellum* collected from the sediment of the Antarctic Penguin Island. Chemical structures were determined using 1D and 2D NMR techniques as well as ESIMS/MS [35].

Next, two highly oxygenated polyketides, penylactones A and B (**26** and **27**) (Figure 8) were isolated and identified from *Penicillium crustosum* PRB-2. These compounds had a similar chemical structure but opposite absolute stereochemistry. Compounds **26** and **27** were tested for their ability to inhibit nuclear factor-κB (NF-κB) via transient transfection and reporter gene expression assays. Of the two compounds, only **27** showed inhibitory activity with a relatively weak effect of 40% inhibitory rate at a concentration of 10 μM [36]. The authors also proposed a biosynthetic pathway for both compounds, shown in Scheme 1 [36]. These penylactones are characterized by a new carbon skeleton formed from two units of 3,5-dimethyl-2,4-diol-acetophenone and γ-butyrolactone. Six compounds were subsequently synthesized through a novel biomimetic synthesis pathway, as shown in Scheme 2 [37].

**Scheme 1.** Proposed biosynthetic pathway to **26** and **27**.

**Scheme 2.** Biomimetic synthesis of *ent*-Penilactone A and Penilactone B.

A study of the Antarctic fungus *Oidiodendron truncatum* GW3-13 isolated two new epipolythiodioxopiperazines, chetrazins B (**28**) and C (**29**), together with five new diketopiperazines, chetracin D (**30**), and oidiooperazines A–D (**31**–**34**) (Figure 9). In vitro studies using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) showed that compound **28** exhibits potent biological activity in the nanomolar range against a panel of five human cancer lines (HCT-8, BEL-7402, BGC-823, A-549, and A-2780) [38].

**Figure 8.** Secondary metabolites isolated from *Penicillium crustosum* PRB-2.

**Figure 9.** Secondary metabolites isolated from the Antarctic fungus *Oidiodendron truncatum*.

In the same study, compounds **29** and **30** exhibited significant cytotoxicity at micromolar concentration. Finally, it was observed that compounds **31**–**34** did not show cytotoxicity at a concentration of 10 μM. This led to the conclusion that the sulfide bridge was a determining factor in the biological activity presented by these compounds. In contrast, the number of sulfur atoms in the bridge did not seem to influence the bioactivity [38].

Organic extracts of several fungi were isolated from samples of Porifera collected on King George Island. Although pure compounds could not be isolated, the presence of biological activities and potential as antimicrobial agents could be investigated. Antimicrobial activity was tested using strains of *Pseudomonas aeruginosa*, *Staphylococcus aureus* ATCC25922, *Clavibacter michiganensis* 807, and *Xanthomonas campestris* 833. Antitumoral activity was assessed using *Agrobacterium tumefaciens* At348 as a model, and antioxidant activity was determined by comparing the absorbance of ascorbic acid obtained from each

extract. Approximately 50% of the 101 extracts showed antibacterial activity against at least one of the bacteria tested, being more active against Gram-positive bacteria such as *Staphylococcus aureus*. Moreover, 43 extracts showed 50% inhibition of crown gall tumor growth on potato. Antioxidant studies revealed that 97 fungal extracts displayed decent activities varying from very low to mild, and only three isolates showed high antioxidant activities [39].

Four new compounds, namely Pseudogymnoascins A–C (**35**–**37**) and 3-nitroasterric acid (**38**) (Figure 10), were characterized from a culture of *Pseudogymnoascus* sp., obtained from an Antarctic marine sponge of the genus *Hymeniacidon* [40]. Remarkably, these compounds were the first nitro derivatives of asterric acid identified. The antimicrobial activity of compounds **35**–**38** was evaluated against *Pseudomonas aeruginosa* PAO1, *Actinobacter baumannii* CL5973, *Escherichia coli* MB2884, *Staphylococcus aureus* EP1167, and *S. aureus* MB5394. Their antifungal activity was also tested against *Candida albicans* MY1055, *C. albicans* ATCC64124, and *Aspergillus fumigatus* ATCC46645. Cytotoxicity against the tested microorganisms was not observed, suggesting that the presence of the nitro group in the structure may negatively influence the biological activity of these compounds [40].

**Figure 10.** Secondary metabolites isolated from *Pseudogymnoascus* sp.

One hundred fungal strains were isolated from 55 samples of maritime Antarctic and classified into 35 fungal taxa within 20 genera. Extracts from these strains were tested against human tumoral cells, parasitic protozoa (*Leishmania amazonensis*, *Trypanosoma cruzi*), fungi, and bacteria. The extracts from *Purpureocillum lilacinum* displayed high trypanocidal, antibacterial, and antifungal activities with moderate toxicity over normal cells [41].

In recent years the chemical compounds of *Penicillium* sp. S-1-18 isolated from Antarctic seabed sediments has been extensively investigated. Butanolide A (**39**), a new furanone derivative, and guignarderemophilane F (**40**), a new sesquiterpene, together with six known compounds: penicyclone A (**41**), xylarenone A (**42**), callyspongidipeptide A (**43**), cyclo-(L-Phe-4R-hydroxyl-L-Pro) (**44**), cyclo-(L-Pro-L-Phe) (**45**), and N-(2-hydroxypropanoyl)-2-aminobenzoic acid amide (**46**), were isolated (Figure 11). The structures of these metabolites were determined using 1D- and 2D-NMR spectroscopic methods. Inhibitory effects against PTP1B activity were tested for all compounds. Only compound **39**

showed activity against PTP1B, which was moderate compared with the positive control oleanolic acid [42].

**Figure 11.** Secondary metabolites isolated from *Penicillium* sp. S-1-18.

An interesting study of the distribution of marine fungi found that *Pseudogymnoascus* sp. and species of the genus *Penicillium* were present in all marine samples. Samples collected at 20 m or more in depth, at temperatures near 0 ◦C, had higher diversity those from the intertidal zone (superficial samples) [43].

The antibacterial activity was assessed for four new compounds, Penixylarins A–D (**47**–**50**), obtained from a culture of the Antarctic fungus *Penicillium crustosum* PRB-2 and the mangrove-derive fungus *Xylaria* sp. HDN12-249 (Figure 12). Compounds **48** and **49** showed antibacterial activity against *B. subtilis*, *M. phlei*, and *V. parahemolyticus*. Compound **49** additionally displayed potential antituberculosis effects against *Mycobacterium phlei* [44].

**Figure 12.** Penixylarins A–D, isolated from *Penicillium crestosum* PRB-2 and the fungus *Xylaria* sp. HDN12-249.

In a 2018 review, Tripathi et al. described more than two hundred natural products isolated from prokaryotes and eukaryotes living in polar regions, including fungi. Their pharmacology, relevant bioactivity, and chemical structures were reported in the review [45]. One year later, anticancer compounds were isolated from seaweed-derived endophytic fungi [46].

Using a different sampling strategy, pieces of excrement from Adelie penguins allowed the isolation of *Penicillium chrysogenum*. Although the sample was not collected from a marine environment per se, the feeding habits of the penguins support the idea that the microorganisms isolated are marine. The exact location of the sample collection site was not stated but is presumably near the Chinese Great Wall Antarctic base. A new compound Chrysonin (**51**) was obtained as a pair of enantiomers 6S- and 6R-chrysonin (**51a** and **51b**) (Figure 13). These compounds display an eight-membered heterocycle fused with a benzene ring. Interestingly, there is no precedent of one natural compound with this structure. Compound 52 was also isolated as a mixture of a new zwitterionic compound chrysomamide (**52a**) and N-[2-trans-(4-hydroxyphenyl) ethenyl] formamide (**52b**) (Figure 13). Compound **53**, shown in Figure 13, contains the unusual isocyanide functional group. This functional group has been found in several marine organisms, such as cyanobacteria, Penicillium fungi, marine sponges, and nudibranchs. Furthermore, there is no precedent of one natural compound with an eight-membered heterocycle fused with a benzene ring. Antibacterial activity of each compound against eight microorganisms was determined. Compound **53** (Figure 13) was active against *Pseudomonas aeruginosa, Klebsiella pneumoniae*, and *Acinetobacter baumannii*. The same metabolite and compound **54** (Figure 13) both showed significant cytotoxicity against four cancer cell lines: *A. baumannii* ATCC 19606, *E. coli* ATCC 25922, *M. luteus* SCSIO MLO1, and MRSA, shhs-A1. Compound **55** (Figure 13) displayed the best alpha glucosidase inhibition [47].

**Figure 13.** Secondary metabolites isolated from *Penicillium chrysogenum*.

*Penicillium echinulatum* was isolated from the surface of the alga *Adenocystis utricularis* collected on a beach close to Comandante Ferraz Brazilian station on King George Island. In this study, photosafety was evaluated using photoreactivity (OECD TG 495) and phototoxicity assays performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and the RHS model. The purification of four alkaloids was achieved in a bio-guided process. Four known metabolites were identified: (−)-cyclopenin (**56**), dehydrocyclopeptine (**57**), viridicatin (**58**), and viridicatol (**59**) (Figure 14), and their photoprotective and antioxidant activities were shown [48].

**Figure 14.** Alkaloids isolated from *Penicillium echinulatum*.

The antibacterial activity of Penicillic acid (**60**) (Figure 15), isolated from *Penicillium* sp. CRM-1540 found in Antarctic marine sediment at King George Island, was evaluated. This compound was obtained as the major bioactive fraction through a bioguided study. Results showed 90% bacterial inhibition in vitro at 25 μg mL−<sup>1</sup> against *Xanthomonas citri* [49].

**Figure 15.** Penicillic acid isolated from *Penicillium* sp. *CRM-1540*.

Talaverrucin A (**61**) (Figure 16), a heterodimeric oxaphenalenone with a rare fused ring system, was isolated from *Talaromyces* sp. HDN151403 (Prydz Bay, Antarctica). The oncogenic Wnt/β-catecin inhibitory effect was tested and showed inhibitory activity in zebrafish embryos in vivo and cultured mammalian cells in vitro [50].

**Figure 16.** Talaverrucin A isolated from *Penicillium* sp. CRM-1540.

The cytotoxic activity of Citromycin (**62**) (Figure 17) was tested against ovarian cancer SKOV3 and A2780 cells. No cytotoxic activity was observed. The compound **62** was obtained from *Sporothrix* sp. and showed inhibition of extracellular signal-regulated kinase (ERK)-1/1 [51].

**Figure 17.** Citromycin isolated from *Sporothrix* sp.

Four new cytotoxic nitrobenzoyl sesquiterpenoids, insulicolides D–G (**63**–**66**) (Figure 18), were isolated from *Aspergillus insulicola* HDN151418, which was obtained from an unidentified Antarctica sponge (Prydz Bay). Compounds **65** and **66** showed selective inhibition against human PDAC cell lines [52].

**Figure 18.** Insucolides (D–G) isolated from *Aspergillus insulicola* HDN151418.

Three new perylenequinone derivatives (Xanalterate A, **67**, Altertoxin VIII, **68** and IX, **69**) together with a known natural product, Stemphyperylenol (**70**) (Figure 19), were isolated from *Alternaria* sp. HDN19-690 associated to an Antarctic sponge. Compound **67** exhibited promising antibacterial activity against methicillin-resistant coagulase negative *Staphylococcus* (MRCNS), *Bacillus subtilis, Proteus mirabilis, Bacillus cereus, Escherichia coli*, and *Mycobacterium phlei* with MIC values ranging from 3.13 to 12.5 μM [53].

**Figure 19.** Perylenequinones isolated from *Aspergillus insulicola* HDN151418.

#### **4. Materials and Methods**

Scifinder database and the repositories of the Pontificia Universidad Católica de Chile and Universidad Técnica Federico Santa Maria were used to search for reports published from 1996 to date. The search criteria focused on marine fungi obtained from Chilean coasts, the South Shetland Islands, and Antarctic peninsula and reports of novel marine NPs that were spectroscopically characterized and presented biological or pharmaceutical properties. Descriptions involving vegetable extracts or primary metabolites were omitted.

#### **5. Conclusions**

Natural products from Chilean marine fungi represent a prolific and yet underexplored source of chemical structures with remarkable biomedical applications (Table 1). Alkaloids, polyketides, terpenoids, isoprenoids, non-isoprenoid compounds, and quinones display the most relevant biological activities. There are few studies on secondary metabolites isolated from marine fungi collected in Chile, highlighting the antimicrobial activity presented by some crude extracts and the antitumor activity of some of the isolated compounds. The dearth of studies may be attributed to the difficulties in cultivating microorganisms, some of which cannot survive under standard laboratory conditions and therefore cannot be cultured using traditional techniques. It is complicated to reproduce the conditions found inside the host marine organisms. The culture medium used is suitable for facultative fungi but probably inadequate for natural marine fungi. Recent advances in chromatographic and spectroscopic techniques now open a world of possibility for isolating secondary metabolites of these organisms that are abundant in Chilean marine ecosystems.





**Author Contributions:** Conceptualization, D.A. and J.R.C.-P.; writing—original draft preparation, D.A.; resources, D.A.; H.C., A.S.-M. and J.R.C.-P.; writing—review and editing, L.T., H.C., J.R.C.-P. and A.S.-M. All authors participated in similar measure in the preparation of this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Instituto Antártico Chileno (INACH) RT\_16-21.

**Acknowledgments:** J.R.C.-P thanks Instituto Antártico Chileno (INACH), for grant RT\_16-21, D.A express thanks to the Dirección de Postgrado y Programas (DPP), Universidad Técnica Federico Santa María. The authors thank Ellen Leffler for critical reading and suggestions.

**Conflicts of Interest:** The authors declare that they have no conflicting interest in the publication.

#### **References**


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### *Review* **Secondary Metabolites, Biological Activities, and Industrial and Biotechnological Importance of** *Aspergillus sydowii*

**Sabrin R. M. Ibrahim 1,2,\*, Shaimaa G. A. Mohamed 3, Baiaan H. Alsaadi 4, Maryam M. Althubyani 4, Zainab I. Awari 5, Hazem G. A. Hussein 6, Abrar A. Aljohani 7, Jumanah Faisal Albasri 8, Salha Atiah Faraj <sup>9</sup> and Gamal A. Mohamed <sup>10</sup>**


**Abstract:** Marine-derived fungi are renowned as a source of astonishingly significant and synthetically appealing metabolites that are proven as new lead chemicals for chemical, pharmaceutical, and agricultural fields. *Aspergillus sydowii* is a saprotrophic, ubiquitous, and halophilic fungus that is commonly found in different marine ecosystems. This fungus can cause aspergillosis in sea fan corals leading to sea fan mortality with subsequent changes in coral community structure. Interestingly, *A. sydowi* is a prolific source of distinct and structurally varied metabolites such as alkaloids, xanthones, terpenes, anthraquinones, sterols, diphenyl ethers, pyrones, cyclopentenones, and polyketides with a range of bioactivities. *A. sydowii* has capacity to produce various enzymes with marked industrial and biotechnological potential, including α-amylases, lipases, xylanases, cellulases, keratinases, and tannases. Also, this fungus has the capacity for bioremediation as well as the biocatalysis of various chemical reactions. The current work aimed at focusing on the bright side of this fungus. In this review, published studies on isolated metabolites from *A. sydowii*, including their structures, biological functions, and biosynthesis, as well as the biotechnological and industrial significance of this fungus, were highlighted. More than 245 compounds were described in the current review with 134 references published within the period from 1975 to June 2023.

**Keywords:** fungi; *Aspergillus sydowii*; metabolites; enzymes; biotechnology; bioremediation; renewable resources; life on land; marine natural products; drug discovery

#### **1. Introduction**

Fungi have so far received substantial attention for enhancing value in agricultural, industrial, pharmaceutical, and health fields [1–4]. During the past few decades, there have been some extremely intriguing advances in the utilization of fungi for new processes,

**Citation:** Ibrahim, S.R.M.; Mohamed, S.G.A.; Alsaadi, B.H.; Althubyani, M.M.; Awari, Z.I.; Hussein, H.G.A.; Aljohani, A.A.; Albasri, J.F.; Faraj, S.A.; Mohamed, G.A. Secondary Metabolites, Biological Activities, and Industrial and Biotechnological Importance of *Aspergillus sydowii*. *Mar. Drugs* **2023**, *21*, 441. https:// doi.org/10.3390/md21080441

Academic Editor: Hafiz M.N. Iqbal

Received: 11 July 2023 Revised: 29 July 2023 Accepted: 2 August 2023 Published: 5 August 2023

**Copyright:** © 2023 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/).

products, and solutions that are crucial for the world. Also, fungi are proven to be a prolific pool of structurally varied bioactive metabolites. Additionally, fungal enzymes have been utilized instead of chemical processes in various industries, including those of textiles, leather, paper, pulp, animal feed, baked goods, beer, wine, and juice, which greatly reduces negative environmental effects [5]. Genus *Aspergillus* (Moniliaceae) is one of the most valuable fungal genera of commercial, biotechnological, and medicinal importance [6–8]. It comprises 400 species and attracts remarkable interest as a wealthy pool of structurally varied metabolites, including terpenoids, alkaloids, peptides, xanthones, and polyketides [7–9]. These metabolites have diverse bioactivities such as antibacterial, cytotoxic, antifungal, and anti-HIV activities.

*Aspergillus sydowii* is a saprotrophic, ubiquitous, and halophilic fungus and represents one of the widely distributed *Aspergillus* species [10–12]. It is commonly found in different habitats all over the world, including diverse soil and marine ecosystems, and possesses a broad range of salinity tolerance [13]. Interestingly, halophilic *A. sydowii* is employed as a model organism for investigating filamentous fungi's molecular adaptation to hyperosmolarity [13]. *A. sydowii* can survive as a food contaminant, as a soil-decomposing saprotroph, and as an opportunistic human pathogen [14]. It causes onychomycosis and aspergillosis in humans, as well as aspergillosis in sea fan corals, on the basis of Koch's postulate and physiological, morphological, and nucleotide sequence analyses [15–17]. Aspergillosis symptoms involve small necrotic lesions of tissues with purple halos, like the pathology of coral bleaching [18]. This leads to sea fan mortality and subsequent changes in coral community structure [18]. It was reported to cause 20–90% mortality in sea fans in the Florida Keys [18].

In addition to its pathogenic potential, *A. sydowii* has captured a considerable number of researchers' attention due to its capacity to create a variety of biotechnologically and industrially significant enzymes, such as lipases, α-amylases, xylanases, cellulases, tannases, and keratinases [19–23]. Additionally, *A. sydowii* biosynthesizes various classes of metabolites, such as sesquiterpenoids, alkaloids, xanthones, monoterpenes, anthraquinones, sterols, triterpenes, diphenyl ethers, pyrones, cyclopentenones, anthocyanins, and polyketides [11,24–38]. These metabolites have drawn remarkable interest because of their prominent bioactivities, including antioxidant, immunosuppression, antiviral, anti-mycobacterial, antimicrobial, cytotoxic, anti-inflammation, protein tyrosine phosphatase 1B (PTP1B) inhibition, anti-nematode, anti-diabetic, and anti-obesity properties [32,34,37,39–48]. Further, this fungus is employed for the synthesis of different types of nanoparticles that could have beneficial pharmaceutical, biotechnological, and industrial applications [49–52]. Recently, the number of articles on *A. sydowii* metabolites and their biotechnological and industrial relevance has risen substantially. It is noteworthy that a review paper discussing *A. sydowii*, particularly the positive aspects of this commercially useful fungus, was not found. Therefore, the current work provided a comprehensive and close insight into this fungus. The published information on the secondary metabolites identified from this fungus and their bioactivities were compiled. Additionally, the research on *A. sydowii*, including applications in industry, biotechnology, and nanotechnology, has been reviewed. Studies published in the literature within the period from 1975 to 2023 were reported. Additionally, the documented biosynthesis routes of the fungus' major metabolites were illustrated.

Searches were conducted in depth on literature databases, namely PubMed, Web of Science, and Scopus, as well as on various websites of publishers (Wiley Online Library, Taylor & Francis, Springer, JACS, Thieme, and Bentham) and scientific websites (Google Scholar, PubMed, and ScienceDirect). The following phrases and keywords were used for the search: "*Aspergillus sydowii*," "*Aspergillus sydowii* + compounds," "*Aspergillus sydowii* + metabolites," "*Aspergillus sydowii* + NMR," "*Aspergillus sydowii* + biological activity," "*Aspergillus sydowii* + Enzymes," "*Aspergillus sydowii* + biotechnology," "*Aspergillus sydowii* + biotechnological importance," and "*Aspergillus sydowii* + nanoparticles".

#### **2. Secondary Metabolites of** *Aspergillus sydowii*

#### *2.1. Sesquiterpenes*

Phenolic bisabolane sesquiterpenoids are among the main constituents reported from this fungus. They are a rare class of terpenes that have a p-alkylated benzene connected with 1C and 8C side chains at C-5 and C-2, respectively. Their structural variability is due to cyclization, reduction, or oxidation at various alkyl chain carbons to yield carboxylic acid, alcohol, lactone, double bond, pyran, and furan functionalities. Besides their fascinating skeletons, they show various bioactivities. It is noteworthy that most of the reported bisabolanes were separated from marine-derived *A. sydowii* as discussed below (Table 1).

**Table 1.** Sesquiterpenoids reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).





In 1978, Hamasaki and his group separated and characterized compounds **1** and **2** as optically inactive metabolites from *A. sydowii* acetone extract by spectral and chemical means. These compounds were soluble in saturated NaHCO3 and positively reacted with bromophenol blue [11] (Figure 1).

**Figure 1.** Structures of sesquiterpenoids (**1**–**21**) reported from *A. sydowii.*

Aspergillusene D (**16**) with a 7*S*-configuration was reported as a new sesquiterpenoid from *Phakellia fusca*-associated *A. sydowii* SCSIO-41301 by Liu et al., along with compounds **1**, **5 8**, **9**, **10**, and **22** that were characterized based on spectral and ECD (electronic circular dichroism) analyses [35]. Xu et al. separated compound **17**, along with compounds **1**, **8**, and **23**, from *A. sydowii* CUGB-F126 isolated from the Bohai Sea, Tianjin, using SiO2 (silica gel)/Sephadex LH-20/HPLC (high-performance liquid chromatography). Compound **17** is a new sydonic acid analog with a glycinate moiety [15].

Sun et al. developed a new approach that integrated computational programs (MS (mass spectrometry)-DIAL and MS-FINDER) and web-based tools (MetaboAnalyst and GNPS) for the identification of *A. sydowii*–*Bacillus subtilis* coculture metabolites, wherein 25 biosynthesized metabolites were detected and purified by SiO2/ODS CC/HPLC. Among them, compounds **1**, **2**, **3**, and **18**–**21** were characterized by spectral and CD (circular dichroism) analyses [58]. Further, Hu et al. separated and characterized new bisabolene-type sesquiterpenoids **24** and **25** as well as the known analogs **2** and **23** from *A. sydowii* EN-434 obtained from *Symphyocladia latiuscula* marine red alga using RP-18 (reversed phase-18)/SiO2 CC (silica gel column chromatography) and spectral and ECD data. Compounds **24** and **25** have 7*S*/8*S* and 7*R*\*/10*R*\* configurations, respectively [32]. Fourteen new phenolic bisabolanes with varied structures, labeled **28**–**41**, were separated and characterized by Niu et al. from the deep-sea sediment-derived *A. sydowii* MCCC-3A00324 (Figure 2).

**Figure 2.** Structures of sesquiterpenoids (**22**–**41**) reported from *A. sydowii*.

Compounds **28** and 29 are the first bisabolanes with a 6/6/6 tricyclic skeleton, whereas compound **30** features a novel *seco*-bisabolane with a rare dioxolane moiety, and compound **38** has an unusual methylsulfonyl moiety [57]. Trisuwan et al. purified—from *A. sydowii* PSUF154 isolated from gorgonian sea fan of genus *Annella*—new bisabolane-type sesquiterpenes **4**, **42**, and **43**, along with **1**. Compound **42** has 2-substituted 6-methyl-2-heptenyl and 1,2,4-trisubstituted benzene. Compound **43**'s benzofuran moiety results from the ether linkage of C-1 OH of the tri-substituted phenyl and 2-substituted 6-methyl-2-heptenyl moieties. Compound **4** is a methyl ether of compound **1** with a 7*S* configuration [56]. The first phenolic bisabolane sesquiterpene glycoside, *β*-D-glucopyranosyl aspergillusene A (**44**), was purified from sponge-derived *A. sydowii* [36] and assigned using spectral and chemical methods [36].

Chung et al. stated that the addition of 5-azacytidine (a DNA methyltransferase inhibitor) to the culture of marine sediment-derived *A. sydowii* obtained from Hsinchu, Taiwan, significantly promoted the production of various metabolites [54]. Investigation

of the EtOAc (ethyl acetate) extract of 5-azacytidine-treated culture broth by SiO2 CC and HPLC yielded new bisabolane sesquiterpenoids **5**, **46**, and **47**, along with **1**, **42**, **45**, and **49**, that were assigned based on spectral analyses. The S-configuration of compounds **5** and **46** was assigned using optical rotation comparison, whereas compound **46** ([α]D +1.87) is a methyl derivative of compound **45** ([α]D +7.2) and compound **5** ([α]D +3.9) is C-12 hydroxy analog of compound **1** ([α]D +23) (Figure 3). On the other hand, compound **47** is closely similar to the previously reported compound **8** except for the absence of the C-3 carboxylic group in compound **47** [54]. Compounds **5**, **46**, and **47** were proposed to be biosynthesized from farnesyl diphosphate (FPP) created from the addition of an IPP (isopentenyl diphosphate) unit to a GPP (geranyl diphosphate) (Scheme 1). Then, cyclization and folding of the carbon chain through an electrophilic attack on double bonds produced the bisabolane nucleus that then underwent a series of carboxylation, hydration, oxidation, and reduction to give compounds **5**, **46**, and **47** [54].

**Figure 3.** Structures of sesquiterpenoids (**42**–**58**) reported from *A. sydowii*.

A new bisabolane sesquiterpenoid, compound **15**, in addition to compounds **1**, **7**, **6**, **42**, **47**, **49**, **50**, and **52**, were purified from *A. sydowii* ZSDS1-F6 EtOAc extract using SiO2/Sephadex LH-20/RP-HPLC by Wang et al. [45]. Compound **51** is a new aromatic bisabolene-type sesquiterpenoid with 11S-configuration purified and characterized from the sea-derived *A. sydowii* SW9 [41]. In 2022, Liu et al. purified a rare iodine- and sulfurcontaining derivative (7*S*)- 4-iodo-flavilane A (**54**) along with compound **53**. Compound **54** is 4-iodinated analog of compound **53** and its absolute S-configuration was proven by ECD analysis [38]. Furthermore, three undescribed cuparene-type sesquiterpenes, labeled **56**–**58**, were isolated from fermented cultured EtOAc extract of the sea sediment-derived *A. sydowii* MCCC-3A00324 using SiO2/RP-18/Sephadex LH-20 CC/HPLC and assigned using spectral and ECD analyses. They represent rare cuparene-type sesquiterpenoids having a C-10 keto group and were discovered for the first time from filamentous fungi [57].

**Scheme 1.** Biosynthetic pathway of compounds **5**, **46**, and **47**: GPP: Geranyl diphosphate; FPP: Farnesyl diphosphate; IPP: Isopentenyl diphosphate [54].

#### *2.2. Mono- and Triterpenoids and Sterols*

In 2020, the chemical investigation of deep-sea sediment-isolated *A. sydowii* MCCC-3A00324 by Niu et al. led to the separation of new osmane-type monoterpenoids aspermonoterpenoids A (**59**) and B (**60**) by SiO2 CC/HPLC and their structures were determined by spectral, ECD, and specific rotation analyses (Table 2, Figure 4). Compounds **59** and **60** are the first osmane monoterpenes reported from fungi, whereas compound **59** features a novel skeleton, which is possibly derived after the cleavage of the cyclopentane ring and oxidation reaction of the osmane monoterpenoid. They have 4*S* and 4*S*/5*R*/6*S* configurations, respectively [60].

**Figure 4.** Structures of mono- (**59** and **60**) and triterpenoids (**61** and **62**) and sterols (**63**–**68**) reported from *A. sydowii.*


**Table 2.** Mono- and triterpenoids and sterols reported from *A. sydowii* (molecular weight and formulae, strain, host, and location).

These metabolites were proposed to be biosynthesized from a GPP that underwent subsequent hydrolysis/oxygenation/cyclization to yield the monocyclic osmane monoterpenoid ring. Then, carbon–carbon bond cleavage of osmane gives intermediate **I** and its further oxygenation yields compound **59**, whilst the osmane oxygenation forms compound **60** [60] (Scheme 2).

**Scheme 2.** Biosynthetic pathway of compounds **59** and **60** [60].

Zhang et al. purified and characterized compound **61**, a new 29-nordammarane-type triterpenoid, in addition to its known analog, compound **62**, from the marine-derived *A. sydowii* PFW1-13 [48]. Compound **61** is structurally similar to compound **62** with a 1,1,2-trisubstituted ethanol unit instead of a trisubstituted ethenyl unit, suggesting that compound **61** is a C24–C25 hydrated derivative of compound **62** [48]. Its configuration

.

was assigned as 4*S*/5*S*/6*S*/8*S*/9*S*/10*R*/13*R*/14*S*/16*S*/17*Z* based on comparing its optical rotation (−118.9) with that of compound **62** (optical rotation −105.1) [48].

Wang et al., in 2019, reported the separation of ergosterol derivatives **63**–**66** from deep-sea water-isolated *A. sydowii* [55], while compounds **68** and **69** were separated by Li et al.; compound **69** was assumed to be a sterol degradation product [44].

#### *2.3. Xanthone and Anthraquinone Derivatives*

Xanthones are commonly found in lichen, fungi, plants, and bacteria [61]. In fungi, xanthones are mostly derived from acetyl-CoA through a series of polyketide synthasecatalyzed chemical transformations [62]. These metabolites were found to demonstrate diverse bioactivities.

Compounds **69** and **71** were reported from the EtOAc extract of 5-azacytidine-treated *A. sydowii* culture broth [54]. Additionally, from liverwort *Scapania ciliate*-accompanied *A. sydowii*, new xanthone derivatives, labeled **72**, **76**, and **77**, and known compounds **74** and **78** were isolated by SiO2/Sephadex LH-20 CC/HPLC and assigned by spectral data. Compounds **76** and **77** are examples of sulfur-containing xanthones; compound **77** has an additional acetyl group at C-13 and compound **72** features C-2-OH instead of the methylthio moiety as in compound **76** [63]. New hydrogenated xanthones, aspergillusones A (**86**) and B (**87**), along with compounds **69**, **71**, **73**, **88**, and **90**, were purified from a strain associated with the gorgonian sea fan of the genus *Annella* by Trisuwan et al. Compound **86** is a 11-deoxy derivative of compound **88** with an optical rotation of −1.6 and the same C-7 and C-8 absolute configuration, whereas compound **87** is a 1-hydroxy analog of compound **90** with 7R/8R and −46.3 optical rotation (Figure 5) [56].

**Figure 5.** Structures of xanthones (**69**–**80**) reported from *A. sydowii.*

In 2019, Wang et al. purified two novel xanthones, labeled **70** and **79**, along with the known xanthones **71**, **72**, **74**, **86**, **88**, and **89** and quinones **91**, **94**, and **96**, from seawaterderived *A. sydowii* C1-S01-A7 using SiO2/Sephadex LH-20/RP-18/HPLC; the compounds were elucidated by spectral analyses (Table 3). Compound **79** is similar to previously reported 2-hydroxyvertixanthone with an additional formyl moiety at C-6, whereas compound **70** is similar to compound **69** with one more acetyl group at C-12 [55].

**Table 3.** Xanthones and quinones reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).



The cultured EtOAc extract of *A. sydowii* SCSIO-41301 associated with *Phakellia fusca* provided new xanthones **80** and **81**. Compound **80** is related to versicone A with 3-OH instead of the isopentene group in versicone A, while compound **81** has an additional 6-OCH3 group compared to compound **80** [35]. The new mycotoxin 6-methoxyl austocystin A (**83**) and the related known compound **82** were isolated from *Verrucella umbraculum*associated *A. sydowii* SCSIO-00305 (Figure 6). Compound **83** is similar to compound **82** except for the presence of an additional C6-OCH3. Their 1 R/2S configuration was assigned based on X-ray analysis [24].

**Figure 6.** Structures of xanthones (**81**–**90**) reported from *A. sydowii.*

Additionally, compounds **92**–**95** are anthraquinones reported by Liu et al. from a *Phakellia fusca*-associated fungal strain [35] (Figure 7). Compounds **98** and **99** are quinone derivatives separated from *A. sydowii* #2B associated with *Aricennia marina* by Wang et al. [64].

#### *2.4. Alkaloids*

Alkaloids have drawn considerable attention because of their unique structural features and varied bioactivities. Interestingly, alkaloids belonging to various classes were reported from *A. sydowii*.

From the culture broth of coral *Verrucella umbraculum*-accompanied *A. sydowii* SCSIO-00305, using bio-guided fractionation, a new indole diketopiperazine member, cyclotryprostatin E (**101**), and compounds **100**, **102**, and **117**–**123** were purified using RP-18 CC/HPLC and characterized by spectral data interpretation [31] (Figure 8). Compound **101** is similar to compound **100** bar the replacement of the tri-substituted double bond in compound **100** with an oxygen-bonded quaternary carbon; compound **117** possesses indolyl, piperazinyl, and 1,2-disubstituted phenyl groups [31].

**Figure 7.** Structures of quinones (**91**–**99**) reported from *A. sydowii.*

**Figure 8.** Structures of alkaloids (**100**–**117**) reported from *A. sydowii.*

In 2008, Zheng et al. purified new diketopiperazines **103**–**105** and a new oxaspiro [4.4]lactam-containing alkaloid, labeled **131**, along with compounds **106**–**109**, **111**, **112**, **130**, and **140**–**143** from the EtOAc extract of *A. sydowii* PFW1-13 isolated from driftwood sourced from Baishamen beach, Hainan, China, using SiO2/Sephadex LH-20 CC/HPLC [48]. The configurations of compounds **103**–**105** and **131** were assigned based on NMR (nuclear magnetic resonance) and CD spectral analyses, and the specific rotation was 3S/12S/18S for compound **103** and 9S/12S for compounds **104** and **105**, while compound **131** was identified as a 14-nor-derivative of compound **130** with 5*S*/8*S*/9*R*/10*S*/11*S*/12*Z* configuration [48].

Biosynthetically, compounds **103**–**105** were postulated to be generated through a mixed mevalonic acid/amino acid pathway. Compound **105** is generated from the oxidation of compound **107**, which results from mevalonic acid, tryptophan, and alanine. A cyclo(Trp-Pro) is formed from proline and tryptophan and is further oxidized and methylated to produce ethoxylated cyclo(Trp-Pro). Then, the latter reacts with mevalonic acid to yield compound **104** and intermediate **I**. An intramolecular aldol reaction of intermediate **I** yields intermediate **III**, which is deoxygenated to produce compound **106**. Additionally, the dehydrogenation of compound **106** gives compound **103** (Scheme 3).

**Scheme 3.** Biosynthetic pathway of compounds **103**–**106** [48].

Kaur et al. separated a new diketopiperazine dimer WIN 64821 (**115**) and the known compound **110** using SiO2 CC and RP-HPLC from the CH3OH/CH3CN extract of *A. sydowii* MSX-19583 obtained from spruce litter; the compounds were assigned by spectral and ECD analyses and Marfey's Method (Table 4). Compound **115** is structurally similar to the ditryptophenaline reported in various *Aspergillus* species and derived from tryptophan and phenylalanine subunits [33].


**Table 4.** Alkaloids reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).


A new quinazolinone alkaloid, labeled **124**, as well as related alkaloid **125** and triazole analog **134** were separated and characterized from the mycelia EtOAc extract of seawaterderived *A. sydowii* SW9 using SiO2/Rp-18/Sephadex LH-20 CC and spectral analyses (Figure 9). Compound **124** is an acetyl derivative of 2-(4-hydroxybenzyl)quinazolin-4(3*H*) one, previously reported from *Cordyceps*-associated *Isaria farinose* [41,66].

**Figure 9.** Structures of quinazoline alkaloids (**118**–**126**) reported from *A. sydowii.*

Acremolins are rare alkaloids with a 5/6/5 tricyclic core, possessing an imidazole moiety fused with a methyl guanine moiety. Interestingly, acremolins were reported from *Aspergillus* species *Aspergillus* sp. S-3-75 and SCSIO-Ind09F01 and *A. sydowii* SP-1 [40,67]. From the Antarctic *A. sydowii* SP-1, a new alkaloid acremolin C (**128**) along with compound **110** were separated using SiO2 CC/ODS/HPLC and characterized by spectral methods. Compound **128** is a regio-isomer of acremolin B previously reported by Tian et al. from the deep-sea-derived fungus *Aspergillus sp.* SCSIO and has a isopropyl group at C-2 instead of C-1 (Figure 10) [40,67]. In 2022, Niu et al. purified and characterized, from the deep-seaderived *A. sydowii* MCCC-3A00324, a new acremolin alkaloid acremolin D (**129**) along with compounds **110**, **126**, **127**, **135**, and **136** using SiO2 CC/HPLC and spectral and ECD data. Compound **129** is closely related to compound **127** in that one CH3 group in **127** has been replaced by an acetoxy methylene group [65].

New hetero-spirocyclic γ-lactam analogs azaspirofurans A (**132**) and B (**133**) were separated from the marine sediment-derived *A. sydowii* D2-6 using SiO2/Sephadex LH-20 CC and were characterized based on spectral and chemical evidence (Figure 10). These compounds featured an ethyl furan ring linked to 1-oxa-7-azaspiro[4,4]non2-ene-4,6-dione core [43].

#### *2.5. Phenyl Ether Derivatives*

Phenyl ethers are a group of simple polyketides that are widely reported in various *Aspergillus* species and have shown significant bioactivities (Table 5).

**Figure 10.** Structures of alkaloids (**127**–**143**) reported from *A. sydowii.*


**Table 5.** Phenyl ether derivatives reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).


A new biphenyl ether derivative diorcinolic acid (**148**) together with compounds **144**–**147**, **152**, and **153** were separated from marine sponge *Stelletta* sp.-associated *A. sydowii* (Figure 11). Compound **149** featured two ether-linked 1,3-dioxy-6-carboxy-5-methylphenyl units. It was assigned as dicarboxylated diorcinol (carboxylated orcinol's ether-linked dimer) [36]. Bioassay-guided separation of the East China Sea sediment-derived *A. sydowii* MF357 yielded new tris-pyrogallol ethers sydowiols A–C (**166**–**168**) and related bis-pyrogall ol ethers **144** and **145** that were characterized based on detailed spectral analysis and symmetry considerations [37]. On the other hand, the LC–UV–MS-guided separation of EtOAc extract of China Sea-derived *A. sydowii* resulted in new diphenyl ethers **155**–**157** and **159**– **161** along with compounds **146**, **147**, **149**, **150**, **152**, and **162**–**164** using SiO2/Sephadex LH-20 CC/HPLC; the compounds were assigned using spectral and chemical methods. Compounds **155** and **156** are rare glycosides, possessing a D-ribose moiety, whereas compound **157** has a D-glucose moiety [9].

**Figure 11.** Structures of phenyl ether derivatives (**144**–**157**) reported from *A. sydowii.*

From cold seep-derived *A. sydowii* 10–31, bisviolaceol II (**165**), a new tetraphenyl ether derivative, was isolated and characterized by Liu et al. using SiO2/Sephadex LH-20 CC/HPLC and spectral tools, respectively [38] (Figure 12).

**Figure 12.** Structures of phenyl ether derivatives (**158**–**168**) reported from *A. sydowii.*

#### *2.6. Chromane and Coumarin Derivatives*

Citrinin is a polyketide-derived mycotoxin that was first reported in *Penicillium citrinum* as lemon-yellow particles. Also, other species of *Monascus*, *Penicillium*, and *Aspergillus* genera are found to be capable of producing this toxin [68].

The coculture of two or more different microbes is a useful approach for activating silent biosynthetic genes to accumulate cryptic compounds. In this regard, an investigation on the EtOAc extract of a coculture of *A. sydowii* EN-534 and *P. citrinum* EN-535 obtained from the marine red alga *Laurencia okamurai* using SiO2/Sephadex LH-20/RP-18 CC/preparative TLC (thin-layer chromatography) resulted in the separation of new citrinin analogs **171** and **172**, in addition to compounds **169**, **170**, and **173**–**176**, that were characterized by spectral, optical rotation, ECD, and X-ray analyses (Table 6, Figure 13). Compounds **171** and **172** are a citrinin dimer and citrinin monomer, respectively. The configurations of compounds **171**–**173** were assigned as 3*R*/4*S*/2 *R*/3 *S*, 3*R*/4*S*/2 *R*, and 3 *S*/1*S*/3*R*/4*S* by X-ray and ECD analyses [69]. Further, asperentin B (**178**), a new asperentin analog, was obtained from the Mediterranean sea sediment-derived *A. sydowii* EN50, which is closely related to compound **177** but with an additional OH at C-6 [46]; it was proposed to be derived from the hydroxylation of PKS (polyketide synthase) precursor at the aromatic ring [46].


**Table 6.** Chromane and coumarin derivatives reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).


**Figure 13.** Structures of chromane and coumarin derivatives (**169**–**192**) reported from *A. sydowii.*

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#### *2.7. Pyrane, Cyclopentene, Cyclopropane, and Lactone Derivatives*

Two new 2-pyrone derivatives **195** and **196** and a new cyclopentenone derivative **208** along with known analog **197** were isolated from the South China Sea gorgonian *Verrucella umbraculum*-derived *A. sydowii* SCSIO-00305 utilizing SiO2/RP-10/Sephadex LH-20 CC/HPLC (Figure 14). The 8*R*/8*S*/5*S* absolute configuration of compounds **195**, **196**, and **208** was established using Mosher's method and ECD spectra [24]. Liu et al. separated pryan analogs **194** and **193** from *A. sydowii* SCSIO-41301 (Table 7) [35]. Two new pyrone derivatives, labeled **189** and **198**, together with compounds **199** and **200** were separated from *Aricennia marina*-inhabiting *A. sydowii* #2B by SiO2/Sephadex LH-20 CC/HPLC. Based on X-ray analysis and optical rotation measurement, compound **189** has 1S-configuration, while compounds **198** and **200** are racemic mixtures. Compounds **198** and **200** are alphapyrone derivatives; however, compound **189** is γ-pyrone [64]. Further, two undescribed α-pyrone derivatives **191** and **201** were separated and characterized from deep-sea-derived *A. sydowii* MCCC-3A00324. Compound **201** bears two phenyl moieties at C-3 and C-5 [60].

**Figure 14.** Structures of pyrane, cyclopentene, cyclopropane, and lactone derivatives (**193**–**216**) reported from *A. sydowii.*


**Table 7.** Chromane and coumarin derivatives reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).


The cyclopropyl moiety is the smallest cycloalkane moiety. It is a strained moiety that usually occurs as a structural subunit of various natural metabolites, particularly alkaloids, steroids, and terpenoids [71,72]. Many polycyclic natural metabolites bearing this ring were reported in higher plants, archaea, fungi, and bacteria, while monocyclic molecules are rarely found [73]. In 1920, the first monocyclic cyclopropane (+)-trans-chrysanthemic acid was reported [42]. In 2022, sydocyclopropanes A–D (**203**–**206**), novel monocyclic cyclopropane acids, along with compound **207** were separated from the deep-sea sedimentassociated *A. sydowii* MCCC-3A00324 using SiO2 CC/Sephadex LH-20/HPLC and were characterized by spectral, ECD, and DP4+ probability analyses by Niu et al. [42]. These metabolites feature a 1,1,2,3-tetrasubstituted cyclopropane moiety with different alkyl side chains. Their established configurations were 1S/2S/3S/12R for compound **203**, 1S/2S/3S for compounds **204** and **205**, and 1S/2R/3S for compound **206**, which was identified as a C-2 epimer of compound **205** [42].

In 2006, Teuscher et al. separated and characterized new hydroxylated, chlorinated diaryl cyclopentenone derivatives **210** and **211** from red alga *Acanthophora spicifera*-associated *A. sydowii* using Sephadex LH-20/HPLC and NMR/CD analyses, respectively. These kinds of metabolites were related to diaryl cyclopentenones reported in order Boletales basidiomycetic fungi and involved in conspicuous bluing reactions of fruiting bodies and reported for the first time from ascomycetes [53]. Compound **215** was isolated as enantiomers, involving (+)-(**215a**) and (−)-(**215b**), using SiO2/RP-18 CC/HPLC from *Rhododendron mole*accompanied *A. sydowii* and elucidated by spectral and CD analyses. They were purified by chiral HPLC and identified to have 7*S* and 7*R* configurations, respectively [26].

#### *2.8. Other Metabolites*

New catechol derivatives **223** and **224** were separated as racemic by Liu et al. and could not be separated into their enantiomers. Compound **224** resembles compound **223**, except for the presence of the C-2 COOH group and a 2-methylpentan-1-ol unit, instead of the 2-CH3 and propionic acid moiety in compound **223** [35] (Table 8, Figure 15). A new chorismic acid analog, labeled **217**, was reported by Liu et al. and its 3R/4S/5R/1 S configuration was assigned based on ECD analysis [41]. The same group separated a dibenzofuran derivative, labeled **234**, from *A. sydowii* SCSIO-41301 [35]. Compounds **228**– **230** and **234** were separated by Niu et al. from the deep-sea sediment-associated *A. sydowii* MCCC-3A00324 [60].

Anthocyanins belong to the flavonoids family and are generally reported from plant sources. These metabolites have various applications in agro-food industries such as in natural dyes; additionally, their substantial therapeutic human health in treating obesity and improving cardiovascular function are of note [74]. In 2020, Bu et al. reported the capacity of *A. sydowii* H-1 to produce anthocyanins using metabolomic and transcriptomic analyses [25]. Compounds **242**–**246** were characterized; compounds **242** and **244** were the most abundant of the identified anthocyanins [25]. Interestingly, cinnamate-4-hydroxylase and chalcone synthase genes were identified as the key genes involved in anthocyanin biosynthesis [25]. This expanded the knowledge of natural anthocyanin biosynthesis by fungi for the first time.


**Table 8.** Other metabolites reported from *Aspergillus sydowii* (molecular weight and formulae, strain, host, and location).


**Figure 15.** Other metabolites (**217**–**246**) reported from *A. sydowii.*

#### **3. Biological Activities of** *A. sydowii* **Extracts and Its Metabolites**

*3.1. Cytotoxic Activity*

*A. sydowii* MSX19583 extract (%cell viability: 54%, conc.: 20 μg/mL) had moderate cytotoxic capacity against MDA-MB-435 (human melanoma cell line) in an MTT assay [33], while a cultured EtOAc extract displayed a marked toxic effect (LD50 (lethal dose 50) 36 μg/mL) in a brine shrimp assay [36].

Vascular endothelial cell growth factor (VEGF) is a tumor-secreted protein that stimulates both the migration and growth of vascular endothelial cells; thus, interference with VEGF signaling suppresses tumor growth or blocks angiogenesis [34].

Compound **88** was found to suppress HUVEC (human umbilical vein endothelial cell) proliferation caused by VEGF, bFGF (basic fibroblast growth factor), or ECGS (endothelial cell growth supplement) (IC50s: 1.4, 2.8 μM, and 6.2 μM, respectively) compared to SU5416 (a tyrosine kinase inhibitor, IC50s: 0.05, 5.3, and 30.5 μM, respectively) [34] and demonstrated selective cytotoxic capacity versus A549 (human lung adenocarcinoma epithelial cell line) (IC50 < 10 μM) [55].

In an MTT assay, compounds **101** and **117** demonstrated a notable cytotoxic capacity toward A375 (human melanoma cell line) (IC50: 5.7 μM), whereas compound **101** had no cytotoxicity towards adenocarcinoma cells A549, A375, and Hela (human cervical epitheloid carcinoma cell line) compared to cis-platin [31] and compounds **1**, **45**, **110**, and **115** were inactive against MDA-MB-435 and HT-29 (human colon cancer cell lin) [33] (Table 9).


**Table 9.** Cytotoxic metabolites reported from *A. sydowii.*

\* IC50, Half maximal inhibitory concentration.

Acremolin D (**129**) had cytotoxic efficacy versus K562 (human erythroleukemic) and Hela-S3 (human cervix adenocarcinoma) cell lines with % inhibition equal to 25.1 and 30.6%, respectively, while compound **127** displayed activity (% inhibition: 20.9–35.5%) versus HepG2 (human hepatocellular liver carcinoma cell line), A549, and K562 in an MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [65]. Azaspirofurans A (**132**) displayed moderate cytotoxic potential versus A549 cell proliferation (IC50: 10 μM) in the MTT method [43] and compounds **103**–**105** (IC50s: 8.29, 1.28, and 7.31 μM, respectively) demonstrated weak capacities [48].

Compounds **44** and **149** were mildly active versus KB (human oral epidermoid carcinoma cell line), HepG2, and HCT-116 (human colon cancer cell line) cells (IC50s: 50–70 μM) compared to doxorubicin (IC50s: 5–6 μM) [36]. Wang et al. reported that compounds **146**, **149**, **152, 155**, **160**, and **161** were found to exhibit cytotoxic potential versus A549, U937 (promonocytic, human myeloid leukaemia cell line), HL-60 (human promyelocytic leukemia cell line), and K562 cells (IC50: 3.36–23.03 μM) [9]. Wang et al. stated that compounds **98**, **99**, **199**, **200**, and **216** possessed cytotoxic capacities versus VCaP (human prostate cancer cell line) (IC50s: 1.92–33.36 μM), but compound **189** was inactive in comparison with docetaxel (IC50: 4.95 nM) in the MTT method [64]. Compounds **83**, **195**, **196**, and **208** possessed toxicity towards brine shrine nauplii (LC50s: 2.9–19.5 μM), whereas compound **83** had a potent efficacy (LC50: 2.9 μM) compared to toosendanin (LC50: 2.2 μM) [24]. On the other hand, compounds **192** and **239** revealed powerful cytotoxic potential versus P388 (menogaril-resistant mouse leukaemia cell line) (IC50s: 0.14 and 0.59 μM, respectively) in a SRB (sulforhodamine B) assay; however, compound **237** was inactive [44].

#### *3.2. Antioxidant and Immunosuppression Activities*

Compound **24** was found to have DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50: 113.5 μM/L), while compound **25** was inactive (IC50 value > 300 μM/L) compared to BHT (butylated hydroxytoluene) (IC50: 30.8 μM/L) in a DPPH assay, suggesting that the OH position and racemization influenced the activity [32]. Also, compound **88** demonstrated antioxidant capacity (IC50: 17.0 μM) compared to butylated hydroxyanisole (IC50: 0.13 μM). Compound **88** differed from compound **71** in lacking C7–C8 double bonds, revealing that the planar structure of compound **71** might reduce its activity [56]. On the other hand, compounds **195** and **196** had more potent antioxidant activity (IC50s: 46.0 and 46.6 μM, respectively) than L-ascorbic acid (IC50: 61.0 μM); however, compounds **83** and **208** were weakly active (IC50s: 98.0 and 86.3 μM, respectively) [24].

Compounds **72**, **74**, **76**–**78**, **91, 94**, **97**, **218**, and **220** were evaluated in vitro for immunosuppression capacity against Con-A (concanavalin A)- and LPS (lipopolysaccharide) induced mouse splenic lymphocyte proliferation. It was noted that compounds **91** and **94** displayed moderate potential (IC50s: 8.45 and 10.10 μg/mL and 10.25 and 14.10 μg/mL, respectively), compared to cyclosporin A (IC50: 0.62 μg/mL for Con-A and 0.53 μg/mL for LPS). Other compounds showed weak or no activity [63].

#### *3.3. Anti-Mycobacterial, Anti-Microalgal, and Antimicrobial Activities*

Infectious illnesses seriously threaten human health worldwide [75,76]. Recently, the increasing recurrence of pathogens' resistance to antimicrobials represents an alarming trend in infectious diseases that results from misuse or overuse of existing antimicrobials and has become a universal health concern [75,76].

*A. sydowii* ethyl acetate extract (conc.: 500 μg/disk) had selective activity against *B. subtilis* and *E. coli* (inhibition zone diameters (IZDs) 12 and 15 mm, respectively); however, it was inactive against *S. aureus*, *C. albicans*, *Cladosporium herbarum*, and *C. cucumerinum* [53]. In another study, the EtOAc extract of *Dactylospongia* sp.-associated *A. sydowii* DC08 revealed antibacterial potential versus *E. coli* and *S. aureus* (IZDs 12.31 and 14.25 mm, respectively) [77]. Wang et al. reported that *A. sydowii* ZSDS1-F6 EtOAc extract displayed significant antimicrobial capacity versus *Aeromonas hydrophila* and *Klebsiella pneumonia* [45].

The antibacterial effectiveness of compounds **1**, **2**, **5**, **11**–**14**, **42**, **43**, and **55** versus phytopathogenic bacteria *Ralstonia solanacarum* and *Pseudomonas syringae* utilizing a broth microdilution method revealed that compound **5** had inhibition potential versus *P. syringae* (MIC (minimum inhibitory concentration): 32 μg/mL), whereas compounds **1**, **14**, and **43** were active versus *R. solanacarum* (MICs: 32 μg/mL) using the broth microdilution method [47] (Table 10). Further, compounds **11** and **14** inhibited *Fusarium oxysporum* spore germination (EC50s: 54.55 and 77.16 μg/mL, respectively), while compounds **1**, **11**, **14**, and **43** inhibited *Alternaria alternata* spore germination (EC50s: 26.02–46.15 μg/mL), suggesting the possible use of bisabolane sesquiterpenoids as anti-phytopathogens [47]. Also, compound **44** revealed antibacterial efficacy versus the human pathogen *S. aureus* and fish pathogens *S. iniae* and *V. ichthyoenteri* [36]. Compounds **71**, **88**, and **91** showed weak potential against *Vibrio rotiferianus* (MICs: 16–33 μg/mL); however, compounds **69**, **79**, **86**, **88**, **91**, and **219** were weakly active versus MRSA (methicillin-resistant *Staphylococcus aureus*) (MICs: 15–32 μg/mL) compared to erythromycin and chloramphenicol [55].


**Table 10.** Anti-mycobacterial, antimicrobial, and anti-microalgal metabolites reported from *A. sydowii.*

Compounds **2**, **3**, and **110** demonstrated notable antibacterial efficacy versus *S. aureus*, MRSA, *S. epidermidis*, and MRSE (MICs: 0.25–1.0 μg/mL) compared to trigecycline (MICs: 0.06–0.12 μg/mL); however, compound **128** displayed moderate-to-weak activity (MICs: 4– 32 μg/mL) [40]. Compounds **42**, **50,** and **146** had moderate effectiveness versus *K. pneumonia* (MICs: 21.4, 10.7, and 21.7 μM, respectively); also, compounds **1** and **42** exhibited moderate

activity against *E. faecalis* (MIC: 18.8 μM) and *A. hydrophila* (MIC: 4.3 μM), respectively, using an agar dilution method [45].

Compounds **53**, **54**, and **165** (conc.: 20 μg/disc) were found to prohibit the growth of bacteria (*V. anguillarum*, *V. harveyi*, *V. parahaemolyticus*, and *V. splendidus)* and harmful microalgae (*P. micans* and *P. minimum*) in a disc diffusion assay [38]. Pathogenic bacteria and harmful algal blooms pose substantial threats to marine aquaculture. Compounds **53** and **54** inhibited *P. micans* and *P. minimum* (IC50 ranging from 1.3 to 11 μg/mL), while compound **165** only had inhibitory efficacy against *P. minimum* (IC50: 5.2 μg/mL). Additionally, these compounds showed inhibition against *Vibrio* species (*V. anguillarum*, *V. harveyi*, *V. parahaemolyticus*, and *V. splendidus)* with IZDs ranging from 6.4 to 8.7 mm. The MICs for compounds **53** and **54** were 8 μg/mL against *V. anguillarum* and *V. parahaemolyticus* and 16 μg/mL against *V. harveyi* [38].

Compounds **61**, **62**, **130**, and **131** displayed notable growth inhibition potential versus *E. coli*, *B. subtilis*, and *M. lysoleikticus* (MICs: 3.74–87.92 μM); compounds **131** and **61** were more powerful than compounds **62** and **130** [48]. Antibacterial testing of compounds **51**, **124**, **125**, and **134** against human pathogenic bacterial strains *E. coli*, *S. aureus*, *S. pneumoniae*, and *S epidermidis* revealed that compounds **51, 124**, and **125** demonstrated selective inhibitory capacities (MICs ranging from 2.0–16 μg/mL), whereas compound **51** had significant activity against *E*. *coli* (MIC: 2.0 μg/mL) that was comparable to chloramphenicol (MIC: 2.0 μg/mL) [41].

Among the pyrogallol ethers, i.e., compounds **144**, **145**, and **166**–**168** reported by Liu et al. in 2013, compounds **166** and **168** (IC50: 14.0 and 24.0 μg/mL, respectively) demonstrated Mt PtpA (protein tyrosine phosphatase A) (*Mycobacterium tuberculosis* protein tyrosine phosphatase A)-inhibitory activity and compound **168** moderately inhibited *S. aureus*(MIC: 12.5 μg/mL) [37]. *M. tuberculosis* secretes PtpA into the infected macrophages' cytosol to avoid devastation by macrophage phagocytosis. Inhibition of PtpA remarkably attenuates *M. tuberculosis* growth in human macrophages; therefore, Mt PtpA is a target for developing anti-tuberculosis drugs [37].

Liu et al. stated that compounds **144**–**146**, **152**, and **153** were moderately effective versus fish pathogens *S. iniae* FP3187, and *V. ichthyoenteri* (Vi0917-1 and Vi099-7) and human pathogen *S. aureus* (SG 511 and SG 503) [36]. Compounds **147** and **149** were inactive, suggesting that methoxy groups increased the antibacterial potential; however, the carboxyl group reduced the activity of diphenyl ether derivatives [36]. Additionally, compounds **138**, **139**, **144**, **209**, and **210** possessed moderate antibacterial effectiveness (MICs: 6.3–25.0 μM) versus series of bacterial strains [28] and compound **84** was moderately active (MICs: 64, 128, 16, 32, and 32 μg/mL, respectively) versus MRSA, MDRPA (multi-drug-resistant *Pseudomonas aeruginosa*), *E. coli*, *S. aureus*, and *P. aeruginosa* in an agar diffusion assay [39].

Compounds **137**, **169**–**172**, **174**–**176**, **221**, and **222** reported from *A. sydowii* EN-534 and *Penicillium citrinum* EN-535 coculture were examined for antibacterial potential versus strains of human and aquatic bacteria. Compounds **137**, **169**, and **170** showed antibacterial capacity against bacteria *E. coli*, *E. ictaluri*, *M. luteus*, *V. parahaemolyticus*, and *V. alginolyticus* (MICs ranged from 4 to 64 μg/mL), while compounds **137**, **171**, **172**, and **174** were active against *V. alginolyticus* and *E. ictaluri* (MICs: 32–64 μg/mL). Additionally, compound **170** had marked activity against *M. luteus* (MIC: 4 μg/mL) compared to chloramphenicol (MIC: 2 μg/mL) [69].

#### *3.4. Anti-Influenza Virus Activity*

The influenza pandemic remains a threat to public health because of its elevated rates of mortality and morbidity. Although vaccination is the primary means for preventing this illness, antiviral medications are an essential adjunct to vaccines for influenza control and prevention [78,79]. In the last several decades, natural products have been subjected to intensive investigations as a possible alternative therapy for the recovery and treatment of influenza. Various reports have demonstrated that developing natural bioactive metabolites has remarkable advantages [78,79]. It is noteworthy that the renowned antiinfluenza oseltamivir was synthesized using natural shikimic and quinic acids as starting materials [78,79]. Some reports assessed the anti-influenza potential of *A. sydowii*-isolated metabolites; these are highlighted below (Table 11).


**Table 11.** Anti-influenza virus metabolites reported from *Aspergillus sydowii.*


Interestingly, compounds **80** and **81** possessed notable selective inhibition versus two influenza A virus subtypes, including A/Puerto Rico/8/34 (H1N1) and A/FM-1/1/47 (H1N1) (IC50s: 2.17–4.70 μM), compared to ribavirin (IC50s: 2.53 to 6.23 μM). Additionally, compounds **92** and **94** had potent efficacy on A/Puerto Rico/8/34 (H1N1) (IC50s: 1.92 and 2.0 μM, respectively). Furthermore, compound **234** demonstrated broad inhibitory potential against A/Puerto Rico/8/34 (H1N1), A/Aichi/2/68 (H3N2), and A/FM-1/1/47 (H1N1) (IC50s: 1.31, 1.24, and 2.84 μM, respectively) compared to ribavirin (IC50s: 2.53, 6.23, and 3.97 μM, respectively) [35]. Compounds **50**, **146,** and **152** demonstrated weak anti-H3N2 potential (IC50s: 57.4, 66.5, and 78.5 μM, respectively) in a CPE (cytopathic effect) inhibition assay compared to Tamiflu (IC50: 0.95 μM) [45]. Further, Yang et al. stated that compounds **137**, **169**–**172**, and **174** demonstrated anti-influenza NA (neuraminidase) activity, with compounds **137** and **174** displaying better efficacy (IC50s: 12.9 nM and 18.5 nM, respectively) compared to oseltamivir (IC50: 3.6 nM) [69]. Additionally, compounds **203**– **207** demonstrated antiviral potential versus the A/WSN/33 virus (H1N1) (IC50s ranged from 26.7 to 77.2 μM), compared to oseltamivir (IC50: 18.1 μM); compounds **203**, **204**, and **207** were the most active (IC50s: 26.7, 29.5, and 35.8 μM, respectively). It was found that the C-1 methyl 2-hydroxy-4-oxobutanoate side chain significantly enhanced the antiviral activity (e.g., compound **203** vs. compound **205**) and C-3 configuration had less influence on activity (e.g., compound **205** vs. compound **206**) [42].

#### *3.5. Anti-Diabetic and Anti-Obesity Activities*

A close relation among between diabetes and obesity has been proven [80]. Insulintriggered cellular glucose uptake is a crucial step in glucose regulation and any defect in this mechanism results in insulin resistance [81]. Enhancement of insulin sensitivity is one of the significant hallmarks of anti-diabetic agents. Lipid accumulation in diabetic patients can result in serious effects such as diabetic cardiomyopathy [82]. Hence, efficient antidiabetics should decrease adipocytes' lipid accumulation and facilitate lipid metabolism and burning [54].

In an anti-diabetic assay, compounds **1**, **5**, **42, 45**, **46**, **47**, **49**, **69**, **71**, and **88** were found to increase differentiated 3T3-L1 (fibroblast embryo mouse cell line) adipocytes' medium glucose consumption. Among them, compound **45** significantly reduced culture medium glucose concentration (324.6 mg/dL) by 24% compared to control (glucose: 427.4 mg/dL). It was noted that the presence of a methylene alcohol and a hydroxy group on C-3 and C-7, respectively, in bisabolane sesquiterpenes is substantial in promoting 3T3-L1 adipocytes' glucose uptake [54]. Additionally, their efficacy on differentiated 3T3-L1 adipocytes' lipid accumulation utilizing oil-red O stain revealed that compound **45** notably prohibited lipid accumulation up to 48% in a 3T3-L1 adipocyte culture medium, indicating the compound **45** promoted glucose consumption and suppressed lipid accumulation in adipocytes [54].

#### *3.6. Protein Tyrosine Phosphatase Inhibition*

Protein tyrosine phosphatases (PTPs) are proven to be substantial new targets for new anti-diabetes [58]. For example, PTP1B (protein tyrosine phosphatase 1B) negatively regulates insulin action in the insulin receptor signaling pathway, SHP1 (SH2-containing protein tyrosine phosphatase 1) negatively controls signaling pathways, which streamlines glucose homeostasis through modulating insulin signaling in muscles and the liver, and CD45 (leukocyte common antigen) is a receptor for some ligands and regulates SHP-1 recruitment [58]. Also, PTP1B has a substantial role in cancer development, inflammation processes, and insulin signaling cascade. Therefore, PTP1B inhibitors are considered drug candidates for treating cancer, diabetes, inflammation processes, and sleeping sickness [46].

Asperentin B (**178**) had potent PTP1B inhibition capacity (IC50: 2.05 μM) compared to suramin (IC50: 11.85 μM). It was sixfold more potent than suramin, suggesting its possible application in anti-diabetes and anti-sleeping sickness therapeutic agents [46]. Furthermore, compounds **1**, **3**, and **18** displayed significant PTP1B-inhibitory potential (IC50s: 7.97, 15.88, and 14.18 μM, respectively), while compounds **1**, **2**, **18**, and **240** had potent activity towards SHP1 (IC50s: 8.35, 15.72, 11.68, and 14.61 μM, respectively). The PTP1B data indicated that the side chains influenced activities [58].

#### *3.7. Anti-Inflammation Activity*

Compounds **42**, **45**, and **88** markedly inhibited fMLP (tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine)/CB (cytochalasin B)-caused superoxide anion generation (IC50s: 5.23, 6.11, and 6.00 μM, respectively) and elastase release (IC50s: 16.39, 8.80, and 6.60 μM, respectively) by neutrophils [54]. It is noteworthy that compounds **1**, **5**, **46**, and **49** had selective inhibition versus fMLP/CB-caused superoxide anion generation [54]. These results demonstrated the importance of C-7 OH (compound **45** vs. compound **46**) and C-3 methylene alcohol (compounds **46**, **45**, and **49** vs. compounds **1** and **5**) on activity (Table 12). On the other hand, compound **71** also revealed a significant superoxide anion generation inhibition capacity (IC50: 21.20 μM) compared to compound **69** [54]. The isolated metabolites, compounds **1**–**3**, **26**–**42**, **45**, **47**, **50**, **56**–**58**, and **214**, showed a dose-dependent inhibition of LPS-induced NO (nitric oxide) secretion (conc.: 10 and 5 μM) in BV-2 microglia cells using a CCK-8 (cell counting kit-8) assay. Compounds **33**, **39**, **42**, **47**, **50**, and **57** revealed an inhibition rate >45% (conc.: 10 μM). The structure–activity relation indicated that the Δ7,8 double bond in sydowic acid derivatives enhanced NO secretion inhibition (e.g., compound **33** vs. compound **26**). Compound **39**, with a 56.8% inhibition rate, was found to exert its

anti-inflammation activity by prohibiting the NF-κB (nuclear factor kappa B)-activated pathway [57].

**Table 12.** Anti-inflammatory metabolites reported from *Aspergillus sydowii.*


It was found that compounds **145** and **153** mildly suppressed NO production induced by LPS-NO in RAW 264.7 cells (IC50: 73 μM) compared to dexamethasone (IC50: 18 μM) [36]. Additionally, compounds **59**, **60**, **146**, **152**, **191**, **201**, **213**, **228**–**230**, and **234** demonstrated an inhibitory capacity of NO production induced by LPS in BV-2 microglia cells without toxicity according to a CCK-8 assay. Interestingly, compound **234** (10 μM) was the most potent (inhibition rate: 94.4%) among these tested compounds (inhibition rate: 10.2–35.4%) [60].

Compounds **98**, **189**, **199**, and **200** possessed inhibitory effectiveness on LPS-boosted NO production in RAW264.7 cells (IC50s: 25.25–43.08 μM), compared to dexamethasone (IC50: 35.17 uM) [64]. Recently, Chen et al. reported that compounds **215** and **236** exhibited weak inhibition of LPS-induced NO production (20.1, 21.5, and 18.1%, respectively), compared to dexamethasone (% inhibition: 99.9%) in RAW 264.7 cells using a Griess reaction assay [26].

#### *3.8. Anti-Nematode Activity*

Globally, parasitic nematodes cause diseases of major socio-economic significance to humans and animals. They have a long-term impact on human health, especially in children [83]. Indeed, nematodes' resistances to available anti-nematode agents are widespread all over the world [84]. Thus, there is an insistent demand to discover new agents for the effective and sustained control of nematodes.

Sun et al. evaluated the anti-nematode activity of compounds **1**–**3**, **18**–**21**, **202, 235**, and **240**. It is noteworthy that only compound **3** showed anti-nematode potential (IC50: 50 μM) [58]. A study by Yang et al. revealed that compounds **1**, **11**, and **14** possessed nematicidal potential versus second-stage juvenile *Meloidogyne incognita* (J2s); compound **1** had the strongest activity (% mortality: 80% at 60 and LC50: 192.40 μg/mL). Furthermore, compounds **1, 11**, and **14** paralyzed the nematode and then impaired its pathogenicity [47].

#### **4. Industrial and Biotechnological Applications**

The discovery and development of effective enzymes for the use of renewable resources as raw materials is a requirement for the transition to a biobased economy. Many enzymes are crucial in efficiently hydrolyzing raw materials by enzymatic means. Exploring the potential of untapped natural habitats is a potent method for overcoming the limited enzymatic toolkit.

*A. sydowii* was found to be a rich source of enzymes with marked industrial and biotechnological potential, including α-amylases, lipases, xylanases, cellulases, keratinase, and tannases, which are discussed here.

#### *4.1. α-Amylase, Tannases, and Lipase Enzymes*

Amylases (AAs) are utilized in multiple manufacturing processes, including fermentation, textile, detergent, paper, and pharmaceutical sectors [85]. Given the low cost and wide availability of the starch feedstock used to make food, bioethanol, textile, paper, detergent, and chemicals, there is a significant demand for α-amylase [86]. However, because of advancements in biotechnology, the use of AAs has increased in a variety of sectors such as those of clinical, pharmaceutical, and analytical chemistry, as well as in the food, textile, and brewing industries [85]. The huge industrial demand for AAs to support economically competitive manufacturing processes is still being severely hampered by the cost and effectiveness of AA cocktails [19]. In this regard, it is imperative to generate effective and affordable AAs by using inexpensive sources such as agricultural wastes.

Adegoke and Odibo produced AAs from *A. sydowii* IMI-502692 utilizing the solid-state fermentation of buffered cassava root fiber. It was found that this activity was enhanced by Ca2+, Cu2+, and Zn2+; however, it was prohibited by Fe2+, Sr2+, Ni2+, and Mn2+ [19].

A study by Elwan et al. reported that *A. sydowii* had a potential for lipase production (lipase yield of 90 μ/mL) in optimum culture conditions, specifically 5.4 pH; 2.0% sucrose, 0.2% corn oil, 0.23% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.05%KCl, and using 0.1 M phosphate–citrate buffer and incubating at 30◦C for 20 h [22].

Tannase, an extracellular enzyme belonging to the hydrolase family, is derived from various species of the *Aspergillus* genus [8,87]. It catalyzes the breakdown of depsides and tannins. Tannase lessens tannins' unwanted effects (astringent and bitter taste), enhancing the flavor qualities of products such as animal feeds and foodstuffs. It is used in various applications, including polyphenolic compound structural elucidation, bioremediating tannin-contaminated wastewaters, gallic acid production, and coffee-flavored soft drink, fruit juice, and instant tea production [20].

In 2020, Albuquerque et al. purified and characterized tannase-acyl hydrolase from *A. sydowii* SIS-25 derived from Caatinga soil (Serra Talhada, Pernambuco, Brazil) utilizing a polyethylene glycol-citrate aqueous two-phase system. This enzyme removed phenolic components and enhanced the sensory qualities of green tea and produced gallic acid [20].

#### *4.2. Bioremediation and Biodegradation*

Sustainable development goals (SDGs) target various concerns in our planet such as food security, health, environmental sustainability, bioremediation, climate change, alternative eco-friendly fuel, improving water quality, sustainable food production, and discovering new drugs [88]. Treatment and measurement of various contaminants in water, soil, and air are complicated issues and are linked to the nature of contaminants and their environmental interactions. Reusing wastewater offers a substitute supply for the irrigation of agricultural land that has been used for decades in many nations. Recycling wastewater adheres to circular economy principles by reducing waste and encouraging ongoing resource reuse [89] which potentially assists various national initiatives in promoting sustainable agriculture methods. Creating agricultural systems with minimal required inputs and zero waste contributes to SDG 2 (End hunger) (via sustainable food production), SDG 12 (Responsible consumption and production), SDG 13 (Climate action), and SDG 15 (Sustainable use of terrestrial ecosystems) [90]. Various researches have focused on

biologically based methods, relying on natural processes to remove contaminants such as the utilization of microorganisms (bioremediation) such as fungi to remarkably contribute to achieving the SDGs [88].

#### 4.2.1. Polycyclic Aromatic Hydrocarbons

PAHs (polycyclic aromatic hydrocarbons) are a heterogeneous class of hydrocarbons having two or more fused aromatic rings. In nature, they are formed as a result of organic matter's incomplete decomposition and human activities such as petroleum spilling, waste incineration, home heaters, and the burning of carbon, oil, gas, or wood [91]. Additionally, PhCs (pharmaceutical compounds), a second class of contaminants, have become more significant in recent years as a result of their durability and abundance in surface water bodies and the ineffectiveness of treatment facilities eliminating them [92]. According to Olicón-Hernández et al., these contaminants are hazardous to aquatic life and contribute to microbial resistance's emergence [93]. Numerous studies have focused on the microbial biodegradation of these contaminants, particularly by fungi [93,94], because these pollutants are known for their high toxicity and persistence [94]. It is noteworthy that halophilic fungi are useful in xenobiotic mycoremediation under high-salinity conditions [94].

González-Abradelo et al. studied the potential of *A. sydowii* EXF-12860 toward the bioremediation of saline wastewaters, containing toxic and persistent PAHs and PhCs. It was stated that *A. sydowii* may be helpful in lowering the amounts of harmful PAHs and PhCs under high-salinity conditions (>1 M NaCl) during the biotechnological downstream processing of diverse industrial wastewater. It removed 100% of fifteen complex PAHs at 500 ppm in biorefinery wastewater at high salt concentrations. Additionally, it has ecotoxic activity as it demonstrated the same capability to eliminate PhCs. This supported its capabilities for xenobiotic biodegradation in low-water activity [94]. A novel piezo-tolerant and hydrocarbon-oclastic deep-sea sediment-derived *A. sydowii* BOBA1 demonstrated a marked degradation potential for PAHs in spent engine oil hydrocarbon fractions (71.2 and 82.5% of spent engine oil, respectively) under high-pressure (0.1 and 10 MPa, respectively) culture conditions with a 21-day retention period. This provided insights into the bioremediation of hydrocarbon-contaminated deep-sea environments [95].

Additionally, Birolli et al. stated that *A. sydowii* CBMAI-935 isolated from a noncontaminated site on the coast of São Sebastião (Brazil) biodegraded anthracene [96]. To biodegrade dieldrin, one of the most widely employed organo-chlorine pesticides, banned due to its long persistence and high toxicity to the environment, Birolli et al. found that *A. sydowii* CBMAI-935 and *A. sydowii* CBMAI-933 were capable of growing in the presence of dieldrin, suggesting its high tolerance. It is noteworthy that no biodegradation byproducts were found in the GCMS, revealing that dieldrin could be converted into polar molecules or mineralized, prohibiting the emergence of harmful or durable derivatives [97].

#### 4.2.2. Heavy Metals and Insecticides

Cadmium (Cd) is often used in the electroplating and metallurgical industries and is found in several pesticides, fertilizers, and fungicides [98]. Upon its absorption by both animals and humans, it accumulates in the kidneys and liver, severely harming the renal tubules and resulting in a variety of symptoms such as proteinuria and hyperglycemia [99]. Trichlorfon (TCF) is a broad-spectrum organic phosphorus pesticide that is utilized for controlling pests on a variety of crops [100]. It is an inhibitor of cholinesterase that causes delayed neuropathy in both animals' and humans' nervous systems [98].

Zhang et al. reported that by inoculating *A. sydowii* into Cd-TCF co-contaminated soil, TCF breakdown was accelerated, and soil enzyme activity was raised. When *Brassica juncea* (Indian mustard) was planted along with *A. sydowii* inoculation, maximum TCF degradation and Cd removal efficacy were noted. *Brassica juncea* is among those hyperaccumulator plant species that are frequently employed for heavy metal phytoextraction from contaminated soil. Thus, using *B. juncea* and *A. sydowii* together is a promising strategy to bioremediate soil that has been contaminated with both TCF and Cd [98]. Tian et al. isolated PAF-2, a new strain of *A. sydowii* from pesticide-contaminated soils, that had potential for the biodegradation of TCF and its degradation [100].

Esfenvalerate (S,S-fenvalerate), is a pyrethroid insecticide that deposits in marine sediments and is extremely harmful to aquatic creatures. Birolli et al. examined its biodegradation by marine-associated *A. sydowii* CBMAI-935. This strain metabolized esfenvalerate into 3-phenoxybenzoic acid, 2-(4-chlorophenyl)-3-methylbutyric acid, and its dihydroxylated derivatives [101].

Alvarenga et al. assessed the biodegradation of a commercial formulation of chlorpyrifos (Lorsban 480 BR), which is one of the most widely utilized organophosphate pesticides, by marine-derived *A. sydowii* CBMAI-935 associated with *C. erecta*. The fungus degraded ≈ 63% of the chlorpyrifos and decreased the concentration of its hydrolysis product 3,5,6-trichloropyridin-2-ol after 30 days [102]. In 2021, Soares et al. reported that this fungus also metabolized chlorpyrifos and profenofos to 3,5,6-trichloro-1-methylpyridin-2(1H) one/2,3,5-trichloro-6-methoxypyridine/tetraethyl dithiodiphosphate/3,5,6-trichloropyridin-2-ol and 4-bromo-2-chlorophenol/4-bromo-2-chloro-1-methoxybenzene/O,O-diethyl Spropylphosphorothioate, respectively [103].

Methyl parathion is an efficient organophosphate acaricide and insecticide that is widely utilized for pest control on a wide variety of crops, but it is extremely toxic. Alvarenga et al. reported the ability of *A. sydowii* CBMAI-935 to biodegrade this pesticide completely after 20 days. This fungus metabolized this pesticide to its more toxic isomerization and oxidation products isoparathion and methyl paraoxon, which were subsequently metabolized to the less toxic product 1-methoxy-4-nitrobenzene/p-nitrophenol/O,O,Otrimethyl phosphorothioate/O,O,S-trimethyl phosphorothioate/trimethyl phosphate, suggesting *A. sydowii* CBMAI-935's efficiency in the bioremediation of this pesticide and its toxic forms [103,104].

#### 4.2.3. Lignocellulosic Biomasses

Due to the acute energy crisis and increased demand for fossil fuels, lignocellulose is widely considered a potential cost-effective, renewable resource for bioethanol production [105,106]. Lignocellulose consists of cellulose, hemicellulose, and lignin. Lignin, which together with hemicellulose and cellulose makes up the majority of a plant's skeleton, is the second-most abundant organic renewable resource on Earth after cellulose [105,106]. The ligninolytic enzymes Lac (laccase), LiP (lignin peroxidase), VP (versatile peroxidase), and Mnp (manganese peroxidase) play a major role in the breakdown of lignin [105,106] and are found among the extracellular enzymes in filamentous fungi. These enzymes play a significant role in bioremediation, as they neutralize or degrade contaminants in the environment [6]. They also have a wide range of uses in the paper, textile, cosmetic, food, chemical, agricultural, and energy industries.

A thermostable, low-molecular-weight xylanase belonging to the glycosyl hydrolase 11 family was purified from *A. sydowii* MG49 by Ghosh et al. and demonstrated specific efficacy only in the presence of xylan and had no activity in the presence of cellulose or carboxymethyl cellulose [23].

*A. sydowii* MS-19 isolated from the Antarctic region produced low-temperature lignindegrading enzymes LiP and Mnp. These results suggested that *A. sydowii* MS-19 could be used as a source of lignocellulosic enzymes [107].

Xylan is the prime constituent of hemicellulose. Its backbone consists of a linear chain of 1,4-linked β-D-xylopyranosyl units, which are substituted with α-L-arabinofuranosyl, 4-O-methyl-α-D-glucuronopyranosyl, or acetyl units. It is degraded by β-D-xylosidases, endo-1,4-β-xylanases, α-glucuronidases, α-l-arabinofuranosidases, acetyl xylan esterases, and ferulic acid esterases [108].

Brandt et al. stated that *A. sydowii* Fsh102 isolated from shrimp shells showed notable xylanase-producing capacity [109]. Two xylanases I and II belonging to GH-11 (glycoside hydrolases) and GH-10 families, respectively, were characterized and expressed in *E. coli.* These enzymes can function in a wide pH range and are tolerant of mesophilic

temperatures. Both xylanases can be characterized as being extremely interesting for the enzymatic breakdown of xylan-containing biomasses in industrial bioprocesses based on their activity and stability [109]. In another study on *A. sydowii* SBS-45 culture filtrate, two xylanases (I and II) were purified. They showed optimum activity at 50 ◦C and 10.0 pH. This activity was boosted by certain metal ions and L-tryptophan [110].

Cellulose breakdown is carried out by cellulases, including β-glucosidase, endoglucanase, and cellobiohydrolase [108,111,112]. Cellulase has wide applications in various fields like oil extraction, agricultural industries, food processing, waste management, carotenoid extraction, animal feed, brewery, textile, bio-stoning, color clarification, paper, laundry, pulp, detergent industry, and deinking [108,111,112].

*A. sydowii* isolated from Indore, India, had the potential to produce cellulases under submerged fermentation. It was found that β-glucosidase, exoglucanase, and endoglucanase were produced at a ratio of 64:27:9, whereas lactose was the best carbon source for inducing cellulase production [113].

#### 4.2.4. Keratinous Wastes

Keratins are components of hooves, wool, horns, nails, hair, and feathers [8,114]. They are insoluble proteins with highly stable polypeptide chains, containing many disulfide bonds [115,116]. According to estimates, the United States, China, and Brazil produce 40 million tons of keratinous waste each year [117]. Also, keratinous waste is produced in millions of tons annually in meat industry slaughterhouses worldwide [115,116]. Normal enzymes such as papain and pepsin that break down proteins cannot break them down. Keratinous waste management utilizing a low-cost solution is needed particularly in underdeveloped nations. These wastes can be broken down by microbial keratinases which are extracellular enzymes secreted by various bacterial and fungal genera [8,114]. They are widely used in different pharmaceutical industries, in treating keratinized skin, calluses, acne, and psoriasis, and in cosmetic products manufacture (e.g., nutritional lotions, anti-dandruff shampoos, and creams) [21,115,116]. Also, they are usually employed in nitrogen fertilizers, feed formulas, and the leather industry, as well as in treating keratin waste-contaminated wastewater [21].

Alwakeel et al. studied the capability of keratinase produced by *A. sydowii* AUMC-10935 isolated from male scalp hair to degrade keratinous materials from chicken feathers. The enzyme had optimal activity (120 IU/mg) at 50 ◦C and pH 8.0, which was notably prohibited by EDTA and certain metal ions [21].

#### *4.3. Biocatalysis*

The pharmaceutical sector is continually looking for new approaches to new therapeutic agent syntheses, which has increased the demand for biocatalytic techniques [118]. Whole microorganism cells are effectively used as catalysts in the stereoselective biotransformation of a variety of chemical molecules. Also, many chemical reactions such as carbonyl ketone reduction, sulfide oxidation, secondary alcohol deracemization, and Baeyer–Villiger reactions were all catalyzed by enzymes from various microorganisms [6]. The whole cell of *A. sydowii* was investigated as a biocatalyst for various chemical reactions. This was highlighted in the current work.

Whole cells of the marine sponge-derived *A. sydowii* Gc12 obtained from the South Atlantic Ocean catalyzed the hydrolysis of (R,S)-benzyl glycidyl ether to produce (R)-benzyl glycidyl ether. Derivatives of glycidyl ether are potentially beneficial intermediates in the manufacture of β-adrenergic blockers. *A. sydowii* Gc12 hydrolases showed regioselectivity in opening the epoxide ring of racemic oxirane [119].

Sponge-associated *A. sydowii* CBMAI-934 derived from *Chelonaplysilla erecta* produced oxidoreductase that catalyzed regioselective mono-hydroxylation of (−)-ambrox® to 1βhydroxy-ambrox. (−)-Ambrox®, a naturally occurring terpene, was separated from ambergris, a pathological substance formed in the blue whale's intestine. This compound is of great commercial value in the perfume industry as a fixative or fragrant agent [120]. de

Paula and Porto investigated progesterone biotransformation by *A. sydowii* CBMAI-935 associated with marine sponge *Geodia corticostylifera*. In a good yield, this fungus was able to oxidize progesterone at the C17-site, resulting in the two major products testololactone and testosterone. Additionally, this Baeyer–Villiger reaction-based bio-oxidation revealed the existence of crucial enzymes in this fungus that can aid in related steroid biotransformation [121]. *A. sydowii* CBMAI-935 only produced 2 ,4-dihydroxy-dihydrochalcone with a yield of 26% from 2 ,4-dihydroxy-dihydrochalcone [122].

Further research was conducted by de Oliveira et al. to assess the potential of *A. sydowii* CBMAI-934 isolated from the marine sponge *Chelonaplysilla erecta* in converting a number of methylphenylacetonitriles into corresponding acids at a high yield. It was found that aryl aliphatic nitrilases were induced by phenyl acetonitrile. Thus, *A. sydowii* CBMAI-934 might serve as a biocatalyst for the production of carboxylic acids from nitriles [123]. Zhou et al. reported that *A. sydowii* PT-2 isolated from Pu-erh tea degraded theobromine to 3-methylxanthine in a liquid culture through N-7 demethylation [124]. Also, Jimenez et al. reported that *A. sydowii* CBMAI 935 associated with *C. erecta* sponge collected from Sao Sebastiao, São Paulo, Brazil, enantioselectively reduced ene of E-2-cyano-3-(furan-2-yl)acrylamide to (R)-2-cyano-3-(furan-2-yl)propanamide with a high yield [125]. In 2018, Morais et al. studied the reduction of α-chloroacetophenones to (S)-alcohols using whole cells of marine-derived *A. sydowii* CBMAI 935 [126]. α-bromoacetophenones' biotransformation by the marine-derived *A. sydowii* Ce19 was studied by Rocha et al. in 2010 [127]. This fungus accelerated α-bromoacetophenones' bioconversion into (R)- 2-bromo-1-phenylethanol (56%), in addition to acetophenone (4%), 1-phenylethan-1,2 diol (26%), phenylethanol (5%) and α-chlorohydrin (9%). The substituted p-nitro- and p-bromoacetophenone's biotransformation produced a low-concentration complex combination of breakdown products [127]. In 2017, Alvarenga and Porto tested the biocatalytic ability of *A. sydowii* CBMAI-935 of marine origin to convert 2-azido-1-phenylethanone and some derivatives to related alcohols for use in the synthesis of enantiomerically bioactive β-hydroxy-1,2,3-triazoles. *A. sydowii* CBMAI 935 displayed extremely high stereoselectivity and conversion values for the bio-reduction of 2-azido-1-phenylethanones to (S)- 2-azido-1-phenylethanols [128]. Further, the marine-derived *A. sydowii* Ce15 converted 1-(4-methoxyphenyl)ethenone to (R)-1-(4-methoxyphenyl)ethanol [129].

#### **5. Nanoparticle Synthesis**

Nanoparticles (NP) have attracted great interest recently because of their apparent applications in different fields such as biosensors, biomedicine, cosmetics, drugs, photocatalysis, animal dietary supplements, biolabeling, etc. [130]. Conventional NP synthesis approaches are not environment-friendly and are cost-intensive. Therefore, the development of biocompatible, environment-friendly, and non-toxic protocols in nanostructure biosynthesis is a wealthy area for scientific research, wherein the use of microbes could be an auspicious alternative [131,132]. Fungi are more effective organisms for these purposes than other microbes because of their special features, including their greater growth capacity, greater potential to produce a variety of enzymes, richness in mycelial branching, ability to accumulate different metals, and capacity to grow in harsh environments [133].

*A. sydowii* derived from Bhavnagar coast water (Gulf of Khambhat, India) had a remarkable intra/extracellular capacity to biosynthesize gold nanoparticles with variable sizes depending on gold ion concentration [52]. Additionally, silver NPs were biosynthesized by Wang et al. using soil-derived *A. sydowii* culture supernatants. These NPs revealed an in vitro antiproliferative capacity against MCF-7 (human breast adenocarcinoma cell line) and HeLa cells and efficient antifungal potential versus various clinical pathogenic fungi [134].

Zhang et al. prepared magnetic chitosan microsphere-immobilized *A. sydowii* by utilizing the cross-linking of γ-Fe2O3 magnetic chitosan nanocomposites with *A. sydowii* through the instant gelation method. This microsphere demonstrated marked Cu adsorption capacity (19.21 mg/g) and good regeneration properties after four cycles, suggesting its potential application as a biosorbent for treating heavy metal-contaminated water [51].

The AgNPs synthesized by Nayak and Anitha from dune-associated *A. sydowii* had significant antimicrobial potential versus selected bacterial stains; its combination with vancomycin and ampicillin showed enhanced activity (by sevenfold against *Shigella* sp. and by sixfold against *B. cereus* and *S. aureus* [50]).

Organic waste and heavy metal removal from wastewater have always been a major concern for the environment. In order to simultaneously remove trichlorfon and cadmium from an aqueous solution, Zhang et al., in 2020, created magnetic chitosan beads-immobilized *A. sydowii* [49]. The beads demonstrated considerable trichlorfon and cadmium removal capabilities, as well as outstanding four-cycle recyclability. As a result, the beads are appropriate and efficient for removing cadmium and trichlorfon simultaneously from wastewater [49].

#### **6. Conclusions**

Fungi have been subjected to much research due to their significance as wealth generators for various enzymes and bio-metabolites, as well as being intriguing for applications in agricultural, industrial, and pharmaceuticals fields.

*A. sydowii* is a globally distributed fungus that was found to have the capacity to biosynthesize diverse classes of metabolites. In the current work, 246 metabolites were separated from *A. sydowii* in the period from 1975 to 2023 (Figure 16). Most of these metabolites were reported from 2017 to 2022.

**Figure 16.** Number of metabolites reported from *A. sydowii* per year.

These metabolites include sesquiterpenoids, alkaloids, xanthones, monoterpenes, anthraquinones, sterols, triterpenes, phenyl ethers, pyrones, cyclopentenones, anthocyanins, coumarins, chromanes, acids, phenols, and other metabolites. Sesquiterpenoids (58 compounds, 24%), phenyl ethers (25 compounds, 10%), alkaloids (44 compounds, 18%), and xanthones (22 compounds, 9%) are the major constituents reported from this fungus (Figure 17).

**Figure 17.** Different classes of metabolites reported from *A. sydowii.* AnTs: anthocyanins; SQT: sesquiterpenes; MT: monoterpenes; OMs: other metabolites; PHs: phenols; TRT: triterpens; ST: sterols: XT: xanthones; QU: quinones; ALK: alkaloids; PhEs: phenyl ethers; CHs: chromanes; COs: coumarins; Pys: pyranes; CPEs cyclopentenes; CyPr: cyclopropane, and lactone derivatives.

This fungus was collected from different sources such as cultures, plants, marine environments (water, sea mud, sediment, gorgonian sea fans, algae, sponge, and driftwood), and liverworts. Most of the reported studies were carried out on *A. sydowii* isolated from marine sources. It is remarkable that this fungus has many enzymatic systems, which may help to explain why its metabolites are so diverse. Future studies will be useful in understanding the enzymes and genes responsible for the manufacture of these metabolites.

It was found that the coculture of this fungus with other microbes, as well as the modification of the culture media, significantly promoted the production of structurally varied metabolites, suggesting avenues of further research using these approaches for activating *A. sydowii*'s silent biosynthetic genes toward the accumulation of various substantial compounds.

These metabolites were assessed for different bioactivities, including cytotoxic, antimicrobial, antioxidant, antiviral, anti-obesity, anti-inflammation, immunosuppression, anti-diabetic, protein tyrosine phosphatase 1B (PTP1B) inhibition, and anti-nematode activities (Figure 18).

Compounds **195** and **196** displayed potent antioxidant activity. Compounds **67**, **187**, **192**, and **239** demonstrated powerful cytotoxic potential. Compounds **2**, **3**, and **110** had notable antibacterial efficacy. Compounds **80**, **81**, **92**, **94**, and **234** displayed potent antiinfluenza virus activity. Furthermore, compound **45** was found to possess anti-diabetic and anti-obesity capacities through promoting glucose consumption and suppressing lipid accumulation, whereas compound **178** had a potent PTP1B inhibition capacity compared to suramin, suggesting its possible application in anti-diabetic and anti-sleeping sickness therapeutic agents.

Despite the large number of metabolites, biological evaluation has only been conducted for a limited number of them, mainly in vitro, and there is a lack of pharmacological investigations that focus on studying the possible action mechanisms of the active metabolites. Therefore, mechanistic and in vivo studies are recommended to clarify and validate potential mechanisms for the active metabolites. Moreover, studies on the structure–activity relationships of these metabolites should be carried out.

**Figure 18.** Number of metabolites evaluated for each bioactivity.

Additionally, molecular dynamic and docking studies could be employed to investigate the possible bioactivities of the untested metabolites.

On the other side, many of the tested metabolites displayed no notable effectiveness in some of the tested activities. Therefore, estimation of other possible bioactivities and molecular dynamic and docking studies, as well as derivatization of these metabolites, should clearly be the target of future research.

For further production of structurally varied metabolites by this fungus, cocultivation techniques should be considered an area for future investigation. In addition, exploring the biosynthetic pathways of these bio-metabolites is required and could enable the rational engineering or refactoring of these pathways for industrial purposes. Further, identification of the biosynthetic genes responsible for these metabolites may provide the opportunity to discover *A. sydowii*'s genetic potential for discovering novel metabolites by metabolic engineering, which could lead to more affordable and novel pharmaceutics.

According to the published reports, *A. sydowii* can produce diverse types of enzymes with potential biotechnological and industrial applications. Research that focuses on engineering enzymes in such a way for maximum activity and stability under appropriate conditions is desirable. Recombinant DNA technology and engineering of proteins are required to improve the industrial production of these enzymes. *A. sydowii* can withstand high-salinity conditions, pointing to its biotechnological and industrial relevance. It was proven that this fungus adsorbed heavy metals and degraded pesticides, agrochemicals, and contaminants. As a result, *A. sydowii* might serve as an environmentally safe tool for bioremediation and for converting hazardous materials into useful products. The minor reports described NP synthesis utilizing this fungus. These biosynthesized NPs possessed antiproliferative and antimicrobial potential as well as biosorbent capacity for treating heavy metal- and pesticide-contaminated water. However, the synthesized NPs using *A. sydowii* are limited to silver, γ-Fe2O3 magnetic chitosan nanocomposites, and chitosan beads-immobilized *A. sydowii*. Therefore, future research should focus on developing protocols for implementing the biosynthesis of other types of NPs such as carbides, metal oxides, and nitrides using this fungus and their bio-evaluation, which could be a promising area for more anticipated beneficial effects.

Despite the large number of published studies on *A. sydowii*, mycologists, biologists, and chemists still need to conduct more extensive research to fully understand the potential of this fungus and its secondary metabolites.

**Author Contributions:** Conceptualization, S.R.M.I., H.G.A.H. and G.A.M.; resources, B.H.A., M.M.A., Z.I.A., A.A.A., J.F.A., S.A.F., and S.G.A.M.; data curation, B.H.A., M.M.A., Z.I.A., A.A.A., J.F.A. and S.A.F.; writing—original draft preparation, S.R.M.I., S.G.A.M., H.G.A.H. and G.A.M.; writing—review and editing, B.H.A., M.M.A., Z.I.A., A.A.A., J.F.A. and S.A.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Article* **Discovery of Anti-MRSA Secondary Metabolites from a Marine-Derived Fungus** *Aspergillus fumigatus*

**Rui Zhang 1,2, Haifeng Wang 1, Baosong Chen 2, Huanqin Dai 2, Jingzu Sun 2, Junjie Han 2,\* and Hongwei Liu 1,2,\***


**Abstract:** Methicillin-resistant *Staphylococcus aureus* (MRSA), a WHO high-priority pathogen that can cause great harm to living beings, is a primary cause of death from antibiotic-resistant infections. In the present study, six new compounds, including fumindoline A–C (**1**–**3**), 12*β*, 13*β*-hydroxyasperfumigatin (**4**), 2-*epi*-tryptoquivaline F (**17**) and penibenzophenone E (**37**), and thirty-nine known ones were isolated from the marine-derived fungus *Aspergillus fumigatus* H22. The structures and the absolute configurations of the new compounds were unambiguously assigned by spectroscopic data, mass spectrometry (MS), electronic circular dichroism (ECD) spectroscopic analyses, quantum NMR and ECD calculations, and chemical derivatizations. Bioactivity screening indicated that nearly half of the compounds exhibit antibacterial activity, especially compounds **8** and **11**, and **33**–**38** showed excellent antimicrobial activities against MRSA, with minimum inhibitory concentration (MIC) values ranging from 1.25 to 2.5 μM. In addition, compound **8** showed moderate inhibitory activity against *Mycobacterium bovis* (MIC: 25 μM), compound **10** showed moderate inhibitory activity against *Candida albicans* (MIC: 50 μM), and compound **13** showed strong inhibitory activity against the hatching of a *Caenorhabditis elegans* egg (IC50: 2.5 μM).

**Keywords:** methicillin-resistant *Staphylococcus aureus*; *Aspergillus fumigatus*; chemical diversity; chemical ecology

#### **1. Introduction**

Methicillin-resistant *Staphylococcus aureus* (MRSA) is recognized as one of the most common bacteria in both community and hospital-acquired infections, causing significant morbidity and mortality [1]. Compared to non-resistant *Staphylococcus aureus* infections, the mortality rate of MRSA infections increases by 64% [2]. Vancomycin is a last-resort treatment for MRSA infections. However, strains that are less susceptible to vancomycin are emerging in clinics [3,4]. As a result, new antibiotics to treat MRSA infections are desperately needed. In 2017, the development of new antibiotics for the treatment of MRSA infections is listed as a high urgency level by the WHO (World Health Organization) [5].

The marine environment is one of the most complex atmospheres on the earth, due to the huge variations in predation, temperature, pressure, light, and nutrient circumstances, etc. [6]. The organisms that thrive in marine environments could produce extremely diverse and complicated functional secondary metabolites that differ from those observed in terrestrial environments [6–8]. In recent decades, an increasing number of bioactive marine natural products (MNPs) have piqued the interest of chemists and pharmacologists for their medicinal values [9,10], such as the earliest marine sponge-derived anticancer drug cytarabine (Cytosar-U®), the marine sponge-derived antiviral drug vidarabine (Arasena A®), the mollusk-derived ziconotide (Prialt®) for the treatment of neuropathic pain, the famous sponge-derived anticancer drugs trabectedin (Yondelis®) and eribulin mesylate

**Citation:** Zhang, R.; Wang, H.; Chen, B.; Dai, H.; Sun, J.; Han, J.; Liu, H. Discovery of Anti-MRSA Secondary Metabolites from a Marine-Derived Fungus *Aspergillus fumigatus*. *Mar. Drugs* **2022**, *20*, 302. https://doi.org/ 10.3390/md20050302

Academic Editor: Hee Jae Shin

Received: 11 April 2022 Accepted: 25 April 2022 Published: 28 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

(Halaven®), and the marine cyanobacterium-derived anticancer drug disitamab vedotin (Aidixi™) and tisotumab vedotintftv (TIVDAK™), etc. [11–17].

Marine fungi have been shown to produce a variety of secondary metabolites with a variety of structures and bioactivities [18], including antibacterial, antiviral, anticancer, and anti-inflammatory characteristics, and have already provided a number of promising leads against MRSA [19,20]. Pestalone is a well-known anti-MRSA compound that was discovered by Fenical and colleagues, after co-culturing a fungus of the genus *Pestalotia* with a unicellular marine bacteria (strain CNJ-328) [21,22].

In our search for new anti-MRSA agents from marine-derived fungi, the EtOAc extract of the fungus *Aspergillus fumigatus* H22 was found to show strong anti-MRSA activity by in vitro anti-MRSA assay. A chemical investigation on its extract led to the identification of 45 secondary metabolites (Figure 1), including six new novel compounds, including fumindoline A–C (**1**–**3**), 12*β*,13*β*-hydroxy-asperfumigatin (**4**), 2-*epi*-tryptoquivaline F (**17**), and penibenzophenone E (**37**). The isolation and structure characterization of the new compounds, as well as the antibacterial activity of all the compounds, are described in this work.

**Figure 1.** Structures of compounds **1**–**45**.

#### **2. Results**

#### *Structure Elucidation of the Isolated Compounds*

Fumindoline A (**1**) was obtained as a chartreuse powder and had the molecular formula of C21H23N3O4, based on HRESIMS data (Figure S8), corresponding to 12 indices of hydrogen deficiency. This molecular formula was corroborated by 1H NMR and 13C NMR spectroscopic data. The 1H NMR data (Table 1) showed characteristic signals for a 1,2,4-trisubstituted benzene ring (*δ* 8.21 (d, *J* = 8.7 Hz), 6.91 (dd, *J* = 8.7 and 2.3 Hz), and 7.05 (d, *J* = 2.3 Hz)), two singlet olefinic protons (*δ*<sup>H</sup> 8.59 (s) and 6.79 (s)), three singlet methyl groups (*δ*<sup>H</sup> 2.08 (s), 2.17 (s), and 3.89 (s)), and two exchangeable (*δ* 8.42 (br. s) and 11.85 (br. s)). The 13C NMR and HSQC data of **1** revealed the presence of twenty-one carbon resonances, including three methyls (*δ*<sup>C</sup> 20.6, 27.3, and 55.4), three methylenes (*δ*<sup>C</sup> 24.9, 31.2, and 38.3), five *sp*<sup>2</sup> methines (*δ*<sup>C</sup> 94.8, 110.2, 111.1, 118.5, and 123.1), and ten nonprotonated carbons (eight *sp*<sup>2</sup> carbons at *δ*<sup>C</sup> 115.0, 128.9, 135.1, 138.7, 142.4, 142.8, 158.3, and 160.7; one amide carbonyl carbon at *δ*<sup>C</sup> 164.7, and one carboxyl carbon at *δ*<sup>C</sup> 174.2).

**Table 1.** 1H (500 MHz) and 13C NMR (125 MHz) data of compounds **1**–**3** in DMSO-*d*6.


"m" means multiplet or overlapped with other signals.

The planar structure of **1** was defined by the 2D NMR spectra, particularly the 1H–1H COSY and HMBC data (Figures S5 and S7). The HMBC correlations from H-9 to C-8, C-11, C-12, and C-13, from H-10 to C-8, and C-11, and from H-12 to C-8, C-10, C-11, and C-13, together with the 1H–1H COSY correlations of H-9/H-10/H-12, which indicated a 1,2,4-trisubstituted benzene. The 1H–1H COSY correlations of *N*H-15/H2-16/H2-17/H2- 18, as well as the HMBC correlations from H2-16 to C-14, C17, and C18, from H2-17 to

C-16, C-18, and C-19, from H2-18 to C-16, C-17, and C-19, led to the identification of the *γ*-aminobutyric acid residue. The HMBC correlations from H-6 to C-2, C-7, C-8, and C-14, H-9 to C-7, and H2-16 to C-14, as well as the chemical shifts of C-2 (*δ* 135.1), C-3 (*δ* 142.4), C-5 (*δ* 158.3), C-6 (*δ* 111.1), and C-14 (*δ* 164.7), supported a 2-pyridinecarboxylic acid moiety that was connected with a *γ*-aminobutyric acid moiety through C-14 and linked with a 1,2,4-trisubstituted benzene moiety through C-7, and completed the assignment of the moiety. The HMBC correlations from H-20 to C-2, C-3, and C-21, together with the 1H–1H COSY correlations of H-20/H3-22/H3-23, suggested that the isobutenyl group was located at C-3 of the 2-pyridinecarboxylic acid moiety. The key HMBC correlations from H3-24 to C-11 indicated that the methoxy group was located at C-11. Furthermore, these data accounted for 11 of the 12 degrees of unsaturation, implying the presence of an additional cycle, attributed to the *N*H bridging between C-2 and C-13 to establish the indole-pyridinecarboxylic acid skeleton (Figure 2). Therefore, the 2D structure of **1** was determined as shown below.

**Figure 2.** Key 1H–1H COSY and HMBC correlations of **1**.

Fumindoline B (**2**) was obtained as a chartreuse powder. Its molecular formula, C22H23N3O4, with 14 degrees of unsaturation, was established on the basis of the HRESIMS data (Figure S15). The UV spectrum showed absorptions at 282 nm and 343 nm, which were similar to those of **1**, indicating that **2** might have the same conjugation system as **1**. The IR spectrum indicated the presence of a secondary amine *N*-H signal (2980 cm<sup>−</sup>1) and an amide carbonyl signal (1628 cm−1). The 1H NMR and 13C NMR spectra indicated the presence of two sets of very similar signals, with the same number of carbons (Figures S9 and S10). The spectra of the two sets of signals are well resolved in pairs at 313K and 298K in DMSO-*d*6, indicating the presence of two relatively stable isomers. From the integrals of the completely resolved signals, a ratio of 1:0.7 was calculated for the two stable isomers. To be better distinguished, we assigned the major isomer as **2a** and the minor one as **2b**, respectively (Figure 3).

**Figure 3.** Scheme of the resonance structure of **2** and the chemical equilibrium between **2a** and **2b**.

The 1H NMR and 13C NMR spectra data of **2** showed close similarity to those of **1**, with the biggest difference in the methine (CH-19). A detailed analysis of the 2D NMR data, including HSQC, HMBC, and 1H–1H COSY spectra, revealed that **2** contained the same indole-pyridinecarboxylic acid skeleton as that of **1** (Figures 4 and S11–S13). The HMBC correlations from H2-16 to C-17 and C-18, H2-17 to C-16, C-18, and C-19, H2-18 to C-17, C-19, and C-20, and the 1H–1H COSY correlations of H2-16/H2-17/H2-18/H-19, together with the molecular formula, indicated the presence of a proline moiety, and this conclusion

was also confirmed by the 14 degrees of unsaturation and the chemical shifts of C-16 (*δ*<sup>C</sup> 49.6 (**2a**); *δ*<sup>C</sup> 47.5 (**2b**)) and C-19 (*δ*<sup>C</sup> 59.8 (**2a**); *δ*<sup>C</sup> 60.4 (**2b**)).

The *E*/*Z* isomer exists in the tertiary amide. In the solution at room temperature, the slow rotation of the C–*N* bond in NMR makes it possess the characteristics of a partial double bond [23]. A comparison of the 1H NMR signals and 13C NMR signals of **2a** and **2b** revealed differences in the proline moiety, including variations in the H-19 (*δ*<sup>H</sup> 4.48 (**2a**); *δ*<sup>H</sup> 5.3 (**2b**)), C-16 (*δ*<sup>C</sup> 49.6 (**2a**); *δ*<sup>C</sup> 47.5 (**2b**)), C-17 (*δ*<sup>C</sup> 25.2 (**2a**); *δ*<sup>C</sup> 21.9 (**2b**)), and C-18 (*δ*<sup>C</sup> 28.6 (**2a**); *δ*<sup>C</sup> 31.2 (**2b**)). As shown in Figure 4, strong NOE effects between H-6 and H-16 for **2a** and between H-6 and H-19 for **2b** were observed in the ROSEY spectrum (Figure S14).

**Figure 4.** Key 1H-1H COSY, HMBC, and ROESY correlations of **2a** and **2b**.

The absolute configuration of the amino acids from compound **2** was determined by the advanced Marfey's method [24]. The mixture obtained after hydrolyzing compound **2** and further derivatization with L-FDAA was analyzed by HPLC-DAD. The HPLC analyses of the mixture of hydrolysates and appropriate amino acid standards confirmed the L configurations for proline in **2** (Figure 5). Consequently, the absolute configuration of **2** was elucidated to be 19*S*.

**Figure 5.** Advanced Marfey's analysis of compound **2**. (**A**): The FDAA derivatives of the hydrolysate of **2**. (**B**,**C**): The retention times for the FDAA derivatives of L-proline and D-proline. The derivatives of the acid hydrolysate and the standard amino acids were subjected to RP HPLC analysis (Kromasil C18 column; 5 μM, 4.6 × 250 mm; 1.0 mL/min; UV detection at 340 nm) with a linear gradient of acetonitrile (30–40%) in water (TFA, 0.01%) over 30 min.

Fumindoline C (**3**) was obtained as a chartreuse powder. The molecular formula of **3** was established to be C23H25N3O4 from its HREIMS data (Figure S22). The 1H and 13C NMR spectra of **3** were similar to those of **2**, possessing two sets of signals (Figures S16 and S17), except for the presence of an additional methoxyl group. The substitution of the methoxyl group was further confirmed by the HMBC correlations from H3-26 to C-20. A further comprehensive analysis of its 1H–1H COSY, HSQC, and HMBC spectra assigned the planar structure of **3** (Figures S18–S20). The relative configuration of **3** was determined to be

the same as that of **2** by their similar structure and ROESY data (Figures S14 and S21). Accordingly, **3** was determined to be a methyl ester of **2**.

12*β*,13*β*-hydroxy-asperfumigatin (**4**) was obtained as a white amorphous solid. Its molecular formula was determined as C27H33N3O7 by HRESIMS data (Figure S31). The 1H NMR spectrum of **<sup>4</sup>** (Figure S25) displayed four singlet methyl groups (*δ*<sup>H</sup> 1.17, 1.25, 2.11, and 2.21), one methoxyl group (*δ*<sup>H</sup> 3.85) and four olefinic/aromatic protons (*δ*<sup>H</sup> 6.40, 6.90, 7.27, and 7.45). The 13C-NMR spectrum (Figure S26) exhibited 27 carbon resonances accounted for the functional groups described above and three amide carbonyl carbons (*δ*<sup>C</sup> 164.7, 165.5, and 165.9). A comprehensive analysis of its 2D NMR spectra, including 1H-1H COSY, HSQC, and HMBC experiments, confirmed the planar structure of **<sup>4</sup>** (Table 2, Figures S27–S29), revealing the presence of the indole moiety and the diketopiperazine moiety in **4** (Figure 6). The planar structure of **4** was determined to be the same as that of asperfumigatin (**5**), by detailed interpretation of the 2D NMR spectra and NMR data comparison between **4** and **5**. Considering the same biosynthesis origin, compound **4** was deduced to share the same absolute configuration at C-3 and C-6 as those of **5**–**13**.

**Table 2.** 1H (500 MHz) and 13C NMR (125 MHz) data for **4** in chloroform-*d*.


**Figure 6.** Key 1H-1H COSY, and HMBC correlations of **4**.

Owing to a lack of sufficient ROESY correlations, the relative configurations of C-12 and C-13 were not determined (Figure S30). The relative configurations of **4** were determined by the DP4+ probability, based on a theoretical NMR calculation that has been proven to be a very powerful tool in natural product structure elucidation [25,26]. The NMR shifts of eight possible relative orientation isomers were calculated with the GIAO method at the MPW1PW91/6-31+G(d,p), and the DP4+ probabilities of each configuration were evaluated based on Boltzmann-averaged theoretical NMR shielding tensors, which provided a 91.55% confidence for the relative configuration 3*S*\*, 6*S*\*, 12*S*\*, 13*R*\* (Tables S1 and S2).

To determine the absolute configurations of **4**, a ECD calculation method was applied. The two configurations (3*S*, 6*S*, 12*S*, 13*R*)-**4** and (3*R*, 6*R*, 12*R*, 13*S*)-**4** were calculated using

time-dependent density functional theory (TDDFT) at PBE1PBE/6-311 G\* level, with the PCM model in methanol, and corrected with a 2 nm blue shift according to UV data. A comparison of the experimental ECD spectrum of **4** and the calculated ECD spectra of (3*S*, 6*S*, 12*S*, 13*R*)-**4** and (3*R*, 6*R*, 12*R*, 13*S*)-**4** showed that the experimental ECD spectrum of **4** is consistent with the calculated ECD spectrum of (3*S*, 6*S*, 12*S*, and 13*R*)-**4** (Figure 7). Thus, the absolute configuration of **4** was assigned as 3*S*, 6*S*, 12*S*, and 13*R*, and named as 12*β*,13*β*-hydroxy-asperfumigatin. The only difference between compound **4** and compound **5** is the orientation of the two hydroxyl groups (12-OH, 13-OH).

**Figure 7.** Experimental ECD spectra of compound **4** and the calculated ECD spectra of (3*S,* 6*S,* 12*S,* 13*R*)-**4** and (3*R,* 6*R,* 12*R,* 13*S*)-**4**.

2-*epi*-tryptoquivaline F (**17**), which was isolated as a white amorphous solid, exhibited the [M + H]<sup>+</sup> peak at *m*/*z* 403.1399 (HRESIMS), corresponding to C22H18N4O4, as well as sixteen degrees of unsaturations (Figure S37). The 1H NMR, 13C NMR and HSQC spectra (Table 3, Figures S32–S34) of **17** revealed the presence of two 1, 2-disubstituted benzene rings (*δ*H*/δ*<sup>C</sup> 7.31 (d, *J* = 7.5 Hz)/124.4, 7.22 (dd, *J* = 8.0 and 7.5 Hz)/125.8, 7.43 (dd, *J* = 8.0 and 7.5 Hz)/131.3, 7.65 (d, *J* = 8.0 Hz)/115.8; *δ*H*/δ*<sup>C</sup> 8.29 (d, *J* = 8.1 Hz)/126.8, 7.58 (dd, *J* = 8.1 and 7.5 Hz)/128.2, 7.85 (dd, *J* = 8.1 and 7.5 Hz)/135.4, 7.78 (d, *J* = 8.1 Hz)/128.0), one methyl (*δ*H*/δ*<sup>C</sup> 1.28 (3H, d, *J* = 6.5 Hz)/17.9), one methylene (*δ*<sup>H</sup> 2.61 (dd, *J* = 13.4 and 10.5 Hz), 3.68 (dd, *J* = 13.4 and 4.3 Hz), *δ*<sup>C</sup> 33.4), four nitrogenated methines (*δ*H*/δ*<sup>C</sup> 4.26 (d, *J* = 6.5 Hz)/58.5, 5.03 (dd, *J* = 4.3 and 10.5 Hz)/58.3, 5.82 (s)/82.6, 8.11 (s)/145.6), eight quaternary carbons including three carbonyls (*δ*<sup>C</sup> 172.4, 170.9, and 161.0), four aromatic or olefinic carbon atoms (*δ*<sup>C</sup> 121.9, 134.4, 138.9, and 148.1), and one oxygenated one (*δ*<sup>C</sup> 91.3). The NMR data of compound **17** were similar to those of tryptoquivaline F [27], indicating the presence of one 6-5-5 gem-methyl imidazoindolone ring and one quinazoline-4-one moiety.

**Table 3.** 1H (500 MHz) and 13C NMR (125 MHz) data of compound **17** in chloroform-*d*.


The partial relative configuration of **17** was confirmed by a ROESY experiment (Figure S36). The ROESY correlations of H-2 (*δ*<sup>H</sup> 5.82, s) with H-15 (*δ*<sup>H</sup> 4.26, q, *J* = 6.5 Hz) indicated that H-2 and H-15 were on the same face, while H3-27 (*δ*<sup>H</sup> 1.28, d, *J* = 6.5 Hz) were on the opposite face (Figure 8). The relative configurations of C-2 and C-15 were assigned as 2*S* and 15*S*. However, owing to a lack of sufficient ROESY correlations, neither the orientation of C-3 nor C-12 could be determined.

**Figure 8.** Key HMBC and NOESY correlations of compound **17**.

Similar to compound **4**, the NMR shifts of four relative configuration isomers (2*S*, 3*S*, 12*R*, 15*S*; 2*S*, 3*R*, 12*S*, 15*S*; 2*S*, 3*R*, 12*R*, 15*S*; 2*S*, 3*S*, 12*S*, 15*S*) were calculated and the DP4+ probability, based on a theoretical NMR calculation, was applied. The 100% DP4+ probability for **17a** revealed that the relative configuration of **17** was 2*S*\*, 3*S*\*, 12*R*\* and 15*S*\* (Tables S3 and S4).

The absolute configurations of **17** were deduced by the comparison of the experimental and simulated ECD spectra generated by TDDFT at B3LYP/6-311+G(2d,p) level with the PCM model in methanol and corrected -5 nm according to the UV data. A comparison of the observed ECD spectra for **17,** with the calculated ECD spectra for the (2*S*, 3*S*, 12*R*, 15*S*)-**17** and (2*R*, 3*R*, 12*S*, 15*R*)-**17** enantiomers, is shown in Figure 9. The overall ECD spectra for (2*S*, 3*S*, 12*R*, 15*S*)-**17** are in good accordance with the experimental ECD for **17**. Thus, compound **17** was determined to be 2*S*, 3*S*, 12*R*, and 15*S*. The differences between **17** and tryptoquivaline F are the configuration of C-2. Therefore, compound **17** was identified as 2-*epi*-tryptoquivaline F.

**Figure 9.** Experimental ECD spectra of compound **17** and the calculated ECD spectra of (2*S,* 3*S,* 12*R,* 15*S*)-**17** and (2*R,* 3*R,* 12*S,* 15*R*)-**17**.

Compound **37** was isolated as a yellowish powder. Its molecular formula was determined as C17H16O7 based on the HRESIMS (Figure S42), implying ten degrees of unsaturation. The 1H NMR spectrum (Figure S38) of **37** showed one hydrogen-bonded phenol

moiety at *δ*<sup>H</sup> 13.55 (s, 6 -OH), four aromatic methine protons at *δ*<sup>H</sup> 7.19 (br. s, H-5), 6.88 (br. s, H-3), 5.90 (d, *J* = 2.2 Hz, H-5 ) and 5.80 (d, *J* = 2.2 Hz, H-3 ) for two sets of AB meta-coupling, two methoxy groups at *δ*<sup>H</sup> 3.64 (s, 8-OMe) and 3.26 (s, 2 -OMe), and a methyl group at *δ*<sup>H</sup> 2.30 (s, 4-Me). A comparison of its 1H NMR and 13C NMR spectra (Table 4) with those of sulochrin (**38**) suggested the same benzophenone skeleton between them [28]. The HMBC correlations from the proton of 6 -OH to C-6 , C-1 and C-5 indicate that 6 -OH was located at C-6 . The HMBC correlations from 2 -OCH3 to C-2 , 4-CH3 to C-3, C-4, C-5, and 8-CH3 to C-7 indicate that the two methoxy groups and one methyl group were located at C-2 , C-7 and C-4, respectively. In addition, the HMBC correlations from H-3 to C-1, C-2, C-5, from H-3 to C-1 , C-2 , C-4 , C-5 , from H-5 to C-1, C-3, C-6 and C-7, and from H-5 to C-1 , C-3 and C-6 confirmed the proposed structure (Figure 10). Therefore, compound **37** was determined as penibenzophenone E.

**Table 4.** 1H (500 MHz) and 13C NMR (125 MHz) data of compound **37** in DMSO-*d*6.


**Figure 10.** Key HMBC correlations of compound **37**.

Other known compounds were identified as asperfumigatin (**5**) [29], demethoxyfumitremorgin C (**6**) [30], fumitremorgin C (**7**) [30], 12,13-dihydroxyfumitremorgin C (**8**) [31], 12*α*-hydroxy-13-oxofumitremorgin C (**9**) [32], fumitremorgin B (**10**) [33], 13-oxofumitremorgin B (**11**) [34], cyclotryprostatin B (**12**) [35], verruculogen (**13**) [36], 6-methoxyspirotryprostatin B (**14**) [37], (−)-spirotryprostatin A (**15**) [38], spirotryprostatin C (**16**) [39], fumiquinazoline C (**18**) [40], (+)-alantrypinone (**19**) [41,42], oxoglyantrypine (**20**) [43], (−)-chaetominine (**21**) [44], 11-epi-chaetominine (**22**) [29], fumigaclavine C (**23**) [45,46], bisdethiobis(methylthio)gliotoxin (**24**) [47], pyripyropene A (**25**) [48], pseurotin F1 (**26**) [49], pseurotin F2 (**27**) [49], pseurotin A (**28**) [50], 11-O-methylpseurotin A (**29**) [51], azaspirofuran B (**30**) [52], azaspirofuran A (**31**) [52], fumagiringillin (**32**) [53], fumagillin (**33**) [54], helvolic acid (**34**) [55], 6-O-propionyl-16-O-deacetylhelvolic acid (**35**) [55], 16-O-propionyl-6-O-deacetylhelvolic acid (**36**) [55], sulochrin (**38**) [28], monomethylsulochrin (**39**) [56], 8 -O-methylasterric acid (**40**) [29], dimethyl 2,3 -dimethylosoate (**41**) [56], questin (**42**) [57], (+)-2 *S*-isorhodoptilometrin (**43**) [58], 6 hydroxy-8-methoxy-3-methylisocoumarin (**44**) [59], and trypacidin (**45**) [60], based on the spectroscopic analyses and in comparison with the literature data.

The antibacterial activities of the isolated compounds were determined against methicillinresistant *Staphylococcus aureus* (MRSA) (clinical isolate strain), vancomycin-resistant enterococci *E. faecalis* (VRE), *Candida albicans* SC5314, *Mycobacterium bovis* ATCC35743 constitutive GFP expression (pUV3583c-GFP), and *Escherichia coli* O57:H7, within 100 μM. The results showed that nearly half of the compounds exhibit antibacterial activity (Table 5), especially compounds **5**, **8**, **10**, **11**, **16**, **21**, **23**, **29**–**38**, and **41** exhibited antimicrobial activities against

MRSA, with minimum inhibitory concentration (MIC) values ranging from 1.25 to 25 μM. Furthermore, compound **8** also exhibited strong activity against *M. bovis* with a MIC of 25 μM, compound **10** showed moderate activity against *C. albicans* with a MIC of 50 μM. Moreover, compound **13** inhibited the egg hatching of *Caenorhabditis elegans* with a IC50 of 2.5 μM.


**Table 5.** Antibacterial assay results of monomeric compounds.

<sup>a</sup> MRSA: methicillin-resistant *Staphylococcus aureus*.

#### **3. Discussion**

The marine environmental stress conditions induce many faunae and symbiont microorganisms to synthesize and release secondary metabolites of unique structures and interesting biological activities [61]. These bioactive compounds can serve as an important source for drug discovery. Marine-derived fungi are important sources for the discovery of new antibacterial natural products. Wang et al. isolated the *Chaetomium* sp. strain NA-S01-R1 from a deep-sea (4050 m) fungus that produced novel chlorinated azaphilone polyketides with antibacterial activity against MRSA [62]. The *Emericellopsis minima* strain A11, isolated from Talcahuano Bay (Chile), produced an antibiotic called emerimicin IV, with moderate activity against clinical isolates of MDR vancomycin-resistant strains of *E. faecalis* and MRSA with MIC of 12.5 μg/mL and 100 μg/mL, respectively [63].

*A. fumigatus* belongs to the filamentous fungi family that is widely distributed in all environments and can cause many diseases and life-threatening conditions in immunocompromised patients [64]. *A. fumigatus* can produce a wide array of secondary metabolites due to its remarkable adaptability to different environmental conditions, such as fumitremorgins, fumagillins, pseurotins, fumigaclavines, gliotoxins, and helvolic acid derivatives.

Inspired by chemical ecology, we found a marine fungus *A. fumigatus* H22 with strong antibacterial activities from the marine fungi library. Through in-depth chemical mining, we found 45 compounds, including 6 new compounds, from the culture of this fungus. A evaluation of biological activity showed that nearly half of the compounds exhibit antimicrobial activity. Fumitremorgins derivatives (**4**-**11**) have very similar structures, but only a few have strong anti-MRSA activity. Compounds **5**, **8** and **11** with strong anti-MRSA activity contain hydroxyl group at C-13, while compounds **6** and **7** without anti-MRSA activity have no hydroxyl group at C-13. In addition, compounds **4** and **5** have the same planar structure, but the 13-OH of compound **4** without anti MRSA activity was α-oriented, while compound **5** and other strongly active compounds were *β*-oriented. Therefore, it is preliminarily speculated that there is a certain correlation between the substituents and stereoconfiguration in C-12 and C-13 and their anti MRSA activity. Fumitremorgin B (**10**) was reported with antifungal activity against a variety of phytopathogenic fungi, but it showed weak activity against vancomycin-resistant *E. faecalis* (VRE), *M. bovis*, and *E. coli* in our in vitro assay, which could be involved in fighting against invasion by other pathogens [65].

Pseurotins, with a unique heterospirocyclic furanone-lactam structure, exhibit a broad range of biological activities. In addition to antifungal and antibiotic activities [66,67], pseurotins were also shown to regulate enzymes of cellular metabolism [68], to possess anti-angiogenic activity, to modulate cell differentiation [69], and to inhibit endothelial cell migration [70–72]. Fumagillin (**33**) have been demonstrated to have antitumor, antibacterial and antiparasitic effects [73]. Previous studies revealed that helvolic acid (**34**) exhibited in vitro antimalarial activity against multidrug resistant *Plasmodium falciparum* [74], antitrypanosomal activity against *Trypanosoma brucei* [75], and antimycobacterial activity against *M. tuberculosis* H37Ra [76]. Our current research showed the strong activities of oxofumitremorgin B (**11**), helvolic acid (**34**), 6-O-propionyl-16-O-deacetylhelvolic acid (**35**), 16-O-propionyl-6-O-deacetylhelvolic acid (**36**), sulochrin (**38**) and 8 -O-methylasterric acid (**40**) against MRSA, with a MIC of 1.25 μM.

From our current findings, it can be found that *A. fumigatus* from marine sources can produce rich bioactive secondary metabolites, especially in anti-MRSA.

#### **4. Materials and Methods**

#### *4.1. General*

UV data, optical rotation, and IR data were recorded on Genesys-10S UV–Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), MCP 200 automatic polarimeter (Anton Paar, Graz, Austria), and IS5 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. NMR spectral data were obtained with a Bruker AVANCE-500 spectrometer (Bruker, Bremen, Germany) (DMSO-*d*6, *δ*<sup>H</sup> 2.50/*δ*<sup>C</sup> 39.52, and CDCl3, *δ*<sup>H</sup> 7.26/*δ*<sup>C</sup> 77.16). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were obtained on an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument (Agilent Technologies, Santa Clara, CA, USA). The CD spectra were measured by JASCO J-815 spectropolarimeter (JASCO, Tsukuba, Japan). Silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200–300 mesh), ODS (octadecylsilyl, 50 μM, YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography. Semi-preparative HPLC was performed on an Agilent 1200 HPLC system equipped with an Agilent DAD UV–vis spectrometric detector (Agilent Technologies Inc., CA, USA), using a reversed-phase Eclipse XDB-C18 column (5 μM, 9.4 × 250 mm; Agilent, MA, USA), with a flow rate of 2.0 mL/min. The biological reagents, chemicals and media were purchased from standard commercial sources, unless stated.

#### *4.2. Fungal Material*

The fungus H22 was isolated from middle seawater from the Western Pacific. The sample (1 mL) was diluted with sterile H2O, 100 μL of which was deposited on a PDA (200 g of potato, 20 g of glucose, 20 g of agar per liter of seawater collected in the Western Pacific) plate containing chloramphenicol (100 μg/mL) and streptomycin (100 μg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and 18*S* rRNA gene sequences. The phylogenetic tree (Figure S1), constructed from the ITS gene sequence, indicated that H22 belonged to the genus of *Aspergillus,* with the highest similarity to *A. fumigatus* (99.86%, accession number NRRL 163 s). In consideration of the morphological features and phylogeny (Figure S2), this fungus was identified as *A. fumigatus*. A reference culture of *A. fumigatus* H22 maintained at −80 ◦C was deposited in our laboratory.

#### *4.3. Fermentation and Extraction*

The isolate was grown for 7 days at 28 ◦C, on slants of a PDA medium. The spores of the strain on the plate were collected using 0.01% sterile Tween 80 (BTL, Warsaw, Poland) and adjusted to 1 × <sup>10</sup><sup>6</sup> CFU/mL to make inoculum. A large-scale fermentation was carried out in 50 × 500 mL Fernbach culture flasks, holding 100 g of rice in 110 mL of distilled water (each with 0.5 mL of spore suspension) and incubated for 4 weeks at 28 ◦C. With the help of ultrasonication, the fermented rice substrates were extracted with ethyl acetate (3 × 5 L), and the organic solvent was filtered and evaporated to dryness under a vacuum to obtain the crude extract (78.0 g).

#### *4.4. Isolation and Characterization Data*

The ethyl acetate (EtOAc) fraction was subjected to silica gel column chromatography (CC), eluted with dichloromethane/acetone (D/A, *v*/*v*, 100:0, 100:1, 50:1, 30:1, 25:1, 20:1, 10:1, 5:1) and dichloromethane/methanol (D/M, *v*/*v*, 5:1, 2:1, 0:100) to give 10 fractions (HS.1–HS.10).

HS.3 (4.94 g eluted with D/A, *v*/*v*, 50:1) was purified by RP-HPLC, using 37% acetonitrile in acidic water (0.01% TFA) to obtain compounds **45** (9.0 mg, *t*<sup>R</sup> = 37.5 min), **42** (103.0 mg, *t*<sup>R</sup> = 44.5 min) and **39** (19.2 mg, *t*<sup>R</sup> = 47.6 min).

Fraction HS.4 (5.98 g from D/A, *v*/*v*, 30:1) was separated by ODS, using a gradient from 20% to 100% methanol in water to afford 12 subfractions (HS.4-1–HS.4-12). HS.4-3 (203.0 mg) was further purified using C8-RP-HPLC on a Agilent Eclipse XDB-C8 (5 μM, 250 × 9.4 mm), with a gradient elution from 30% to 40% acetonitrile in 60 min to give compounds **21** (102.1 mg, *t*<sup>R</sup> = 22.5 min), **18** (12.0 mg, *t*<sup>R</sup> = 30.6 min), **17** (7.0 mg, *tR* = 39.6 min) and **41** (2.0 mg, *t*<sup>R</sup> = 50.2 min). HS.4-4 (77 mg) was further purified using C8-RP-HPLC with 35% acetonitrile to give compounds **8** (3.5 mg, *t*<sup>R</sup> = 33.6 min), **7** (3.5 mg, *t*<sup>R</sup> = 41.5 min) and **31** (3.0 mg, *t*<sup>R</sup> = 66.2 min). Compounds **13** (79.0 mg, *t*<sup>R</sup> = 17.4 min), **10** (7.0 mg, *t*<sup>R</sup> = 23.6 min) and **33** (5.0 mg, *t*<sup>R</sup> = 25.8 min) were obtained from HS.4-9 (252 mg) by RP-HPLC, using 55% acetonitrile in acidic water.

Fraction HS.5 (5.43 g, from D/A, *v*/*v*, 25:1) was first separated by ODS, using a gradient from 30% to 100% methanol in water to afford HS.5-1–HS.5-11. Subfraction HS.5-4 (30 mg) was purified using RP-HPLC on a Agilent Eclipse XDB-C8 column (5 μM, 250 × 9.4 mm) with 40% acetonitrile in 20 min to give compounds **23** (5.0 mg, *t*<sup>R</sup> = 4.1 min) and **39** (4.2 mg, *t*<sup>R</sup> = 7.5 min). Compound **24** (5.0 mg, *t*<sup>R</sup> = 24.8 min) was obtained from subfraction HS.5-4-5 (32 mg) by RP-HPLC, using 28% acetonitrile in acidic water (0.01% TFA). Compounds **22** (2.2 mg, *t*<sup>R</sup> = 28.1 min) and **21** (3.0 mg, *t*<sup>R</sup> = 29.7 min) were obtained from HS.5-4-8 (144 mg) by RP-HPLC, using 29% acetonitrile in acidic water (0.01% TFA).

Fraction HS.6 (6.72 g, from D/A, *v*/*v*, 20:1) was first separated by ODS, using a gradient from 20% to 100% methanol in water to afford HS.6-1–HS.6-17. HS.6-2 (289 mg) was purified using C8-RP-HPLC eluting with 50% to provide compound **44** (2.0 mg, *t*<sup>R</sup> = 16.3 min). Compounds **37** (3.0 mg, *t*<sup>R</sup> = 19.1 min) and **38** (1.5 mg, *t*<sup>R</sup> = 20.2 min) were obtained from HS.6-4 (326.0 mg) by RP-HPLC, using 45% acetonitrile in acidic water. HS.6-5 (522 mg) was purified using RP-HPLC eluting with 50% acetonitrile to give compounds **15** (9.0 mg, *t*<sup>R</sup> = 10.2 min), **9** (15.0 mg, *t*<sup>R</sup> = 13.2 min), and **12** (100.0 mg, *t*<sup>R</sup> = 14.6 min). Compound **16** (3.0 mg, *t*<sup>R</sup> = 25.2 min) was obtained from subfraction HS.6-9 (17.5mg) by RP-HPLC, using 60% acetonitrile in acidic water. Compounds **33** (19.8 mg, *tR* = 10.7 min), **34** (2.0 mg, *t*<sup>R</sup> = 12.3 min) and **35** (2.0 mg, *t*<sup>R</sup> = 13.2 min) were obtained from HS.6-17 (365 mg) by RP-HPLC, using 70% acetonitrile in acidic water.

Fraction HS.7 (10.63 g, D/A, *v*/*v*, 10:1) was first separated by ODS, using a gradient from 35% to 100% methanol in water to afford HS.7-1–HS.7-13. Compounds **40** (6.0 mg) and **29** (2.0 mg) were obtained from HS.7-2 and HS.7-3 by recrystallization in methanol, respectively. Compounds **30** (2.0 mg, *t*<sup>R</sup> = 9.1 min), **20** (2.0 mg, *t*<sup>R</sup> = 11.1 min) and **6** (2.1 mg, *t*<sup>R</sup> = 11.9 min) were obtained from HS.7-4 (11.2 mg) by RP-HPLC, using 65% acetonitrile in acidic water. Compounds **4** (3.2 mg, *t*<sup>R</sup> = 14.9 min) and **5** (2.8 mg, *t*<sup>R</sup> = 16.1 min) were purified from HS.7-7, using RP-HPLC with 50% acetonitrile. HS.7-9 (420.0 mg) was purified using C8-RP-HPLC with 65% methanol to give compounds **43** (2.0 mg, *t*<sup>R</sup> = 12.5 min), **32** (20.2 mg, *t*<sup>R</sup> = 16.2 min) and **19** (37.3 mg, *t*<sup>R</sup> = 18.1 min).

Fraction HS.8 (8.82 g, D/A, *v*/*v*, 5:1) was first separated by ODS, using a gradient from 20% to 100% methanol in water to afford 22 subfractions (HS.8-1–HS.8-22). HS.8-3 (100.0 mg) was further purified on C8-RP-HPLC eluting with 35% acetonitrile in acidic water to give compounds **28** (11.8 mg, *t*<sup>R</sup> = 19.3 min), **27** (3.1 mg, *t*<sup>R</sup> = 22.4 min), and **26** (2.8 mg, *t*<sup>R</sup> = 24.3 min). HS.8-18 (124.0 mg) was purified on a C8-RP-HPLC eluting with a gradient elution from 70% methanol to give compounds **3** (15.0 mg, *t*<sup>R</sup> = 12.5 min), **2** (5.0 mg, *t*<sup>R</sup> = 15.2 min), **11** (20.0 mg, *t*<sup>R</sup> = 18.4 min), **1** (5.0 mg, *t*<sup>R</sup> = 19.2 min) and **25** (8.0 mg, *t*<sup>R</sup> = 22.5 min).

Fumindoline A (**1**). UV (MeOH) *λ*max (log *ε*) 286 (1.62), 345 (0.48). 1H NMR and 13C NMR, see Table 1, 2D NMR spectra, see Supplementary Figures S3–S7. Positive HRESIMS: *m/z* 382.1768 [M + H]+ (calcd for C21H24N3O4, 382.1761, Figure S8).

Fumindoline B (**2**). Chartreuse powder; (α)<sup>25</sup> <sup>D</sup> −34.99 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *ε*) 282 (2.82), 343 (0.82); 1H NMR and 13C NMR, see Table 1, 2D NMR spectra, see Supplementary Figures S9–S14; Positive HRESIMS: *m/z* 394.1765 [M + H]<sup>+</sup> (calcd for C22H24N3O4, 394.1761, Figure S15).

Fumindoline C (**3**). Chartreuse powder; (α)<sup>25</sup> <sup>D</sup> −21.00 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *ε*) 286 (1.62), 345 (0.50); 1H NMR and 13C NMR, see Table 1, 2D NMR spectra, see Supplementary Figures S16–S24; Positive HRESIMS: *m/z* 408.1916 [M + H]<sup>+</sup> (calcd for C23H26N3O4, 408.1918, Figure S22).

12*β*,13*β*-hydroxy-asperfumigatin (**4**). White amorphous solid; (α)<sup>25</sup> <sup>D</sup> +26.00 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *ε*) 222 (1.51), 270 (0.60); 1H NMR and 13C NMR, see Table 2, 2D NMR spectra, see Supplementary Figures S25–S30; Positive HRESIMS: *m/z* 494.2720 [M + H − H2O]<sup>+</sup> (calcd for C27H32N3O6, 494.2726, Figure S31).

2-*epi*-tryptoquivaline F (**17**). White amorphous solid; (α)<sup>25</sup> <sup>D</sup> +221.96 (*c* 0.1, CH2Cl2); UV (CH2Cl2) *λ*max (log *ε*) 212 (2.21), 233 (1.69); 1H NMR and 13C NMR, see Table 3, 2D NMR spectra, see Supplementary Figures S32–S36; Positive HRESIMS: *m/z* 403.1399 [M + H]+ (calcd for C22H19N4O4, 403.1401, Figure S37).

Penibenzophenone E (**37**). Yellowish powder; UV (MeOH) *λ*max (log *ε*) 205 (3.28), 303 (1.63); 1H NMR and 13C NMR, see Table 4, 2D NMR spectra, see Supplementary Figures S38–S41; Positive HRESIMS: *m/z* 355.0789 [M + Na]<sup>+</sup> (calcd for C17H16O7Na, 355.0788, Figure S42).

#### *4.5. Marfey's Analysis of Compound 2*

Compound **2** (2.0 mg) was dissolved in 6 *N* HCl (2.0 mL) and heated at 100 ◦C for 24 h. The solutions were then evaporated to dryness and placed in a 4 mL reaction vial and treated with a 10 mg/mL solution of FDAA (200 μL) in acetone, followed by 1 M NaHCO3 (40 μL). The reaction mixtures were heated at 45 ◦C for 90 min, and the reactions were quenched by the addition of HCl (1 *N*, 40 *μ*L). In a similar fashion, the standard L-proline and D-proline were derivatized separately. The derivatives of the acid hydrolysate and the standard amino acids were subjected to RP HPLC analysis (Kromasil C18 column; 5 μM, 4.6 × 250 mm; 1.0 mL/min; UV detection at 340 nm), with a linear gradient of acetonitrile (30–40%) in water (TFA, 0.01%) over 30 min. The retention times for the authentic standards were as follows: L-proline derivative (8.91 min) and D-proline derivative (9.88 min). The absolute configuration of the chiral amino acid in **2** was determined by comparing the retention times.

#### *4.6. Computational Details for NMR and ECD*

The GMMX software tool was used to undertake the systematic conformational evaluations for **4** and **17,** utilizing the MMFF94 molecular mechanics force field. Gaussian 16 software was used to further improve the MMFF94 conformers, utilizing the M062X/6- 31G(d) basis set level in gas for NMR calculations and B3LYP/6-31+G(d,p) basis set level in methanol, with a PCM model for ECD calculations. The shielding constants were calculated using the GIAO technique in chloroform, using the SMD solvent model and Gaussian function at mPW1PW91/6-31+G(d,p). A previously documented approach was used to calculate the 1H and 13C chemical shifts for the DP4+ probability analysis [77]. ECD spectra were stimulated in methanol with a Gaussian function at the B3LYP/6-311+G(2d,p) level using the PCM model, and 60 NStates were calculated. Boltzmann statistics were used to compute the equilibrium populations of the conformers at 298.15 K, based on their respective free energies (**Δ**G). The Boltzmann weighting of the key conformers was then used to construct the overall ECD spectra. UV correlation was used to correct the systematic mistakes in predicting the wavelength and excited-state energy [78].

#### *4.7. Antimicrobial Assay*

An antimicrobial assay was performed according to the Antimicrobial Susceptibility Testing Standards, outlined by the Clinical and Laboratory Standards Institute against MRSA (clinical strain from Chaoyang Hospital, Beijing, China), *Pseudomonas aeruginosa* (ATCC 15692), *Escherichia coli* (O57:H7), *Mycobacterium bovis* (ATCC35743), vancomycinresistant *Enterococci faecalis* (VRE) (clinical strain from 309 Hospital, Beijing, China), and pathogen fungi *Candida albicans* SC5314. The protocol was performed as previously reported [58,59]. The positive controls were vancomycin against MRSA, *E. faecalis*, ciprofloxacin against *P. aeruginosa* and *E. coli*, amphotericin B for *C. albicans*, and rifampicin for *M. bovis*. All the experiments were performed in triplicate.

#### **5. Conclusions**

In summary, we isolated forty-five compounds from *A. fumigatus* H22, including six new compounds **1–4**, **17**, and **37**. The stereochemistry of the new compounds was determined by quantum calculations of NMR, ECD calculations and chemical derivatizations. Bioactivity screening indicated that compounds **5**, **8**, **10**, **11**, **16**, **21**, **23**, **29**–**38**, and **41** exhibited antimicrobial activities against MRSA, with MIC values ranging from 1.25 to 25 μM. Compound **8** also exhibited strong activity against *M. bovis,* with a MIC of 25 μM. To the best of our knowledge, this is the first report for the antimicrobial activities of compounds **5**, **10**, **11**, **16**, **30**, **31**, and **37**. The strains of *A. fumigatus* from ocean environments are a good source of antibacterial natural products, deserving further exploitation.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20050302/s1. Table S1: NMR calculation of **4**; Table S2: sDP4+, uDP4+ and DP4+ probabilities (%) for **4**; Table S3: NMR calculation of **17**; Table S4: sDP4+, uDP4+ and DP4+ probabilities (%) for **17**; Figures S1 and S2: phylogenetic tree and morphology of *A. fumigatus* H22; Figures S3–S8: 1D, 2D NMR, and HRESIMS of **1**; Figures S9–S15: 1D, 2D NMR, and HRESIMS of **2**; Figures S16–S24: 1D, 2D NMR, and HRESIMS of **3**; Figures S25–S31: 1D, 2D NMR, and HRESIMS of **4**; Figures S32–S37: 1D, 2D NMR, and HRESIMS of **17**; Figures S38–S42: 1D, 2D NMR, and HRESIMS of **37**; Figure S43: eight possible stereoisomers of **4** (**4a**–**4h**); Figure S44: four possible stereoisomers of **17** (**17a**–**17d**).

**Author Contributions:** Conceptualization, R.Z., H.W. and J.H.; methodology, J.H. and B.C.; validation and data curation, R.Z., J.S. and H.D.; formal analysis, R.Z. and J.H.; investigation, J.H. and H.L.; resources, H.D.; writing—original draft preparation, R.Z. and J.H.; writing—review and editing, J.H. and H.L.; supervision and project administration, J.H. and H.L.; funding acquisition, H.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was supported by the National Natural Science Foundation (Grant No. 82073723).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Exophilone, a Tetrahydrocarbazol-1-one Analogue with Anti-Pulmonary Fibrosis Activity from the Deep-Sea Fungus** *Exophiala oligosperma* **MCCC 3A01264**

**Ming-Jun Hong 1,†, Meng-Jiao Hao 1,†, Guang-Yu Zhang 1, Hou-Jin Li 2, Zong-Ze Shao 3, Xiu-Pian Liu 3, Wen-Zhe Ma 4, Jun Xu 1, Taifo Mahmud <sup>5</sup> and Wen-Jian Lan 1,\***


**Abstract:** A new compound, exophilone (**1**), together with nine known compounds (**2**–**10**), were isolated from a deep-sea-derived fungus, *Exophiala oligosperma*. Their chemical structures, including the absolute configuration of **1,** were elucidated using nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and electronic circular dichroism (ECD) calculation. Compounds were preliminarily screened for their ability to inhibit collagen accumulation. Compounds **1**, **4**, and **7** showed weaker inhibition of TGF-β1-induced total collagen accumulation in compared with pirfenidone (73.14% inhibition rate). However, pirfenidone exhibited cytotoxicity (77.57% survival rate), while compounds **1**, **4**, and **7** showed low cytotoxicity against the HFL1 cell line. Particularly, exophilone (**1**) showed moderate collagen deposition inhibition effect (60.44% inhibition rate) and low toxicity in HFL1 cells (98.14% survival rate) at a concentration of 10 μM. A molecular docking study suggests that exophilone (**1**) binds to both TGF-β1 and its receptor through hydrogen bonding interactions. Thus, exophilone (**1**) was identified as a promising anti-pulmonary fibrosis agent. It has the potential to be developed as a drug candidate for pulmonary fibrosis.

**Keywords:** *Exophiala oligosperma*; marine fungus; pulmonary fibrosis (PF); molecular docking

#### **1. Introduction**

Deep-sea is one of the extreme ecological environments on earth, with high salinity, high pressure, low temperature, low oxygen concentration, darkness, and other characteristics [1]. Therefore, organisms, including microbes, that live in deep-sea are normally equipped with certain physical and biochemical traits that help them survive that extreme environment [2]. In addition, many of them have the ability to produce specialized metabolites which are different from those produced by terrestrial organisms. Recent studies have shown that fungi from extreme environments have great potential as a source of clinically important compounds [1,3].

Tetrahydro carbazole derivatives have been isolated from microorganisms of terrestrial and marine origin and exhibit a variety of activities, including anti-*Candida albicans* activity [4], anti-*Bacillus subtilis* activity, and anti-*Micrococcus luteus* activity [5], etc. In

**Citation:** Hong, M.-J.; Hao, M.-J.; Zhang, G.-Y.; Li, H.-J.; Shao, Z.-Z.; Liu, X.-P.; Ma, W.-Z.; Xu, J.; Mahmud, T.; Lan, W.-J. Exophilone, a Tetrahydrocarbazol-1-one Analogue with Anti-Pulmonary Fibrosis Activity from the Deep-Sea Fungus *Exophiala oligosperma* MCCC 3A01264. *Mar. Drugs* **2022**, *20*, 448. https:// doi.org/10.3390/md20070448

Academic Editor: Hee Jae Shin

Received: 10 June 2022 Accepted: 4 July 2022 Published: 9 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

particular, sorazolons D2, E, and E2 from *Sorangium cellulosum* strain Soce375 exhibited anti-fibrosis activity [5]. Pulmonary fibrosis (PF) is a lung disease in which scarring of the lungs increases over time [6]. The progression of PF is related to environmental pollution, certain drug use, connective tissue diseases, infections (including COVID-19 and the related SARS virus), and/or interstitial lung disease [7]. To date, only two drugs, nintedanib and pirfenidone, have been approved by the FDA for the treatment of idiopathic pulmonary fibrosis (IPF). Nintedanib can significantly slow disease progression compared to placebo in IPF patients [8,9]. However, its clinical applications are somewhat limited due to poor oral bioavailability, metabolic instability, and off-target side effects [10]. Clinical trials have shown that pirfenidone alleviates the decline in lung function in patients with IPF, but 24.3% of patients stopped pirfenidone treatment due to adverse drug reactions in Japan [11]. Although lung transplantation is considered the most effective treatment for PF, it is limited by the lack of suitable donor organs [12]. Therefore, there is still an urgent need to identify and discover new agents to treat PF. PF is characterized by excessive collagen deposition in the lung; therefore, an in vitro cell screening assay that is based on deposition of collagen in cells has been established [13].

As part of an effort to discover anti-PF compounds from extremophilic fungi, we investigated the metabolites of the fungus *Exophiala oligosperma* MCCC 3A01264, a "blackyeast" isolated from seawater collected at a depth of 3300 m in the northern basin of the South China Sea. While *E. oligosperma* has been reported to cause infections in humans, particularly in immunocompromised patients [3], little is known about its secondary metabolism or production of natural products. In this study, we focused our effort on bioactive compounds that have potential to be developed as drugs for the treatment of pulmonary fibrosis (PF). Here, we report the isolation, structure characterization, and collagen accumulation inhibitory activity of a new compound, exophilone (**1**), together with eleven known compounds (**2**–**10**) from *E. oligosperma* (Figure 1).

**Figure 1.** Chemical structures of compounds **1**–**10**.

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

#### *2.1. Structural Elucidation*

Exophilone (**1**) was isolated as a pale yellow solid, and its molecular formula was established to be C13H13NO3 by HRESIMS (*m/z* 232.0967 [M + H]+, calcd. 232.0968) (Supplementary Figure S1), suggesting eight degrees of unsaturation. Analyses of its 1H, 13C, and HMQC NMR spectra (Table 1 and Supplementary Figures S2–S5) revealed the presence of one carbonyl (C-1), two aromatic quaternary (C-4a, C-5a), two tertiary amines (C-8a, C-1a), and four aromatic methines (C-5 to C-8), as well as one tertiary alcohol (C-2), one secondary alcohol (C-3), one methylene (C-4), and one methyl (C-10). The carbonyl and four double bonds accounted for five out of the eight degrees of unsaturation required by the molecular formula, and the remaining three suggested a structure with three rings in **1**. Based on the COSY correlations between H-5 (δ<sup>H</sup> 7.66, d, *J* = 7.8 Hz) and H-6 (δ<sup>H</sup> 7.08, ddd, *J* = 7.8, 7.2, 1.2 Hz), between H-6 and H-7 (δ<sup>H</sup> 7.30, ddd, *J* = 7.8, 7.2, 1.2 Hz), between H-7 and H-8 (δ<sup>H</sup> 7.38, d, *J* = 7.8 Hz), as well as HMBC correlations between H-5 and C-5a (δ<sup>C</sup> 125.3), C-8a (δ<sup>C</sup> 138.9), and C-4a (δ<sup>C</sup> 123.6), between H-8 and C-5a (δ<sup>C</sup> 125.3) and C-8a (δ<sup>C</sup> 138.9), between H-9 (δ<sup>H</sup> 11.59, s) and C-4a (δ<sup>C</sup> 123.6), C-5a (δ<sup>C</sup> 125.3), C-8a (δ<sup>C</sup> 138.9), and C-1a (δ<sup>C</sup> 129.5), the presence of 2,3-substituted indole moiety was confirmed (Figure 2 and Supplementary Figure S6–S7). The third ring was confirmed by C-1 (δ<sup>C</sup> 192.4) to C-4 (δ<sup>C</sup> 27.5), where C-1 attaches to C-1a and C-4 attaches to C-4a. This was supported by the HMBC correlation between C-4 methylene protons and C-4a, C-1a, C-2, and C-3, between the methylene protons and the carbonyl C-1, between the methyl protons CH3-10 (δ<sup>H</sup> 1.24, s) and C-2, C-1, and between H-3 (δ<sup>H</sup> 4.03, m) and C-4a suggested that C-1 is connected to C-2 and C-2 is connected to C-3 and C-10. Further, the HMBC spectrum allowed the assignment of the position of the OH groups (δ<sup>H</sup> 5.25 and 5.16 ppm) to C-11 and C-12 from their correlations with C-1 and C-10 as well as C-2 and C-4, respectively (Figure 2 and Supplementary Figure S7). Hence, the structure of **1** was elucidated as shown (Figure 1).


**Table 1.** 1H (400 Hz) and 13C (100 Hz) NMR data for compound **1** in DMSO-*d*6.

Since the NOESY spectrum of **1** (Supplementary Figure S8) did not provide enough information to determine its configuration, the absolute configuration of **1** was elucidated to be 2*S*, 3*R* by comparisons of the experimental and calculated electronic circular dichroism (ECD) spectra (Figure 3). Since the structure of **1** has not been reported previously, it is named exophilone (**1**).

**Figure 2.** 1H-1H COSY and key HMBC correlations of compound **1**.

**Figure 3.** Comparisons of the experimental and calculated ECD spectra of **1**.

The other eleven compounds were determined to be flazine (**2**) [14], perlolyrine (**3**) [15], (1H-indol-3-yl) oxoacetamide (**4**) [16], N-acetyl tryptamine (**5**) [17], indole-3 methylethanoate (**6**) [18], 3-(hydroxyacetyl)-indole (**7**) [19], indole-3-acetic acid (**8**) [20], N-acetyl-tyramine (**9**) [21], and uracil (**10**) [22] by comparing their NMR data (Supplementary Figures S9–S26) with those reported in the literature.

#### *2.2. Effect of Compounds* **1**–**10** *on HFL1 Cell Viability*

To assess the cytotoxicity of compounds **1**–**10**, we performed cell viability assay with the HFL1 cell line. The cells were treated with compounds **1**–**10** as well as with pirfenidone as a positive control for 48 h, and the cell viability was measured and compared with the untreated control group (control) (Figure 4A and Table 2). Among the compounds tested, compound **8** and pirfenidone are somewhat toxic to HFL1 cells at 10 μM, with cell survival rates of 78.49% and 77.57%, respectively. On the other hand, compounds **1**, **2**–**7**, **9**, and **10** did not significantly affect cell viability at the same concentration. Particularly, compounds **1**, **4**, and **7** had no cytotoxicity at 10 μM, with the cell survival rates of above 98%.


**Table 2.** Collagen accumulation inhibition rate (IR) and cell survival rate (SR) of **1**-**10**.

Inhibitory effect against TGF-β1 induced total collagen accumulation in HFL1 cells at a concentration of 10 μM. Cell survival rate is calculated by CCK8 assay. The results are the mean ± SD of at least three independent experiments.

#### *2.3. Effect of Compounds* **1**–**10** *on HFL1 Cell Collagen Accumulation*

To evaluate the compounds' inhibitory activity on TGF-β1-induced total collagen accumulation, the Sirius red dye staining, which has been accepted to be an effective and convenient method for the anti-fibrotic screening model in vitro [13,23], was used. Among the compounds tested, compounds **1**, **4**, and **7** showed good inhibition of collagen

accumulation (60.44%, 57.37%, and 44.96%) in HFL1 cells (Table 2 and Figure 4B). While they are somewhat less active than pirfenidone, their toxicity profiles are less than pirfenidone (77.57% survival rate) toward HFL1 cells. More significantly, exophilone (**1**) showed a respectable collagen deposition inhibition effect (60.44% inhibition rate) and low toxicity toward HFL1 cells (98.14% survival rate) at a concentration of 10 μM. The cells were observed with Picro-Sirius Red staining and visualized (Figure 5).

**Figure 5.** Picro-Sirius Red (PSR) staining for the total collagen accumulation induced by TGF-β1 in HFL1 cells. The representative images are the cells induced by TGF-β1 and treated with 10 μM of compounds **1**, **4**, **7**, pirfenidone, and the control group (untreated normal cells). Scale bar: 200 μM.

#### *2.4. Molecular Docking Study*

The inhibitory effect of compound **1** on TGF-β1-induced total collagen accumulation in HFL1 cells might be due to its competitive binding with TGF-β1 (PDBID: 1KLS) or with its receptors (PDBID: 3KFD). In order to investigate the binding mode of compound **1,** molecular docking experiments were performed using Autodock software 1.56 [24]. The results are shown in Figure 6.

**Figure 6.** Molecular docking studies of compound **1**. (**A**) Docking mode of compound **1** to 1KLS; (**B**) Docking mode of compound **1** to 3KFD (Yellow dotted lines represent hydrogen bonds, and numbers represent bond distances).

The docking study showed three hydrogen bonds between compound **1** and the active site residues of TGF-β1 (1KLS) (Figure 6A); a strong hydrogen bond (distance: 1.7 Å) between the indole nitrogen atom and the Cys-78 residue of TGF-β1, and two hydrogen bonds (distance: 2.2, 1.8 Å) between the two hydroxyl groups and Cys-78 and Gly-46, respectively. The data suggest that compound **1** may inhibit TGF-β1-induced total collagen accumulation in HFL1 cells by directly binding to TGF-β1. However, the study also showed three hydrogen bonds between compound **1** and the TGF-β1 receptor (3KFD) (Figure 6B); one hydrogen bond (distance: 1.7 Å) between the indole nitrogen atom and Cys-76, a hydrogen bond (distance: 2.9 Å) between the C-1 ketone and Cys-76, and another hydrogen bond between the C-3 hydroxyl group and Cys-62 (distance: 1.9 Å). The results suggest that compound **1** may bind to the active site of the TGF-β1 as well as to its receptor by hydrogen bonding interactions. These may preliminarily explain why compound **1** inhibits the accumulation of collagen induced by TGF-β1 similar to pirfenidone in HFL1 cells. The interactions of compound **1** with TGF-β1 and its receptor will be a subject of our future investigations.

#### **3. Discussion**

Exophilone (**1**) is a tetrahydro carbazole derivative that is structurally very similar to Sorazolon A [5], which was previously found in soil-derived *Sorangium cellulosum* strain soce375 and thus presumably has a similar biosynthetic pathway. The main differences between exophilone (**1**) and Sorazolon A are the carbonyl C-1 and the secondary alcohol C-3 replacing the tertiary alcohol and carbonyl. Natural tetrahydro carbazole derivatives, including 3-hydroxy-1,2-dimethyl-1,2,3,9-tetrahydrocarbazol-4-one isolated from *Streptomyces ehimensis* strain JB201 [4] and carbazomycin dimers and 3-hydroxy-1,2 dimethyl-2,3-dihydro-1*H*-carbazol-4-one isolated from *Streptomyces* sp. BCC26924 [25], showed antifungal activity and antituberculosis activity. Furthermore, synthetic tetrahydro carbazole protects DNA against oxidative stress [26]. Natural products are a rich source of lead molecules for anti-fibrosis drug discovery. Current pulmonary fibrosis treatment drugs (e.g., colchicine, cyclophosphamide, cyclosporine A, pirfenidone, and nitinol) have therapeutic effects but also significant side effects. Therefore, it is crucial to screen drugs with progressive therapeutic effects to treat pulmonary fibrosis [27].

In this study, a new compound, exophilone (**1**), together with nine known compounds (**2**–**10**), were isolated from a deep-sea-derived fungus, *Exophiala oligosperma*. Among them, exophilone (**1**) showed the best anti-pulmonary fibrosis activity, with low toxicity in HFL1 cells (98.14% survival rate) at a concentration of 10 μM. Exophilone (**1**) has the potential of anti-pulmonary fibrosis and may bind to both TGF-β1 and its receptor through hydrogen bonding interactions.

#### **4. Materials and Methods**

#### *4.1. General Procedures*

The PerkinElmer Spectrum Two spectrometer (PerkinElmer, Waltham, MA, USA) was used for IR spectra measurement. ECD spectra were measured on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., Leatherhead, UK). UV spectra were obtained on a Shimadzu UV-vis-NIR spectrophotometer (Shimadzu Corporation, Nakagyoku, Kyoto, Japan). 1D and 2D NMR spectra were recorded in CDCl3 or DMSO-*d*6 on Bruker Avance II 400, Bruker Avance IIIT 500HD, Bruker Avance IIIT 600AV spectrometers (Bruker Bio Spin AG, Industriestrasse 26, Fällanden, Switzerland). The chemical shifts are relative to the residual solvent signals (CDCl3: δ<sup>H</sup> 7.260 and δ<sup>C</sup> 77.160; DMSO-*d*6: δ<sup>H</sup> 2.500, δ<sup>C</sup> 39.520). The high-resolution ESI-MS spectra were obtained on a Thermo Fisher LTQ Orbitrap Elite High-Resolution liquid chromatography-mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Preparative HPLC was performed using a Shimadzu LC-15C HPLC pump (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) supplied with an SPD-15C dual λ absorbance detector (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), and a Shim-pack PRC-ODS HPLC column I (250 × 20 mm i.d., 5 μm, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). Silica gel (SiO2, 200–300 mesh, Qingdao Puke parting Materials Co., Ltd. Qingdao, China) and Sephadex LH-20 (green herbs, Beijing, China) were used for column chromatography.

#### *4.2. Fungal Strain and Culture Method*

The marine fugus *Exophiala oligosperma* MCCC 3A01264 was obtained from Marine Culture Collection of China (MCCC). It was originally isolated from seawater collected at a depth of 3300 m in northern basin of the South China Sea. A voucher specimen was stored in the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, P.R. China. Analysis of the internal transcribed spacer (ITS) rDNA by BLAST database screening provided a 99.9% match to *Exophiala oligosperma*.

The fermentation medium contained glucose (15 g/L), peptone (10 g/L), yeast extract (2 g/L), L-tryptophan (2 g/L), L-phenylalanine (2 g/L), L-methionine (2 g/L), L-threonine (2 g/L), sea salt (25 g/L), and H2O (1 L), and was adjusted to pH 7.5. Fungal mycelia were cut uniformly and transferred aseptically to 1 L Erlenmeyer flasks with each containing 600 mL liquid medium sterilized at 120 ◦C for 30 min. The flasks were incubated at 28 ◦C for 60 days.

#### *4.3. Extraction and Isolation*

Two hundred liters of culture broth were filtered through the cheesecloth. The culture broth was extracted three times using EtOAc and then concentrated under reduced pressure to afford an EtOAc extract (31.68 g).

The EtOAc extract was chromatographed on a silica gel column (diameter: 80 mm, length: 610 mm, silica gel: 400 g) with a gradient of petroleum ether–EtOAc (10:0–0:10, *v*/*v*) followed by EtOAc–MeOH (10:0–0:10, *v*/*v*) to afford 10 fractions (coded Fr.1–Fr.10). Compound **8** (320 mg) was crystallized from Fr.6 severally. Fr.7 (1.2 g) was subjected to a silica gel column (7 g) eluted with petroleum ether-EtOAc (100:0−0:100) (total volume 2 L) with increasing polarity to obtain ten subfractions (Fr.7-1−Fr.7-10) after pooling the similar fractions as monitored by TLC (petroleum ether−EtOAc = 4:1). Compound **6** (4.2 mg) was obtained from Fr.7-4 directly. Fraction 8 (143 mg) was chromatographed on Sephadex LH-20 (10 g) and eluted with MeOH (total volume 500 mL) to give five subfractions (Fr.8- 1−Fr.8-5). Fr.8-4 was further fractionated by preparative HPLC (MeOH–H2O, 55:45, *v*/*v*, column I) to yield compound **1** (2.1 mg, TR = 22 min), compound **7** (3.0 mg, TR = 16.5 min), and compound **4** (4.2 mg, TR = 25 min). Compound **2** was filtered from Fr.10 directly. The rest of Fr.10 was separated by silica gel column using a step gradient elution with petroleum ether–EtOAc (10:0–0:10) to get 5 subfractions (Fr.10-1–Fr.10-5). Compound **5** (RT = 40.2 min, 3 mg), compound **3** (RT = 23 min, 8 mg), and compound **9** (RT = 36 min, 6 mg) were obtained from Fr.10-2 by preparative HPLC (MeOH–H2O, 80:20, *v*/*v*, column I). Compound **10** (RT = 15 min, 2 mg) was isolated by preparative HPLC (MeOH–H2O, 80:20, *v*/*v*, column I).

Exophilone (**1**). UV (MeOH) λmax (logε) 307 (1.20), 233 (1.29), 206 (2.02). ECD (0.3 mM, MeOH) λmax (Δε) 211 (+3.05) nm. IR υmax 3287, 2922, 2852, 1716, 1651, 1456, 1374, 1330, 1237, 1152, 1097, 1070, 1044, 998, 920, 744, 554 cm−1. 1H and 13C NMR data, shown in Table 1; HR-ESI-MS *m/z* 232.0967 [M + H]<sup>+</sup> (calcd. for C13H13NO3, 232.0968).

#### *4.4. Cell Culture and Cytotoxicity Assay*

The human fetal lung fibroblasts (HFL1) were purchased from Procell Life Science & Technology Co., Ltd. (Cat. No.: CL-0106 Wuhan, China). Cells were cultured in Ham's F-12K medium (PM150910, Procell Life Science & Technology, Wuhan, China) supplemented with 10% fetal bovine serum (FBS) (#10270-106, GIBCO, Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin in an incubator at 37 ◦C with 5% CO2. The cell viability was assayed using the Cell Counting Kit-8 (CCK8) according to the manufacturer's protocol. The cells were treated with 10 μM compounds **1**-**10**, or pirfenidone (TargetMol, Wellesley Hills, MA, USA) for 48 h. The absorbance of the solution was

then measured at 450 nm using a microplate reader (Thermo Fisher, Waltham, MA, USA). Survival rate = (Administration A value − Blank A value)/(Control A value − Blank A value) × 100%. All assays were repeated in triplicate.

#### *4.5. Collagen Accumulation Inhibition In Vitro*

The anti-fibrosis activities of the compounds were tested in HFL1 cells. The cells were treated with medium containing TGF-β1 (5 ng/mL) and 10 μM compounds **1**-**10**, and pirfenidone for 48 h. Subsequently, the supernatant was removed, and 4% paraformaldehyde was added to fix for 30 min at room temperature. Next, the cells were washed with PBS twice and then were added the 0.1% Sirius red dye with saturated picric acid. After 4 h of staining protected from light, the collagenous fiber was dyed red. Then, the cells were washed three times with 0.1% acetic acid and visualized under a cell imaging system (EVOS FL Auto, Life Technologies, Carlsbad, CA, USA). For the quantitative determinations of the accumulated collagen, the stained cells were destained with 0.1 M NaOH (100 μL/well) for 10 min. Then, the absorbance was measured at 540 nm with a spectrophotometer. Total collagen accumulation inhibition = 1 − (Administration A value − control A value)/(model A value − control A value) × 100%. All assays were repeated in triplicate.

#### *4.6. Molecular Docking*

Protein structure was obtained from the Protein Data Bank (https://www.pdbus.org/, accessed on 29 May 2022). The X-ray crystal structure of TGF-β1 (PDB ID: 1LKS) and its receptor (PDB ID: 3KFD) were chosen for the molecular docking analysis in this study. Compound **1** was prepared with Avogadro 1.1.1, with a 5000 steps Steepest Descent as well as 1000 steps Conjugate Gradients geometry optimization using MMFF94 force field. Docking experiments were performed using AutoDock 1.56 Vina and Pymol 2.4.

#### *4.7. Statistical Analysis*

The data are represented as the mean ± SD. Statistical analysis was performed using the GraphPad Prism 8.0 software (San Diego, CA, USA). The significant differences between groups were statistically analyzed using the one-way analysis of variance (ANOVA) followed by a post hoc test (LSD). All differences were considered statistically significant at *p* < 0.05.

#### **5. Conclusions**

A new tetrahydrocarbazol-1-one analogue, exophilone (**1**), together with nine known compounds (**2**–**10**), were isolated from a deep-see-derived fungus *Exophiala oligosperma*. Among all compounds, exophilone (**1**) showed the most significant inhibition of collagen accumulation with low toxicity in HFL1 cells. Further molecular docking experiments showed that exophilone (**1**) may act through hydrogen bonding to the stimulation site of TGF-β1 and its receptor. Given the limitations of the available anti-pulmonary fibrosis drugs, exophilone (**1**) and its analogs could be developed as candidates for the treatment of pulmonary fibrosis.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md20070448/s1. Figures S1–S26: The HR-(+)ESI-MS and NMR spectra of compounds **1**–**10**; Figures S27–S30: NMR spectra of Compounds **11** and **12**.

**Author Contributions:** Conceived and designed the experiments: W.-J.L. Performed the experiments: M.-J.H. (Ming-Jun Hong), M.-J.H. (Meng-Jiao Hao) and G.-Y.Z. Wrote the paper: M.-J.H. (Ming-Jun Hong), M.-J.H. (Meng-Jiao Hao) and G.-Y.Z. Revised the paper: W.-J.L., T.M. Guided experiments: W.-J.L., H.-J.L., Z.-Z.S., X.-P.L., W.-Z.M. and J.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Science Foundation of China (No. 81872795), Guangdong Basic and Applied Basic Research Foundation (Nos. 2021A1515011761, 2018A030313157), and the Key Research and Development Program of Guangdong Province (No. 2020B1111110003).

**Data Availability Statement:** All relevant data are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Anti-inflammatory Polyketides from the Marine-Derived Fungus** *Eutypella scoparia*

**Ya-Hui Zhang 1,2,3, Hui-Fang Du 2, Wen-Bin Gao 4, Wan Li 2, Fei Cao 2,\* and Chang-Yun Wang 1,3,\***


**Abstract:** Three new polyketides, eutyketides A and B (**1** and **2**) and cytosporin X (**3**), along with four known compounds (**4**–**7**), were obtained from the marine-derived fungus *Eutypella scoparia*. The planar structures of **1** and **2** were elucidated by extensive HRMS and 1D and 2D NMR analyses. Their relative configurations of C-13 and C-14 were determined with chemical conversions by introducing an acetonylidene group. The absolute configurations of **1**–**3** were determined by comparing their experimental electronic circular dichroism (ECD) data with their computed ECD results. All of the isolated compounds were tested for their anti-inflammatory activities on lipopolysaccharideinduced nitric oxide production in RAW 264.7 macrophages. Compounds **5** and **6** showed stronger anti-inflammatory activities than the other compounds, with the inhibition of 49.0% and 54.9% at a concentration of 50.0 μg/mL, respectively.

**Keywords:** marine-derived fungus; *Eutypella scoparia*; polyketide; absolute configuration; antiinflammatory activity

#### **1. Introduction**

*Eutypella* species, which are one genus of the ubiquitous fungi, are widely distributed in many extreme environments, including Antarctica, tropical forests, and marine organisms [1–3]. Chemical investigations of *Eutypella* species have resulted in diverse metabolites, including *γ*-lactones, benzopyrans, cysporins, terpenoids, and nitrogen-containing compounds [4,5]. Among them, many bioactive secondary metabolites were obtained, such as antibacterial scoparasin B [5], cytotoxic phenochalasin B [6], and antitumor diaporthein B [7]. The *Eutypella* genus has become an attractive target for discovering leading compounds due to its remarkable biological activity and novel complex structures. In recent years, a lot of work has been carried out on the isolation, total synthesis, pharmacological research, and drug development for the genus *Eutypella* [8–10].

As part of our ongoing investigation of bioactive natural products from marine-derived fungi [11–16], the strain *Eutypella scoparia* HBU-91 attracted our attention because the EtOAc extract of the culture showed anti-inflammatory activity. As a result, the new eutyketides A and B (**1** and **2**) and cytosporin X (**3**), together with four known compounds (**4**–**7**) (Figure 1), were obtained by using silica gel and LH-20 column chromatography and semipreparative HPLC. Structurally, compounds **1** and **2** were a pair of epimers with *vic*-diol unit on their side chain, while **3** exhibited a skeleton characterized by a polyketide moiety and a terpenoid part. All of the isolated compounds were tested for their anti-inflammatory activities. Herein, we report their isolation, structure elucidation, and biological activities.

**Citation:** Zhang, Y.-H.; Du, H.-F.; Gao, W.-B.; Li, W.; Cao, F.; Wang, C.-Y. Anti-inflammatory Polyketides from the Marine-Derived Fungus *Eutypella scoparia*. *Mar. Drugs* **2022**, *20*, 486. https://doi.org/10.3390/md20080486

Academic Editor: Hee Jae Shin

Received: 8 July 2022 Accepted: 27 July 2022 Published: 28 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

**Figure 1.** Chemical structures of compounds **1**−**7**.

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

#### *2.1. Structural Elucidation*

Eutyketide A (**1**) was obtained as a pale yellow oil. The molecular formula of **1** was determined to be C18H26O5 based on the HRESIMS of the pseudomolecular ion (*m*/*z* 345.1669 [M + Na]+, calcd for C18H26O5Na, 345.1672), indicating six degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3385 cm<sup>−</sup>1), double bond (1558 cm−1), and ester carbonyl (1683 cm−1) functionalities. The 1H NMR spectrum (Table 1) showed resonances for five olefinic protons [*δ*<sup>H</sup> 7.13 (dd, *J* = 15.0, 11.5 Hz), 6.42 (dd, *J* = 15.0, 11.5 Hz), 6.08 (d, *J* = 15.0 Hz), 6.07 (s), and 6.05 (dd, *J* = 15.0, 6.0 Hz)], two oxymethines [*δ*<sup>H</sup> 4.23 (dd, *J* = 6.0, 4.2 Hz) and 3.72 (m)], a methoxy [*δ*<sup>H</sup> 3.87 (s)], and two methyls [*δ*<sup>H</sup> 1.93 (s) and 0.88 (t, *J* = 6.6 Hz)]. 13C NMR combined with HSQC spectra (Table 1) of **1** displayed 18 carbon resonances that could be assignable to 9 sp2 deshielded carbons, including a *α*,*β*-unsaturated carbonyl [*δ*<sup>C</sup> 165.0, (C-1)] and 8 olefinic carbons [*δ*<sup>C</sup> 157.3 (C-3), 96.1 (C-4), 165.8 (C-5), 103.2 (C-6), 122.8 (C-9), 134.8 (C-10), 130.8 (C-11), and 137.5 (C-12)], and 9 sp<sup>3</sup> deshielded carbons, including a methoxy [*δ*<sup>C</sup> 56.4, (C-7)], 2 oxymethines [*δ*<sup>C</sup> 75.2 (C-13) and 74.6 (C-14)], 4 methylenes [*δ*<sup>C</sup> 32.2 (C-15), 31.9 (C-17), 25.7 (C-16), and 22.7 (C-18)], and 2 methyls [*δ*<sup>C</sup> 8.9 (C-8) and 14.1 (C-19)]. The 1H and 13C NMR data revealed that **1** shares the same carbon framework as graphostrin I, a polyketide obtained from the Atlantic hydrothermal fungus *Graphostroma* sp. MCCC 3A00421 [17]. The main differences between them were the presence of a methyl at C-6 and a *vic*-diol [−OHCH−CHOH−] substructure at C-13/14 in **1** instead of the group [−CH2−CH2−] and the absence of the hydroxy group at C-18 in graphostrin I. The above differences were confirmed by the COSY cross-peaks of H-12/H-13/H-14/H2-15 and H2-17/H2-18/H3-19 and the HMBC correlations from H-8 to C-1, C-5, and C-6, from H-13 to C-11 and C-15, and from H-14 to C-12 and C-16, respectively (Figure 2). In addition, *trans* geometries at C-9−C-10 and C-11−C-12 double bonds were assigned by the large coupling constants (*J*9,10 = 15.0 Hz and *J*11,12 = 15.0 Hz) [17]. By detailed analysis of its 2D NMR spectra, the planar structure of **1** was assigned.

Eutyketide B (**2**) was also obtained as a pale yellow oil. It exhibited the same molecular formula as **1**, C18H26O5, according to the pseudomolecular ion at *m*/*z* 345.1669 [M + Na]+ in the HRESIMS spectrum. Detailed analysis of the 1H and 13C NMR spectra of **2** (Table 1) revealed that its 1D NMR data were similar to those of **1**. The differences were attributable to the signals [*δ*<sup>H</sup> 4.05 (m, H-13) and 3.51 (m, H-14); *δ*<sup>C</sup> 138.7 (C-12), 74.4 (C-13), and 33.2 (C-15) in **2** vs. *δ*<sup>H</sup> 4.23 (dd, *J* = 6.0, 4.2 Hz, H-13) and 3.72 (m, H-14); *δ*<sup>C</sup> 137.5 (C-12), 75.2 (C-13), and 32.2 (C-15) in **1**], indicating that the structural differences between them

should be located in this part of the structure (C-13 and C-14). Thus, it was deduced that **1** and **2** were either C-13 or C-14 epimers.


**Table 1.** 1H (600 MHz) and 13C (150 MHz) NMR Data of **1** and **2** in CDCl3.

**Figure 2.** COSY and key HMBC correlations of compounds **1** and **2**.

The structural differences between **1** and **2** and their relative configurations were elucidated on the basis of chemical conversions and 1D NOE experiments. Treatment of **1** and **2** with 2,2-dimethoxypropane in the presence of TsOH afforded **1a** and **2a** as the acetonide products. In the selective NOE of **1a** (Figure 3), irradiation of H-13 at *δ*<sup>H</sup> 4.59 and H-14 at *δ*<sup>H</sup> 4.17 resulted in the enhancement of H3-21, indicating that H-13 and H-14 should be placed on the same face of **1**. In the selective NOE of **2a** (Figure 3), irradiation of H-13 at *δ*<sup>H</sup> 4.09 led to the enhancement of H3-21, while irradiation of H-14 at *δ*<sup>H</sup> 3.70 caused the enhancement of H3-22, suggesting that H-13 and H-14 should be placed on the opposite side of **2**.

**Figure 3.** Structures and 1D NOE correlations of the acetonide products of **1a** and **2a**.

The calculation of the solution conformers is the most time-demanding part of the ECD calculation in conformationally flexible molecules and may be aided by simplifying the input geometry to reduce the number of conformers and save computational time [18]. For example, alkyl side chains and unsaturated side chains with isolated chromophores in an achiral environment could be simplified by truncation [18]. The absolute configuration of the hydroxyl group at C-13 was affected by the conjugate system, which can be determined by ECD calculation [19]. For **1** and **2**, the absolute configurations of C-13 and C-14 were determined by comparing their experimental electronic circular dichroism (ECD) results with the computed results of their simplified model compounds. The group of C-15 to C-19 was a saturated alkyl side chain with no chromophore and had a negligible effect on the ECD spectrum. Thus, the C-15 to C-19 alkyl substituent was truncated to a methyl group as model compound **1b** (Figure 4)**.** Molecules of (13*S*,14*S*)-**1b**, (13*R*,14*R*)-**1b**, (13*R*,14*S*)- **1b**, and (13*S*,14*R*)-**1b** were chosen for ECD calculations, which were carried out at the B3LYP/6-311+G(d,p) level in MeOH using the PCM model. The predicted ECD spectrum of (13*R*,14*R*)-**1b** matched well with the experimental ECD curve of **1**, and the predicted ECD spectrum of (13*R*,14*S*)-**1b** was in good agreement with the experimental ECD data of **2** (Figure 4). Therefore, the absolute configurations of **1** and **2** could be defined as 13*R*,14*R* and 13*R*,14*S*, respectively.

**Figure 4.** Calculated ECD spectra of (13*R*,14*R*)-**1b**, (13*S*,14*S*)-**1b**, (13*R*,14*S*)-**1b**, and (13*S*,14*R*)-**1b** and the experimental ECD spectra of **1** and **2**.

To the best of our knowledge, compounds **1** and **2** are very similar to prosolanapyrones and their congeners [20]. They share the same pyranone framework with long alkyl side chains. In addition to the conjugate double bonds, compounds **1** and **2** also contain a *vic*-diol unit on their side chains, while prosolanapyrones just possess double bonds on their side chains.

Cytosporin X (**3**) was obtained as a colorless oil. The molecular formula C19H32O5 was determined for **3** from the pseudomolecular ion peak at *m*/*z* 363.2132 [M + Na]<sup>+</sup> (calcd 363.2142 for C19H32O5Na), which is consistent with four degrees of unsaturation. The IR spectrum of **3** at 3402 and 1652 cm-1 suggested the presence of hydroxyl and double bond groups. The 1H NMR spectrum of **3** displayed resonances for four oxygenated methine protons [*δ*<sup>H</sup> 4.40 (s), 4.26 (d, *J* = 3.6 Hz), 3.67 (d, *J* = 12.0 Hz), and 3.24 (d, *J* = 3.6 Hz)], an oxygenated methylene proton [*δ*<sup>H</sup> 4.24 (d, *J* = 12.0 Hz) and 4.04 (d, *J* = 12.0 Hz)], two singlet methyl protons [*δ*<sup>H</sup> 1.32 (s) and 1.30 (s)], a terminal methyl proton [*δ*<sup>H</sup> 0.87 (t, *J* = 6.6 Hz)], and a series of multiplet signals (Table 2). The 13C NMR spectrum of **3** revealed 19 resonances, including 2 olefinic carbons [*δ*<sup>C</sup> 128.3 (C-8) and 138.1 (C-9)], 2 oxygenated quaternary carbons [*δ*<sup>C</sup> 56.1 (C-5) and 77.2 (C-2)], 4 oxygenated methine carbons [*δ*<sup>C</sup> 60.1 (C-6), 67.2 (C-7), 68.6 (C-10), and 73.5 (C-3)], an oxygenated methylene carbon [*δ*<sup>C</sup> 62.2 (C-13)], 7 methylene carbons [*δ*<sup>C</sup> 22.7 (C-19), 29.0 (C-15), 29.2 (C-17), 29.8 (C-16), 30.6 (C-14), 31.9 (C-18), and 35.6 (C-4)], and 3 methy carbons [*δ*<sup>C</sup> 28.0 (C-12), 16.3 (C-11), and 14.2 (C-20)] (Table 2). The NMR data revealed that **3** belongs to the family of hexahydrobenzopyrane

skeletons and is characterized by a polyketide moiety and a terpenoid part (the red part in Figure 5) with a tricyclic structure containing a hexahydrobenzopyrane moiety fused with an oxirane ring [1]. Careful comparison of the NMR data of **3** with those of the known hexahydrobenzopyrane cytosporin D (**4**) indicated that the structure of **3** is closely related to **4**. The notable difference between them lay in the presence of two methylene signals [*δ*<sup>H</sup> 2.27 and 2.16 (H-14), 1.33 and 1.42 (H-15); *δ*<sup>C</sup> 30.6 (C-14) and 29.0 (C-15) in **3**] and the absence of two olefinic methine signals [*δ*<sup>H</sup> 6.48 (H-14), 6.15 (H-15); *δ*<sup>C</sup> 124.8 (C-14) and 135.9 (C-15) in **4]**. The COSY cross-peaks of H-14/15/16 and the key HMBC correlations from H-14 to C-7/C-9 and from H-15 to C-8/C-17 (Figure 5) confirmed the above difference. Therefore, the planar structure of **3** was established.


**Table 2.** 1H (600 MHz) and 13C (150 MHz) NMR Data of **3** in CDCl3.

**Figure 5.** COSY and key HMBC correlations of **3**.

The relative configuration of **3** was determined by analysis of the NOESY data (Figure 6). The NOESY correlations of H-3/H3-12, H3-11/H-10, H-10/H-7, H-10/H-4*β*, and H-4*α*/H-6 indicated that H-3 and H-6 were situated on the same side of the molecule with an *α*-orientation, while C-5, C-6, H-7, and H-10 were accordingly assigned to be *β*-configured. In addition, the observed NOEs are consistent with the structure and relative configuration of **4**.

**Figure 6.** Key NOESY correlations of **3**.

The absolute configuration of **3** was determined on the basis of ECD calculations. Compound **3** had a long flexible side chain with no chromophore. Thus, the side chain was truncated to two methyl groups attached at C-8, and model compound **3a** was used for ECD calculations. The calculations were carried out for (3*S*,5*R*,6*S*,7*R*,10*S*)-**3a** and (3*R*,5*S*,6*R*,7*S*,10*R*)-**3a** at the B3LYP/6-311+G(d,p) level using the PCM model (MeOH). The calculated ECD curve of (3*S*,5*R*,6*S*,7*R*,10*S*)-**3a** matched well with the experimental ECD data of **3** (Figure 7). Therefore, the absolute configuration of **3** was defined as 3*S*,5*R*,6*S*,7*R*,10*S*.

**Figure 7.** Calculated ECD spectra of (3*S*,5*R*,6*S*,7*R*,10*S*)-**3a** and (3*R*,5*S*,6*R*,7*S*,10*R*)-**3a** and the experimental ECD spectrum of **3**.

The known compounds **4**−**7** were identified as cytosporin D (**4**) [1], 4,8-dihydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochroman-1-one (**5**) [21], banksialactone A (**6**) [22], and 4,8-dihydroxy-3-(hydr-oxymethyl)-6-methoxy-4,5-dimethylisochroman-1-one (**7**) [23], respectively, by comparing their NMR and MS data with reported values.

#### *2.2. Anti-Inflammatory Activity*

The anti-inflammatory activities of **1**−**7** were tested by evaluating their influence on nitric oxide (NO) production in RAW264.7 cells induced by lipopolysaccharide (LPS). Compounds **5** and **6** showed stronger anti-inflammatory activities than other compounds, with inhibition rates of 49.0%, 32.1%, and 27.4% for **5** and 54.9%, 35.9%, and 21.1% for **6** at concentrations of 50.0, 25.0, and 12.5 μg/mL, respectively. Moreover, **5** was also active at 6.25 μg/mL with 24.1% inhibition. In addition, **1** exhibited 20.3% inhibition when tested at 6.25 μg/mL (Table S2).

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

The OR data were recorded on a JASCO P-2000 spectrometer (Jasco Corp., Tokyo, Japan) in MeOH. ECD and UV spectra were measured by MOS450-SFM300 (Biologic, Grenoble, France) and a Perkin-Elmer model 241 spectrophotometers (Perkin-Elmer Corp., Waltham, MA, USA), respectively, with samples dissolved in MeOH. IR spectra were acquired on an FTIR-8400 spectrometer (Shimadzu, Kyoto, Japan) using KBr pellets. NMR data were recorded on a Bruker AV-600 spectrometer (Bruker Corp., Rheinstetten, Germany) with TMS as the internal standard. HRESIMS spectra were obtained from a Bruker apexultra 7.0T spectrometer (Bruker Corp., Rheinstetten, Germany). HPLC separation was performed on the Shimadzu LC-20AT system (Shimadzu, Kyoto, Japan) using an RP-18 HPLC column (Waters, Worcester, MA, USA, 10 × 250 mm, 5 μm).

#### *3.2. Isolation of Fungal Material*

#### 3.2.1. Fungal Material

The fungal strain *Eutypella scoparia* HBU-91 (GenBank, OM892669) was collected from the Bohai Sea (Huanghua, China, Apr. 2017). The strain was deposited in the College of Pharmaceutical Sciences, Hebei University, Baoding, China.

#### 3.2.2. Fermentation and Purification

Fermentation was carried out for the fungus *E. scoparia* using rice medium (170 mL water and 200 g rice in 1 L Erlenmeyer flasks, 200 flasks) at 28 ◦C for 40 days. After cultivation, the fermented rice substrate was extracted with a mixture of CH2Cl2/MeOH (1:1, 500 mL for each flask) five times and EtOAc five times successively to produce a residue (40.0 g), which was further subjected to silica gel column chromatography (CC), eluting with EtOAc−petroleum ether (PE) stepped gradient elution (0%–100%), to afford six fractions, Fr.1−Fr.6. Fr.2 was separated by Sephadex LH-20 CC (PE−CH2Cl2−MeOH (*v*/*v*, 2:1:1)) to afford four subfractions, Fr.2-1−Fr.2-4. Then, Fr.2-2 was fractionated by silica gel CC (PE−EtOAc, 5:1) and further purified by semipreparative HPLC (MeOH−H2O, 70:30, 2.0 mL/min) to afford **6** (2.3 mg) and **5** (3.9 mg). Fr.4 was separated by Sephadex LH-20 CC (MeOH−CH2Cl2 (*v*/*v*, 1:1)) to give subfractions Fr.4-1−Fr.4-3. Fr.4-1 was fractionated by silica gel CC (PE−Acetone, 3:1) and further purified by semipreparative HPLC (MeOH−H2O, 70:30, 2.0 mL/min) to give **4** (15.9 mg). Compound **3** (15.5 mg) was obtained from Fr.4-2 under the same conditions as **4.** Fr.4-3 was fractionated by semipreparative HPLC (MeOH−H2O, 60:40, 2 mL/min) to provide **1** (4.0 mg) and **2** (3.2 mg). Fr.5 was separated by Sephadex LH-20 CC (MeOH−CH2Cl2 (*v*/*v*, 1:1)) to give subfractions Fr.5-1 and Fr.5-2. Fr.5-2 was fractionated by silica gel CC (CH2Cl2/MeOH, 80:1) and further purified by semipreparative HPLC (MeOH−H2O, 50:50, 2.0 mL/min) to afford **7** (4.8 mg).

Eutylactone A (**1**): pale yellow oil; [*α*] *D* <sup>20</sup> +105.8 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *ε*) 225 (2.85), 396 (2.36) nm; ECD (1.04 mM, MeOH) *λ*max (Δ*ε*) 221 (−3.5), 251 (+0.9) nm; IR (KBr) *v*max 3385, 2932, 2341, 1683, 1558, 1027 cm−1; 1H and 13C NMR data see Table 1; HRESIMS *m*/*z* 345.1669 [M + Na]+ (calcd for C18H26O5Na, 345.1672 [M + Na]+).

Eutylactone B (**2**); pale yellow oil; [*α*] *D* <sup>20</sup> +9.5 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *ε*) 223 (2.85), 392 (2.39) nm; ECD (1.04 mM, MeOH) *λ*max (Δ*ε*) 221 (−1.3), 319 (+0.4) nm; IR (KBr) *v*max 3300, 2929, 2359,1679, 1556, 1027 cm−1; 1H and 13C NMR data see Table 1; HRESIMS *m*/*z* 345.1669 [M + Na]+ (calcd for C18H26O5Na, 345.1672 [M + Na]+).

Cytosporin X (**3**); pale yellow oil; [*α*] *D* <sup>20</sup> −314.5 (*c* 0.1, MeOH); UV (MeOH) *λ*max (log *ε*) 254 (2.64), 335 (2.04) nm; ECD (0.98 mM, MeOH) *λ*max (Δ*ε*) 199 (−2.15) nm; IR (KBr) *v*max 3402, 2918, 2351, 1652, 1018 cm−1; 1H and 13C NMR data see Table 2; HRESIMS *m*/*z* 363.2132 [M + Na]<sup>+</sup> (calcd for C19H32O5Na, 363.2142 [M + Na]+); 379.1871 [M + K]+ (calcd for C19H32O5K, 379.1881 [M + K]+).

3.2.3. Acetonide Formation of **1** and **2**

A mixture of **1** (1.0 mg), 2,2-dimethoxypropane (2.0 mL), and *p*-TsOH (0.2 mg) was stirred at room temperature for 0.5 h. Saturated aqueous NaHCO3 (6.0 mL) was then added, and the reaction mixture was extracted with EtOAc (24 mL × 3). The organic solvents were removed with a high-vacuum pump, and the crude mixture was subjected to preparative HPLC to obtain acetonide product **1a** (0.96 mg). Acetonide product **2a** (0.91 mg) was obtained from **2** under the same conditions as **1a.**

Compound **1a**: 1H NMR (600 MHz, CDCl3) *δ* 7.17 (1H, dd, *J* = 15.1, 10.9 Hz, H-10), 6.74 (1H, dd, *J* = 15.0, 5.6 Hz, H-11), 6.37 (1H, dd, *J* = 15.0, 10.9 Hz, H-9), 6.06 (1H, s, H-4), 5.96 (1H, dd, *J* = 15.0, 7.2 Hz, H-12), 4.59 (1H, m, H-13), 4.35 (1H, s, H-14), 3.88 (3H, m, H-7), 1.94 (3H, s, H-8), 1.38 (3H, s, H-21), 1.34 (3H, s, H-22), 1.30-1.50 (8H, m, H-15/16/17/18), 0.88 (3H, t, *J* = 6.6 Hz, H-19).

Compound **2a**: 1H NMR (600 MHz, CDCl3) *δ* 7.15 (1H, dd, *J* = 15.0, 11.1 Hz, H-10), 6.43 (1H, dd, *J* = 15.0, 11.1 Hz, H-11), 6.09 (1H, d, *J* = 15.0 Hz, H-9), 6.06 (1H, s, H-4), 5.94 (1H, dd, *J* = 15.0, 7.2 Hz, H-12), 4.09 (1H, m, H-13), 3.88 (3H, s, H-8), 3.70 (1H, m, H-14), 1.95 (3H, s, H-8), 1.26 (3H, s, H-21), 1.25 (3H, s, H-22), 1.30-1.50 (8H, m, H-15/16/17/18), 0.89 (3H, t, *J* = 6.6 Hz, H-19).

#### *3.3. Computational Section*

A conformational search for the molecules was carried out using the MMFF94S force field and Compute VOA software, with relative energies ranging from 0−10.0 kcal/mol energy, respectively. The conformers were optimized at the B3LYP/6-31G(d)//B3LYP/6- 311+G(d) levels with Gaussian 09 software [24]. Then, stable conformers with relative energy within a 2.5 kcal/mol energy window were chosen for ECD calculations at the B3LYP/6-311+G(d,p) level in methanol using the PCM model, with a total of 60 excited states. A standard deviation of 0.3 eV was used for ECD simulations. Boltzmann statistics were applied for the final simulations of the ECD spectra by the software SpecDis 1.64 [25].

#### *3.4. Cell Culture and Viability Assay*

Compounds **1**−**7** were first tested for their cytotoxic effects on RAW264.7 cells at 3.13, 6.25, 12.5, 25.0, and 50.0 μg/mL. Murine monocytic RAW264.7 macrophages were cultivated at 37 ◦C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM), which was added with 10% (*v*/*v*) fetal bovine serum (FBS) as well as 1% (*v*/*v*) penicillin/streptomycin. RAW264.7 cells were grown in 96-well plates and then incubated with the tested compounds for 24 h. A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at a concentration of 5.0 mg/mL was substituted for the culture medium. After incubation at 37 ◦C for 4 h, the MTT solution was removed, and DMSO was chosen for the dissolution of the formazan crystals. The absorbance was measured at 540 nm with a microplate reader [26]. Compounds **3**−**7** exhibited no toxicity at a concentration of 50.0 μg/mL, **1** showed no toxicity at a concentration of 25.0 μg/mL, and **2** displayed no toxicity at a concentration of 6.25 μg/mL.

#### *3.5. Inhibition of NO Production Assay*

The Griess assay was applied to evaluate the production of NO through the level of nitrite (NO2) in the medium [27]. RAW264.7 cells were inoculated into 96-well plates, and then LPS at a concentration of 1.0 μg/mL was added to induce inflammation. The tested compounds at different concentrations were added to the above mixture. The Griess reaction was used for the quantification of NO production in the supernatant. The absorbance was measured at 540 nm with a microplate reader. All of the experiments were carried out in triplicate.

#### **4. Conclusions**

In summary, three new polyketides (**1**−**3**), together with four known compounds (**4**−**7**), were isolated from the marine-derived fungus *Eutypella scoparia*. Chemical conversions and TDDFT ECD calculations were used to determine the absolute configurations of **1**−**3**. Compounds **1**, **5**, and **6** exhibited certain anti-inflammatory activities on nitric oxide (NO) production in RAW264.7 cells induced by lipopolysaccharide (LPS). Our findings will contribute to the diversity of these fungal metabolites.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20080486/s1. Figures S1−S29: 1D and 2D NMR, HRESIMS, and IR and UV spectra of compounds **1**−**3** and 1D NOE of **1a** and **2a**; Table S1: Cytotoxic activity data of compounds **1**−**7**; Table S2: Anti-inflammatory activity data of compounds **1**−**7**; Tables S3−S8: The coordinates for the lowest-energy conformers of **1b** and **3a** in ECD calculations.

**Author Contributions:** Conceptualization, Y.-H.Z. and F.C.; methodology, Y.-H.Z.; software, H.-F.D.; validation, Y.-H.Z. and H.-F.D.; formal analysis, Y.-H.Z. and W.-B.G.; investigation, Y.-H.Z.; resources, F.C.; data curation, Y.-H.Z. and W.L.; writing—original draft preparation, Y.-H.Z.; writing—review and editing, F.C. and C.-Y.W., visualization, F.C.; supervision, F.C. and C.-Y.W.; project administration, F.C. and C.-Y.W.; funding acquisition, F.C. and C.-Y.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (No. 41830535), Shandong Provincial Natural Science Foundation (Major Basic Research Projects) (ZR2019ZD18), the Program of Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory for Marine Science and Technology (Qingdao, China) directed by Kai-Xian Chen and Yue-Wei Guo, and the Taishan Scholars Program, China, Natural Science Foundation of Hebei Province of China (No. H2021201059).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Material.

**Acknowledgments:** We would like to thank the High Performance Computer Center of Hebei University for providing computational services.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **New Alkylpyridinium Anthraquinone, Isocoumarin, C-Glucosyl Resorcinol Derivative and Prenylated Pyranoxanthones from the Culture of a Marine Sponge-Associated Fungus,** *Aspergillus stellatus* **KUFA 2017**

**Fátima P. Machado 1,2, Inês C. Rodrigues 1, Luís Gales 1,3, José A. Pereira 1,2, Paulo M. Costa 1,2, Tida Dethoup 4, Sharad Mistry 5, Artur M. S. Silva 6, Vitor Vasconcelos 2,7 and Anake Kijjoa 1,2,\***


**Abstract:** An unreported isocoumarin, (*3S*,4*R*)-4-hydroxy-6-methoxymellein (**2**), an undescribed propylpyridinium anthraquinone (**4**), and an unreported C-glucosyl resorcinol derivative, acetyl carnemycin E (**5c**), were isolated, together with eight previously reported metabolites including *p*-hydroxybenzaldehyde (**1**), 1,3-dimethoxy-8-hydroxy-6-methylanthraquinone (**3a**), 1,3-dimethoxy-2,8-dihydroxy-6-methylanthraquinone (**3b**), emodin (**3c**), 5[(3*E*,5*E*)-nona-3,5-dien-1-yl]benzene (**5a**), carnemycin E (**5b**), tajixanthone hydrate (**6a**) and 15-acetyl tajixanthone hydrate (**6b**), from the ethyl acetate extract of the culture of a marine sponge-derived fungus, *Aspergillus stellatus* KUFA 2017. The structures of the undescribed compounds were elucidated by 1D and 2D NMR and high resolution mass spectral analyses. In the case of **2**, the absolute configurations of the stereogenic carbons were determined by comparison of their calculated and experimental electronic circular dichroism (ECD) spectra. The absolute configurations of the stereogenic carbons in **6a** and **6b** were also determined, for the first time, by X-ray crystallographic analysis. Compounds **2**, **3a**, **3b**, **4**, **5a**, **5b**, **5c**, **6a**, and **6b** were assayed for antibacterial activity against four reference strains, viz. two Gram-positive (*Staphylococcus aureus* ATCC 29213, *Enterococcus faecalis* ATCC 29212) and two Gram-negative (*Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 27853), as well as three multidrug-resistant strains. However, only **5a** exhibited significant antibacterial activity against both reference and multidrug-resistant strains. Compound **5a** also showed antibiofilm activity against both reference strains of Grampositive bacteria.

**Keywords:** *Aspergillus stellatus*; Trichocomaceae; marine sponge-associated fungus; anthraquinones; isocoumarin; C-glucosyl resorcinols; antibacterial activity; antibiofilm activity

#### **1. Introduction**

The genus *Aspergillus* (family Aspergillaceae) is one of the most extensively studied genera of filamentous fungi, mainly due to the medical relevance, food spoilage, and industrial application of some of its species. Aspergilli can grow in a wide range of niches, mainly in soils and on dead matter, but some are also capable of colonizing living animal or plant hosts. However, the increasing numbers of *Aspergillus* species have been found in

**Citation:** Machado, F.P.;

Rodrigues, I.C.; Gales, L.; Pereira, J.A.; Costa, P.M.; Dethoup, T.; Mistry, S.; Silva, A.M.S.; Vasconcelos, V.; Kijjoa, A. New Alkylpyridinium Anthraquinone, Isocoumarin, C-Glucosyl Resorcinol Derivative and Prenylated Pyranoxanthones from the Culture of a Marine Sponge-Associated Fungus, *Aspergillus stellatus* KUFA 2017. *Mar. Drugs* **2022**, *20*, 672. https://doi.org/ 10.3390/md20110672

Academic Editor: Hee Jae Shin

Received: 22 September 2022 Accepted: 26 October 2022 Published: 27 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

different niches in the marine environment, from tropical waters [1] to the Arctic [2], and from shallow waters [3] to the deep-sea [4]. In total, approximately 350 *Aspergillus* species have been identified [5].

In the last two decades, marine-derived *Aspergillus* species have attracted tremendous attention from researchers working on marine natural products since they were responsible for a production of the highest numbers of marine fungal metabolites as demonstrated by two review articles, one covering the period of January 1992 to December 2014, reporting 512 metabolites, and another covering the period of 2015 to December 2020, which reported 361 compounds, isolated from marine-derived *Aspergillus* species [6]. It is also worth mentioning that the specialized metabolites produced by marine-derived *Aspergillus* species possess not only structural diversity such as indole alkaloids, diketopiperazine derivatives, meroterpenoids, anthraquinones, isocoumarins, xanthones, *p*-terphenyl derivatives, and peptides, but also a myriad of biological activities [6].

In our pursuit of antibiotic and antibiofilm compounds from marine-derived fungi from tropical seas, we focused our attention on the marine sponge-associated *Aspergillus stellatus* since this fungus has not been extensively investigated. A literature search revealed that a mycotoxin, asteltoxin, was isolated from toxic maize meal cultures of *A. stellatus* Curzi (MRC 277) [7]. In another work, Kamal et al. reported the isolation of tajixanthone, shamixanthone, ajamxanthone, shahenxanthone, najamxanthone, radixanthone, and mannitol from the mycelium of *A. stellatus* Curzi [8]. For this reason, we investigated secondary metabolites from the culture of *A. stellatus* KUFA 2017, isolated from a marine sponge, *Mycale* sp., which was collected from the coral reef at the Samaesan Island, in the Gulf of Thailand.

Fractionation of the ethyl acetate (EtOAc) extract of the culture *A. stellatus* KUFA 2017 by column chromatography of silica gel, followed by purification by preparative TLC, Sephadex LH-20 column and crystallization, led to the isolation of undescribed (*3S*,4*R*)-4 hydroxy-6-methoxymellein (**2**), stellatanthraquinone (**4**), and acetyl carnemycin E (**5c**), as well as the previously reported *p*-hydroxybenzaldehyde (**1**) [9], 1,3-dimethoxy-8-hydroxy-6-methylanthraquinone (**3a**) [10], 1,3-dimethoxy-2,8-dihydroxy-6-methylanthraquinone (**3b**) [11], emodin (**3c**) [12] and 5[(3*E*,5*E*)-nona-3,5-dien-1-yl]benzene (**5a**) [13], carnemycin E (**5b**) [13], tajixanthone hydrate (**6a**) [14,15], and 15-acetyl tajixanthone hydrate (**6b**) [16]. (Figure 1). The structures of the undescribed compounds were established on the basis of an extensive analysis of their 1D and 2D NMR as well as HRMS spectra. In the case of **2**, the absolute configurations of their stereogenic carbons were established by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra. Additionally, the absolute structures of tajixanthone hydrate (**6a**), and 15-acetyl tajixanthone hydrate (**6b**) were also unambiguously determined by X-ray analysis for the first time.

**Figure 1.** Structures of *p*-hydroxybenzaldehyde (**1**), (3*S*,4*R*)-4-hydroxy-6-methoxymellein (**2**), 1,3-dimethoxy-8-hydroxy-6-methylanthraquinone (**3a**), 1,3-dimethoxy-2,8-dihydroxy-6-methylanthraquinone (**3b**), emodin (**3c**), stellatanthraquinone (**4**), 5[(3*E*,5*E*)-nona-3,5-dien-1-yl]benzene-1,3-diol (**5a**), carnemycin E (**5b**), acetyl carnemycin E (**5c**), tajixanthone hydrate (**6a**), 15-acetyl tajixanthone hydrate (**6b**).

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

The structures of *p*-hydroxybenzaldehyde (**1**) [9], 1,3-dimethoxy-8-hydroxy-6-methylanthraquinone (**3a**) [10], 1,3-dimethoxy-2,8-dihydroxy-6-methylanthraquinone (**3b**) [11], emodin (**3c**) [12], 5[(3*E*,5*E*)-nona-3,5-dien-1-yl]benzene-1,3-diol (**5a**) [13], carnemycin E (**5b**) [13], tajixanthone hydrate (**6a**) [14,15], and 15-acetyl tajixanthone hydrate (**6b**) [16] were elucidated by the analysis of their 1D and 2D NMR spectra as well as HRMS data (Figures S1, S2, S9–23, S29–S38, S47–S56, S59 and S60, Tables S1–S3) and by the comparison of their NMR spectral data with those reported in the literature.

Compound **2** was isolated as white crystals (mp 116–118 ◦C), and its molecular formula C11H12O5 was established based on the (+)-HRESIMS *m/z* 225.0765 [M + H]+ (calculated for C11H13O5, 225.0763) (Figure S57), requiring six degrees of unsaturation. The 13C NMR spectrum (Table 1, Figure S4), exhibited eleven carbon signals, which in combination with DEPT and HSQC spectra (Figure S6), can be categorized as one conjugated ester carbonyl (*δ*<sup>C</sup> 169.1), two oxygen-bearing non-protonated sp<sup>2</sup> (*δ*<sup>C</sup> 166.2 and 164.5), two non-protonated sp2 (*δ*<sup>C</sup> 142.1 and 100.0), two protonated sp<sup>2</sup> (*δ*<sup>C</sup> 106.8 and 101.3), two oxymethine sp3 (*δ*<sup>C</sup> 77.8 and 67.5), one methoxy (*δ*<sup>C</sup> 55.8) and one methyl (*δ*<sup>C</sup> 15.9) carbon, respectively. The 1H NMR spectrum (Table 1, Figure S3), in combination with the HSQC spectrum (Figure S6), displayed a singlet of the hydrogen-bonded hydroxyl proton at *δ*<sup>H</sup> 11.20 (OH-8), a multiplet of a hydroxyl proton at *δ*<sup>H</sup> 2.28 (OH-4), two doublets of metacoupled aromatic protons at *δ*<sup>H</sup> 6.48 (*J* = 2.3 Hz, H-5/*δ*<sup>C</sup> 106.8) and *δ*<sup>H</sup> 6.45 (*J* = 2.3 Hz, H-7/*δ*<sup>C</sup> 101.3), a double quartet at *δ*<sup>H</sup> 4.63 (*J* = 6.6, 2 Hz, H-3/*δ*<sup>C</sup> 77.8), which was coupled with a double doublet at *δ*<sup>H</sup> 4.50 (*J* = 5.6, 1.5 Hz, H-4/*δ*<sup>C</sup> 67.5), a methoxy singlet at *δ*<sup>H</sup> 3.85 (*δ*<sup>C</sup> 55.8) and a methyl doublet at *δ*<sup>H</sup> 1.55 (*J* = 6.6 Hz, Me-9/*δ*<sup>C</sup> 15.9). The HMBC spectrum (Table 1, Figure S7) exhibited correlations from OH-8 to the carbons at *δ*<sup>C</sup> 164.5 (C-8), 101.3 (C-7) and 100.0 (C-8a), H-5 to the carbons at *δ*<sup>C</sup> 166.2 (C-6), C-7, C-8a, C-4, H-7 to C-5, C-6, C-8a, H-3 to Me-9, H-4 to C-4a (*δ*<sup>C</sup> 142.1), C-5, C-8a, OMe-6 to C-6, Me-9 to C-3, C-4, and a weak correlation from OH-4 to C-3 and C-4.


**Table 1.** 1H and 13C NMR data (CDCl3, 300 and 75 MHz), COSY, HMBC, and NOESY for **2**.

The 1H and 13C NMR chemical shift values, together with COSY and HMBC correlations, revealed that the planar structure of **2** is the same as that of the enantiomeric mixture of (3*R*,4*R*)- and (3*S*,4*S*)-4-hydroxy-6-methoxymellein, previously isolated from the culture extract of the mycobionts of *Graphis* sp. Since the mixture showed a negative sign of Cotton effect at 267 nm, the authors proposed that the (3*R*,4*R*)-enantiomer was predominant [17]. Surprisingly, the NOESY spectrum (Table 1, Figure S8) of **2** exhibited a strong correlation from H-4 to Me-9, but not from H-3 to H-4, suggesting that H-4 and Me-9 are on the same face, which is contrary to (3*R*,4*R*)- and (3*S*,4*S*)-4-hydroxy-6-methoxymellein where H-3 and H-4 are on the same face.

The absolute configurations of C-3 and C-4 were thus determined by comparison of the experimental ECD spectrum with a quantum-mechanically simulated spectrum derived from the most significant conformations of the computational models of (3*S*,4*R*)-**2** and (3*R*,4*S*)-**2** (Figure 2). Figure 3 shows a very good match between the experimental ECD spectrum and the calculated ECD spectrum for (3*S*,4*R*)-**2**, thus confirming that **2** is (3*S*,4*R*)-4-hydroxy-6-methoxymellein. Compound **2** is, therefore, a diastereomer of (3*R*,4*R*) and (3*S*,4*S*)-4-hydroxy-6-methoxymellein [17] and has never been previously reported.

**Figure 2.** Model of the most abundant conformation of **2** (lowest APFD/6-311+G(2d,p)/acetonitrile energy conformer), accounting for 25% of conformer population, in its ECD-assigned (3*S*,4*R*) configuration.

**Figure 3.** Experimental acetonitrile ECD spectrum of **2** (solid black line) and theoretical ECD spectra of its (3*R*,4*S*) (dot-dashed blue line) and (3*S*,4*R*) (dashed red line) computational models. Of the two theoretical spectra, the (3*R*,4*S*) was the one actually simulated and the (3*S*4,*R*) was obtained by changing the sign of every point of the (3*R*,4*S*) spectrum.

Compound **4** was isolated as a red solid (mp. 228–229 ◦C), and its molecular formula C23H19NO5 was established on the basis of (+)-HRESIMS *m/z* 390.1340 [M + H]<sup>+</sup> (calculated for C23H20NO5, 390.1341) (Figure S58), requiring fifteen degrees of unsaturation. The 13C NMR spectrum (Table 2, Figure S25) displayed 23 carbon signals which, in combination with DEPT and HSQC spectra (Figure S27), can be classified as two conjugated ketone carbonyls (*δ*<sup>C</sup> 184.5 and 183.3), three oxygen-bearing non-protonated sp<sup>2</sup> (*δ*<sup>C</sup> 171.9, 161.2, 159.5), seven non-protonated sp2 (*δ*<sup>C</sup> 146.7, 142.8, 134.4, 133.2, 123.2, 114.6, 100.0), seven protonated sp<sup>2</sup> (*δ*<sup>C</sup> 147.2, 146.2, 145.6, 127.5, 124.4, 120.4, and 118.6), two methylene sp3 (*δ*<sup>C</sup> 33.8 and 23.7), and two methyl (*δ*<sup>C</sup> 21.9 and 13.7) carbons. The 1H NMR spectrum (Table 2, Figure S24) showed two singlets of hydrogen-bonded phenolic hydroxyls at *δ*<sup>H</sup> 12.50 and 13.44, seven aromatic protons appearing as a broad singlet at *δ*<sup>H</sup> 8.91, two singlets at *δ*<sup>H</sup> 7.11 and 6.80, three doublets at *δ*<sup>H</sup> 8.57 (*J* = 8.0 Hz), 8.85 (*J* = 6.1 Hz), 7.46 (*J* = 1.0 Hz), one double doublet at *δ*<sup>H</sup> 8.16 (*J* = 8.0, 6.1 Hz), one methylene triplet at *δ*<sup>H</sup> 2.83 (*J* = 7.4 Hz), one methylene sextet at *δ*<sup>H</sup> 1.70 (*J* = 7.4 Hz), one methyl singlet at *δ*<sup>H</sup> 2.40, and one methyl triplet at *δ*<sup>H</sup> 0.94 (*J* = 7.3 Hz). The presence of the 1,8-dihydroxy-6-methyl-1,2,3,6,8-pentasubstitututed anthraquinone scaffold was supported by COSY correlations (Table 2, Figures 4 and S26) from H-7 (*δ*<sup>H</sup> 7.11, s/*δ*<sup>C</sup> 124.4) to H-5 (7.46, d, *J* = 1.0 Hz)/*δ*<sup>C</sup> 120.4) and Me-11 (2.40, s/*δ*<sup>C</sup> 21.9), as well as by HMBC correlations (Table 2, Figures 4 and S28) from OH-1 (*δ*<sup>H</sup> 13.44, s) to C-1 (*δ*<sup>C</sup> 159.5), C-2 (*δ*<sup>C</sup> 123.2), C-9a (*δ*<sup>C</sup> 100.0), OH-8 (*δ*<sup>H</sup> 12.50, s) to C-7 (*δ*<sup>C</sup> 124.4), C-8

(*δ*<sup>C</sup> 161.2), C-8a (*δ*<sup>C</sup> 114.6), H-5 (*δ*<sup>H</sup> 7.46, d, *J* = 1.0 Hz) to C-10 (*δ*<sup>C</sup> 183.3), C-7, C-8a, Me-11 (*δ*<sup>C</sup> 21.9), H-7 (*δ*<sup>H</sup> 7.11, s) to C-8, C-5 (*δ*<sup>C</sup> 120.4), C-8a, Me-11, H-4 (*δ*<sup>H</sup> 6.80,s) to C-10, C-2 (*δ*<sup>C</sup> 123.2), and C-9a. That another portion of the molecule is 3-propylpyridinium was corroborated by COSY correlations (Table 2, Figures 4 and S26) from H-4' (*δ*<sup>H</sup> 8.57, d, *J* = 8.0 Hz) to H-5' (*δ*<sup>H</sup> 8.16, dd, *J* = 8.0, 6.1 Hz) and H-2' (*δ*<sup>H</sup> 8.91, brs), H-5' to H-4' and H-6' (8.85, d, *J* = 6.1 Hz), which was confirmed by HMBC correlations (Table 2, Figures 4 and S28) from H-2' to C-3' (*δ*<sup>C</sup> 142.8), C-4' (*δ*<sup>C</sup> 146.2) and C-1" (*δ*<sup>C</sup> 33.8), H-4' to C-2' (*δ*<sup>C</sup> 147.2), C-6' (*δ*<sup>C</sup> 145.6) and C-1", H-5' to C-3', and C-6' and H-6' to C-4' and C-5' [18]. That the propyl group was on C-3' of the pyridinium ring was supported by the spin system from H2-1" (*δ*<sup>H</sup> 2.83, t, *J* = 7.4 Hz/*δ*<sup>C</sup> 33.8) through H2-2" (*δ*<sup>H</sup> 1.70, sex, *J* = 7.4 Hz/*δ*<sup>C</sup> 23.7) to H3-3" (*δ*<sup>H</sup> 0.94, t, *J* = 7.4 Hz/*δ*<sup>C</sup> 13.7) as well as by HMBC correlations from H-3" to C-1" and C-2", H-2" to C-1", and C-3" and C-2'. Since H-2' and H-6' showed strong and weak cross peaks, respectively, to C-2 (Table 2, Figures 4 and S28), the 3-propylpyridinium moiety is linked to the anthraquinone scaffold through the nitrogen atom for the former and C-2 of the latter.

**Table 2.** 1H and 13C NMR data (DMSO-*d6*, 500 and 125 MHz), COSY and HMBC assignment for **4**.


Since 1,8-dihydroxy-6-methyl anthraquinone and the 3-propylpyridinium moiety account for C23H19NO4, the only oxygen atom left must be on C-3 of the anthraquinone nucleus to produce a molecular formula C23H19NO5. Therefore, the oxygen atom on C-3 should bear a negative charge (**4**). This was supported by a high chemical shift value of C-3 (*δ*<sup>C</sup> 171.9). This phenoxide ion can establish an ionic interaction with the positive-charged nitrogen of the pyridinium ring. Interestingly, although alkyl pyridiniumcontaining compounds have never been reported from marine-derived fungi, cyclic 3 alkylpyridinium alkaloids are common secondary metabolites from sponges of the order Haplosclerida [19,20]. Therefore, **4** is the first 3-alkylpyridinium anthraquinone reported from nature, and it was named stellatanthraquinone.

Compound **5b** was isolated as a pale-yellow viscous mass, and its molecular formula C21H30O7 was established on the basis of the (+)-HRESIMS *m/z* 395.2076 [M + H]<sup>+</sup> (calculated for C21H31O7, 395.2070 (Figure S60), requiring seven degrees of unsaturation. Analysis of its 1H, 13C NMR, DEPT, COSY, HSQC, and HMBC spectra (Table 3, Figures S34–S38) revealed the presence of a 5 [(3*E*,5*E*)-nona-3,5-dien-1-yl]benzene-1,3-diol moiety, identical to **5a**, with a substitution on C-2. The presence of five oxymethine sp3 (*δ*<sup>C</sup> 81.5, 79.1, 75.0, 72.1, 70.3), one oxymethylene sp3 (*δ*<sup>C</sup> 61.2) carbons, two hydroxyl groups at *δ*<sup>H</sup> 4.90 dd (*J* = 10.7, 2.9 Hz) and 4.59 d (*J* = 5.5 Hz), and the molecular formula C6H11O5 of the substituent on C-2 revealed the presence of a pyranosyl moiety. However, since four oxymethine protons of the sugar moiety appeared as complex multiplets at *δ*<sup>H</sup> 3.20–3.22 and 3.74, it was not possible to identify the sugar moiety of **5b**. Although Wen et al. [13] identified the sugar moiety in carnemycin E as glucopyranosyl, it was not possible to compare its 1H and 13C chemical shift values with those of the sugar moiety **5b** since the 1H and 13C NMR spectra of carnemycin E were obtained in pyridine-*d5*, while those of **5b** were obtained in DMSO-*d6*. Moreover, carnemycin E was obtained as an amorphous reddish gum, while **5b** was obtained as a pale-yellow viscous mass. In order to unravel the identity of the sugar moiety in **5b**, we tried to compare its carbon chemical shift values with those of the C-glycosides from the 13C NMR spectra obtained in DMSO-*d6*. The chemical shift values of C-1', C-2', C-3', C-4', C-5' and C-6' of the sugar moiety of **5b** (Table 3, Figure S36) were nearly identical to those of C-glucosyl moiety of tricetin 6,8-di-C-glucoside [21]. Moreover, the chemical shift value and coupling constant of H-1' were also identical with those of the corresponding proton in tricetin 6,8-di-C-glucoside [21]. The value of the coupling constant of H-1' (*J* = 9.6 Hz) confirmed the presence of a β-D-glucopyranosyl moiety. Therefore, **5a** was elucidated as carnemycin E, previously isolated from the culture extract of *Aspergillus* sp., which was isolated from superficial mycobiota of the brown alga, *Saccharina cichorioides* f. *sachalinensis,* collected from the South China Sea [13].


**Table 3.** 1H and 13C NMR data (DMSO-*d6*, 300 and 75 MHz), COSY, and HMBC assignment for **5b**.

Compound **5c** was also isolated as a pale-yellow viscous mass and its molecular formula C23H32O8 was established on the basis of the (+)-HRESIMS *m/z* at 437.2175 [M + H]+ (calculated for C23H33O8, 437.2175), and *m/z* 459.1989 [M + Na]+ (calculated for C23H32O8Na, 459.1995) (Figure S61).The 1H and 13C NMR spectra of **5c** (Table 4; Figures S39 and S40) resembled those of **5b** (Table S2 and Figures S34 and S35) except for CH2-6', which appeared at higher frequencies (*δ*<sup>H</sup> 4.32 d*, J* = 11.6 Hz, and 3.98 dd, *J* = 11.6, 3.9 Hz/*δ*<sup>C</sup> 64.8) than those of **5b** (*δ*<sup>H</sup> 3.50, dd, *J* = 11.0, 5.5 Hz, and 3.65, dd*, J* = 11.0, 5.2 Hz)/*δ*<sup>C</sup> 61.2) as well as the appearance of an acetyl group (*δ*<sup>H</sup> 2.00, s/*δ*<sup>C</sup> 21.2, CH3; *δ*<sup>C</sup> 170.9, CO), suggesting that **5c** is a C-21 acetate of **5b**.

Contrary to other proton signals, the signals of OH-3, OH-3', and OH-4' appeared as broad signals in the 1H NMR spectrum at 500 MHz (Figure S39). Moreover, they did not show any COSY and HMBC correlations with any protons (Table 4, Figures S41 and S43), which made it impossible to assign them. Interestingly, in the 1H NMR spectrum at 300 MHz (Figure S44), the signal of OH-3 appeared as a sharp singlet at *δ*<sup>H</sup> 8.70, whereas those of OH-3' and OH-4' appeared as two well-resolved doublets at *δ*<sup>H</sup> 4.59, d *(J* = 6.5 Hz) and 5.15, d (*J* = 4.4 Hz), respectively. Furthermore, in the 300 MHz spectra, OH-3 displayed HMBC correlations to C-2 (*δ*<sup>C</sup> 109.9) and C-3 (*δ*<sup>C</sup> 157.2) (Figure S46), while OH-3' and OH-4' showed COSY correlations to H-3' (*δ*<sup>H</sup> 3.20) and H-4' (*δ*<sup>H</sup> 3.18) (Figure S45), respectively. The coupling constant of H-1' (*J* = 9.8 Hz) confirmed the β-anomer of the glucosyl moiety. Since **5c** has never been previously reported, it was named acetyl carnemycin E.


**Table 4.** 1H and 13C NMR data (DMSO-*d6*, 500 and 125 MHz), COSY, and HMBC assignment for **5c**.

The 1H and 13C NMR spectra of **6a** and **6b** (Table S3. Figures S47, S48, S52 and S53) are in agreement with those reported for tajixanthone hydrate [14] and 15-acetyl tajixanthone hydrate [16]. However, Pornpakakul et al. [14] assigned the configurations of C-15, C-20 and C-25 of tajixanthone hydrate, based on the coupling constant between H-14 and H-15 and NOESY correlations of the protons in tajixanthone methanoate, and also referred its stereochemistry to the previous study by Chexal et al. [22], who elegantly determined the absolute configurations of C-15 and C-25 of tajixanthone as 15*S* and 25*R* by chemical transformation (the method of Boar and Damps) while the relative configuration of C-20 was suggested by the preferred axial conformation of the isopropyl substituent in the hydrogenated derivatives [22]. Later on, the same group [23], described the isolation of tajixanthone hydrate which they obtained in a small quantity. The structure and stereochemistry of tajixanthone hydrate were identified on the basis of the same optical rotation of the acid-catalyzed hydrolysis product of tajixanthone and of the natural product. On the other hand, the absolute stereochemistry of 15-acetyl tajixanthone hydrate was concluded

to be the same as that of tajixanthone hydrate, which was obtained by hydrolysis of 15 acetyl tajixanthone hydrate. However, neither optical rotation nor absolute configurations of their stereogenic carbons were provided [16].

Literature search revealed that the absolute configurations of C-15, C-20, and C-25 of both tajixanthone hydrate and 15-acetyl tajixanthone hydrate have never been established by either X-ray crystallographic or chiroptical methods. Fortunately, we were able to obtain suitable crystals of both **6a** and **6b** for X-ray analysis using an X-ray diffractometer equipped with CuKα radiation. The Ortep views of **6a** and **6b** are shown in Figures 5A and 5B, respectively, revealing that the absolute configurations of C-15, C-20, and C-25 in both compounds are 15*S*, 20*S*, and 25*R*. Moreover, both compounds are levorotatory.

(**A**)

(**B**)

**Figure 5.** Ortep views of **6a** (**A**) and **6b** (**B**).

The antimicrobial activity of **2**, **3a**, **3b**, **4**, **5a**, **5b**, **5c**, **6a**, and **6b** was evaluated against four reference bacterial species and three multidrug-resistant strains. However, only **5a** exhibited antibacterial activity against all Gram-positive strains, viz. *E. faecalis* ATCC 29212, vancomycin-resistant *E. faecalis* B3/101, and methicillin-resistant *S. aureus* 74/24, with a MIC value of 16 μg/mL, and *S. aureus* ATCC 29213, with a MIC value of 32 μg/mL (Table 5). The minimal bactericidal concentration (MBC) for **5a** was equal to or one-fold higher than the MIC, indicating its bactericidal effect towards *E. faecalis* ATCC 29212*, S. aureus* 74/24, and *S. aureus* ATCC 29213. For *E. faecalis* B3/101, its MBC was more than two-fold higher than the MIC, suggesting its bacteriostatic effect.


**Table 5.** Antibacterial activity of **2**, **3a**, **3b**, **4**, **5a**, **5b**, **5c**, **6a**, and **6b** against Gram-positive reference and multidrug-resistant strains. MIC and MBC are expressed in μg/mL.

MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentration; VAN, vancomycin; OXA, oxacillin.

Although **5a** was found to significantly inhibit NO production in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells [13], this compound has never been tested for antibacterial acivity. Interestingly, some alkenylresorcinols, such as 9-(3,5 dihydroxy-4-methylphenyl)nona-3(*Z*)-enoic acid, isolated from the methanolic extract of fruits of *Hakea sericea*, significantly inhibited the growth of *E. faecalis*, *Listeria monocytogenes* and *Bacillus cereus*, and showed good MIC values against *S. aureus* strains, including the clinical isolates and MRSA strains [24]. Intriguingly, **5b** and **5c**, analogs of **5a** which possess a β-glucosyl moiety on C-2 of the benzene ring, were void of antibacterial activity in our assays. We speculate that the polar and bulky glucosyl moiety might have negatively affected the antibacterial activity, possibly by preventing the compounds from penetrating the bacterial cell wall.

Another interesting aspect is that even though there were several reports on the antibaterial activity of anthraquinones from marine-derived fungi [25], neither of the three anthraquinones tested, i.e., **3a**, **3b**, and **4**, showed antibacterial activity in our assays. This is not surprising since we also found in our previous report that the anthraquinone purnipurdin A, isolated from the culture extract of the marine sponge-associated fungus, *Neosartorya spinosa* KUFA 1047, did not exhibit any antibacterial activity against the same bacterial strains tested [26].

Compounds **2**, **3a**, **3b**, **4**, **5a**, **5b**, **5c**, **6a***,* and **6b** were also investigated for their potential synergy with clinically relevant antibiotics on multidrug-resistant strains, by both disk diffusion method and checkerboard assay; however, no interactions were found with cefotaxime (ESBL *E. coli*), methicillin (MRSA *S. aureus*), and vancomycin (VRE *E. faecalis*).

The inhibitory effect of **2**, **3a**, **3b**, **4**, **5a**, **5b**, **5c**, **6a**, and **6b** on biofilm production was also evaluated in all reference strains. However, only **5a** showed an extensive ability to significantly inhibit biofilm formation in two of the four reference strains used in this study (Table 6). Indeed, **5a** was able to completely inhibit biofilm formation in *S. aureus* ATCC 29213 and *E. faecalis* ATCC 29212, at both MIC and 2xMIC concentrations.


**Table 6.** Percentage of biofilm formation in the presence of **5a** after 24 h incubation.

Data are shown as mean ± SD of three independent experiments. One-sample *t*-test: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 significantly different from 100%.

#### **3. Experimental Sections**

#### *3.1. General Experimental Procedures*

The melting points were determined on a Stuart Melting Point Apparatus SMP3 (Bibby Sterilin, Stone, Staffordshire, UK) and are uncorrected. Optical rotations were measured on an ADP410 Polarimeter (Bellingham + Stanley Ltd., Tunbridge Wells, Kent, UK). 1H and 13C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA) operating at 300 or 500 and 75 or 125 MHz, respectively. High resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Aquity UPLC system. A Merck (Darmstadt, Germany) silica gel GF254 was used for preparative TLC, and a Merck Si gel 60 (0.2–0.5 mm) was used for column chromatography. LiChroprep silica gel and Sephadex LH 20 were used for column chromatography.

#### *3.2. Fungal Material*

The fungus was isolated from the marine sponge *Mycale* sp., which was collected by scuba diving at a depth of 5–10 m, from the coral reef at Samaesan Island (12◦34 36.64 N 100◦56 59.69 E), in the Gulf of Thailand, Chonburi province, in May 2015. The sponge was washed with 0.01% sodium hypochlorite solution for 1 min, followed by sterilized seawater three times, and then dried on a sterile filter paper under a sterile aseptic condition. The sponge was cut into small pieces (*ca*. 5 × 5 mm) and placed on Petri dish plates containing 15 mL potato dextrose agar (PDA) medium mixed with 300 mg/L of streptomycin sulfate and incubated at 28 ◦C for 7 days. The hyphal tips emerging from sponge pieces were individually transferred onto a PDA slant and maintained as pure cultures at Kasetsart University Fungal Collection, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. The fungal strain KUFA 2017 was identified as *Aspergillus stellatus*, based on morphological characteristics such as colony growth rate and growth pattern on standard media, namely Czapek s agar, Czapek yeast autolysate agar, and malt extract agar. Microscopic characteristics including size, shape, and ornamentation of conidiophores and spores were examined under light microscope. This identification was confirmed by molecular techniques using internal transcribed spacer (ITS) primers. DNA was extracted from young mycelia following a modified Murray and Thompson method [27]. Primer pairs ITS1 and ITS4 were used for ITS gene amplification [28]. PCR reactions were conducted on Thermal Cycler and the amplification process consisted of initial denaturation at 95 ◦C for 5 min, 34 cycles at 95 ◦C for 1 min (denaturation), at 55 ◦C for 1 min (annealing), and at 72 ◦C for 1.5 min (extension), followed by final extension at 72 ◦C for 10 min. PCR products were examined by Agarose gel electrophoresis (1% agarose with 1× Tris-Borate-EDTA (TBE) buffer) and visualized under UV light after staining with ethidium bromide. DNA sequencing analyses were performed using the dideoxyribonucleotide chain termination method [29] by Macrogen Inc. (Seoul, South Korea). The DNA sequences were edited using FinchTV software and submitted to the BLAST program for alignment and compared with that of fungal species in the NCBI database (http://www.ncbi.nlm.nih.gov/, accessed on 18 May 2021). Its gene sequences were deposited in GenBank with the accession number MZ331807.

#### *3.3. Extraction and Isolation*

The fungus was cultured in five Petri dishes (i.d. 90 mm) containing 20 mL of PDA per dish at 28 ◦C for one week. The mycelial plugs (5 mm in diameter) were transferred to two 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose broth (PDB), and incubated on a rotary shaker at 120 rpm at 28 ◦C for one week. Thirty 1000 mL Erlenmeyer flasks, each containing 300 g of cooked rice, were autoclaved at 121 ◦C for 15 min. After cooling to room temperature, 20 mL of mycelial suspension of the fungus was inoculated per flask and incubated at 28 ◦C for 30 days, after which 500 mL of EtOAc was added to each flask of the moldy rice and macerated for 7 days, and then filtered with Whatman No. 1 filter paper.

The EtOAc solutions were combined and concentrated under reduced pressure to yield 280.4 g of a crude EtOAc extract, which was dissolved in 300 mL of CHCl3, washed with H2O (3 × 500 mL) and dried with anhydrous Na2SO4, and filtered and evaporated under reduced pressure to obtain 134.9 g of a crude CHCl3 extract. The crude CHCl3 extract (57.1 g) was applied on a silica gel column (385 g) and eluted with mixtures of petrol-CHCl3 and CHCl3-Me2CO, wherein 250 mL fractions (frs) were collected as follows: frs 1–61 (petrol-CHCl3, 1:1), 62–129 (petrol-CHCl3, 3:7), 130–231 (petrol-CHCl3, 1:9), 232–397 (CHCl3-Me2CO, 9:1), 398–524 (CHCl3-Me2CO, 7:3). Frs 40–45 were combined (241.9 mg) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 37 subfractions (sfrs) of 2 mL were collected. Sfrs 30–35 were combined (102.9 mg) and precipitated in MeOH to produce 13.6 mg of **3a**. Frs 74–77 were combined (851.6 mg) and precipitated in Me2CO to produce 20.3 mg of **3b**. Frs 78–89 were combined (396.5 mg) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 28 sfrs of 2 mL were collected. Sfrs 21–25 were combined to produce 11.4 mg of **3c**. Frs 95–118 were combined (535.5 mg) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 47 sfrs of 2 mL were collected. Sfrs 16–33 were combined (231.4 mg) and applied over another Sephadex LH-20 column (5 g), and eluted with CHCl3, wherein 22 sub-subfractions (ssfrs) of 1 mL were collected. Ssfrs 9–10 were combined to produce 69.4 mg of **2**, while ssfrs 20–22 were combined to produce 45.0 mg of **1**. Frs 126–140 were combined (274.9 mg) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 31 sfrs of 2 mL were collected. Sfrs 4–15 were combined (108.4 mg) and precipitated in Me2CO to produce 26.7 mg of **6b**. Frs 149-173 were combined (1.02 g) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 49 sfrs of 1 mL were collected. Sfrs 20–28 were combined (305.2 mg) and applied over another Sephadex LH-20 column (5 g), and eluted with CHCl3, wherein 20 ssfrs of 0.5 mL were collected. Ssfrs 15–19 were combined to produce 153.0 mg of **5a**. Frs 178–206 were combined (509.4 mg) and precipitated in MeOH to produce 46.8 mg of **6a**. Frs 237–238 were combined (209.3 mg) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 20 sfrs of 2 mL were collected. Sfrs14-20 were combined (182.4 g) and applied over another Sephadex LH-20 column (5 g), and eluted with CHCl3, wherein 13 ssfrs of 1 mL were collected. Ssfrs 8–9 were combined to produce 7.3 mg of **5c**. Frs 437-455 were combined (323.3 mg) and applied over a Sephadex LH-20 column (15 g), and eluted with MeOH, wherein 24 sfrs of 2 mL were collected. Sfrs 11–15 were combined (211.9 mg) and applied over another Sephadex LH-20 column (5 g), and eluted with CHCl3, wherein 18 ssfrs of 0.5 mL were collected. Ssfrs 17–18 were combined to produce 141.7 mg of **5b**. Frs 460–513 were combined (297.3 mg) and applied over a Sephadex LH-20 column (15g), and eluted with MeOH, wherein 25 sfrs of 2 mL were collected. Sfrs 15–18 were combined (20.9 mg) and applied over another Sephadex LH-20 column (5 g), and eluted with CHCl3, wherein 13 ssfrs of 0.5 mL were collected. Ssfrs 4–7 were combined to produce 5.6 mg of **4**.

#### 3.3.1. (3*S*,4*R*)-4-Hydroxy-6-Methoxymellein (**2**)

White crystal. Mp 116–118 ◦C. [α] 23 *<sup>D</sup>* −200 (*<sup>c</sup>* 0.05, MeOH); For 1H and 13C spectroscopic data (CDCl3, 300 and 75 MHz), see Table 1; (+)-HRESIMS *m/z* 225.0765 [M + H]<sup>+</sup> (calculated for C11H13O5, 225.0763).

#### 3.3.2. Stellatanthraquinone (**4**)

Red solid. Mp. 228–229 ◦C. For 1H and 13C spectroscopic data (DMSO-*d6*, 500 and 125 MHz), see Table 2; (+)-HRESIMS *m/z* 390.1340 [M + H]+ (calculated for C23H20NO5, 390.1341).

#### 3.3.3. Carnemycin E (**5b**)

Pale-yellow viscous mass. [α] 20D +60 (*c* 0.05, MeOH). 1H and 13C spectroscopic data (DMSO-*d6*, 300 and 75 MHz), see Table 3; (+)-HRESIMS *m/z* 395.2076 [M + H]+ (calculated for C21H331O7, 395.2070).

#### 3.3.4. Acetyl Carnemycin E (**5c**)

Pale-yellow viscous mass. [α] 20D +260 (*c* 0.05, MeOH). 1H and 13C spectroscopic data (DMSO-*d6*, 500 and 125 MHz), see Table 4; (+)-HRESIMS *m/z* 437.2175 [M + H]+, (calculated for C23H33O8, 437.2175); *m/z* 459.1989 [M + Na]+ (calculated for C23H32O8Na, 459.1995).

#### *3.4. X-ray Crystal Structures*

Single crystals were mounted on cryoloops using paratone. X-ray diffraction data were collected at 290 K with a Gemini PX Ultra equipped with CuK<sup>α</sup> radiation (λ = 1.54184 Å). The structures were solved by direct methods using SHELXS-97 and refined with SHELXL-97 [30]. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were either placed at their idealized positions using appropriate HFIX instructions in SHELXL and included in subsequent refinement cycles or were directly found from difference Fourier maps and were refined freely with isotropic displacement parameters.

#### 3.4.1. X-ray Crystal Structure of **6a**

Crystal was orthorhombic, space group *P*212121, cell volume 2178.1(4) Å<sup>3</sup> and unit cell dimensions *a* = 6.2933(5) Å, *b* = 17.9862(18) Å and *c* = 19.243(3) Å (uncertainties in parentheses). Flack *x* [31] was refined parameter by means of TWIN and BASF in SHELXL to 0.0(5). The refinement converged to R (all data) = 8.88% and wR2 (all data) = 17.80%. Full details of the data collection and refinement and tables of atomic coordinates, bond lengths and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre (CCDC 2206108).

#### 3.4.2. X-ray Crystal Structure of **6b**

The crystal was orthorhombic, space group P212121, cell volume 2454.0(4) Å3, and unit cell dimensions *a* = 5.9772(6) Å, *b* = 13.8321(13) Å and *c* = 29.682(2) Å (uncertainties in parentheses). Calculated crystal density was 1.306 g/cm−3. The structure was solved by direct methods using SHELXS-97 and refined with SHELXL-97 [30]. The refinement converged to R (all data) = 8.45% and wR2 (all data) = 12.84% and Flack parameter = 0.0(3). Full details of the data collection and refinement and tables of atomic coordinates, bond lengths and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre (CCDC 2204631).

#### *3.5. Electronic Circular Dichroism (ECD)*

The experimental ECD spectrum of **2** (*ca.* 2 mg/mL in acetonitrile) was obtained in a Jasco J-815 CD spectropolarimeter (Jasco Europe S.R.L., Cremella, Italy) with a 0.1 mm cuvette and 6 accumulations. The simulated ECD spectra were obtained by first determining all the relevant conformers of the computational model. Its conformational space was developed by rotating all the single, non-ring, bonds for each of the two possible bends of the non-aromatic ring. The resulting 24 molecular mechanics conformers were minimized using the quantum mechanical DFT method B3LYP/6-31G with Gaussian 16W (Gaussian Inc., Wallingford, USA). The lowest 95% of the conformer Boltzmann populations (11 models) were subjected to a final minimization round using the method APFD/6- 311+G(2d,p)/acetonitrile method (Gaussian 16W), which was also used, coupled with a TD method, to calculate its first 50 ECD transitions. The line spectrum for each one of the

11 models was built by applying a Gaussian line broadening of 0.15 eV to each computed transition with a constant UV-shift of 5 nm. The final ECD spectrum was obtained by the Boltzmann-weighted sum of the 11 line spectra [32].

#### *3.6. Antibacterial Activity Bioassays*

#### 3.6.1. Bacterial Strains and Testing Conditions

Four reference strains, obtained from the American Type Culture Collection (ATCC), viz. two Gram-positive (*Staphylococcus aureus* ATCC 29213, *Enterococcus faecalis* ATCC 29212), and two Gram-negative (*Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 27853), were included in this study. Additionally, three multidrug-resistant strains including an extended-spectrum β-lactamase (ESBL)-producing *E. coli* (clinical isolate SA/2), and two environmental isolates, i.e., a methicillin-resistant isolate (MRSA) *S. aureus* 74/24 [33], and a vancomycin-resistant (VRE) isolate *E. faecalis* B3/101 [34]. All bacterial strains were cultured in MH agar (MH-BioKar Diagnostics, Allone, France) and incubated overnight at 37 ◦C before each assay. Stock solutions of each compound (4 mg/mL for the less soluble compounds, **3a** and **4**, and 10 mg/mL for the others) were prepared in dimethyl sulfoxide (DMSO-Alfa Aesar, Kandel, Germany), kept at −20 ◦C, and freshly diluted in the appropriate culture media before each assay. In all experiments, in-test concentrations of DMSO were kept below 1%, as recommended by the Clinical and Laboratory Standards Institute [35].

#### 3.6.2. Antimicrobial Susceptibility Testing

The Kirby–Bauer method was performed to screen the antimicrobial activity of the compounds according to CLSI recommendations [36]. Briefly, sterile blank paper discs with 6 mm diameter (Oxoid/Thermo Fisher Scientific, Basingstoke, UK) were impregnated with 15 μg of each compound and placed on MH plates, previously inoculated with a bacterial inoculum equal to 0.5 McFarland turbidity. After 18–20 h incubation at 37 ◦C, the diameter of the inhibition zones was measured in mm. Blank paper discs impregnated with DMSO were used as a negative control. Minimal inhibitory concentrations (MIC) were determined by the broth microdilution method, as recommended by the CLSI [37]. Two-fold serial dilutions of the compounds were prepared in cation-adjusted Mueller– Hinton broth (CAMHB-Sigma-Aldrich, St. Louis, MO, USA). With the exception of **3a** and **4**, the tested concentrations ranging from 1 to 64 μg/mL were used to keep in-test concentrations of DMSO below 1% to avoid bacterial growth inhibition. For **3a** and **4**, the highest concentration tested was 32 μg/mL. Colony forming unit (CFU) counts of the inoculum were conducted to ensure that the final inoculum size closely approximated the <sup>5</sup> × 105 CFU/mL. The 96-well U-shaped untreated polystyrene plates were incubated for 16–20 h at 37 ◦C, and the MIC was determined as the lowest concentration of the compound that prevented visible growth. During the essays, vancomycin (VAN-Oxoid/Thermo Fisher Scientific, Basingstoke, UK) and oxacillin sodium salt monosulfate (OXA-Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls for *E. faecalis* ATCC 29212 and *S. aureus* ATCC 29213, respectively. The minimal bactericidal concentration (MBC) was determined by spreading 10 μL of the content of the wells with no visible growth on MH plates. The MBC was defined as the lowest concentration to effectively reduce 99.9% of the bacterial growth after overnight incubation at 37 ◦C [38]. At least three independent assays were conducted for reference and multidrug-resistant strains.

#### 3.6.3. Antibiotic Synergy Testing

The Kirby–Bauer method was also used to evaluate the combined effect of the tested compounds with clinically relevant antibacterial drugs, as previously described [39]. A set of antibiotic discs (Oxoid/Thermo Fisher Scientific, Basingstoke, UK), to which the isolates were resistant, was selected: cefotaxime (CTX, 30 μg) for *E. coli* SA/2, vancomycin (VAN, 30 μg) for *E. faecalis* B3/101, and oxacillin (OXA, 1 μg) for *S. aureus* 74/24. Antibiotic discs impregnated with 15 μg of each compound were placed on seeded MH plates. The controls used included antibiotic discs alone, blank paper discs impregnated with 15 μg of each compound alone, and blank discs impregnated with DMSO. Plates with CTX were incubated for 18–20 h and plates with VAN and OXA were incubated for 24 h at 37 ◦C [35]. Potential synergy was considered when the inhibition halo of the antibiotic disc impregnated with compound was greater than the inhibition halo of the antibiotic or compound-impregnated blank disc alone.

The MIC method was also performed in order to evaluate the combined effect of the compounds and clinically relevant antimicrobial drugs. Briefly, when it was not possible to determine a MIC value for the tested compound, the MIC of CTX (Duchefa Biochemie, Haarlem, The Netherlands), VAN (Oxoid, Basingstoke, England), and OXA (Sigma-Aldrich, St. Louis, MO, USA) for the respective multidrug-resistant strains was determined in the presence of the highest concentration of each compound tested in previous assays (64 μg/mL or 32 μg/mL for compounds **3a** and **4**). The tested antibiotic was serially diluted whereas the concentration of each compound was kept fixed. Antibiotic MICs were determined as described above. Potential synergy was considered when the antibiotic MIC was lower in the presence of compound [40]. Fractional inhibitory concentrations (FIC) were calculated as follows: FIC of the compound = MIC of the compound combined with antibiotic/MIC of the compound alone, and FIC antibiotic = MIC of antibiotic combined with compound/MIC of antibiotic alone. The FIC index (FICI) was calculated as the sum of each FIC and interpreted as follows: FICI ≤ 0.5, 'synergy'; 0.5 < FICI ≤ 4, 'no interaction'; 4 < FICI, 'antagonism' [41].

#### 3.6.4. Biofilm Formation Inhibition Assay

In order to evaluate the antibiofilm activity of the compounds, the crystal violet method was used to quantify the total biomass produced [39,42]. Briefly, the highest concentration of the compound tested in the MIC assay was added to bacterial suspensions of <sup>1</sup> × 106 CFU/mL prepared in unsupplemented Tryptone Soy broth (TSB-Biokar Diagnostics, Allone, Beauvais, France) or TSB supplemented with 1% (*w/v*) glucose (D-(+)-glucose anhydrous for molecular biology, PanReac AppliChem, Barcelona, Spain) for Gram-positive strains. When it was possible to determine the MIC, concentrations between 2× MIC and 1/4 MIC were tested, while keeping in-test concentrations of DMSO below 1%. When it was not possible to determine the MIC value, the concentration tested was 64 μg/mL (or 32 μg/mL for compounds **3a** and **4**). Controls with appropriate concentration of DMSO, as well as a negative control (TSB or TSB + 1% glucose alone) were included. Sterile 96-well flat-bottomed untreated polystyrene microtiter plates were used. After a 24 h incubation at 37 ◦C, the biofilms were heat-fixed for 1 h at 60 ◦C and stained with 0.5% (*v/v*) crystal violet (Química Clínica Aplicada, Amposta, Spain) for 5 min. The stain was resolubilized with 33% (*v/v*) acetic acid (Acetic acid 100%, AppliChem, Darmstadt, Germany) and the biofilm biomass was quantified by measuring the absorbance of each sample at 570 nm in a microplate reader (Thermo Scientific Multiskan® FC, Thermo Fisher Scientific, Waltham, MA, USA). The background absorbance (TSB or TSB + 1% glucose without inoculum) was subtracted from the absorbance of each sample and the data are presented as percentage of control. Three independent assays were performed for reference strains, with triplicates for each experimental condition.

#### **4. Conclusions**

The EtOAc extract of the culture of a marine-derived fungus, *Aspergillus stellatus* KUFA 2017, isolated from a marine sponge *Mycale* sp., which was collected in the Gulf of Thailand, furnished three previously unreported secondary metabolites viz. (3*S*,4*R*)-4 hydroxy-6-methoxymellein (**2**), a structurally unique propylpyridinium anthraquinone, stellatanthraquinone (**4**), and acetyl carnemycin E (**5c**), in addition to eight previously reported compounds, including *p*-hydroxybenzaldehyde (**1**), 1,3-dimethoxy-8-hydroxy-6-methylanthraquinone (**3a**), 1,3-dimethoxy-2,8-dihydroxy-6-methylanthraquinone (**3b**), emodin (**3c**), 5[(3*E*,5*E*)-nona-3,5-dien-1-yl]benzene (**5a**), carnemycin E (**5b**), tajixanthone

hydrate (**6a**), and 15-acetyl tajixanthone hydrate (**6b**). While the absolute configurations of the stereogenic carbons in **2** were established unambiguously by a combination of NOESY correlations and a comparison of experimental and calculated ECD spectra, the stereostructures of **6a** and **6b** were established by X-ray analysis for the first time.

All the compounds, except **1** and **3c**, were evaluated for their antibacterial activity against four reference strains: two Gram-positive (*Staphylococcus aureus* ATCC 29213, *Enterococcus faecalis* ATCC 29212) and two Gram-negative (*Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 27853), as well as three multidrug-resistant strains including an extended-spectrum β-lactamase (ESBL)-producing *E. coli* (clinical isolate SA/2), a methicillin-resistant isolate (MRSA) *S. aureus* 74/24 and a vancomycin-resistant (VRE) isolate *E. faecalis* B3/101. However, only **5c** exhibited antibacterial activity against all Gram-positive strains with a MIC value of 16 μg/mL toward *E. faecalis* ATCC 29212, vancomycin-resistant *E. faecalis* B3/101, and methicillin-resistant *S. aureus* 74/24, but with a higher MIC value (32 μg/mL) toward *S. aureus* ATCC 29213. Since **5a** displayed the minimal bactericidal concentration (MBC) equal to or one-fold higher than the MIC, it was suggested that **5a** exerted a bactericidal effect towards *E. faecalis* ATCC 29212*, S. aureus* 74/24, and *S. aureus* ATCC 29213. On the contrary, the MBC of **5a** was more than two-fold higher than the MIC toward *E. faecalis* B3/101; therefore, this compound was suggested to have a bacteriostatic effect against this multidrug-resistant species. Interestingly, **5b** and **5c**, which are C-glucosylated **5a**, were void of antibacterial activity against all the tested organisms. These results lead to a conclusion that the polar and bulky glucosyl substituent on the benzene ring can negatively affect the antibacterial activity of this series of compounds. Finally, **5a** was also able to completely inhibit biofilm formation in *S. aureus* ATCC 29213 and *E. faecalis* ATCC 29212 at both MIC and 2× MIC concentrations. Since **5a** possesses interesting antibacterial and potent antibiofilm activities, this compound can be considered as an interesting model for the development of a new type of antibiotics.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md20110672/s1. Figures S1–S56: 1D and 2D NMR spectra of compounds **1**, **2**, **3a**, **3b**, **3c**, **4**, **5a**, **5b**, **5c**, **6a**, **6b**. Figures S57–S61: HRMS data for compounds **2**, **4**, **5a**, **5b**, and **5c**. Table S1: 1H and 13C NMR data (CDCl3, 300 and 75 MHz) of compounds **3a** and **3b**. Table S2: 1H and 13C NMR data of **5b** (DMSO-*d6*, 300 and 75 MHz), **5c** (DMSO-*d6*, 500 and 125 MHz), and **5a** (CDCl3, 300 and 75 MHz). Table S3: 1H and 13C NMR data of **6a** and **6b** (CDCl3, 300 and 75 MHz).

**Author Contributions:** A.K. conceived, designed the experiments, and elaborated the manuscript; F.P.M. performed isolation, purification, and part of structure elucidation of the compounds; T.D. collected, isolated, identified, and cultured the fungus; J.A.P. performed calculations and measurement of ECD spectra and interpretation of the results; L.G. performed X-ray analysis; I.C.R. and P.M.C. performed antibacterial and antibiofilm assays; S.M. provided HRMS; A.M.S.S. provided NMR spectra; V.V. assisted in the preparation of a manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is partially supported by the national infrastructure PT-OPENSCREEN (NORTE-01-0145-FEDER-085468) and the national funds through the FCT-Foundation for Science and Technology with the scope of UIDB/04423/2020 and UIDP/04423/2020.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data sharing is not applicable.

**Acknowledgments:** The authors acknowledge the support of the Biochemical and Biophysical Technologies i3S Scientific Platform with the assistance of Frederico Silva and Maria de Fátima Fonseca for the access of the Jasco J-815 CD spectropolarimeter.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Noonindoles A–F: Rare Indole Diterpene Amino Acid Conjugates from a Marine-Derived Fungus,** *Aspergillus noonimiae* **CMB-M0339**

**Sarani Kankanamge 1, Zeinab G. Khalil 1, Paul V. Bernhardt <sup>2</sup> and Robert J. Capon 1,\***


**\*** Correspondence: r.capon@uq.edu.au; Tel.: +61-7-3346-2979

**Abstract:** Analytical scale chemical/cultivation profiling prioritized the Australian marine-derived fungus *Aspergillus noonimiae* CMB-M0339. Subsequent investigation permitted isolation of noonindoles A–F (**5**–**10**) and detection of eight minor analogues (**i**–**viii**) as new examples of a rare class of indole diterpene (IDT) amino acid conjugate, indicative of an acyl amino acid transferase capable of incorporating a diverse range of amino acid residues. Structures for **5**–**10** were assigned by detailed spectroscopic and X-ray crystallographic analysis. The metabolites **5**–**14** exhibited no antibacterial properties against G-ve and G+ve bacteria or the fungus *Candida albicans*, with the exception of **5** which exhibited moderate antifungal activity.

**Keywords:** indole diterpene; noonindole; marine-derived fungus; *Aspergillus noonimiae*; antifungal; Australia; microbial biodiscovery; molecular network chemical profiling; MATRIX cultivation profiling

#### **1. Introduction**

Since the indole diterpene (IDT) paxilline (**1**) (Figure 1) was first described in 1975 as a tremorgenic agent of *Penicillium paxilli* [1], a number of fungal natural products sharing this fused hexacyclic skeleton have been reported (i.e., paxillines, paspalines, paspalinines, paspalicine, paspalitrems, penijanthine A, penitrems, aflatrem, penerpenes, sulpinines, and terpendoles) from a wide range of genera, including from terrestrially sourced *Acremonium* [2], *Albophoma* [3–5], *Aspergillus* [6–10], *Chaunopycnis* [11], *Claviceps* [12], *Emericella* [13,14], *Eupenicillium* [15], *Neotyphodium* [16], *Penicillium* [2,17–21], *Phomopsis* [22], and marine-derived *Penicillium* [23–27]. In addition to tremorgenic properties [1,2,6,7,13,14,17,22,28], members of this IDT family have been reported to exhibit anticancer [23,29], anti-insectan [8,9,15] and anti-H1N1 [24] activity, with Merck researchers reporting potent and selective potassium ion channel antagonist properties [28] prompting patent protection for the treatment of neurodegenerative diseases (i.e., Alzheimer's disease) [30] and glaucoma [31]. Paxilline (**1**) has been noted as one of the most potent and selective nonpeptidergic inhibitors of largeconductance, voltage, and Ca2+ dependent BK-type potassium channels [32], stimulating interest in chemical synthesis [33–35] and biosynthesis [36–42] across this structure class. For example, a 2022 report [43] described two genes encoding monomodular nonribosomal peptide synthetase (NRPS)-*like* enzymes that catalyse the acylation of 14-hydroxypaspalinine (**2**) (Figure 1) to the only two known natural product examples of amino acid conjugated IDTs: 14-(*N*,*N*-dimethyl-L-valyloxy)paspalinine (**3**) (Figure 1) reported [9] in 1993 from *Aspergillus nominus* NRRL 13,137 and patented in 1993 as an anti-insectan for controlling Coleopteran and Lepidopteran insects [44], and in 2003 for the treatment of glaucoma [31]; and 14-(*N*,*N*-dimethyl-l-leucyloxy)paspalinine (**4**) patented in 2003 for the treatment of glaucoma [31] and subsequently optimised for production from *Aspergillus alliaceus* [10].

**Citation:** Kankanamge, S.; Khalil, Z.G.; Bernhardt, P.V.; Capon, R.J. Noonindoles A–F: Rare Indole Diterpene Amino Acid Conjugates from a Marine-Derived Fungus, *Aspergillus noonimiae* CMB-M0339. *Mar. Drugs* **2022**, *20*, 698. https:// doi.org/10.3390/md20110698

Academic Editor: Hee Jae Shin

Received: 18 October 2022 Accepted: 4 November 2022 Published: 7 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

**Figure 1.** Known fungal IDTs highlighting the paxilline scaffold (blue) and rare amino acid acyl functionality (tan).

This current report describes our discovery of new IDT amino acid conjugates produced by the Australian marine-derived fungus *Aspergillus noonimiae* CMB-M0339. Using an integrated strategy of chemical and cultivation profiling to both prioritize and facilitate production, we successfully detected, isolated, characterised, and identified five new IDT amino acid conjugates and a key hydroxy precursor, noonindoles A–F (**5**–**10**), together with the four known analogues paspaline (**11**) [12], paspaline B (**12**) [18], the carboxylic acid (**13**) [24], and emindole SB (**14**) [13]. Structures for **5**–**14** were assigned by detailed spectroscopic and X-ray analysis as summarised below, along with a plausible biogenetic relationship. We also made use of biosynthetic considerations and diagnostic MS/MS fragmentations to tentatively assign structures to a series of eight minor IDT amino acid co-metabolites (**i**–**viii**).

#### **2. Results**

An EtOAc extract was prepared from a 3.3% saline M1 agar plate cultivation of the marine-derived fungus *Aspergillus noonimiae* CMB-M0339. UPLC-DAD (Figure S6) was subjected to a global natural product social (GNPS) [45] molecular network (Figure 2) analysis to reveal peaks/nodes for three prominent and structurally related metabolites: **5** (*m*/*z* 579 (M+H), C34H46N2O6); **6** (*m*/*z* 565 (M+H), C33H44N2O6); and **7** (*m*/*z* 593 (M+H), C35H48N2O6). Online database searching suggested these metabolites were unprecedented in the natural products literature. Subsequent cultivation profiling using a miniaturized 24-well plate microbioreactor methodology (MATRIX) [46] employing ×11 different media under solid agar (2 mL), as well as static and shaken broth (1.5 mL) conditions (Figure S7) supported by UPLC-DAD and GNPS chemical profiling (Figures 3 and S8) confirmed D400 solid phase agar as the optimal culture condition. The EtOAc extract of a ×300 plate D400 solid phase 12 day cultivation was subjected to solvent trituration and reversed-phase HPLC (Figure S9) to yield **5**–**14** (Figure 4). Detailed spectroscopic analysis successfully identified the known natural products paspaline (**11**) [12], paspaline B (**12**) [18], the carboxylic acid **13** [24], and emindole SB (**14**) [13] (Tables S8–S11, Figures S50–S68). Further spectroscopic analysis identified the new noonindoles A–F (**5**–**10**) as summarised below.

HRESIMS analysis of **5** revealed a molecular formula (C34H46N2O6, Δmmu +2.7) requiring thirteen double bond equivalents (DBE). The NMR (methanol-*d*4) data for **5** (Tables 1 and S2, and Figures S10–S15) disclosed resonances for ten sp2 olefinic carbons and two sp<sup>2</sup> ester/lactone carbonyls, accounting for seven DBE and requiring that **5** be hexacyclic, while diagnostic 2D NMR correlations (Figure 5) established a carbon/hetero skeleton in common with **1** further annotated by an *N*,*N*-dimethyl-valinyloxy ester pendant to C-14. The structure and absolute configuration for noonindole A (**5**) were confirmed by a single crystal X-ray analysis (Figure 6 and Table S13).

**Figure 2.** GNPS molecular network of an inhouse fungal extract library, revealing a unique cluster of metabolites (expansion) associated exclusively with strain CMB-M0339.

**Figure 3.** GNPS molecular network of a set of MATRIX extracts of CMB-M0339 showing production of noonindoles A–E (**5**–**9**) and related minor metabolites under selected culture conditions.

HRESIMS analysis of **6**–**8** revealed molecular formulae (C33H44N2O6, Δmmu +2.0; C35H48N2O6, Δmmu +2.6; C33H44N2O6, Δmmu +1.5) consistent with lower/higher homologues of **5**. Comparison of the NMR (methanol-*d*4) data for **6** (Tables 1 and S3, Figures S17–S22) with **5** allowed the principle differences to be attributed to replacement of the *N*,*N*-dimethyl-valinyloxy side chain in **5** (δ<sup>H</sup> 2.90, s; δ<sup>C</sup> 43.1) with an *N*-methylvalinyloxy side chain in **6** (δ<sup>H</sup> 2.72, s, NH(CH3); δ<sup>C</sup> 33.8, NH(CH3)). Likewise, comparison of the NMR (methanol-*d*4) data for **7** (Tables 1 and S4, and Figures S24–S29) and **8** (Tables 2 and S5, and Figures S31–S35) with **5** allowed the principle differences to be attributed to replacement of the *N*,*N*-dimethyl-valinyloxy moiety in **5** with an *N*,*N*-dimethylleucinyloxy moiety in **7** and an *N*,*N*-dimethyl-homoalaninyloxy in **8**. These conclusions were reinforced by diagnostic 2D NMR correlations (Figure 5) which, together with biogenetic considerations, allowed assignment of structures to noonindoles B–D (**6**–**8**).

**Figure 4.** New noonindoles A–F (**5**–**10**) and known **11**–**14**.

**Table 1.** 1D NMR (methanol-*d*4) data for noonindoles A–C (**5**–**7**) #.



**Table 1.** *Cont.*
